The Role of the Skin in Carbohydrate Metabolism

JOHN A. JOHNSON* AND RAMON M. FUSAROt

The Role of the Skin in Carbohydrate Metabolism I. Introduction II. Properties of Skin A. Structure and Mass B. Characteristics of the Dermis C. Characteristics of the Epidermis D. Oxygen Tension in Skin III. The Role of the Dermis in Glucose Homeostasis A. Historical Aspects B. The Cutaneous Glucose Tolerance Test (CutGTT) C. Fates of Excess Dermal Glucose IV. Carbohydrate Metabolism of the Epidermis A. Enzymes Present in Epidermis B. Glucose Metabolism in the Epidermis V. Sources of Energy in the Epidermis A. Introduction B. Energy from Glucose Utilization C. Energy from Fatty Acid Oxidation D. Comments VI. Glucose/Lactate Balance in the Intact Animal A. The Skin Cori Cycle B. Glucose/Lactate Interconversion in the Intact Animal . . . . C. Lactate Balance in Oligemic Shock D. Lactate Production in the Gastrointestinal Tract E. Comments VII. The Skin as an Indicator of Impaired Carbohydrate M e t a b o l i s m . . . A. Introduction B. The Cutaneous Glucose Tolerance Test C. Glycogenoses

2 3 3 4 6 7 10 10 11 18 19 19 23 34 34 35 36 37 40 40 40 43 44 44 45 45 45 46

* Departments of Dermatology and Biochemistry, University of Nebraska Medical Center, Omaha, Nebraska. t Department of Dermatology, University of Nebraska Medical Center, Omaha, Nebraska. 1

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John A. Johnson and Ramon M. Fusaro

VIII. Concluding Remarks Addendum References

47 49 50

I. Introduction The skin is a relatively large organ, constituting about 10% of the normal body weight. In the past the importance of this organ has been assessed in a physical sense. Thus, skin was shown to serve such important functions as protection of the animal from the environment, regulation of body temperature, and storage and release of water. The major portion of the skin mass consists of dermis, which in turn contains a large extracellular space. Hence, a fourth physical role may be assigned to skin: Temporary storage of glucose when blood glucose is elevated. On the other hand, the metabolically active portion of skin, epidermis, derives much of its energy from the energetically inefficient process of anaerobic glycolysis. Consequently, skin converts large amounts of glucose to lactate which is discharged into the blood. Thus it can be seen that the skin may play a passive (dermal) as well as an active (epidermal) role in the carbohydrate metabolism of the intact animal. Although skin exhibits features of carbohydrate metabolism other than utilization and turnover of glucose (e.g., synthesis of mucopolysaccharides), these functions are of little quantitative importance in overall carbohydrate balance. We will therefore restrict our remarks to those aspects of skin carbohydrate metabolism that involve glucose and such metabolic derivatives as lactate and glycogen. The red blood cell, because of its ready availability, is useful in the study of metabolic disturbances (Beutler, 1971), particularly in man whose internal tissues are usually not accessible for investigational purposes. Red cell metabolism, however, is almost exclusively glycolytic; and the tissue is therefore not useful as an indicator of the in vivo status of other metabolic pathways. In contrast, although skin relies heavily on glycolysis for its energy needs, the tissue displays most of the metabolic pathways observed in other organs. Pertinent information on the subject is not in the literature, but it seems likely that, in the intact animal, skin responds to hormonal and homeostatic influences as do other tissues. These features, coupled with ready accessibility, render skin the ideal tissue for physiological and biochemical studies in the intact animal, especially man. Unfortunately, this concept has not gained wide acceptance, and in vivo investigations of skin metabolism are consequently rare. A primary goal of this report is to alert

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the reader to the importance of skin in one aspect of body function, carbohydrate metabolism. To accomplish this we will review the physical role of skin in glucose homeostasis, the carbohydrate metabolism of skin, and the relationship of skin metabolism to carbohydrate balance in the intact animal. It is hoped that awareness of the interaction of skin with bodily function will prompt some readers to employ this useful tissue for meaningful in vivo investigations of many areas of human metabolism. Such studies might greatly supplement our knowledge in the etiology of disease, early diagnosis, and rationale of therapy.

II. Properties of Skin A. Structure and Mass The detailed anatomy of the skin has been presented by others (Montagna, 1962; Marples, 1965; Pillsbury, 1971), and it is sufficient here to briefly describe its major structural features. The skin consists of a massive, relatively inert dermis underlying the metabolically active epidermis. Though the dermis constitutes perhaps 95% of the mass of human skin, the epidermis accounts for most of the metabolic activity of the tissue. Several appendages—the hair follicle, sebaceous gland, and sweat gland—are metabolically important. These structures originate deep in the dermis and penetrate the epidermis to reach the skin surface. Metabolic studies are often performed with epidermal slices, which contain portions of the various appendages. However, since the latter share a common ectodermal origin with the epidermis (Flaxman, 1971) and appear to metabolize glucose in similar fashion, they may be considered part of the epidermal mass. Therefore, in subsequent discussions, comments on epidermal metabolism will often refer to the combined activities of the epidermis and its appendages. The dermoepidermal interface is characterized by epidermal cones and ridges which project into the dermis. Corresponding dermal papillae project away from the dermis, and fit snugly into the valleys between the epidermal projections so that no empty space occurs between the two structures. The undulating interface provides a much greater contact area between dermis and epidermis than would be the case if they joined in a flat surface. The significance of this anatomic feature is apparent when one recognizes that the abundant blood supply of the skin is confined to the dermis. The epidermis therefore must obtain its nutrients and discharge its waste products by diffusion across the dermoepidermal interface.

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Skin thickness varies with body site, and measures 2-3 mm in man. Of this, only a small portion (about 0.1 mm) is occupied by epidermis (Freeman et al, 1962). Various estimates of skin density and mass were reviewed earlier (Fusaro and Johnson, 1970). For the approximate calculations which will be employed in this report, we will use a figure of 10% of body weight (7 kg for a 70-kg man) for total skin mass. With a total skin surface area of 1.7 m2 and density of 1.1 gm/cm 3 , the mass of epidermis (0.1 mm thick) is 190 gm.

B. Characteristics of the Dermis 1. BLOOD SUPPLY

The blood supply of the skin (dermis) far exceeds the nutritional needs of the epidermis. Each dermal papilla contains a capillary loop which arises from a subcapillary arterial plexus and returns to a corresponding venous plexus. Arteriovenous anastomoses are found, especially in areas where thermorégulation occurs. Opening and closing of these shunts provides a means of varying the cutaneous blood flow over a wide range, while blood flowing through the capillary loops provides uninterrupted nourishment of the epidermis. Champion (1970) noted that cutaneous blood flow rates from slightly more than zero to 2.5 ml per 100 gm of tissue per minute are sufficient for nutritional needs, whereas the rate may exceed 100 ml/100 gm per minute in the fingers when the body is transferring excess heat to its environment. Burton (1961) calculated that a flow of 0.8 ml/100 gm per minute would provide sufficient oxygen for the skin's metabolic needs. Evans and Naylor (1966-1967b) used implanted electrodes to measure saturation and desaturation times for dermal oxygen in human forearms, and calculated a blood flow rate of 36 ml/100 gm per minute. However, they considered this value too high and suggested that the presence of the electrodes may have increased the local blood flow. The use of radioactive microspheres enabled Neutze et al (1968) to observe that human skin received 7.4% of the cardiac output, at a flow rate of 13.7 ml/100 gm per minute. Larsen and Sejrsen (1968) described a nontraumatic technique for measuring skin blood flow, based on the disappearance of epicutaneously applied 133 Xe. They obtained a value of 5.7 ml/100 gm per minute. Nyfors and Rothenborg (1970) administered the radioisotope subcutaneously and observed flow rates of 6.5 ml/100 gm per minute in normal skin and in uninvolved psoriatic skin, and about 12 ml/100 gm per minute for psoriatic tissue.

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2. COMPOSITION OF THE DERMIS

The dermis has been described by Gersh and Catchpole (1960) as a heterogeneous system containing a colloid-rich, water-poor phase in equilibrium with a water-rich, colloid-poor phase. Eisele and Eichelberger (1945) reported that a kilogram of fat-free human skin contained 546 gm of connective tissue which has 330 gm of water associated with it. This water is available to the body during water deprivation, and the skin was therefore considered to be an important water-storage organ of the body. Flemister (1941-1942) observed that rabbit skin contributes 53% of the water lost during dehydration and accepts 43% of the water gained after increased water intake. Because of the viscous, semifluid nature of the ground substance which fills the dermal extracellular space, even small molecules move slowly in their passage to or from the blood capillaries. This ground substance contains macromolecules such as hyaluronic acid, chondroitin sulfates, and mucopolysaccharides. The sulfate and carboxylic-acid groups of these polyanions impart ion-exchange qualities which retard the passage of cations through the dermis. 3. EXTRACELLULAR SPACE

Walser and Bodenlos (1954) measured sodium, chloride, and potassium levels in rat skin and calculated extracellular water as 137 gm per 100 gm of fat-free dry tissue. This value corresponds to 42% based on wet weight of skin. Determination of extracellular space by equilibration of skin samples in a medium containing an extracellular marker is difficult to perform because of the difficulty of removing excess medium without squeezing out skin fluid. Some years ago (unpublished results) we determined the raffinose space of rat skin to be 58% of wet weight, a value which intuitively does not appear extreme. In Section III, B, 3, we will discuss the use of an endogenous marker (glucose) to calculate the extracellular space. For the present it is sufficient to note that the extracellular volume of human skin (dermis) probably lies between 30 and 50%. 4. RELATIONSHIP OF THE DERMIS TO THE EPIDERMIS

As already noted, exchange of nutrients and waste products between the blood and epidermis occurs by diffusion through the dermis. It was also pointed out that because of the gel nature of the dermal space, exchange occurs slowly. The large bulk of the dermis in comparison to that of epidermis partially offsets the slow transfer of materials, because the dermis provides a large glucose and oxygen reserve for the epidermis, and similarly offers a large storage volume for metabolic products such as lactate. This buffering capacity of the dermis may be of

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John A. Johnson and Ramon M . Fusaro

critical importance in maintaining epidermal integrity during anoxic stress.

C. Characteristics of the Epidermis 1. METABOLIC ACTIVITY

The function of the epidermis is the continuous replacement of the tough, impervious outer layer of the skin, the stratum corneum; thus maintaining an intact barrier between the animal and his environment. In order to accomplish this, epidermal cells are produced and differentiated at a rate such that the human epidermis replaces itself every 3 to 4 weeks. Therefore, despite its reputation as an inert covering of the body, the epidermis is a metabolically active tissue. 2. NATURE OF EPIDERMAL GROWTH

The most active cells in the epidermis are those of the basal layer, adjoining the dermal interface. These cells contain nuclei and mitochondria and are capable of dividing. As a new daughter cell is produced, another cell of the basal layer is forced into the layer above it. With continued division of basal cells, overlying epidermal cells migrate toward the skin surface and undergo systematic changes. These alterations involve loss of nuclei and mitochondria, production of a unique skin protein (keratin), and progressive flattening of the cells. It is appropriate that the basal cells lie closest to the dermis, since the intense metabolic activity involved in cell division requires an adequate oxygen supply. The oxygen tension of the epidermis decreases with increasing distance from the dermis, and one must assume that the outward-migrating cell is exposed to a steadily increasing degree of anaerobiosis. Little is known about the biochemistry of keratinization, but the comments of Bradfield (1951) are of interest. Glycogen is absent in basal cells but appears in cells above the basal layer and gradually disappears as cells migrate outward and keratinization becomes complete. Bradfield suggested that as a cell leaves the basal layer its oxygen supply becomes limited and its metabolism shifts to anaerobic glycolysis. Initially, sufficient glucose is available that the cell can store glycogen. As the cell moves further from its source of nutrient, glycogen provides the energy necessary for completion of the keratinization process. The ultimate fate of the epidermal cell is conversion to a tough, flattened, overlapping element of the stratum corneum. As portions of the surface of the corneum become "weathered," they are sloughed off, to be replaced from below by an equal number of newly transformed epidermal cells. In this manner the

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epidermis, operating like a "holocrine gland," presents a fresh, constantly renewed interface between the animal and his environment.

D. Oxygen Tension in Skin 1. INTRODUCTORY REMARKS

The relative importance of aerobic and anaerobic processes in a tissue must be assessed in terms of the physiological oxygen tension of the system; until recently such data were not available for skin. Lack of this basic information, however, did not prevent the development of considerable controversy over the relative contributions of aerobic and anaerobic metabolism to the energy needs of the epidermis. Numerous in vitro studies were performed to resolve this question, with specimens incubated in air, oxygen, or anaerobic atmospheres. Yet without knowledge of the in vivo state of oxygénation of skin, one cannot assess the validity or relevance of the results of these investigations. Recently Evans and Naylor developed micro oxygen electrodes which could be calibrated to measure absolute oxygen tensions. These investigators have measured skin oxygen levels under various experimental conditions, and in so doing have performed a valuable service to students of skin metabolism. 2. OXYGEN TENSION IN THE DERMIS

Evans and Naylor (1966-1967a) measured the dermal oxygen tension of human forearm skin and obtained a value of 51 mm Hg for subjects breathing air. By the use of multicathode electrodes, they observed that the tension was constant at several skin depths between 0.3 and 2.5 mm. Mean capillary tension was assumed to be as high as 95 mm Hg. When the subjects breathed pure oxygen, the dermal tension rose to 352 mm Hg and again was independent of skin depth. The authors analyzed their data by a model postulating that oxygen diffusion from each skin capillary was not influenced by the other capillaries; they obtained anomalous results. Use of the other extreme model, assuming that oxygen diffused so rapidly between the capillaries that the tissue oxygen was in equilibrium with venous blood, also yielded contradictory information. The best fit with the experimental data was obtained with an intermediate system with an assumed arteriovenous difference of 0.6 vol % in blood oxygen content, and a difference of 50 mm Hg between mean capillary tension and that at some points between the capillaries. In a second study (Evans and Naylor, 1966-1967b) the authors measured saturation and desaturation times and noted that they did not vary

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with skin depth. The data obtained in this study permitted calculation of blood volume in dermal capillaries, 8% ; respiration rate, 0.07 μ\ 0 2 per milligram wet weight per hour (mid-dermis) ; blood flow, 36 ml/100 gm per minute; and arteriovenous oxygen difference, 0.3 vol %. The values for capillary blood volume and skin blood flow were considered high, due possibly to local tissue disturbances caused by insertion of the electrode. 3. OXYGEN TENSION IN THE EPIDERMIS

Evans and Nay lor (1967a) reported that the oxygen tension at the surface of human forearm skin was less than 3.5 mm Hg for subjects breathing air or oxygen. The corresponding value for inflamed skin was about 30 mm Hg. The oxygen gradient from the subpapillary capillaries to the surface of noninflamed skin was determined from two sets of data and found to be 40 mm Hg and 45 mm Hg. The results of occlusion experiments suggested that epidermis (about 100 μ thick) had little influence on the rate of oxygen decrease and that the decisive element was a layer of dermis several hundred microns deep. They calculated the oxygen consumption rate of epidermis and upper dermis to be 0.11 μ\ per milligram wet weight per hour. The calculated arteriovenous oxygen difference immediately below the epidermis (0.7 vol %) was compared with dermal values obtained with surface electrodes (0.27 vol %) and with intradermal measurements (0.34 vol % ) . The authors suggested that the higher figure may reflect greater oxygen demand by the epidermis upon the superficial blood supply. In another study, Evans and Naylor (1967b) measured oxygen tension across the epidermis of suction blisters. The oxygen tension at the blister surface was 33 mm Hg, similar to that observed earlier for inflamed skin, and the pressure at the base of the blister was 68 mm Hg. The difference, 35 mm Hg, was somewhat lower than the value reported for uninjured epidermis (40 mm), and may reflect a lower metabolic rate in the blister tissue. An important observation was that, after occlusion, the rate of oxygen decrease was the same on the blister surface as at the blister base. According to the authors, this suggests that the rate of fall is controlled mainly by oxygen utilization and solubility in the dermis rather than in the epidermis. The actual oxygen gradient across the epidermis was calculated to be 28 mm Hg. 4. UPTAKE OF ATMOSPHERIC OXYGEN BY SKIN

The older literature contains many references to absorption of oxygen by skin, and several investigators considered this to be an important factor in skin metabolism. The subject has been reviewed by Rothman

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(1954), Fitzgerald (1957), and Tregear (1966). Fitzgerald (1957) calculated the distance oxygen could diffuse into the skin and obtained a value of 48 μ. This suggests that all the oxygen absorbed by skin would be utilized in the epidermis. Similar calculations revealed that, if no oxygen were present at the skin surface, the oxygen available from the blood would be utilized by the epidermis before it could reach the surface. This result explained the failure of others to detect outward diffusion of oxygen, and is consistent with the low surface oxygen tension observed by Evans and Naylor (1967a). There appears to be no recent information concerning oxygen uptake through skin, and Fitzgerald doubted the reliability of early reports. The most recent studies cited by him (Lanari, 1953; Kihn and Rackow, 1954) reported values of 30-50 ml oxygen absorbed per square meter of skin per hour. For an epidermal thickness of 0.1 mm, density 1.1 gm/ml, and 30% dry weight, these values correspond to about 1 μ\ of oxygen absorbed per milligram dry weight of epidermis per hour. This value is comparable to those cited for in vitro oxygen utilization by epidermal slices; and indeed, Fitzgerald used a value of 7.8 μ\ per milliliter of tissue per minute for his calculations, which equals 1.4 μ\ per milligram dry weight per hour. Thus, if one accepts the available data, it appears that the epidermis could obtain most of its oxygen needs from the environment. Fitzgerald suggested that intake of exogenous oxygen may protect the skin during periods of anoxia due to reduced cutaneous blood flow. Gruber et al. (1970a) examined the feasibility of avoiding the toxic effects of conventional hyperbaric oxygen therapy by topical application of oxygen at high pressure. Rabbits were placed in a chamber at 3-5 atm abs of oxygen while they breathed room air. Little oxygen penetrated the skin, as evidenced by only a slight rise in venous tension. In another study (Gruber et ai., 1970b), oxygen electrodes were implanted in excised human abdominal skin, and the specimens were exposed to oxygen at 1 atm abs. No increase in tension was observed in superficial dermis (0.3 mm deep) or in deep dermis (2 mm). Full-thickness skin slices were used in order to preclude oxygen entry from the dermal side. The oxygen tensions of freshly excised specimens were 20-50 mm Hg, and decreased to near zero in 10 minutes. The studies of Gruber and coworkers appear to rule out net penetration of oxygen through the skin but, of course, provide no information on the possibility of uptake and utilization within the epidermis. 5. CONCLUDING COMMENTS

The conclusion reached by Evans and Naylor (1967a,b) that the rate of oxygen decrease in the occluded forearm depends mainly on dermal

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activity, seems to negate our earlier statement that most of the metabolic activity of skin occurs in the epidermis. One might attempt to explain this anomaly by pointing out that the epidermal appendages penetrate deep into the dermis, and must consume oxygen from the dermal space. However, the constancy of oxygen tension and of saturation and desaturation times throughout the dermal depth suggests that the effect of the appendages is insignificant. A more productive approach is simply to recognize that, because of its great mass and relatively high level of oxygénation, the dermis may very well consume a substantial portion of the oxygen utilized by skin. In contrast, epidermal oxygen consumption is probably restricted mainly to the cells of the basal layer. These cells contain nuclei and mitochondria and engage in metabolic activities (protein synthesis, cell division, etc.) which require efficient energy production. The basal cells may be among the most active cells of the body, but they constitute such a small fraction of the total epidermal mass that overall aerobic metabolism is low. The important point to be recognized is that most of the epidermal metabolism is anaerobic; and when total skin metabolism is considered, epidermal activity overshadows that of the dermis. In this context, the observations of Evans and Taylor confirm that aerobic processes constitute only a minor part of the total activity of the epidermis. The physiological significance of external oxygen in skin metabolism is impossible to assess; indeed, it is difficult to visualize experiments that would shed light upon this question. The fact that skin divers spend long intervals with their bodies exposed to the low oxygen levels of water or wet suits without harm to their skin suggests that epicutaneous oxygénation is not vital. Similarly, the healing effects of occlusive skin dressings testify against the importance of exogenous oxygen. One might also speculate that since the mode of entry of gases into the skin is not known, oxygen may penetrate the epidermis via extracellular channels rather than through the cells. If this were true, external oxygen would not be readily available to the superficial layers of epidermal cells.

III. The Role of the Dermis in Glucose Homeostasis A . Historical Aspects The history of skin carbohydrate studies has been reviewed by Pillsbury (1931), Cornbleet (1940), Rothman (1954), and Fusaro and Johnson (1970). As early as 1917, Palmer reported that the skin of the dog

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served as a temporary storehouse for elevated blood glucose. Urbach and Fantl (1928) performed similar studies with humans and arrived at the same conclusion. Folin et al. (1927) assigned a temporary storage function to skin in dogs and considered skin glucose influx to be due to passive diffusion. Although early kinetic investigations supported the concept of passive exchange of blood and skin glucose, reports of fasting skin glucose levels higher than those of blood were at odds with this concept. With the development of highly specific techniques for assay of skin glucose, it now appears that equilibrium glucose content of skin never exceeds that of blood; and therefore passive diffusion suffices to explain glucose exchange between the tissues. In this context, one must note an important distinction between the concentration units employed for glucose levels of blood and skin: The former is recorded as milligrams of glucose per 100 ml blood; the latter as milligrams per 100 gm of skin. Since the glucose distribution space of skin is less than that of blood, it follows that skin glucose based on weight must be lower than blood glucose per unit volume. To illustrate this point, assume that the glucose space of blood is 80% (glucose diffuses freely into the red cell), and that of skin (extracellular space) is 50%. Then a "true" blood glucose level of 110 mg/100 ml water would appear as 88 mg/100 ml blood. Similarly, if extracellular skin glucose content were 110 mg/100 ml H 2 0 and skin density is 1.1 gm/ml, the observed skin value would be 50 mg/100 gm. Thus, in the fasted animal when glucose is in equilibrium in the body's extracellular spaces, the ratio of skin glucose to blood glucose should not exceed 57%. This conclusion is supported for human skin by the results of several investigators (reviewed by Peterka and Fusaro, 1965a). B. The Cutaneous Glucose Tolerance Test (CutGTT) 1. INTRODUCTION

Although many investigators monitored the rise and fall of skin glucose after administration of a glucose load, only Urbach and colleagues (1928, 1929) studied humans. Their work is of great historical importance but they were hampered by several unavoidable drawbacks: (1) Analytical techniques were neither sufficiently precise nor sensitive enough to provide accurate estimates of glucose contents of small skin specimens. (2) The concepts of the intravenous glucose tolerance test (IVGTT), which permit disappearance of excess glucose to be expressed in mathematical form, had not been developed. (3) Reliable estimates of human skin mass were not available. Despite these handicaps, the investigators

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obtained valuable information on human skin glucose kinetics, and provided the inspiration for us to apply present-day techniques and knowledge to the development of the CutGTT. 2. TECHNIQUE

Performance of the CutGTT has been described in detail (Fusaro and Johnson, 1965) and will be outlined only briefly here. The test is an extension of the IVGTT, with the additional feature that skin glucose concentration is also monitored. Triplicate blood and skin specimens were obtained from a fasting subject, and 35 gm of glucose was administered intravenously. At appropriate intervals blood specimens were collected and lysed in 30 vol of water. Likewise, skin specimens were obtained from the back with a motor-driven, 3-mm rotary punch, quickly weighed, and immersed in 1% aqueous ZnS04. After 1.5 hours, an equimolar quantity of 0.06 N Ba(OH) 2 was added to each specimen, and the mixtures were centrifuged. The four aliquots obtained from each clear supernatant fluid were employed in two separate glucose assays (in duplicate), with glucose oxidase reagent. Blood samples were deproteinized with 0.3 N Ba(OH) 2 and 5% ZnS04, and aliquots were assayed with the microvolumetric procedure employed for skin. Excess blood and skin glucose values were calculated by subtracting average fasting glucose from the postinjection levels. The plots of logarithm excess glucose versus postinjection time were fitted with straight lines by the method of least squares; and from the slopes of the corresponding lines a disappearance rate constant (percentage per minute) for blood glucose and for skin glucose was calculated. 3. RESULTS AND DISCUSSION

a. Disappearance Rate. The mathematical relationship of blood glucose to postinjection time was detailed by Amatuzio et al. (1953) and is illustrated in Fig. lb: log excess glucose is proportional to time. Skin excess glucose content (Fig. lc) attained a maximum at 30 minutes after glucose injection and thereafter decreased in an exponential manner. The similarity to blood glucose disappearance is apparent in the straight-line semilog plot of Fig. Id. By analogy with the mathematical treatment of blood glucose data, we represent skin glucose disappearance as follows: S = S0e-kt where S is excess skin glucose at time t minutes, S0 is the extrapolated zero-time value (Fig. Id), and k is the glucose disappearance constant. The constant is readily obtained from the slope of the semilog plot and represents the percentage of excess glucose disappearing per minute.

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The Role of the Slpn in Carbohydrate Metabolism

Log Excess Glucose

Excess Glucose mg%

y ! \

mg%

Slope of line is related to k

\x .

15 30 45 Time (minutes)

l

1

. 1 .1 1 15 30 45 Time (minutes)

Log Excess Glucose

Excess Glucose mg%

mg %

30

45

60

Time (minutes)

l·r

15

30 Time

45

60

(minutes)

FIG. 1. Blood and skin glucose kinetics after an intravenous glucose load. Excess blood glucose is plotted against postinjection time in (a) rectangular and (b) logarithmic coordinates, (c and d) Corresponding plots for excess skin glucose. Ordinates : milligrams per 100 ml (blood) ; milligrams per 100 gm (skin) ; k, glucose disappearance constant. (Reprinted from Fusaro and Johnson, 1970, by permission of Apple ton-Century-Crofts.)

Average k values for 32 young males were 3.8% per minute for blood and 2.0% per minute for skin (Fusaro, 1965). In a separate study, ten healthy subjects were tested three times with glucose loads of 35, 40, and 45 gm of glucose. The disappearance rates were 4.0, 3.8, and

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John A. Johnson and Ramon M. Fusaro

3.9, respectively, for blood; and 2.0, 2.0, and 1.9% per minute, respectively, for skin (Fusaro, 1965). b. Fasting Glucose Content of Human Skin. Because the fasting skin glucose is subtracted from each postinjection value, we analyzed triplicate fasting specimens in order to ensure accuracy. We therefore collected a great deal of information on skin glucose levels of fasting humans (Peterka and Fusaro, 1965a,b; 1966; Fusaro and Johnson, 1970). On the basis of these studies, several conclusions may be drawn: (1) Individual fasting levels varied widely within an experimental group of subjects (31 to 63 mg/100 gm), but average glucose contents of the backs of different groups were constant (46 to 48 mg/100 gm). (2) Average glucose contents were constant over intervals of several months. (3) Average glucose contents of the back, arm, abdomen, and thigh were essentially identical. c. Extracellular Space of Human Skin. Some time after we stopped performing the CutGTT, it occurred to us that, with little additional effort, one can employ fasting glucose levels as a measure of extracellular volume of skin. Assuming that skin and blood glucose are in equilibrium in the fasting subject and that skin glucose is extracellular, one need only collect an additional blood specimen and determine its water content to make the necessary calculations. The subject of whether glucose occurs intracellularly will be discussed in Section IV, B, 5; but in an operational sense, the extraction procedure employed by us (equilibration of the specimen in ZnS04) ensures that most of the glucose detected is extracellular. Unfortunately, we have not had the opportunity to verify our assumptions by direct comparison of individual fasting glucose levels and blood water content. Calculations based on previously obtained fasting levels were therefore performed employing the average water content of several random blood specimens, 77.9%. The data obtained for the 14 subjects examined in the study of skin glucose content at different body sites, were analyzed as follows: The fasting blood glucose of each subject was divided by 0.779 to yield a value which was considered a measure of glucose concentration, milligram per 100 ml of tissue water. The quotient of skin glucose content (mg/100 gm) divided by the calculated tissue-water value (mg/100 ml) is considered a reasonably good indicator of skin glucose (extracellular) space (ml/100 gm). The average values so obtained for back, arm, abdomen, and thigh were 43, 46, 45, and 44 ml/100 gm, respectively. The practical significance of calculations such as those just cited remains to be determined. In view of the facility with which skin reacts to fluid excess or deficit, one might postulate some utility in the assessment of edema or dehydration in patients. To be honest, however, this kind of information can probably be obtained

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more directly by simply determining skin water content. It appears then, that information concerning skin extracellular space is mainly of academic interest for the moment, and the calculations cited are offered in that context. d. Glucose Storage Capacity of Skin. If one estimates skin mass and determines the maximum excess skin glucose attained after an intravenous glucose load, one can calculate the amount of glucose temporarily stored in the skin. These calculations were performed with the results of the CutGTT performed on 10 nondiabetic young men. Skin glucose uptakes in percent of the administered dose (35 gm), ranged from 11 to 19% with a mean of 14%. This value was also independent of glucose load: 14, 13, and 14% for 35, 40, and 45 gm of glucose, respectively. Curtis-Prior et al. (1969) pointed out that although the glucose tolerance test has been widely studied in man and other animals, little information is available concerning the quantitative roles played by various organs and tissues in assimilating a glucose load. Accordingly, these authors administered physiologic doses of glucose containing glucose-14C to rats, and measured the uptake of radioactivity in various organs. When glucose was administered intravenously, 28% of the radioactivity was detected in skin after 5 minutes and 11% after 40 minutes. After oral loading, the skin contained 7.5, 7.1, and 5.4% of the radioactivity at 15, 90, and 180 minutes respectively. These results are in general agreement with our data for humans, although the rapid uptake of intravenous glucose by rat skin deserves additional study. In contrast to this rapid accumulation, Gaitonde (1965) performed a "reverse" skin tolerance test in rats by injecting glucose-14C subcutaneously. The total activity of blood, as well as the specific activities of blood glucose and lactate, peaked at 30 minutes after administration. 4. SOURCES OF ERROR IN SKIN GLUCOSE DETERMINATION

a. Introduction. Much controversy has centered around the determination of "true" skin glucose levels, and only since the recent development of enzymatic glucose assays has this problem been at least partially resolved. Kansky (1966) employed the hexokinase, glucose-6-phosphate dehydrogenase system to demonstrate that the Hagedorn and Jensen chemical method yielded erroneously high skin glucose values. Schulze (1961, 1968) evaluated optimum conditions for extraction of skin glucose and compared several chemical assays with the glucose oxidase method. These authors analyzed large skin samples, and consequently had to resort to homogenization to obtain adequate extraction of glucose. b. Analysis of CutGTT Extracts. Early in our studies we decided to collect small skin specimens (3 mm in diameter, 2-3 mm deep) in

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order to avoid undue trauma and scarring of the subject. The use of small samples (4-9 mg wet weight) required the development of a micro glucose assay at a time when such techniques were not common; but in retrospect, small sample size proved to be an advantage. We made numerous attempts to homogenize skin in a variety of agents (water, dilute HC1, dilute acetic acid, dimethyl sulfoxide, dimethyl formamide) with limited success in terms of accuracy and reproducibility of glucose recovery. Finally, as a "last resort," we decided to allow the specimens to remain in aqueous ZnS04 for a sufficient interval to allow extracellular glucose to equilibrate into the medium. The choice of ZnS04 was fortuitous; glucose content of the medium attained a maximum within 0.5 hour and remained constant for several hours. A control experiment performed with added glycogen demonstrated a fact not too well known at the time, that commercial glucose oxidase preparations contain a carbohydrase contaminant that hydrolyzes glycogen to glucose. This problem appeared solved when we noted (Johnson and Fusaro, 1963) that supernatant fluids obtained by treatment of aqueous glycogen solutions with ZnS04 and Ba(OH) 2 contained no detectable glycogen. Furthermore, glucose was recovered quantitatively from glycogen/glucose mixtures after "deproteinization." Lest the reader place too much confidence in this mode of glycogen removal, we hasten to add that in later investigations of muscle carbohydrate we recovered over 90% of the glycogen contained in KOH/phosphate buffer, after similar treatment (Johnson and Fusaro, 1972). A more serious source of error, however, is the well known in vitro breakdown of endogenous tissue glycogen to glucose and oligosaccharides. These compounds are not precipitated from aqueous solution by ZnS04 and Ba(OH) 2 , and if present in skin extracts, would cause high glucose oxidase assay values. By incubation of large quantities of fresh rat skin, deproteinization, and paper chromatography, we readily demonstrated the formation of glucose, maltose, maltotriose, and maltotetraose. However, it must be emphasized that these experiments were performed with skin pulverized in a Dry-Ice cooled mill (Smith and Johnson, 1965), and the relatively large area of broken cell surfaces allowed diffusion of intracellular material into the medium. Since glycogen breakdown presumably occurs intracellularly and since the ratio of cut-cell surface to intact-cell volume in the cylindrical CutGTT specimens is low, one would not expect much leakage of oligoglucosides into the equilibration medium. On the other hand, Dahlqvist (1964) described a way of avoiding glycogen interference by preparing the glucose oxidase reagent in Tris buffer (TGO) instead of water (GO). We verified that neither maltose nor glycogen cause high glucose yields when the assay is performed with TGO (Johnson and Fusaro, 1966).

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17

Rather belatedly, we tested TGO versus GO on CutGTT skin extracts (Johnson and Fusaro, 1972). In four CutGTT, GO glucose values exceeded TGO levels by (4^9%). However, in any given test, all glucose levels (including fasting) were elevated to the same extent in GO assay so that excess values (experimental minus fasting) were equivalent to those of TGO assay. Skin glycogen levels are low (less than 100 mg/100 gm), and whatever breakdown products occurred in an extract, they would be only partially hydrolyzed by the carbohydrase contaminant during GO assay. When carbohydrase enzyme was added to GO reagent in order to convert all glycogen and oligoglucosides present to glucose, the assay values were the same as those obtained with GO alone. Thus, we cannot say at the moment which assay (GO or TGO) provides the more accurate skin glucose values. In an unrelated study with muscle preparations (Johnson and Fusaro, 1970a), we observed that hexokinase/glucose-6phosphate dehydrogenase assay agreed best with TGO values. However, the higher reliability of TGO was in the opposite sense (TGO greater than GO) from that observed with skin extracts. c. Skin Surface Glucose. Several reports in the literature (e.g., Zaias et al.y 1963; Schragger, 1962) claim a close correlation between skin surface glucose and hyperglycemia. Before the start of a CutGTT, the sampling site (back) of the subject was wiped with 70% isopropanol. This would remove any surface glucose, but the possibility existed that during the hyperglycémie period after glucose loading, glucose might spill on the skin surface and thus be detected in subsequent assay. In order to check this possibility, in 1964 we monitored 10 subjects during performance of the CutGTT by placing moistened Tes-Tape (Eli Lilly and Co.) on the back. The fact that no positive glucose reactions occurred at 20 minutes and 60 minutes after glucose administration was reassuring. Yet, persistence of reports of detection of surface glucose prompted us to perform a more definitive study in 1967 (Mclntyre et al.). Accordingly, the washed backs and palms of nondiabetic subjects were monitored with Tes-Tape during the entire course of 15 CutGTT. Again no positive reactions were encountered, despite the fact that blood glucose levels at 8 minutes postinjection were 240-348 mg/100 ml and skin glucose maxima attained 106-144 mg/100 gm. Furthermore, although much has been written about detection of skin surface glucose with moistened test strips, no one to our knowledge has determined the sensitivity of the procedure. We therefore applied glucose solutions of various concentrations on the skin of a subject, tested for glucose with Tes-Tape, and assayed for glucose with glucose oxidase. In this way we noted that as little glucose as 1.2 /*g/cm2 produced an unequivocal color change with moist Tes-Tape. This surface concentration (if present) would con-

18

John A . Johnson and Ramon M . Fusaro

stitute less than 4% of the fasting glucose content of the smallest specimen obtained in the CutGTT. d. Blood Content of Skin. The question of how much of the skin glucose arises from the blood contained in the specimen may be answered in several ways. First, histochemical examination of skin sections of the back reveals that the ratio of total vascular cross section to skin area is low. Second, studies with 131 I-labeled albumin provided plasma volumes of 1% (Humphrey et al, 1957) and 2% (Olsson and Plantin, 1970) in rabbit skin, and of less than 5% in guinea pig skin (Song et al, 1966). And perhaps the most convincing argument against significant influence of blood glucose is consideration of the time sequence of blood and skin glucose change. After glucose loading, skin glucose rises steadily for 30 minutes while blood glucose decreases rapidly. Such a relationship, of course, could not exist if most of the glucose detected in skin specimens arose from blood. e. Concluding Remarks. Although there is need for additional evaluation of GO and TGO assay results, we believe we have anticipated the major possible sources of error in determination of extracellular glucose content of the small skin specimens that can be readily collected from humans without anesthesia. The absolute value of fasting human skin glucose concentration probably lies between those found by TGO and GO, i.e., 40-50 mg/100 gm. For studies such as the CutGTT which involve changes in skin glucose content after stress (glucose loading), the two assay systems yield equivalent results (excess glucose values).

C. Fates of Excess Dermal Glucose Dermal glucose arising from a glucose load can leave the dermis by several routes: (1) diffuse back to the blood, (2) return via the lymph, (3) enter the epidermis. The exponential rate of fall of skin glucose in the CutGTT is consistent with our belief that most of the excess diffuses directly back to the blood. In an experiment in which cutaneous lymph glucose was monitored during a CutGTT, we observed that the lymph and dermal compartments were in equilibrium. Skin and lymph glucose levels peaked at the same time and thereafter disappeared at the same rate. The lymph route, although slow, operates steadily and thereby removes an unknown, but perhaps significant, amount of excess dermal glucose. The third fate of dermal glucose, utilization in the epidermis, is of major importance in skin metabolism and will be discussed next.

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IV. Carbohydrate Metabolism of the Epidermis A. Enzymes Present in Epidermis 1. GENERAL COMMENTS

It can be stated that, with few exceptions, those enzymes that have been looked for in epidermis have been detected. Some, for example isocitric dehydrogenase, exhibit low activity in skin and required sensitive assays for detection. Perhaps the presence of a few others may be in doubt due to inadequate control studies. Both histochemical and biochemical detection techniques have been employed for localization and quantitation of skin enzymes. It seems certain, from investigations of enzyme activities, substrate utilization and product formation, that all the major metabolic pathways except gluconeogenesis are operative in epidermis. The question of whether epidermis synthesizes its own enzymes was considered in detail by Weber and colleagues (reviewed by Weber, 1962), and resolved in favor of synthesis of endogenous enzymes in the basal layer. 2. ENZYMES OF GLYCOGEN METABOLISM

a. Degradative Enzymes. (1) Glycogen phosphorylase has been demonstrated repeatedly in epidermis by histochemical techniques. More pertinent, several investigators have quantitated the activity of the enzyme (Adachi, 1961; Halprin and Ohkawara, 1966b; Mier and Cotton, 1967a; Leathwood and Ryman, 1971). Adachi, and Mier and Cotton, distinguished between phosphorylase a and b activities. Halprin et al. (1968) reported that most of the phosphorylase activity of human epidermis is present in the absence of AMP (phosphorylase a), and detected two isozymes by starch gel electrophoresis. One band stained for phosphorylase only in the presence of AMP (phosphorylase b ) . {2) Amylo-1,6-glucosidase {débrancher) and acid a-glucosidase were assayed by Leathwood and Ryman (1971) in human epidermis. The latter enzyme was reported earlier by Black and Anglin (1967) in guinea pig skin. {8) a-Amylase. Weber (1964) cited several early references to detection of amylase in skin. Kiverin (1957) reported that extracts of skin hydrolyze starch or glycogen by means of amylase. We detected amylolytic products (oligoglucosides) in incubated rat skin; and detected the enzyme histochemically on starch-coated slides and biochemically via glycogen breakdown (unpublished results). Halprin and Ohkawara 1966a)

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John A . Johnson and Ramon M . Fusaro

reported that breakdown of endogenous glycogen to glucose in human epidermis was one-tenth as active as phosphorylase activity. b. Synthetic Enzymes. (1) Glycogen synthetase was assayed in the presence of glucose 6-phosphate by Adachi (1961) in dog skin, and by Halprin and Ohkawara (1966a,b) in human epidermis. Sasai (1968) reported the histochemical detection of synthetase in human epidermis and interpreted his results to mean that the enzyme was mostly in the D form. Smith (1970) distinguished the I and D forms histochemically in monkey sweat glands. {2) Branching enzyme. Sasai (1968) implied that ethanol in his incubation medium inhibited branching enzyme and thereby enhanced the sensitivity of the staining agent (iodine) for newly formed glycogen. Presumably this occurred because the blue-black color of the iodine-straight chain complex is more intense than the orange-brown color obtained with normal, branched glycogen. Endogenous skin glycogen stains orange-brown with iodine, thus implying the in vivo activity of branching enzyme. (S) UDPG-pyrophosphorylase was assayed by Halprin and Ohkawara (1966a) in human epidermis. c. Conclusion. Glycogen content of human epidermis is low, 44 mg/100 gm (Halprin and Ohkawara, 1966a) ; it is therefore of little consequence in overall carbohydrate balance. However, the rapid appearance and disappearance of the carbohydrate during wound injury (Montagna, 1962), hair growth (Adachi, 1961), and sweating (Dobson, 1962) suggests that glycogen is important in epidermal metabolism. Adachi's study (1961) with perfused surviving dog skin provided evidence that in vivo glycogen synthesis and degradation are in dynamic equilibrium; and the demonstrated presence of the necessary enzymes strengthens this belief. 3. GLYCOLYTIC ENZYMES

a. Introduction. Several investigators have assayed the activities of skin glycolytic enzymes (Rippa and Vignali, 1965; Rassner, 1965; Halprin and Ohkawara, 1966a,b; Mier and Cotton, 1966a,b, 1967a,b,c; 1970a,b; Hammar et al., 1968). It may be stated with confidence that all enzymes required in the conversion of glucose to lactate have been detected in the epidermis of several species, including man. Three of the more important enzymes will receive separate attention below. b. Key Glycolytic Enzymes. (1) Hexokinase has been assayed in human skin by Mier and Cotton (1966a) and by Halprin and Ohkawara (1966a,b). Katzen (1967) has described the multiple forms of hexokinase and their relation to insulin action. Three isozymes (I—III) are common

The Role of the S\in in Carbohydrate

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21

to most tissues ; IV (glucokinase) is found only in liver. Katzen proposed that if a tissue contains high levels of I I and low I, it will be insulin sensitive; if all three forms (especially I) are present, the tissue glucose uptake will not be affected by insulin. He classified rat skin as insulin insensitive (high I and I I ) . Halprin et al. (1968) detected one (possibly two) forms of hexokinase in extract of human epidermis, and interpreted their data to mean that the main isozyme was type I. This conclusion is consistent with the direct finding (Halprin and Ohkawara, 1966c) that insulin did not affect hexokinase activity of epidermal extract. (#) Phosphofructokinase in human skin has been reported by Rassner (1965), Halprin and Ohkawara (1966a,b), and Mier and Cotton (1967c). Unfortunately, although the central rate-controlling role of this enzyme is well established for many glycolytic systems, the properties of skin phosphofructokinase have not been studied. {&) Lactic dehydrogenase activity is high in epidermis and has been reported by many investigators. Hershey et al. (1960) employed quantitative histochemical techniques to determine the distribution of activity in human epidermis. Most of the enzyme was located in areas of high metabolic activity—epidermis, hair follicles, sebaceous glands, and sweat glands. Mier and Cotton (1970b) assayed the enzyme in extracts of human and mouse skin and obtained values similar to those reported by several other investigators. The authors observed a flat pH optimum, from 5.5 to 7.0, which they considered similar to that of previous reports with enzymes from other sources. Halprin and Ohkawara (1966d) examined the properties of lactic dehydrogenase in human epidermal extracts, and obtained a sharp pH optimum at 7.0-7.5. The authors also investigated kinetic parameters of the enzyme and endogenous levels of substrates and products; and concluded that these factors would not explain the fact that lactic dehydrogenase activity of intact epidermis is only 1% of the activity determined by in vitro assay. The LDH isozyme pattern of a tissue often correlates with the degree of aerobic or anaerobic metabolism the tissue undergoes. A documented discussion of this relationship was pesented by Tushan et al. (1969), who demonstrated that the isozyme pattern of articular cartilage is consistent with their hypothesis that the tissue derives most of its energy from anaerobic glycolysis. Several investigators have examined the isozyme pattern of epidermis; one of the most significant studies was described by Weber (1962). The epidermal isozyme distribution was similar to that of other tissues which undergo anaerobic metabolism; and the pattern was so different from that of serum LDH that one could conclude that the epidermal enzyme is indeed synthesized in the epidermis. c. Enzymes of "Reverse Glycolysis." As is well known, reversal of

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John A . Johnson and Ramon M . Fusaro

glycolysis (i.e., gluconeogenesis) requires that three essentially irreversible glycolytic steps be bypassed: those catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase. These reverse roles are played, respectively, by glucose-6-phosphatase, fructose diphosphatase, and pyruvate carboxylase plus PEP-carboxykinase. Pyruvate carboxylase has not, to our knowledge, been looked for in skin. Halprin and Ohkawara (1966b) reported the activity of PEP-carboxykinase, in the direction of oxaloacetate formation, in human epidermis. The assay conditions were not detailed. They also reported that fructose diphosphatase activity as measured by fructose 6-phosphate formation, was 20% of that of phosphofructokinase, and proposed that the former may serve to recycle triose phosphates arising from the hexose monophosphate pathway. Again, assay conditions were not described. Halprin and Ohkawara (1966a) cited unpublished observations for the presence of glucose-6~ phosphatase in human epidermis. In contrast, Leathwood and Ryman (1971) found no activity with an assay involving correction for nonspecific phosphatases. In this context, it may be noted that histochemists can readily demonstrate a variety of phosphatases in skin sections, and the enzymatic "specificity" to some extent relates to the substrate that is added to the incubation medium. Thus, Hashimoto and Ogawa (1963) described an alkaline phosphatase that can be visualized with glucose 1-phosphate, fructose 6-phosphate, or glucose 6-phosphate. In conclusion, it may be inferred that lack of at least one key enzyme precludes gluconeogenesis in epidermis. The study of Halprin and Chow (1961) with perfused dog skin supports this opinion. They observed that acetate- 14 C was incorporated into succinate and fumarate, but not into glycogen or pyruvate. 4. ENZYMES OF THE HEXOSE MONOPHOSPHATE PATHWAY

Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase have been detected in guinea pig skin by Rassner (1965) and in human epidermis by Rippa and Vignali (1965) and Halprin and Ohkawara (1966a,b). Halprin and Ohkawara cited indirect eivdence to support the conclusions that transaldolase and transketolase were elevated in psoriatic skin, although these enzymes were not assayed directly. 5. ENZYMES OF GLUCURONATE METABOLISM

Glucuronic acid and various related compounds are found in the mucopolysaccharides of connective tissues, and one might therefore expect to find the appropriate interconverting enzymes in skin. Jacobson and Davidson (1962a) purified UDPG dehydrogenase 18-fold from whole rabbit skin and verified that the enzyme converts UDPG to U D P glucuronic acid. They noted (1962b) that the major sulfated mucopoly-

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23

saccharide of skin contains L-iduronate, the 5-epimer of the more usual component, D-glucuronate. They separated a crude fraction of rabbit skin extract that had UDP-glucuronate-6-epimerase activity—i.e., a fraction that converted the substrate to UDP-L-iduronate. They also mentioned the presence of a third enzyme in rabbit skin extract, f/DP-iVacetyl-v-glucoBamine-It-epimerase. Fukui and Halprin (1970) assayed human epidermal slices and blister lid, and detected UDPG-dehydrogenase, Ώ-glucuronate reductase, h-xylulose reductase, and xylitol dehy~ drogenase. They noted that the enzyme activities were low, one-hundredth that of glycolytic enzymes; and that activities in pure epidermis (blister) were lower than those in slices that contained some dermis. We interpret these observations to suggest that the enzymes may be concentrated in their expected locale, the dermis; and that only slight activity resides in the epidermis. 6. TBICARBOXYLIC ACID CYCLE ENZYMES

Cruickshank et al. (1958) employed a sensitive assay procedure to detect isocitric dehydrogenase, and thereby provided a deciding piece of evidence that the enzymes necessary for operation of the Krebs cycle are present in skin. Halprin and Chow (1961) obtained practical verification of this conclusion when they observed that acetate-14C was incorporated into Krebs cycle intermediates by perfused dog skin. Although Krebs cycle activity can be readily demonstrated in skin, surprisingly little of the glucose utilized by skin is oxidzed via tricarboxylic acids. Despite this apparent anomaly, little attention has been directed to the detection of the enzyme system catalyzing the last step before entry of pyruvate into the cycle: pyruvate decarboxylase. This enzyme complex regulates the oxidative conversion of pyruvate to acetyl coenzyme A; and it would seem axiomatic to evaluate its activity if Krebs-cycle oxidation of glucose were low. One can infer efficient conversion of pyruvate to acetyl coenzyme A from the study performed by Booij (1965) with rat skin. He reported that one-third of the respiratory C0 2 arose from pyruvate-2-14C; and when malonate was added to inhibit Krebs cycle activity, incorporation of label into the C-l position of acetoacetate increased 10-fold. B. Glucose Metabolism in the Epidermis 1. LACTATE PRODUCTION IN SKIN

Perhaps the most important single biochemical observation made of skin is its high lactate concentration with respect to blood: 30 mg or

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John A. Johnson and Ramon M . Fusaro

more per 100 gm of human skin versus about 10 mg/100 ml blood. This concentration difference exists despite the abundant cutaneous blood supply; and indeed, the "excessive" blood flow in skin may be a useful adaptation which serves to promote efficient removal of lactate. Several mechanisms could account for the lactate concentration gradient: (1) active transport from the blood; (2) formation in the epidermis and retention in the dermis; (3) formation in the epidermis at a rate sufficiently high to maintain the dermal/blood gradient. Since lactate is thought to diffuse freely into and out of cells, an active transport mechanism seems unlikely. The polyanionic nature of dermal substances could be invoked for retention of cations, but there appears no similar rationale for retardation of anions such as lactate. The last mechanism, lactate formation faster than it can diffuse away, is supported by numerous in vitro experiments attesting to the ability of the epidermis to rapidly convert glucose to lactate. Because the epidermis metabolizes large amounts of glucose to lactate, the skin has considerable influence on overall carbohydrate balance. To our knowledge, the role of the skin has never been considered in glucose/lactate balance studies; yet some simple calculations will illustrate the magnitude of lactate production in this organ. Halprin and Ohkawara (1966d) cited a value from the literature of 0.7 jumoles of lactate per minute per gram wet weight, produced by epidermal sheets floating on a medium containing glucose. This value corresponds to 17 gm of lactate produced daily by the epidermis (190 gm) of a 70-kg man. An in vivo calculation can be made from the observed skin lactate level. If this value is 30 mg/100 gm and skin mass is 7 kg (10% of body weight), the skin lactate pool is 2100 mg. If one then makes the conservative estimate that lactate diffuses from the dermis at the same rate as does glucose, 2% per minute .(CutGTT), then it follows that the skin loses lactate at the rate of 42 mg per minute, or over 60 gm per day; to be replaced by a like quantity synthesized in the epidermis. These calculations illustrate two biochemical features of skin which in our opinion cannot be ignored in future glucose/lactate balance studies: (1) a large lactate pool (4 times that of blood) ; (2) active, continuous lactate synthesis. 2. LACTATE/PYRUVATE RATIOS IN TISSUES

a. Noncutaneous Tissues. The lactate/pyruvate ratio (L/P) of a tissue may provide a measure of its redox state, and it is of interest to examine some aspects of this concept. Hohorst et al. (1965) reported that the lactate levels of various rat tissues (blood, plasma, skeletal muscle, liver, brain) ranged from 1 to 3 jumoles per gram wet weight; pyruvate concentrations were 0.1-0.2 /miole/gm. With the exception of muscle, the

The Role of the Sfyn in Carbohydrate Metabolism

25

L/P of these tissues was 10 to 13. Muscle exhibited a ratio of 21 arising not from elevated lactate, but rather from low pyruvate. Jensen sarcoma had high lactate (10.6 /xmoles/gm) and normal pyruvate for an L/P of 56; reflecting perhaps, the anaerobic conditions existing within the tumor. Surprisingly (at first consideration), the erythrocyte L/P is not higher than that of plasma, despite the dependence of the cell on glycolysis. This is apparent from Hohorst's data (plasma L/P was 13; blood, 12) and from the report of Mandelli et al. (1966), who observed that erythrocytes and whole blood had the same L/P, 18. Huckabee (1956) calculated the ratio of plasma lactate to erythrocyte lactate in 47 samples of human arterial blood and showed that the value was identical with that predicted by the Gibbs-Donnan equilibrium theory. This conclusion is consistent with the rate of lactate diffusion being much greater than its rate of production in the cell. The red cell can generate high L/P ratios, as shown by Minakami et αί. (1965) : The value rose from 22 to 156 after 1 hour of incubation at 37°. The L/P of rat thoracic duct lymph (Vogel et al, 1963) is higher than that of plasma, 22 versus 8, owing mainly to elevated lactate (3.4 vs 1.7 ftmole/ml). The increased lymph lactate probably reflects drainage from such glycolytic tissues as the skin and the gastrointestinal tract. b. Skin and Epidermis. Our few assays for lactate content of whole human skin averaged 33 mg/100 gm (3.7 /miole/gm) ; unfortunately, pyruvate was not determined. Halprin and Ohkawara (1966b) reported lactate levels of 7 to 8 /xmole/gm for epidermis of uninvolved and psoriatic skin, and pyruvate values of 0.2-0.3 ^mole/gin; which yielded L/P ratios of about 30. In a later report (1966d), the authors cited a physiological ratio for normal epidermis of 20. Rabbiosi and Giannetti (1967, 1968) and Giannetti and Rabbiosi (1967) described various aspects of carbohydrate metabolism of rat skin. Average lactate content of whole skin of rats on a normal diet was 10 ^mole/gm; pyruvate was 0.4 ftmole/gm; and L/P, 24. The L/P of rats on a high carbohydrate diet was elevated to 30; and was decreased to 17 after 5 days of starvation. The normal blood ratio of 15 was unchanged on the high carbohydrate diet. c. Sweat. To our knowledge, no information is available concerning lactate and pyruvate concentrations of skin appendages. However, the metabolic wastes of one of them, the eccrine sweat gland, can be readily sampled as sweat. The eccrine sweat gland is marvelously adapted to producing copious quantities of sweat during heat stress. According to Cage (1971), the average man can produce sweat at a rate of 1 to 2 liters per hour under appropriate stimulus, corresponding to 30-60 ml per hour per gram of gland. The relatively high level of glycogen

26

John A . Johnson and Ramon M . Fusaro

in the gland decreases rapidly after onset of sweating, and further secretory activity appears to be sustained by metabolism of glucose supplied by the blood. Sweat lactate varies from 4 to 40 mM ; and pyruvate, from 0.1 to 0.8 mM (Carruthers, 1962). These values correspond to an L / P range of 40 to 50. Emrich and Zwiebel (1966) showed that lactate and pyruvate levels vary with sweating rate. Pyruvate decreased from 1.5 to 0.2 mM, whereas lactate varied from 16 to 37 mM. Lactate concentration decreased at high sweat output, but total amount excreted was proportional to sweating rate. And significantly, the L / P increased from 10 (extrapolated to zero sweat output) to over 100 at high sweat rate. d. Comments. To the extent that lactate/pyruvate ratios reflect the redox state of tissues, many organs seem to be in equilibrium with blood in the resting animal. Skin is a notable exception to this generalization; as illustrated by the elevated L / P . Also, the progressive lactate gradient between epidermis, dermis, and blood (approximately 8, 4, and 2 /xmoles/gm, respectively) supports the conclusion arrived at in the previous section, that high skin lactate is due to rapid synthesis in the epidermis relative to the diffusion rate of the acid. The striking increase in L / P of sweat with elevated output demonstrates the versatility of the sweat gland in switching to anaerobic glycolysis during periods of sudden, high energy demand. In this respect, the organ resembles exercising skeletal muscle in oxygen debt. 3. SOURCE OF SKIN LACTATE

Our far-reaching conclusion that skin metabolism exerts a strong influence on glucose/lactate balance in the intact human is based at this point of our discussion, on the high lactate content of skin. One might wonder whether this elevated level is due to epidermal metabolism or to intermittent sweat gland activity. Several lines of evidence suggest that the epidermis is the normal site of lactate production in human skin. Blank (1971) pointed out that very little water is lost as sweat at environmental temperatures less than 30° ; i.e., when there is no need for temperature regulation. Our skin specimens were obtained from the washed backs of humans, as were the epidermal sheets assayed by Halprin and co-workers; the glands at this site are normally quiescent and do not respond to emotional stimuli (Cage, 1971). It is therefore likely that the lactate in these specimens originated in the epidermis. Furthermore, the secretory apparatus of the sweat gland lies deep in the dermis ; yet thin epidermal slices readily convert glucose to lactate in vitro. In addition, nonprimates have only a few sweat glands which are restricted to the paws (Montagna, 1962), yet have elevated skin lactate. Rat skin lactate, for example, was 88 mg/100 gm, while the blood level

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27

was 9 mg/100 ml (Rabbiosi and Gianneti, 1967). And last, Weber (1962) examined an anhydrotic patient and verified histologically that the sweat pores were occluded and the sweat glands were atrophied. Despite his 'inability to sweat, the patient's skin lactate level was twice that of his blood. 4. GLUCOSE UTILIZATION BY EPIDERMIS

A voluminous literature exists on the subject of skin glucose metabolism, and it is not our intent to present an exhaustive review. However, we will discuss the historical background and some of the pertinent experiments which have been performed, in order to give the reader some insight into this important aspect of skin metabolism. Pillsbury (1931) was one of the first investigators to demonstrate the presence of lactate in skin, and showed that in vitro production of lactate was increased when glucose was added to the medium. Despite this early recognition of skin glycolysis, few investigators studied glucose utilization by skin for the next 25 years. Cruickshank and Trotter (1956) found that conversion of glucose to lactate by guinea pig epidermis was decreased in the presence of oxygen, thus demonstrating the Pasteur effect. In a later study (Cruickshank et al, 1957) it was reported that glucose utilization and lactate formation were constant over a glucose concentration of 5-20 milf in the incubation medium. These authors (1962) also monitored endogenous glucose and glycogen and measured formation of urea and ammonia in epidermal slices incubated in the absence of glucose. They calculated that the oxygen utilization of the tissue could not be accounted for by disappearance of glucose, glycogen, and protein; and concluded, " . . . it is very likely that, by exclusion, the main energy source for the endogenous respiration of skin is lipid." Freinkel (1960) incubated human epidermal slices with glucose-U-14C, glucose-l-14C, and glucose-6-14C and followed the incorporation of activity into lactate, C0 2 , fatty acids, and sterols. From the ratio of C0 2 arising from C-l and C-6 of glucose, she calculated that activity of the hexose monophosphate (HMP) pathway exceeded that of the Krebs cycle, and that less than 2% of the glucose utilized was oxidized via the latter path. Up to 70% of the assimilated glucose appeared as lactate; and the author concluded that, if Krebs cycle activity is indeed low, the Embden-Meyerhof pathway may be the major source of epidermal ATP. She also demonstrated that epidermal slices were more than twice as active as dermal specimens and that dermal activity was higher when sebaceous glands were present. Pomerantz and Asbornsen (1961) performed a similar experiment with rat skin and glucose labeled uniformly and at C-l and C-6. They pointed out that the specific activity

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John A. Johnson and Ramon M. Fusaro

of the respired C0 2 was considerably lower than it would be had the radioactive glucose been the sole precursor, and therefore concluded that much of the C0 2 arose from oxidation of endogenous fat. The authors observed C-l/C-6 ratios for C0 2 radioactivity similar to those reported by Freinkel, and commented that her calculations did not account for glucose recycling or involvement of intermediates in exchange reactions. Consequently, HMP activity was unduly emphasized at the expense of Krebs cycle contribution. Katz and Wood (1960) were cited in support of the contention that C0 2 data alone do not permit quantitative estimates of the relative pathways of glucose; and Pomerantz and Asbornsen interpreted their C0 2 results as simply a qualitative indication of operation of both the HMP pathway and the Krebs cycle. The radioactivity of lactate arising from glucose-l-14C and glucose-6-14C was located in the methyl carbon as expected from operation of the Embden-Meyerhof cycle; and total incorporation of activity was essentially the same from either precursor. The latter observation was somewhat unexpected, because operation of the HMP pathway would siphon off the C-l carbon of glucose as C0 2 ; and therefore incorporation of activity from the C-1-labeled compound into glycolytic products would be reduced. However (again citing Katz and Wood), the authors noted that if EmbdenMeyerhof activity were high compared to that of the HMP pathway (as it is in skin), operation of the latter cycle would have little effect on C-l/C-6 ratios of incorporation. The following distribution of radioactivity was observed after 3 hours of incubation: unused hexose, 28%; carboxylic acids (lactic, acetic, and formic), 36%; C0 2 , 2%; amino acids, 5%; phosphorylated compounds, 5%. Lactate and acetate accounted for about 80% and 10%, respectively, of the activity of the carboxylic acid fraction. The specific activity of lactate was nearly that of the glucose precursor, indicating endogenous synthesis accounted for little of the lactate formed. On the other hand, the relatively low specific activity of acetate suggested dilution caused by breakdown of endogenous fat. It might also be mentioned that incorporation of glucose radioactivity into acetate adds to the skimpy evidence alluded to earlier (Section IV, A, 6), for the presence of pyruvate decarboxylase activity in skin. Additional information obtained in this valuable study concerns conversion of glucose to amino acids. Radioactivity was incorporated into alanine, serine, glutamate, and aspartate. The specific activities of alanine fractions were the same as those of the corresponding lactate samples, while the specific activities of the other amino acids were much lower. Little radioactivity occurred in glycine, and none was detected in threonine, valine, isoleucine, lysine, or tyrosine. Firschein and Bell (1961) incubated whole rat skin in the presence of glucose and fluoroace-

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täte and observed an increase in citrate, thus demonstrating functional operation of the Krebs cycle. Yardley and Godfrey (1961) reported that respiration of guinea pig skin in the absence of "substrate" (i.e., glucose) is supported by oxidation of fatty acids arising from endogenous phospholipids. In a later study (1962), they investigated the effect of hypoglycin, a specific blocker of fatty acid oxidation, on skin respiration. In the absence of glucose, 0.1 mikf hypoglycin caused a 14% decrease in oxygen uptake; the decrease in the presence of 20 mM glucose (4%) was not significant. In 1964 the authors reported that oxygen uptake of guinea pig skin was increased in the presence of glucose. When skin was incubated in a medium containing phosphate-32P and glucose, many labeled sugar phosphates were detected by two-dimensional paper chromatography; almost none were produced if glucose was omitted from the medium. These observations established that the endogenous substrate of guinea pig skin was not glucose. Gilbert (1962) demonstrated the Crabtree effect (inhibition of oxygen uptake by glucose) in cattle skin, and reported that the loss of respiratory ATP was compensated for by the ATP arising from increased conversion of glucose to lactate. He concluded tentatively that the endogenous substrate of cattle skin was fat and protein, that the in vitro conditions did not permit efficient utilization of exogenous substrate (glucose), and that respiration was limited by availability of ADP and inorganic phosphate. In 1964 he considered the consequences of control by ADP and phosphate, and predicted the Pasteur effect (reported by others), the Crabtree effect (reported by him for cattle skin), and an increase in respiration and glycolysis in the presence of dinitrophenol. Gilbert verified the last two predictions for human skin. In contrast to his observation with cattle skin that overall energy yield was the same with and without glucose, he noted that with human skin, the ATP derived from glycolysis was slightly greater than that lost by reduction of oxygen consumption. He also reported that 0.1 M oxamate (an inhibitor of lactic dehydrogenase) caused a 50% reduction of glycolysis. The work of Herdenstam (1962) deserves mention for his exhaustive study of the fate of glucose in normal human skin, and in uninvolved and psoriatic skin. His report provides a thorough description of techniques ; operation of glycolysis, HMP pathway, and Krebs cycle; and conversion of glucose to numerous metabolic compounds. Booij (1965), in contrast to Gilbert's observations with cattle and human skin, detected no Crabtree effect with the skin of young rats and stated his finding was consistent with those for rat, mouse, and guinea pig skin. He also reported that the activity of C0 2 arising from pyruvate-2-14C corresponded to about one-third of the total C0 2 produced. Im and Hoopes (1970) found that 85% of the glucose utilized

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in guinea pig skin was converted to lactate; they estimated that 2% was metabolized by the H M P pathway and 8% by the Krebs cycle. Adachi and Uno (1968) studied glucose metabolism of growing and of resting human hair follicles and observed that 95% of the glucose utilized was converted to lactate in each instance. Krebs cycle activity was minimal, and operation of the H M P pathway consumed 2.5% of the glucose with growing hair and 0.6% with resting follicles. The growing follicle consumed nearly twice as much glucose as did the resting one; and the 4-fold increase in H M P activity was consistent with higher glucose-6-phosphate dehydrogenase activity (1.5-3.5 times) in the former. Wolfe et al. (1970) dissected eccrine sweat glands from human surgical or biopsy specimens and incubated them in 5.6 milf glucose, in the presence or absence of methacholine or epinephrine (the latter compounds induce sweating in vivo when injected intradermally). By considering in vitro lactate production of stimulated (epinephrine) glands and the number of glands in a normal man, the authors calculated that anaerobic glycolysis of glycogen and glucose is sufficient to account for the lactate present in the copious amounts of sweat that an adult male can produce (1 liter/hour, 8-12 mM lactate). They also measured oxygen consumption and estimated that less than 1% of the glucose utilized is metabolized aerobically. Sato and Dobson (1971) reported rates of lactate production in human glands similar to those observed by Wolfe et al. and also obtained stimulation with the pharmacologie agents. Monkey sweat glands exhibited lower activity per gland but were stimulated by methacholine and epinephrine in the same manner as human glands. K. Sato (1971) personal communication reported that Wolfe's data on low aerobic metabolism conflict with Sato's unpublished observations with monkey tissue, and commented that C 0 2 production of human glands is one-third to one half that of monkey glands. 5. CONTROL OF GLYCOLYSIS IN EPIDERMIS

a. Introduction. As pointed out by Hess and Boiteux (1968), several glycolytic enzymes exhibit the allosteric properties essential for rapid, coordinated control: hexokinase, phosphofructokinase, glyceraldehyde3-phosphate dehydrogenase, pyruvate kinase, pyruvate decarboxylase, and ATPase. None of the appropriate skin enzymes have been isolated and examined for their in vitro characteristics, and only a few studies have been performed with crude extracts. The scanty information available on control of skin glycolysis is reviewed below. b. Phosphofructokinase (PFK). Kondo and Adachi (1971) cited references validating the importance of PFK as a glycolytic control factor in brain, heart, diaphragm, skeletal muscle, liver, ascites tumor, and

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microorganisms. To this list, one can add erythrocytes (Rose, 1971), the ocular lens (Lou and Kinoshita, 1967; Chylack, 1971), and intestinal mucosa (Srivastava and Hubscher, 1966). Yet, in spite of the well documented importance of this enzyme in a wide variety of systems, its function in skin has been largely ignored. The notable exception to this statement is the recent work of Kondo and Adachi. These investigators applied the classic crossover theorem of Chance et al. (1958) to data obtained with anoxic human hair bulbs and microdissected monkey skin. The increased glycolysis resulting from anoxia was accompanied by decreased hexose phosphate levels and increased fructose diphosphate. This depletion/accumulation pattern of substrate and product implicates PFK as a control point. Two groups of tissue were distinguished: (I) human hair bulb and monkey epidermis—PFK was fully activated during anoxia; (II) monkey eccrine and sebaceous glands—the enzyme appeared to be only partially activated. c. Hexokinase (HK). Hexokinase activity is intimately related to that of PFK, because of the susceptibility of the former to product inhibition. In most tissues, glucose 6-phosphate (G6P) is in rapid equilibrium with fructose 6-phosphate, which in turn serves as substrate for PFK. Alterations in PFK activity are therefore reflected in reciprocal changes in G6P levels; and thus, control of PFK activity is amplified by a corresponding effect on HK. In the ocular lens, the absolute amount of HK appears to limit the maximum glycolytic rate (Lou and Kinoshita, 1967). Halprin and Ohkawara (1966c) cited Schragger's (1962) report of intracellular epidermal glucose in support of their belief that HK controls the rate of glucose utilization by the cell. Schragger's work suffers from two sources of error, both of which caused overestimation of glucose: homogenization of epidermal slices in buffer at 0° permitted glycogenolysis to occur, and use of glucose oxidase reagent allowed interference from glycogen and oligoglucosides. Halprin and Ohkawara (1967) and Kahlenberg and Kalant (1966) have reported the presence of intracellular glucose in human epidermal slices after incubation in the presence of glucose. We have some questions about the interpretation of data in these studies, and it must also be noted that they provide no information on the in vivo distribution of epidermal glucose. Halprin et al. (1967) measured glucose content of human epidermis, and with the assumption that serum glucose was 60-100 mg per 100 ml of water, concluded that epidermal glucose was 35-67% of serum glucose. Since this glucose space was far greater than the epidermal extracellular compartment, it was presumed that some of the glucose was intracellular. We recalculated their data for average glucose content of epidermal slices,

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and on the basis of an assumed blood glucose content of 110 mg per 100 ml of water, obtained a glucose distribution space of 20%. This agrees with the measured extracellular volume (15-18%) reported by Halprin and Ohkawara (1967). Whatever the distribution of epidermal glucose, it should be recognized that the occurrence of free hexose inside the cell does not implicate H K as the major or sole glycolytic control. This point is well illustrated by the erythrocyte, which readily takes up glucose, yet is under the joint control of PFK and HK. I t may be true in some tissues that low quantities of HK may control the maximum attainable glycolytic rate; yet the actual in vivo expression of enzyme function is controlled by G6P concentration, which in turn is dependent on P F K activity. Halprin and Ohkawara (1966c) focused their attention on HK alone for the control of epidermal glycolysis; as a consequence, they proposed control mechanisms that act in opposition to the energy needs of the cell: increased glycolysis when ATP concentration is high; decreased rate when ADP is elevated. There is some evidence that skin HK is not saturated in vivo under normal conditions. Thus, intravenous glucose loading was followed by increased epidermal glycolysis, as evidenced by increased skin lactate in rats (Rabbiosi and Giannetti, 1967) and in a human (Fusaro and Johnson, 1970). d. Glyceraldehyde-8-phosphate Dehydrogenase (GA3P-DH). Im et al. (1966) found high GA3P-DH activity in epidermis, hair follicle, and sweat gland of monkey skin. They postulated that the enzyme may regulate glycolysis through mediation of the Pasteur effect, a role assigned to it by others in ascites tumor cells and muscle. The only other evaluation of GA3P-DH as a control enzyme of skin glycolysis is a brief comment made by us (Johnson and Fusaro, 1970b) in reviewing a study by Halprin et al. (1969). e. Conclusion. The available evidence is scanty, but there is no reason to believe that control of epidermal glycolysis is different from that established for other tissues. The central role of P F K and auxiliary function of HK seem likely; and GA3P-DH may provide additional control in special circumstances. 6. ROLE OF LACTIC DEHYDROGENASE (LDH)

IN GLYCOLYSIS

Huckabee (1958) described the mechanism by which a cell adapts to anerobiosis. Since epidermal cells are subjected to varying degrees of anaerobiosis, it is approprate to discuss Huckabee's comments and to expand somewhat upon them. As the intracellular oxygen tension falls, the oxidation potential of the cell decreases and the redox couples

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of the electron transport chain shift toward the reduced state, in the order: cytochrome oxidase, cytochromes, flavoprotein, and finally NAD. Of the various NAD-linked systems in the cell, the one with the highest potential (and therefore the one to be affected first by decreasing cell potential) is the pyruvate/lactate couple. The presence of high LDH activity keeps pyruvate and lactate in rapid equilibrium, and thus enables the cell to adjust quickly to anaerobiosis. It is significant that the LDH-catalyzed reaction is "dead-end"; that is, lactate suffers no fate other than conversion back to pyruvate. Since the products of other NAD-coupled systems in the cell are reactants for succeeding reactions, control at one of those points could cause serious imbalance of an entire reaction sequence. It is therefore obligatory for the well-being of the cell that redox control be exerted at the LDH site. Furthermore, since the electron-transport chain has been rendered inoperative by oxygen lack, it is imperative that the cell maintain glycolysis as its sole mechanism for production of ATP. Maintenance of glycolysis, in turn, requires reoxidation of the NADH formed in the glyceraldehyde-3-phosphate dehydrogenase step. This function is performed admirably in the LDHcatalyzed conversion of pyruvate to lactate. The epidermal cell is well supplied with LDH ; Halprin and Ohkawara (1966d) reported that the activity of epidermal extract is 100-fold greater than that required for lactate production in epidermal slices in vitro. Mier (1969) was baffled that although skin produces much pyruvate (lactate), little glucose is oxidized via the Krebs cycle (Clark and Sherratt, 1967, cited the same "mystery" for intestinal mucosa.) However, no mystery need exist if one considers that pyruvate must be oxidatively decarboxylated to acetyl-CoA before entering the Krebs cycle and that the first step of this conversion is catalyzed by pyruvate dehydrogenase. If little pyruvate traverses the Krebs cycle, the simplest explanation is that LDH (known to be present in great excess) competes more effectively for pyruvate than does pyruvate dehydrogenase. Another explanation, based on presumed formation of acetyl-CoA, will be presented in Section V, D. 7. ALTERNATE FATES OF GLUCOSE 6-PHOSPHATE IN EPIDERMIS

It is apparent that glycolytic rate can be influenced not only by controlling individual glycolytic enzymes, but also by altering the ability of other pathways to compete for G6P. Some of these alternate fates are of little importance in epidermis. Thus, glucose-6-phosphatase is probably absent and the activities of enzymes involved in mucopolysaccharide synthesis are low. Glycogen synthesis occurs readily, but the total amount of the polysaccharide stored in skin is small compared

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John A. Johnson and Buimon M. Fusaro

to glucose turnover. The HMP pathway therefore emerges as the primary alternate fate for G6P. Pentose cycle activity increases in rapidly growing epidermis, in keeping with the teleological argument that this provides the pentose required for increased nucleic acid synthesis, and NADPH for other synthetic processes. Psoriasis is characterized by accelerated epidermal growth, and the activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are 2- to 3-fold higher in psoriatic plaque than in uninvolved skin (Halprin and Ohkawara, 1966b). In a later study, Halprin et al (1969) reported that although occlusive steroid treatment provided clinical improvement of psoriasis, the enzymatic abnormalities were not corrected. We examined some of their data and concluded that the therapy may have accomplished precisely what is necessary to inhibit epidermal proliferation: reduce the activity of all the enzymes involved in glucose utilization, but lower HMP activity to a greater extent than that of glycolysis. This would enable the cell to obtain sufficient energy for normal growth while restricting the amounts of pentose and NADPH available for abnormal processes. Im and Hoopes (1970) reported an interesting rearrangement in glucose metabolism in regenerating guinea pig skin. The amount of glucose converted to lactate increased from 85% in normal skin to 90% in wounded skin; Krebs cycle activity decreased from 8% (normal) to 4% (wounded) ; and HMP activity increased from 2% (normal) to 3% (wounded). Another indication of increased pentose cycle function in growing epithelial cells was mentioned earlier (Section IV, B, 4) : the HMP activity of growing hair was four times that of resting follicles (Adachi and Uno, 1968).

V. Sources of Energy in the Epidermis A . Introduction The epidermis is constantly renewing itself and therefore needs a continuous supply of energy. Sims (1970) estimated that 20% of the protein requirements of the adult are used for epidermal replacement. At a turnover rate of 3-4 weeks, the 190 gm of epidermis of a 70-kg man is replaced at a rate of 8 gm per day. To be sure, little of this material is lost from the surface of the skin, 0.5 to 1 gm per day (Kligman, 1964). Nevertheless, the energy expended in cell division, progressive differentiation of outward-migrating cells, and résorption and reuse of metabolic products, is considerable.

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Like other tissues, the skin may satisfy its energy needs in several ways. In terms of nutrient turnover, anaerobic glycolysis is the most important catabolic process in skin ; yet this pathway is so poor a source of energy (2 ATP per glucose molecule) that other energy-producing mechanisms may be important. Thus, the potentially high energy yield of the Krebs cycle compensates for the fact that little glucose is metabolized by this route. Furthermore, as mentioned in Section IV, B, 4, several investigators reported that skin metabolizes endogenous lipid or fatty acid in vitro. Since the ultimate fate of fatty acid is oxidation via the Krebs cycle, the catabolism of this nutrient provides much more energy than does the conversion of glucose to lactate. The major controversy in skin metabolism today centers around the relative contributions of anaerobic glycolysis and fatty acid oxidation to the energy needs of the epidermal cell. That the epidermis converts large amounts of glucose to lactate is accepted without question; the point of contention lies in how much lipid, if any, is catabolized by the tissue in the intact animal.

B. Energy from Glucose Utilisation Freinkel (1960) appears to have been the first to suggest that anaerobic glycolysis may provide most of the ATP synthesized in skin; later, however, she proposed that lipid is the major endogenous fuel. We have believed for some time (Fusaro, 1965; Fusaro and Johnson, 1966) that the most important energy-producing pathway in skin is anaerobic glycolysis; and Mier (1969), after reviewing the literature, arrived at the same conclusion. There is much qualitative evidence to support this premise; some of the quantitative aspects will be discussed here. Kristensen and Schousboe (1968) calculated that the energy derived from conversion of endogenous glycogen to lactate in frog skin was sufficient to account for the observed transport of sodium through the tissue. Im and Hoopes (1970) reported that 62% of the energy produced by glucose utilization in normal guinea pig skin arose from anaerobic glycolysis, and 38% from Krebs cycle activity. The corresponding values for wounded skin were 74% and 26%. The sweat gland is well supplied with blood (see Montagna, 1962, p. 315, for a spectacular illustration of the vascular pattern), and is therefore presumably well oxygenated. Furthermore, cells of the gland consume oxygen, are well supplied with mitochondria, and contain oxidative enzymes such as cytochrome oxidase (Hubbard and Weiner, 1969). Yet, despite the sweat gland's ability

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John A. Johnson and Ramon M. Fusaro

to undergo aerobic metabolism, during intervals of intense sweating when energy demand is high, copious amounts of lactate are excreted. Emrich and Zwiebel (1966) reported that anaerobic glycolysis provides at least 25-30% of the energy required for sodium excretion and reabsorption, and noted that their calculations did not include the energy associated with the unknown amount of lactate formed in the gland but removed by the blood. Hubbard and Weiner (1969) observed that ischemia produced by occlusion of the forearm caused the expected reduction in sweating rate and in the output of lactate and sodium. If the subject breathed hyperbaric oxygen before occlusion, lactate and sodium excretion decreased at the same rates as in the control, but the reduction in sweat output was less abrupt. The increased tissue oxygen level produced by breathing hyperbaric oxygen had no effect on sweat rate, or on lactate and sodium output, of the unoccluded arm. The authors concluded that lactate production is not simply a result of hypoxia, and that glycolysis provides the energy for sodium reabsorption in the sweat duct. The results of this investigation make it possible to distinguish two metabolic components of sweat gland function: an anaerobic one involving lactate and sodium excretion; and an aerobic process associated with sweat (water) output.

C. Energy from Fatty Acid Oxidation The concept that lipids may be a natural substrate for skin arose from the observations that in vitro oxygen consumption could not be accounted for by the amounts of endogenous carbohydrate and protein oxidized. To the credit of early investigators such as Cruickshank and co-workers, they qualified their remarks about skin burning lipid by specifying in vitro conditions in the absence of glucose. However, later speculations appear to ignore the unphysiological conditions inherent in the original concept; and the persistence of statements in the recent dermatologie literature to the effect that lipids serve as the major endogenous fuel for skin (Freinkel, 1971) must not go unchallenged. It perplexes us that some investigators attach much importance to the ability of the epidermis to oxidize lipids in the absence of glucose. One need only consider the reaction of the diabetic animal to glucose deprivation due to insulin lack, to realize that cells possess survival mechanisms allowing them to utilize alternate nutrients such as fatty acids when their primary substrate is unavailable. It is regrettable that few experiments have been performed that examine the mutual effect of glucose

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and fatty acids on skin respiration. The observation of Yardley and Godfrey (1962) that hypoglycin (an inhibitor of fatty acid oxidation) reduces the in vitro respiration of guinea pig skin, is cited by others as evidence that skin metabolizes endogenous lipids. However, the authors also reported that the inhibitory effect of hypoglycin was blocked by glucose. Thus, guinea pig skin functions well in the presence of glucose, even when fatty acid oxidation is inhibited. Herndon and McGuire (1967) noted that guinea pig epidermis oxidized palmitate and glucose about equally well, and that dZ-carnitine stimulated fatty acid oxidation. Significantly, they found that 0.02 mM palmitate inhibited glucose oxidation 50%, and 0.3 mM glucose reduced palmitate oxidation to 60% of the control level. In a similar study, Sansone et al. (1970) examined the oxidation of a variety of substrates by human skin. Their results are available only in abstract form, but the authors reported that epidermis oxidized short-chain fatty acids much more rapidly than glucose. One of the unresolved questions of that project (G. Sansone-Bazzano, 1971, personal communication) is that of the physiological availability of such compounds as octanoic acid. Both of the above-mentioned investigations were performed by monitoring production of radioactive C0 2 from appropriately labeled glucose and fatty acids. Such experiments provide valuable information, but it must be recognized that C0 2 formation alone does not allow one to judge the metabolic implications of fatty acid oxidation. Specifically, measurements should be made in the presence of physiological concentrations of glucose, and should be accompanied by determination of the amount of glucose converted to lactate during the observation period. Experiments of this nature would provide quantitative data concerning the relative contributions of anaerobic glycolysis and fatty acid oxidation to the energy needs of the epidermis, at least in vitro. Unfortunately, it appears that such elementary studies have not been reported.

D. Comments It is not pertinent to the main theme of this communication whether the epidermis derives most of its energy from anaerobic or aerobic processes—the fact that it produces much lactate and therefore influences overall carbohydrate balance, is undisputed. We are concerned, however, that some investigators have attached great significance to fatty acid oxidation on the basis of inadequate evidence, while definitive experiments have yet to be performed. We can hope that our comments may stimulate some reader (s) to conduct the appropriate experiments to re-

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solve this important question; and with that thought in mind, we offer the following additional remarks. If we were to select an alternate fuel for skin, we would choose acetoacetate (AcAc). The production of this compound in the liver and its utilization in peripheral tissue is well documented, and the topic was thoroughly reviewed by Wieland (1968). The liver is capable of producing up to 150 gm of AcAc per kilogram per day, but because of the low activity of the activating system, is unable to metabolize the ketone body. The most important activating system in peripheral tissue is succinyl-CoA:3-oxo-acid CoA-transferase, which catalyzes the formation of acetoacetyl-CoA from succinyl-CoA and AcAc. The CoA derivative is readily cleaved by thiolase enzyme to acetyl-CoA, which is then available for oxidation via the Krebs cycle. Wieland pointed out that the liver is unique in producing AcAc instead of C 0 2 as an end product of aerobic metabolism, and considers the ketone body to represent a transport form of the energy contained in fatty acids. In this manner, the liver performs a "digestive" function by converting fatty acids into a readily metabolized nutrient which can be utilized by peripheral tissue for rapid energy production. Circulating AcAc concentration is on the order of 0.1 mJli in the fasting human, and is therefore available to peripheral tissue in significant amounts. Our thoughts concerning acetoacetate as a nutrient for skin were inspired by a report by Suie et al. (1970), who demonstrated that human skin and guinea pig epidermis utilize the compound. High substrate levels (3 m l ) were required, and added CoA (0.2 m l ) and ATP (0.6 m l ) were necessary for utilization. Unfortunately, glucose was not present in the incubation medium. It is significant that guinea pig epidermis and skeletal muscle utilized AcAc to the same extent. The authors reported another resemblance to muscle, in that they observed no 3-hydroxybutyrate:NAD oxidoreductase activity in human skin or guinea pig epidermis. On the other hand, succinyl-CoA:3-oxo-acid CoA-transferase activity was detected. If it can be demonstrated that epidermis utilizes AcAc under physiological conditions, one will then have another answer to the "mystery" (Section IV, B, 6) that although the tissue converts large amounts of glucose to lactate (pyruvate), little hexose is oxidized via the Krebs cycle. Each molecule of acetoacetyl-CoA, under the influence of thiolase, yields 2 acetyl-CoA moieties. Acetyl-CoA, in turn, exerts feedback inhibition on pyruvate dehydrogenase, the first enzyme in the complex which converts pyruvate to acetyl-CoA. Thus, AcAc catabolism exerts a direct block on entrance of glucose carbons into the Krebs cycle. I t should be mentioned that this explanation does not suffice for results

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obtained with in vitro studies performed in the absence of added AcAc. However, one would expect that acetyl-CoA arising from endogenous lipid or from added fatty acid would similarly inhibit glucose oxidation. The inhibitory effect of palmitate reported by Herndon and McGuire (mentioned in the preceding section) is consistent with this rationale. One further topic merits discussion: as pointed out by Wheatley et al. (1971), a unique cutaneous function is the synthesis of lipids which help maintain the integrity of the skin surface. These investigators noted (1970) that although the fundamental building unit of fatty acids is acetyl-CoA, glucose is considered the major physiological precursor in most tissues. Whereas glucose added to an incubation medium serves as the substrate for fatty acid synthesis by skin specimens, radioactive acetate performs a tracer function by labeling the endogenous acetate pool. Wheatley and co-workers have perhaps defined the primary physiological relationship between glucose and fatty acids in skin metabolism with the following comments (1971) : "They (the present studies) have provided convincing evidence of the important role played by glucose. It is almost certainly the major exogenous precursor of lipids and the only compound studied able to stimulate lipogenesis." Viewed in this context, one must assume that conversion of glucose to lipids is of greater metabolic importance than is oxidation of fatty acids for energy, under physiological conditions. While this manuscript was in preparation, Long and Yardley (1971) described a study which helps resolve the question of whether epidermis derives significant amounts of energy from the oxidation of endogenous fatty acids. Oxygen consumption of skin slices obtained from guinea pig ear was monitored in the presence of 2-bromostearic acid (an inhibitor of fatty acid oxidation), with and without exogenous glucose. A similar experiment was performed with dinitrophenol (DNP). The effect of 2-bromostearic acid was similar to that noted earlier (Section V, C) for hypoglycin: Respiration was inhibited in the absence of glucose, and unchanged in its presence. An the other hand, DNP inhibited endogenous respiration and stimulated oxygen uptake in the presence of glucose. The latter effect is consistent with the known uncoupling effect of DNP on other tissues and probably arose from increased availability of inorganic phosphate, which allowed glycolysis to proceed at an accelerated rate. The inhibitory effect when substrate was lacking presumably occurred because insufficient ATP was available for activation of fatty acids prior to their oxidation. The results of this investigation provide two additional examples of the ability of skin to function well in the presence of glucose, even though oxidation of endogenous fatty acids was blocked.

John A. Johnson and Ramon M. Fusaro

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Nicolaides (1965) noted that lipid synthesis and subsequent oxidation may be uniquely beneficial to the epidermal cell. Thus, mitotic activity in the basal layer requires synthesis of lipids to build membranes for new cellular elements, and the energy for this synthesis is derived from glucose. As the epidermal cell migrates away from its glucose supply and undergoes keratinization, cell organelles disintegrate and the liberated lipid may provide the energy necessary for completion of the keratinization process.

VI. Glucose/Lactate Balance in the Intact Animal A. The Skin Cori Cycle For some time (Fusaro, 1965; Fusaro and Johnson, 1966, 1970), we have promoted the concept of a skin Cori cycle: Lactate produced in the skin enters the blood, returns to the liver, and is transformed back to glucose. We claim no great intellectual achievement for this idea; indeed, we have been chided by a biochemist colleague for belaboring an obvious point. Yet this simple concept has focused our attention on the skin as a large, metabolically active organ and has led us, in halting steps, to our present belief that the skin exerts a significant influence on overall carbohydrate balance. Others interested in skin carbohydrate metabolism seem not to have grasped the physiological significance of cutaneous lactate production. Only recently, Mier (1969) remarked that the liver presumably removes the large amounts of lactate produced by the skin and described the latter as being "parasitic" upon the liver. (Indeed, if acetoacetate is confirmed as an alternate nutrient, the skin will prove to be even more "parasitic.") At any rate, it appears certain that the skin must be ranked with the red cell and the gastrointestinal tract as the major producers of lactate in the nonexercising animal.

B. Glucose/Lactate Interconversion in the Intact Animal Cahill and Owen (1968) reviewed glucose turnover and carbohydrate balance in man. They listed the results of various studies performed with glucose-14C, which provided glucose turnover rates of 100-200 gm/day in a 70-kg man. The use glucose-l-14C enabled Reichard et al. (1963) to calculate the amount of glucose recycled as lactate (42 gm/day), and total turnover (270 gm/day). Cahill et al (1966) per-

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formed similar studies with fasted subjects and obtained values of 36 gm/day recycled and 147 gm/day turned over. Cahill and Owen enumerated the "so-called obligatory-glycolytic tissues" as red cells, leukocytes, platelets, and minor tissues, such as renal medulla. The recycling value of 36 gm/day was nicely balanced by estimated lactate production of red cells (26 gm/day), leukocytes (4 gm/day), platelets (4 gm/day), and renal medulla (2 gm/day). The cumulative lactate production of these tissues exactly matches the observed recycling rate of 36 gm/day, and therefore leaves no room for contributions of the skin or gut. The answer to this quandary may lie in recent turnover studies conducted by Forbath and Hetenyi (1970) with dogs and Kreisberg et al (1970) with humans. Forbath and Hetenyi infused lactate-14C and glucose-6-3H and measured the specific activities of plasma glucose and lactate. They reported that lactate production was about 2.5 mg/kg body weight per minute, corresponding to 250 gm/day for a "70-kg dog." Kreisberg et al. infused lactate-14C and monitored the specific activities of expired C0 2 and of blood glucose and lactate. The same experiment, employing glucose-l-14C, was performed the next day. The average conversion of glucose to lactate (5 normal subjects on a standard diet) was 81.3 mg/kg per hour, or 137 gm/day for a 70-kg man. This figure accounted for 66% of the glucose turned over (207 gm/day). The authors distinguished between glucose recycling (22 gm/day) and conversion of glucose to lactate. The low recycling value apparently arose from inefficient conversion of lactate to glucose: Only 21% of the lactate arising from glucose was converted back to glucose. The data obtained with labeled lactate provided values for lactate turnover, conversion to glucose, and oxidation. Lactate turnover was 137 gm/day, the same as the glucose-to-lactate conversion obtained from the glucose-14C experiments. However, the combined values for lactate oxidation and conversion to glucose accounted for only one-third of the turnover rate; and thus the fate of about 90 gm/day of lactate was undetermined. It would be presumptous of us to attempt a rigorous analysis of this complex study, and we must restrict ourselves to general comments. The recycling rate for glucose (22 gm/day) is of the order of that found by Cahill et al (1966) ; but it is worth emphasizing that Kreisberg et al. interpreted their value to represent the amount of glucose converted to lactate and back to glucose, not total amount of lactate produced in the body. We are at a loss to understand the low conversion of lactate to glucose; intuitively one would expect the liver to be able to handle large quantities of the acid. Yet Rowell et al. (1966) observed that one-half of the lactate produced in exercising humans was removed from the circulation by extrahepatic tissues, and that hepatic-splanchnic removal was 0.77% of the total body lactate per minute, or 1110% per

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day. Kreisberg et ai. calculated the lactate pool to be 36 mg/kg or 2.5 gm per 70-kg man. Combining this value with that for hepatic lactate extraction, one can calculate that the liver handles 28 gm of lactate per day. This calculation agrees with observed glucose recycling values, but an important point must be made: Both groups of investigators assumed that lactate is uniformly distributed throughout the body, whereas we have already mentioned (Section IV, B, 1) that skin lactate concentration exceeds that of blood and that the skin lactate pool alone is about 2 gm. Our limited background in kinetic studies forbids further speculation, but it is to be hoped that qualified investigators will reevaluate the data of glucose/lactate interconversion experiments, with the additional skin-lactate parameter in mind. It might be noted that recognition of a skin lactate pool may resolve an anomaly described by Kreisberg et al. They noted that the data obtained after injection of lactate-14C provided a lactate space which exceeded the volume of body water. We also suggest that lactate production by the gut be given due consideration, with the qualification that much of this lactate pool may be removed by the liver from the portal system before it can influence lactate kinetics in the general circulation. Cahill (1970) provided a clear picture of readjustment of body carbohydrate balance during starvation in man. He pointed out that alanine is an efficient precursor of glucose in the liver; and proposed in analogy to the Cori cycle, a glucose-alanine-glucose cycle. This mechanism could serve to transport ammonia (arising from protein catabolism) from the muscle to the liver. Since the energy required by the liver for gluconeogenesis is derived from oxidation of depot fat, the Cori cycle and the alanine cycle provide means for peripheral tissue to obtain energy indirectly from fat. Cahill noted that the glucose-pyruvate-alanine sequence provides more energy than does glucose/lactate conversion. We suggest as pure speculation, that a lactate-pyruvate-alanine sequence may remove part of the lactate unaccounted for by Kreisberg et al. Possible involvement of skin is suggested by a single piece of evidence, noted earlier (Section IV, B, 4) : Incubation of rat skin with glucose-14C yielded alanine which had the same specific activity as that of the lactate isolated (Pomerantz and Asbornsen, 1961). Considerable protein degradation must take place during the continuous differentiation of epidermal cells as they move toward the surface of the skin, and the alanine cycle would provide an efficient mechanism for removal of the ammonia arising from deamination of amino acids. After this manuscript was completed, we became aware of a recent report by Kreisberg et al. (1971). By the use of a refined purification technique for lactate, the authors observed that conversion of glucose

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to lactate was only 68 gm/day per 70-kg man (40.7 mg/kg per hour), rather than the value of 137 gm/day reported earlier (1970). Lactate turnover was still 137 gm/day. We were concerned that the revised value for glucose conversion did not provide sufficient lactate production to encompass the contributions of skin and gut. However, R. A. Kreisberg (1971, personal communication) pointed out that the observed in vitro glucose conversion rates of various tissues were obtained with glucose as the only exogenous substrate, whereas in the intact animal the tissues are presented with lactate precursors (e.g., alanine, stored glycogen) in addition to glucose. Thus, the important figure is total lactate production (i.e., turnover, 137 gm/day) ; and one should tally cumulative tissue lactate yields on that basis.

C. Lactate Balance in Oligemic Shock Shock arising from blood loss is characterized by decreased circulation in skin, muscle, and gastrointestinal tract; which in turn results in anaerobiosis and increased lactate production (Schumer, 1968). The adaptation of the cell to anerobiosis was described in Section IV, B, 6; and was reviewed by Schumer and Sperling (1967). Reduced perfusion of the so-called "nonvital" tissues causes pooling of lactate in these organs, and subsequent restoration of blood volume is accompanied by "washout" of excess lactate into the circulation. If this recovery occurs in the early stages of shock when the liver is well oxygenated, the organ efficiently removes the extra lactate (Schumer et al, 1969). However, as the duration of shock is prolonged, decreased hepatic perfusion impairs the liver's ability to convert lactate to glucose; and the increased lactate output of peripheral tissues accumulates in the blood. Washout of pooled lactate at this stage can add an intolerable load to an incapacitated liver which is already burdened with elevated blood lactate. The magnitude of lactate pooling is demonstrated in the study reported by Schumer (1968). Bleeding of 65% of the blood volume of dogs caused the lactate concentrations of muscle and gastrointestinal tract to rise to 231% and 309% of normal, respectively. W. Schumer (1971, personal communication) reported that monkey skin lactate increased severalfold after 120 minutes of shock. We are not aware of data concerning skin lactate levels of humans in shock, but one might gain some insight by considering the available substrate if skin blood flow were stopped. The normal lactate concentration of 30 mg/100 gm could be augmented by glycolysis of glucose (50 mg/100 gm) and glycogen (90 mg/100 gm; J. A. Johnson and

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R. M. Fusaro, 1965, unpublished data). Thus, 140 mg excess lactate per 100 gm skin, or 9.8 gm (109 mmoles), could pool in the skin of a 70-kg man during extended shock. If this quantity suddenly flooded into an extracellular space of 15 liters, a significant lactate increment (7 meq/ liter) would be added to the elevated blood level. Since an important factor in shock is that lactic acid causes most of the metabolic acidosis which occurs, it is apparent that skin plays an important role in this metabolic disorder.

D. Lactate Production in the Gastrointestinal Tract This topic does not properly fall within the scope of the present communication, except we have referred to lactate production by the gut on several occasions. Also, since this tissue, like the skin, has been ignored in glucose/lactate balance studies, we feel some brief comments are in order. Much has been written about glycolysis in intestinal mucosa, but a search of the literature revealed no pertinent information about humans. Hohenleitner and Senior (1969) perfused dog intestine and observed lactate outputs of 0.17 to 3.73 mmoles/100 gm tissue per hour. At an output of 1 mmole/100 gm per hour and an intestinal mass of 37 gm/kg body weight, one can calculate a daily lactate production of 56 gm for a 70-kg dog. Although one hesitates to extrapolate such calculations directly to man, one must assume that in humans, lactate production in the gut is large enough to be reckoned with in balance studies. It is worth noting that less than 20% of the peripheral circulation passes into the portal venous system (Grim, 1963; extrapolated from data on dogs). Since the gastrointestinal tract drains into the portal system, the liver removes much of the lactate produced in that region before it can enter the general circulation. Consequently, though lactate production by the gut must certainly affect overall carbohydrate balance, its effect on glucose-lactate isotope exchange in the general circulation may go unnoticed.

E. Comments To sum up, glucose/lactate interconversion studies to date have not considered the role of the skin and gut as lactate producers. Failure to do so may have led early investigators to accept abnormally low values for lactate production in man and has led to underestimation of the total lactate pool. Since the gastrointestinal tract drains into

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the portal system whereas the skin discharges into the peripheral circulation, the latter organ probably has a greater influence on isotope exchange of intravenously administered glucose or lactate. Except for Schumer, investigators of shock have not considered the effect of skin glycolysis on lactic acidosis. The outpouring of pooled skin lactate may explain the "washout" phenomenon observed during recovery from shock, when peripheral circulation is restored. The preceding remarks dramatize our conclusion that skin lactate production is a potent factor in carbohydrate homeostasis, in sickness and in health.

VII. The Skin as an Indicator of Impaired Carbohydrate Metabolism A. Introduction The epidermis contains the enzymes involved in most biochemical pathways of the body, and it is therefore likely that many metabolic disorders characterized by enzyme defects or disturbances in biochemical sequences will be reflected in corresponding biochemical lesions of the skin. Since this tissue is readily accessible, it behooves the investigator to consider using skin specimens for diagnostic and investigational studies of humans. Obviously, examination of skin specimens may be of value in any area of metabolism, but for the purposes of this report our comments will be limited to carbohydrate turnover.

B. The Cutaneous Glucose Tolerance Test 1. DIABETES

The original impetus for our skin glucose studies arose from the observation that diabetics are prone to skin infections. We reasoned that the high skin glucose levels of diabetics would provide abundant nutrient for invasive organisms, and would thereby promote the establishment of pathogenic colonies. This rationale has been discussed elsewhere (Fusaro and Goetz, 1971) ; we will describe here the results of the CutGTT performed on diabetics. The average skin glucose disappearance rate of 20 diaibetics was very low: 0.3% per minute versus 2.0% per minute for nondiabetics (Fusaro, 1965). This result seemed consistent with impaired glucose transport; but upon reflection, we realized that

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it simply mirrored the low blood disappearance rate (1.2% per minute versus 3.8% per minute for nondiabetics). We therefore concluded that in diabetics, as in normal individuals, simple diffusion provides an adequate explanation for glucose transport into and out of the dermal compartment. 2. ACNE

Two observations suggest that acne patients may have slightly impaired carbohydrate metabolism: (1) Some patients improve on a low carbohydrate diet. (2) The hypoglycémie agent, phenformin, has been reported to provide clinical improvement. In order to check the possibility of impaired carbohydrate metabolism, we examined blood and skin glucose kinetics of acne patients before and after 3 months treatment with DBI (USV Pharmaceutical Corp.). Although blood and skin disappearance rates were slightly low (3.0 and 1.7% per minute, respectively), no change was observed after placebo or phenformin therapy. Significantly, 3 of the 5 persons in the DBI group showed clinical improvement, whereas only one of the placebo-treated subjects was slightly improved (Mclntyre et al., 1968). It thus appears that acne patients have little or no impairment in ability to handle a glucose load; and that the beneficial effect of phenformin is not due to improvement of blood or skin glucose kinetics. 3. CONCLUSION

Our original high expectations for the diagnostic utility of the CutGTT have not been fulfilled: Every experiment performed to evaluate such an application yielded results consistent with the premise that glucose transport between blood and skin occurs by simple diffusion. These experimental efforts have not been futile, however. Besides establishing beyond doubt the passive nature of blood/skin glucose exchange, the results dramatize the need to examine the skin enzymes and metabolites involved in carbohydrate turnover.

C. Glycogenoses Several investigators have explored the feasibility of employing tissues more readily available than liver and muscle, for the differentiation of glycogen storage diseases. Leathwood and Ryman (1971) analyzed epidermal blister specimens and reported ranges of normal values for glycogen, amylo-l,6-glucosidase (débrancher), acid a-glucosidase (acid maltase), and glycogen phosphorylase. No glucose-6-phosphatase activ-

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ity was detected in normal epidermis; therefore the glycogenosis (type I, von Gierke's disease) characterized by lack of this enzyme in liver cannot be diagnosed with skin specimens. Epidermis from a patient with type V storage disorder (McArdle's disease, lack of muscle phosphorylase) had a normal level of phosphorylase activity, as did the subject's platelets. The presence of skin phosphorylase in this instance contrasts with Adachi's observation (1961) that skin phosphorylase b resembled the muscle enzyme in its susceptibility to activation by adenylic acid. On the other hand, 2 patients with type II disease (Pompe's, lack of acid α-glucosidase) exhibited no glucosidase activity in liver, muscle, or epidermis. In the one patient examined further, skin glycogen was 10 times normal and leukocytes had normal glucosidase activity. The results described by Leathwood and Ryman demonstrate the utility of epidermal specimens for the detection of at least one type of glycogenosis; and as suggested by the authors, their techniques may be applicable to the diagnosis of other inborn errors of metabolism.

VIII. Concluding Remarks Our concepts of the passive and active roles of the skin in carbohydrate homeostasis have developed slowly; and indeed, if recent evidence of production of large amounts of lactate in humans were not available, we would not now be promoting the active role so vigorously. It may be instructive to review the chronological evolution of our thoughts concerning the relationship of skin function to carbohydrate balance. In 1965 (Fusaro) and 1966 (Fusaro and Johnson) we discussed a passive and an active role for skin after intravenous administration of glucose. As mentioned earlier (Section III, A), the idea of a passive role (temporary glucose storage) was not new; our contribution to the concept was quantitation of the amount of a glucose load which was temporarily deposited in the skin. To our knowledge, an active role had never been postulated; and it must be confessed that our early comments were directed mainly to a homeostatic function for skin in converting excess glucose to lactate during periods of elevated blood glucose. We reasoned that since blood glucose levels of nondiabetic subjects return to normal within 3 hours after ingestion of carbohydrate, the amount of glucose metabolized by skin during intervals of hyperglycemia was quantitatively insignificant. Therefore, skin played a relatively minor active role in control of elevated blood glucose. At a symposium in 1968 (Fusaro

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and Johnson, 1970), we amplified our views and presented additional quantitative data concerning the passive role of skin, but our thoughts about an active role were still restricted to the handling of excess glucose. About this time we calculated the approximate daily output of skin lactate, and W. Schumer (1968, personal communication) had alerted us to the possible importance of skin lactate in shock. Yet the time was not right; the concept of the skin as a major lactate producer seemed unrealistic when one considered that the accepted value for daily lactate production in man was accounted for by the combined outputs of noncutaneous glycolytic tissues. Only now, with recent estimates revising body lactate production upward, can one state with assurance that the skin produces significant amounts of lactic acid, and therefore must be reckoned with in carbohydrate balance studies. It is worth emphasizing that our original concept of the skin playing a minor, intermittent, active role during periods of elevated blood glucose ; has been superseded by the premise that the organ exerts an important, continuous influence on the body's carbohydrate metabolism. In our attempts to relate skin carbohydrate metabolism to that of the intact animal, we have strayed from our field of competence into areas best handled by the physiologist. We make no apology for this. The skin as an active metabolic unit has been too long ignored, and we are thankful that the nature of our glucose kinetic studies forced

FIG. 2. Present concepts of the passive and active roles of skin in glucose and lactate homeostasis. Passive role is played by the dermis in temporarily storing excess glucose during periods of hyperglycemia ; active role is performed by epidermis in the continuous conversion of glucose to lactate.

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us to examine the metabolic interrelationships of the skin and the body. It is to be hoped that persons interested in the fundamental aspects of glucose/lactate interconversion will expand or modify our conjectures, and will revise their interpretations of prior studies to accommodate the influence of lactate production by the skin and possibly the gut. Future experiments, designed with these new parameters in mind, should provide exciting information concerning carbohydrate balance in the intact animal. Our present concepts of the skin's role in carbohydrate homeostasis are illustrated in Fig. 2, which admittedly exaggerates skin function at the expense of other tissues. In our defense we cite a comment on skin carbohydrate metabolism which has sustained and inspired us during the preparation of this report: "This is a difficult area, left in obscurity, and neglected by students of metabolism" (Levine and Haft, 1970). In this context, we may perhaps be excused for overreacting somewhat in championing the skin as an integral, active organ of the body.

Addendum Correspondence with R. A. Kreisberg alerted us to the need for further justification of our belief that skin lactate levels are elevated in vivo. Although the indirect evidence for high skin lactate (Sections IV, B, 1-3) is quite convincing, it must be noted that there is little direct proof of such an assumption. Several investigators have measured lactate content of human epidermis, but to our knowledge the only reliable data on whole skin specimens are the results of a few assays performed by us. It is therefore appropriate to review our analytical technique in order to convince the reader that our observed value of 33 mg/100 gm accurately reflects the in vivo lactate concentration of human skin. Specimens were removed from the back of fasting subjects, quickly weighed, immersed in perchloric acid, and frozen on Dry Ice. The frozen mixtures were allowed to thaw and equilibrate (1.5 hours) at 4°; they were then neutralized with K2 C0 3 , cooled, and centrifuged. Aliquots of the clear supernatants were assayed for lactate with lactic dehydrogenase; other aliquots were assayed for glucose with glucose oxidase. This procedure afforded uniform lactate and glucose values for replicate specimens. It might be objected that the time required to weigh the specimen (30 seconds or less) would permit considerable glycolysis to occur before enzyme inactivation. However, one must note that the meta-

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bolically active component (epidermis) constitutes only 5% of the specimen; and the remainder (dermis) provides a large glucose and oxygen reserve, as well as a reservoir for lactate. Thus, contrary to the situation in other tissues, the epidermis does not become anoxic as soon as the specimen is severed from the donor; nor does ongoing epidermal glycolysis rapidly change the glucose or lactate content. For example, even at an assumed total lactate production of 60 gm/day, the amount formed in 60 seconds by the epidermis of a skin specimen would be less than 5% of the total lactate present. We therefore believe our observed value of 33 mg/100 gm is a realistic estimate of human skin lactate content in vivo. Another aspect of skin lactate turnover requires clarification. The calculation of in vivo lactate production (Section IV, B, 1) was based on the assumption that the lactate diffusion rate was equal to the observed value for skin glucose (2% per minute). This value wTas obtained from semilogarithmic plots of excess glucose levels (glucose content minus fasting value) ; and it can be argued that this figure should be applied not to the total skin lactate pool, but to "excess" lactate (skin concentration minus blood level). Better yet, one should use the disappearance rate constant derived from plots of total glucose levels, a value which will be less than that obtained for excess glucose. For example, when a series of CutGTT which provided an average excess glucose disappearance of 2% per minute were recalculated on the basis of total glucose, the rate was 1.1% per minute. Calculation of in vivo lactate synthesis with this value provides a figure of 33 gm/day, rather than the aforementioned 60 gm/day. It should be noted, however, that the assumption that lactate diffuses only as rapidly as glucose may itself be a conservative estimate. ACKNOWLEDGMENTS

We are grateful to Dr. William Schumer for helpful advice and comments concerning glucose/lactate balance, and the influence of lactate on shock. We are also indebted to Dr. Robert A. Kreisberg for stimulating and informative correspondence concerning glucose/lactate interconversion. The personal investigations described in this report were supported in part by USPHS Grants AM-03964 and AM-06841.

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