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Lipid Metabolism in Human Skin
Vol. 47, No. 5
THE JOURNAL 01' INVESTIGATIVE DERMATOLOGY
Copyright
Printed in U.S.A.
1966 by The Williams & Wilkins Co.
LIPID METABOLISM IN HUMAN SKIN II. A STUDY OF LIPOGENESIS IN SKIN OF DIABETIC PATIENTS*
S. L. HSIA, PH.D., M. A. DREIZE AND M. C. MARQUEZ
In our recent studies a method was developed which allowed evaluation of lipogenic activity in
trol subjects and 6 patients in diabetic acidosis. After the patients were treated with insulin and
lipogenic activity was indicated by the amounts of "C incorporated into fatty acids (Acidic frac-
the time of admission. Their ages were 21—65, their fasting blood sugar levels were 348—666 mg%; serum cholesterol 315—1314 mg%; urinary sugar 4+ and urinary ketones 4+. One of the patients (D.H.) had skin xanthomas and had been treated with DBI° at the time of the removal of
acidosis was brought under control, specimens small specimens of human skin (1). After in- the were again taken from 4 of these patients. None cubation of the specimen with acetate-1-"C, of these patients had been treated with insulin at
tion) and nonsaponifiable lipids. Since the amount of skin needed for an experiment was small (12—25 mg), punch biopsy specimens could
suffice. This paper reports a study in which the method was applied to evaluate lipogenesis in the skin of patients in diabetic acidosis. Impairment of lipogenesis under diabetic con-
ditions has been repeatedly demonstrated in animal tissues (2-11). Our study revealed a similar impairment in human skin. After the acidosis in these patients was brought under control with insulin therapy, a reversal towards normal was noticed. The effect of fasting, and the in vitro effects of glucose and insulin were also explored in this study. EXPERIMENTAL
Incubation and Analysis Immediately after excision, the skin specimens were trimmed of subcutaneous fat, and 12—50 mg of the specimen were dropped into a vial contain-
ing the incubation medium. The weight of the specimen was determined by weighing the vial with contents on an analytical balance before
and after addition of the skin. The incubation medium contained streptomycin (200 ig), penicillin
(200 units), gentamicin sulfate (200 g) and
other additions as specified, in 1.9 ml of KrebsRinger phosphate buffer (pH 7.4). Sodium acetate-1-14C (2 oc in 0.17 mole) was then added in 0.1 ml of water and the mixture was incubated at 37°C in air with gentle shaking for 6 hours. As
in previous experiments (1), a drop of the in-
Materials Sodium acetate-1-14C (specific activity 29 mc/
mmole) was purchased from Nuclear Chicago. Reagents were of A.R. grade and solvents were distilled before use. Unmodified Squibb insulin was used in experiments where the in vitro effect of insulin was tested.
Skin Specimens
Skin biopsy specimens (4 and 6 mm punches) were taken under local anesthesia (Xylocaine) from the lower abdomen of 10 non-diabetic con* From the Departments of Dermatology and
Biochemistry, University of Miami School of Medicine, Miami, Florida.
the first biopsy specimen.
cubate was tested for bacterial growth by culturing on three media. No growth was detected after
48 hours from any of the incubates reported in this study. Details of the methods for hydrolysis of lipids, extraction and separation of the Nonsaponifiable and Acidic fractions were the same as reported in a previous communication (1). Briefly, the incubation was terminated with 2 g KOH and 15 ml ethanol. The mixture was kept at 60CC overnight to allow hydrolysis of fatty acid esters and disintegration of tissue. After acidifica-
tion to pH 1, the lipids were extracted four times with 100 ml of dicholomethane. Acetic acid-1-°'C was removed by the addition and evaporation of
non-radioactive acetic acid and benzene. The residue was subjected to paper chromatography
in the system of benzene :methanol :5 N NH4OH This study was supported in part by Career De- (2:1:2), and the amounts of '4C in the Nonvelopment Award 5-K-3AM-28,095 and Grant saponifiable and Acidic fractions were determined AM09497 from the Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service by a Vanguard Autoscanner 880 with automatic data system. The amounts of "C (dpm) incorand by the Dermatology Foundation of Miami. Abbreviations used in this paper are: ATP, porated into the two fractions per mg of skin were adenosine triphosphate; CoA, coensyme A; and then calculated. TPNH, reduced triphosphopyridine nucleotide. 1 DBI is a registered trademark of U.S. Vitamin Presented at the Twenty-seventh Annual Meeting of the Society for Investigative Dermatology, & Pharmaceutical Corp. for the oral hypoglycemic Inc., Chicago, Illinois, June 26, 1966.
agent N1-p-phenethylbiguanide HC1.
443
444
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY RESULTS
TABLE II Incorporation of 14C into the Non-saponifiable
Non-diabetic Controls
fraction bn skin of diabetic patients
The UC incorporated into the Non-saponifiable and Acidic fractions by skin specimens of the 10 non-diabetic subjects are shown in Table
'C Incorporated, dpm/mg skin Name
194; and from 900 to dence interval of 382 1900 in the Acidic fraction with a 95% confi293. There was no dence interval of 1511 apparent correlation between values of the Nonsaponifiable and Acidic fractions in each individual specimen.
therapy
Increase after therapy
77 215 468 256
127
—
119 (124%) 273 (140%) 246 (2460%)
51
—
—
84
284
173
In acidosis After insulin
I. The values varied from 125 to 888 in the Non-saponifiable fraction, with a 95% confi-
D.H. G.H.
24 96
C.J.
195 10
W.T. M.M. C.W.
Mean
53 (221%)
—
Diabetic Patients Standard devia70 162 104 Values obtained from specimens of the 6 pation tients in diabetic acidosis were significantly 166 lower than those of the non-diabetic controls. 95% confidence 84 73 254 258 173 interval After treatment with insulin, rises were noticed in both the Non-saponifiable and Acidic fractions of the 4 patients studied. Table II shows 194), these values the results of the Non-saponifiable fraction be- confidence interval, 382 fore and after insulin therapy. The values be- were significantly lower. After the insulin therfore therapy varied from 10 to 195, with a 95% apy, the values were from 77—468, and an inconfidence interval of 84 73. When compared crease was noticed in each case varying from with values of the non-diabetic controls (95% 124 to 2460%. A statistical analysis indicated that the increases after insulin therapy could be significant (95% confidence interval of the inTABLE I crease = 173 166). But because only four Incorporation of "C by skin of non-diabetic patients were available for the study, the evicontrol subjects dence derived from these data could not be con'C Incorporated, dpm/mg skin
sidered conclusive.
Nonsaponifiable
ACId1C
The results obtained in the Acidic fraction are compared in Table III. The values before insulin therapy ranged from 159 to 985 with a
Name
Si.
255
1096
95% confidence interval of 594
I.M.
163
1800
ll.B. Z.F.
636 555
1600 1000
G.M.
the high values in this group overlapped with the low values of the non-diabetic group, these values were statistically lower than those of the
125
1900
J.M.
145
1873
M.D. M.K. W.A.
888
1222
625
1825
275 150
1892
J.W.
non-diabetics (95% confidence interval, 1511
293). After the treatment with insulin, the values were from 592 to 1346, and there was an increase in each case varying from 24 to 272%
900
(97.5% 419
Mean
382
1511
Standard deviation
271
409
95% confidence inter-
val
382
194
1511
322. Although
293
confidence
interval of the increase =
252).
In vitro Effects of Glucose and Insulin The stimulatory effect of glucose on in vitro lipogenesis in human skin was previously observed by Griesemer and Thomas (12). The effects of glucose and insulin in enhancing lipo-
445
LIPID METABOLISM IN HUMAN SKIN II.
TABLE III Incorporation of 14C into Acidic fraction by skirt of diabetic patients '4C Incorporated, dpm/mg skin
Name
. n an OSIS
370 or 233% 620 or 125% 446 or 50% 241 or 24% — —
1070
419
glucose and insulin by the two non-diabetic subjects in fasting and in post-absorptive state was
332
158
therapy
496 900 985
M.M. C.W.
433
— —
Mean
594
Standarddeviation
307
529 1116 1346 1226
590
95% confi- 594 dence interval
subject had a meal. The experiment was repeated with specimens obtained before breakfast, from another non-diabetic subject (WA.), and the results are shown in Table VI. The increases with the addition of glucose and glucose plus insulin were much greater than in postprandial specimens studied in the previous experiment. The observed difference in the response to
After insulin
D.H. G.H. C.J. W.T.
159
The lack of response in skin specimens of nondiabetic subjects to stimulations by glucose and insulin was puzzling. It was later realized that the specimens studied were obtained after the
322 1070
Increase after therapy
528 419
252
genesis in rat skin were reported by Gaylor (13). The following experiment was designed to test if lipogenesis in the skin of diabetic patients could also be similarly enhanced. Biopsy specimens were taken from a patient (M.M.) who was in diabetic acidosis. The specimens were incubated with and without the addition of glucose (5mg/mi) and/or insulin (1 unit/mi). The results are shown in Table IV. Significant increases in lipogenic activity were observed with either glucose or insulin, and the greater increases were found with glucose and insulin
interesting. To explore further, the experiment was repeated with specimens from a third nondiabetic volunteer (J.W.). Skin biopsy specimens were taken after an eighteen hour fast and TABLE IV In vitro effects of glucose and insulin on lipo genesis
in, skin of a patient in diabetic acidosis Acidic
Non-saponifiable
Addition
dpm) Increase dpmf Increase over over mg mg
None (control) Glucose, 5 mg/mi
skin
control
skin
control
127 233
—
590 1426
—
106
400
Insulin, 1 unit/mi
273
1166
738
611
576
(98%)
(215%)
Glucose, 5 mg/mi + insulin, 1 unit/mi
836
(142%)
(83%) 2210
1620
(275%)
(481%)
combined.
For comparison, a similar experiment was carried out with specimens from a non-diabetic subject (G.M.) The results are shown in Table V. An increase in the Acidic fraction was noticed, but only slight or insignificant increases were noticed with the addition of glucose and insulin, although the level of glucose concentration (10mg/mi) in the incubation medium was twice that in the previous experiment. In several
other experiments with skin of non-diabetic subjects, the results indicated slight rises with glucose but the effect of insulin was irregular.
In several instances, a slight decrease in the incorporation of 14C was noticed with the addi-
TABLE V In vitro effects of glucose and insulin on lipo genesis
in skin of a non-diabetic subject Non-saponifiable
Addition
the acetate-1-14C.
'C in-
14C in-
skin
skin
corpo- Increase corpo- Increase rated over rated over dpm/mg control dpm/mg control
None (control) Glucose, 10 mg/mi
125 300
Insulin, 1 unit/mi
150
tion of insulin. This might indicate increased 10mg/mi + conversion of glucose to acetate which diluted Glucose, insulin, 1 unit/mi
Acidic
125
— 175
1900 2100
— 200
(140%) (11%) 25 2000 100 (20%) (5%) 0 2500 600
(32%)
446
THE JOTJRNAL OF INVESTIGATIVE DERMATOLOGY
TABLE VI In vitro effects of glucose and insulin on lipo genesis
in skin of a non-diabetic fasting subject Acidic
Non-saponifiable
'C
Addition
14c
incor- Increase incor- Increase over porated over poratd dpm/ control pm/ control ing skin nig skin
None (control)
—
240 480
Glucose, 10 mg/mi
Glucose, 10mg/mi + 600 insulin, 1 unit/mi
800 3300
240
— 2410
(100%) (271%) 360 835 1725 (150%) (88%)
again one hour after a full meal. Several levels of glucose were tested, and the results are presented in Figure 1. The data indicate that lipogenesis was depressed during fasting, and that the in vitro stimulatory effect of glucose reached the maximum at the concentration of 5 mg/ml. The stimulation was more pronounced on the tissue obtained during fasting than on the post-
prandial specimens; it is also of interest to
from acetate-1-14C was impaired in diabetic acidosis. Improvement of this defect was indicated after the acidosis was brought under control with insulin therapy. Also observed were the decrease in incorporation of '4C in the skin of non-diabetic subjects during fasting, and the increase in activity following a meal.
Impairment of lipogenesis under diabetic conditions has long been recognized (2), and has been substantiated by observations on liver preparations (3—6), adipose tissue (7, 8), aorta (9), skin (10) and uterus (11) of alloxan diabetic rats. Similarly, decreased synthesis of fatty acids has been observed in liver of starved rats (14—16). Recently, impairment of lipogenesis in blood elements of diabetic patients has been
reported (17). Thus the observations made in this study on human skin under diabetic and fasting conditions are in agreement with previous findings on other tissues. It is generally accepted that the primary bio-
chemical abnormality in diabetes mellitus is impaired glucose utilization due to lack of insulin. Consequently the body responds to meet the
note that even with the maximum stimulation energy needs by increasing mobilization and by glucose in vitro, fasting values were lower catabolism of fatty acids. Similar biochemical responses are noted during starvation, when the than post-prandial values. energy needs are met mainly by catabolism of DISCUSSION
It was observed in this study that the ability of human skin to incorporate 140 into lipids FAST/NC
3p00 4 2,500 C, E
fatty acids. The observed decrease in lipogenesis in the skin under these conditions is therefore a
manifestation of the homeostatic response in POSTPRAND/AL
3P00
Acidic Fraction • Nonsaponifiable
2,000
Fraction
-S..
E
a-
500
1,500
Acidic Fraction
a)
C
0 a-
00 C
lpo0.
000
500
500
0 0!
2.5
5
7.5
Glucose conc. mg /ml
0
Jo
• Nonsaponifiable Fraction
2.5
5
7.5
0
Glucose conc. mg /ml
Fia. 1. The effect of glucose in vitro. Glucose was added to the incubation medium at various concentrations. Stimulation of lipogenesis was noticed in skin specimens obtained from a fasting non-diabetic subject. The effect on post-prandial specimens from the same
subject was less pronounced.
LIPID METABOLISM IN HUMAN SKIN II.
which decreased carbohydrate utilization is com-
pensated by increased fatty acid catabolism
447
explored by various investigators, but has not been fully elucidated.
In studies with rat liver, Lynen et at. (18) and decreased lipid syntheses. Lipid syntheses are endergonic processes and it is conceivable demonstrated that acetyl-CoA carboxylase that such processes be curtailed when the energy which catalyzes the initial step in fatty acid supply from carbohydrate utilization is dimin- synthesis is the rate-limiting enzyme, and that ished. this enzyme is activated by citric acid and other The data presented in Table TV, Table VI intermediates of the citric acid cycle. This reguand Figure 1, show increased lipogenesis with latory mechanism also appears to exist in the the addition of glucose and/or insulin to the in- adipose tissue as evidenced by findings of Martin cubation medium. The stimulatory effects were and Vagelos (19). However, it was pointed out observed in skin specimens of diabetic patients that the loss of acetyl-CoA carboxylase and the and of fasting non-diabetics. Low levels of glu- lower citric acid level in rat liver under diacose utilization could be expected in these tis- betic or fasting conditions could not quantitasues, and the addition of glucose and insulin tively account for the observed decrease in fatty could theoretically increase glucose utilization. acid synthesis (15, 18). Numa €t at. (20) gave Since evidence is lacking for a direct effect of evidence that inhibition of acetyl-CoA carboxylinsulin on lipogenesis, the observed stimulation ase by long chain acyl-CoA derivatives may be of lipogenesis from acetate-1-14C by insulin is likely to be an effect secondary to increased an additional controlling mechanism, and higher carbohydrate utilization. It is interesting that levels of long-chain acyl-CoA derivatives were the stimulatory effects of glucose and insulin found in liver of fasted rats (21) and in liver were slight or insignificant on post-prandial and heart of alloxan-diabetic rats (22). The stimulatory effect of glycerol 3-phosphate specimens of non-diabetic subjects (Table V and Figure 1). It is reasonable to assume in this on fatty acid synthesis was reported (23, 24). case, that the skin might have been already This product of the glycolytic pathway is stimulated to the maximum in vivo, after in- needed for the synthesis of glycerides and phospholipids. The mechanism of its enhancement gestion of food prior to excision. An alternative interpretation of the decreased of fatty acid synthesis was proposed as to faincorporation of 'C into lipids from acetate-i- cilitate the transfer of acyl groups from acyl 140 may be that an increased endogenous acetate carrier protein so that more free acyl carrier pooi existed in skin of the diabetic and fasting protein becomes available for fatty acid synindividuals. A larger acetate pool would result thesis (24). in greater dilution of the acetate-1-14C and hence
Recently Wakil et at. (25) reported that
less amounts of 140 incorporated. The actual size of acetate pooi in skin under the various conditions is not known, but studies on liver of diabetic rats by Elwood and Van Bruggen (4) indicated a decreased rather than an increased acetate pool. If the pool size of acetate were to increase in acidosis and during fasting, the ob-
phosphorylated sugars, particularly fructose 1, 6-diphosphate can stimulate fatty acid syn-
served decrease of 14C incorporation must be in-
terpreted with caution as to indicate decrease of lipogenesis. On the other hand, the increased incorporation of 14C in the presence of added glucose and insulin (Tables IV, VI and Figure 1) undoubtedly indicated increase of lipogenesis.
thesis using enzyme preparations of pigeon liver
and E. coli. Considerations were given to the cofactors, ATP and TPNH which are needed in lipogenesis
and can be furnished by glucose utilization. The controversies concerning the supply of TPNH as a regulatory mechanism were discussed by Masoro (26). The role of co-enzymes and 02 in the control of lipogenesis in adipose
tissue was discussed by Flatt and Ball (27). From these studies with animal and microbial
The findings of this study thus corroborate systems, it appears that lipogenesis may be previous findings, suggesting that lipogenesis regulated by carbohydrate metabolism at multimay be in some way regulated by carbohydrate ple points. Whether any or all of these mechautilization. The exact mechanism by which this nisms, or still other mechanisms operate in huhomeostatic regulation is accomplished has been man skin can only be clarified by further studies.
448
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY SUMMARY
Skin biopsy specimens from 10 non-diabetic subjects and 6 patients in diabetic acidosis were compared with regard to their ability to incorporate 14C into lipids from acetate-1-'4C. Specimens from the diabetics were found to incorporate significantly lower amounts of 14C. After insulin therapy, skin specimens taken from 4 of these patients were again studied, and increased incorporation was noted in each case. Specimens of non-diabetic subjects in fasting incorporated less amounts of '4C into lipids than post-prandial specimens. Stimulatory effects on
lipogenesis by the addition of glucose and insulin in vitro were demonstrated in skin specimens of diabetics and fasting non-diabetics. The controlling mechanisms of lipogenesis in
animal and microbial systems were briefly reviewed.
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THE JOURNAL 01' INVESTIGATIVE DERMATOLOGY
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LIPID METABOLISM IN HUMAN SKIN II. A STUDY OF LIPOGENESIS IN SKIN OF DIABETIC PATIENTS*
S. L. HSIA, PH.D., M. A. DREIZE AND M. C. MARQUEZ
In our recent studies a method was developed which allowed evaluation of lipogenic activity in
trol subjects and 6 patients in diabetic acidosis. After the patients were treated with insulin and
lipogenic activity was indicated by the amounts of "C incorporated into fatty acids (Acidic frac-
the time of admission. Their ages were 21—65, their fasting blood sugar levels were 348—666 mg%; serum cholesterol 315—1314 mg%; urinary sugar 4+ and urinary ketones 4+. One of the patients (D.H.) had skin xanthomas and had been treated with DBI° at the time of the removal of
acidosis was brought under control, specimens small specimens of human skin (1). After in- the were again taken from 4 of these patients. None cubation of the specimen with acetate-1-"C, of these patients had been treated with insulin at
tion) and nonsaponifiable lipids. Since the amount of skin needed for an experiment was small (12—25 mg), punch biopsy specimens could
suffice. This paper reports a study in which the method was applied to evaluate lipogenesis in the skin of patients in diabetic acidosis. Impairment of lipogenesis under diabetic con-
ditions has been repeatedly demonstrated in animal tissues (2-11). Our study revealed a similar impairment in human skin. After the acidosis in these patients was brought under control with insulin therapy, a reversal towards normal was noticed. The effect of fasting, and the in vitro effects of glucose and insulin were also explored in this study. EXPERIMENTAL
Incubation and Analysis Immediately after excision, the skin specimens were trimmed of subcutaneous fat, and 12—50 mg of the specimen were dropped into a vial contain-
ing the incubation medium. The weight of the specimen was determined by weighing the vial with contents on an analytical balance before
and after addition of the skin. The incubation medium contained streptomycin (200 ig), penicillin
(200 units), gentamicin sulfate (200 g) and
other additions as specified, in 1.9 ml of KrebsRinger phosphate buffer (pH 7.4). Sodium acetate-1-14C (2 oc in 0.17 mole) was then added in 0.1 ml of water and the mixture was incubated at 37°C in air with gentle shaking for 6 hours. As
in previous experiments (1), a drop of the in-
Materials Sodium acetate-1-14C (specific activity 29 mc/
mmole) was purchased from Nuclear Chicago. Reagents were of A.R. grade and solvents were distilled before use. Unmodified Squibb insulin was used in experiments where the in vitro effect of insulin was tested.
Skin Specimens
Skin biopsy specimens (4 and 6 mm punches) were taken under local anesthesia (Xylocaine) from the lower abdomen of 10 non-diabetic con* From the Departments of Dermatology and
Biochemistry, University of Miami School of Medicine, Miami, Florida.
the first biopsy specimen.
cubate was tested for bacterial growth by culturing on three media. No growth was detected after
48 hours from any of the incubates reported in this study. Details of the methods for hydrolysis of lipids, extraction and separation of the Nonsaponifiable and Acidic fractions were the same as reported in a previous communication (1). Briefly, the incubation was terminated with 2 g KOH and 15 ml ethanol. The mixture was kept at 60CC overnight to allow hydrolysis of fatty acid esters and disintegration of tissue. After acidifica-
tion to pH 1, the lipids were extracted four times with 100 ml of dicholomethane. Acetic acid-1-°'C was removed by the addition and evaporation of
non-radioactive acetic acid and benzene. The residue was subjected to paper chromatography
in the system of benzene :methanol :5 N NH4OH This study was supported in part by Career De- (2:1:2), and the amounts of '4C in the Nonvelopment Award 5-K-3AM-28,095 and Grant saponifiable and Acidic fractions were determined AM09497 from the Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service by a Vanguard Autoscanner 880 with automatic data system. The amounts of "C (dpm) incorand by the Dermatology Foundation of Miami. Abbreviations used in this paper are: ATP, porated into the two fractions per mg of skin were adenosine triphosphate; CoA, coensyme A; and then calculated. TPNH, reduced triphosphopyridine nucleotide. 1 DBI is a registered trademark of U.S. Vitamin Presented at the Twenty-seventh Annual Meeting of the Society for Investigative Dermatology, & Pharmaceutical Corp. for the oral hypoglycemic Inc., Chicago, Illinois, June 26, 1966.
agent N1-p-phenethylbiguanide HC1.
443
444
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY RESULTS
TABLE II Incorporation of 14C into the Non-saponifiable
Non-diabetic Controls
fraction bn skin of diabetic patients
The UC incorporated into the Non-saponifiable and Acidic fractions by skin specimens of the 10 non-diabetic subjects are shown in Table
'C Incorporated, dpm/mg skin Name
194; and from 900 to dence interval of 382 1900 in the Acidic fraction with a 95% confi293. There was no dence interval of 1511 apparent correlation between values of the Nonsaponifiable and Acidic fractions in each individual specimen.
therapy
Increase after therapy
77 215 468 256
127
—
119 (124%) 273 (140%) 246 (2460%)
51
—
—
84
284
173
In acidosis After insulin
I. The values varied from 125 to 888 in the Non-saponifiable fraction, with a 95% confi-
D.H. G.H.
24 96
C.J.
195 10
W.T. M.M. C.W.
Mean
53 (221%)
—
Diabetic Patients Standard devia70 162 104 Values obtained from specimens of the 6 pation tients in diabetic acidosis were significantly 166 lower than those of the non-diabetic controls. 95% confidence 84 73 254 258 173 interval After treatment with insulin, rises were noticed in both the Non-saponifiable and Acidic fractions of the 4 patients studied. Table II shows 194), these values the results of the Non-saponifiable fraction be- confidence interval, 382 fore and after insulin therapy. The values be- were significantly lower. After the insulin therfore therapy varied from 10 to 195, with a 95% apy, the values were from 77—468, and an inconfidence interval of 84 73. When compared crease was noticed in each case varying from with values of the non-diabetic controls (95% 124 to 2460%. A statistical analysis indicated that the increases after insulin therapy could be significant (95% confidence interval of the inTABLE I crease = 173 166). But because only four Incorporation of "C by skin of non-diabetic patients were available for the study, the evicontrol subjects dence derived from these data could not be con'C Incorporated, dpm/mg skin
sidered conclusive.
Nonsaponifiable
ACId1C
The results obtained in the Acidic fraction are compared in Table III. The values before insulin therapy ranged from 159 to 985 with a
Name
Si.
255
1096
95% confidence interval of 594
I.M.
163
1800
ll.B. Z.F.
636 555
1600 1000
G.M.
the high values in this group overlapped with the low values of the non-diabetic group, these values were statistically lower than those of the
125
1900
J.M.
145
1873
M.D. M.K. W.A.
888
1222
625
1825
275 150
1892
J.W.
non-diabetics (95% confidence interval, 1511
293). After the treatment with insulin, the values were from 592 to 1346, and there was an increase in each case varying from 24 to 272%
900
(97.5% 419
Mean
382
1511
Standard deviation
271
409
95% confidence inter-
val
382
194
1511
322. Although
293
confidence
interval of the increase =
252).
In vitro Effects of Glucose and Insulin The stimulatory effect of glucose on in vitro lipogenesis in human skin was previously observed by Griesemer and Thomas (12). The effects of glucose and insulin in enhancing lipo-
445
LIPID METABOLISM IN HUMAN SKIN II.
TABLE III Incorporation of 14C into Acidic fraction by skirt of diabetic patients '4C Incorporated, dpm/mg skin
Name
. n an OSIS
370 or 233% 620 or 125% 446 or 50% 241 or 24% — —
1070
419
glucose and insulin by the two non-diabetic subjects in fasting and in post-absorptive state was
332
158
therapy
496 900 985
M.M. C.W.
433
— —
Mean
594
Standarddeviation
307
529 1116 1346 1226
590
95% confi- 594 dence interval
subject had a meal. The experiment was repeated with specimens obtained before breakfast, from another non-diabetic subject (WA.), and the results are shown in Table VI. The increases with the addition of glucose and glucose plus insulin were much greater than in postprandial specimens studied in the previous experiment. The observed difference in the response to
After insulin
D.H. G.H. C.J. W.T.
159
The lack of response in skin specimens of nondiabetic subjects to stimulations by glucose and insulin was puzzling. It was later realized that the specimens studied were obtained after the
322 1070
Increase after therapy
528 419
252
genesis in rat skin were reported by Gaylor (13). The following experiment was designed to test if lipogenesis in the skin of diabetic patients could also be similarly enhanced. Biopsy specimens were taken from a patient (M.M.) who was in diabetic acidosis. The specimens were incubated with and without the addition of glucose (5mg/mi) and/or insulin (1 unit/mi). The results are shown in Table IV. Significant increases in lipogenic activity were observed with either glucose or insulin, and the greater increases were found with glucose and insulin
interesting. To explore further, the experiment was repeated with specimens from a third nondiabetic volunteer (J.W.). Skin biopsy specimens were taken after an eighteen hour fast and TABLE IV In vitro effects of glucose and insulin on lipo genesis
in, skin of a patient in diabetic acidosis Acidic
Non-saponifiable
Addition
dpm) Increase dpmf Increase over over mg mg
None (control) Glucose, 5 mg/mi
skin
control
skin
control
127 233
—
590 1426
—
106
400
Insulin, 1 unit/mi
273
1166
738
611
576
(98%)
(215%)
Glucose, 5 mg/mi + insulin, 1 unit/mi
836
(142%)
(83%) 2210
1620
(275%)
(481%)
combined.
For comparison, a similar experiment was carried out with specimens from a non-diabetic subject (G.M.) The results are shown in Table V. An increase in the Acidic fraction was noticed, but only slight or insignificant increases were noticed with the addition of glucose and insulin, although the level of glucose concentration (10mg/mi) in the incubation medium was twice that in the previous experiment. In several
other experiments with skin of non-diabetic subjects, the results indicated slight rises with glucose but the effect of insulin was irregular.
In several instances, a slight decrease in the incorporation of 14C was noticed with the addi-
TABLE V In vitro effects of glucose and insulin on lipo genesis
in skin of a non-diabetic subject Non-saponifiable
Addition
the acetate-1-14C.
'C in-
14C in-
skin
skin
corpo- Increase corpo- Increase rated over rated over dpm/mg control dpm/mg control
None (control) Glucose, 10 mg/mi
125 300
Insulin, 1 unit/mi
150
tion of insulin. This might indicate increased 10mg/mi + conversion of glucose to acetate which diluted Glucose, insulin, 1 unit/mi
Acidic
125
— 175
1900 2100
— 200
(140%) (11%) 25 2000 100 (20%) (5%) 0 2500 600
(32%)
446
THE JOTJRNAL OF INVESTIGATIVE DERMATOLOGY
TABLE VI In vitro effects of glucose and insulin on lipo genesis
in skin of a non-diabetic fasting subject Acidic
Non-saponifiable
'C
Addition
14c
incor- Increase incor- Increase over porated over poratd dpm/ control pm/ control ing skin nig skin
None (control)
—
240 480
Glucose, 10 mg/mi
Glucose, 10mg/mi + 600 insulin, 1 unit/mi
800 3300
240
— 2410
(100%) (271%) 360 835 1725 (150%) (88%)
again one hour after a full meal. Several levels of glucose were tested, and the results are presented in Figure 1. The data indicate that lipogenesis was depressed during fasting, and that the in vitro stimulatory effect of glucose reached the maximum at the concentration of 5 mg/ml. The stimulation was more pronounced on the tissue obtained during fasting than on the post-
prandial specimens; it is also of interest to
from acetate-1-14C was impaired in diabetic acidosis. Improvement of this defect was indicated after the acidosis was brought under control with insulin therapy. Also observed were the decrease in incorporation of '4C in the skin of non-diabetic subjects during fasting, and the increase in activity following a meal.
Impairment of lipogenesis under diabetic conditions has long been recognized (2), and has been substantiated by observations on liver preparations (3—6), adipose tissue (7, 8), aorta (9), skin (10) and uterus (11) of alloxan diabetic rats. Similarly, decreased synthesis of fatty acids has been observed in liver of starved rats (14—16). Recently, impairment of lipogenesis in blood elements of diabetic patients has been
reported (17). Thus the observations made in this study on human skin under diabetic and fasting conditions are in agreement with previous findings on other tissues. It is generally accepted that the primary bio-
chemical abnormality in diabetes mellitus is impaired glucose utilization due to lack of insulin. Consequently the body responds to meet the
note that even with the maximum stimulation energy needs by increasing mobilization and by glucose in vitro, fasting values were lower catabolism of fatty acids. Similar biochemical responses are noted during starvation, when the than post-prandial values. energy needs are met mainly by catabolism of DISCUSSION
It was observed in this study that the ability of human skin to incorporate 140 into lipids FAST/NC
3p00 4 2,500 C, E
fatty acids. The observed decrease in lipogenesis in the skin under these conditions is therefore a
manifestation of the homeostatic response in POSTPRAND/AL
3P00
Acidic Fraction • Nonsaponifiable
2,000
Fraction
-S..
E
a-
500
1,500
Acidic Fraction
a)
C
0 a-
00 C
lpo0.
000
500
500
0 0!
2.5
5
7.5
Glucose conc. mg /ml
0
Jo
• Nonsaponifiable Fraction
2.5
5
7.5
0
Glucose conc. mg /ml
Fia. 1. The effect of glucose in vitro. Glucose was added to the incubation medium at various concentrations. Stimulation of lipogenesis was noticed in skin specimens obtained from a fasting non-diabetic subject. The effect on post-prandial specimens from the same
subject was less pronounced.
LIPID METABOLISM IN HUMAN SKIN II.
which decreased carbohydrate utilization is com-
pensated by increased fatty acid catabolism
447
explored by various investigators, but has not been fully elucidated.
In studies with rat liver, Lynen et at. (18) and decreased lipid syntheses. Lipid syntheses are endergonic processes and it is conceivable demonstrated that acetyl-CoA carboxylase that such processes be curtailed when the energy which catalyzes the initial step in fatty acid supply from carbohydrate utilization is dimin- synthesis is the rate-limiting enzyme, and that ished. this enzyme is activated by citric acid and other The data presented in Table TV, Table VI intermediates of the citric acid cycle. This reguand Figure 1, show increased lipogenesis with latory mechanism also appears to exist in the the addition of glucose and/or insulin to the in- adipose tissue as evidenced by findings of Martin cubation medium. The stimulatory effects were and Vagelos (19). However, it was pointed out observed in skin specimens of diabetic patients that the loss of acetyl-CoA carboxylase and the and of fasting non-diabetics. Low levels of glu- lower citric acid level in rat liver under diacose utilization could be expected in these tis- betic or fasting conditions could not quantitasues, and the addition of glucose and insulin tively account for the observed decrease in fatty could theoretically increase glucose utilization. acid synthesis (15, 18). Numa €t at. (20) gave Since evidence is lacking for a direct effect of evidence that inhibition of acetyl-CoA carboxylinsulin on lipogenesis, the observed stimulation ase by long chain acyl-CoA derivatives may be of lipogenesis from acetate-1-14C by insulin is likely to be an effect secondary to increased an additional controlling mechanism, and higher carbohydrate utilization. It is interesting that levels of long-chain acyl-CoA derivatives were the stimulatory effects of glucose and insulin found in liver of fasted rats (21) and in liver were slight or insignificant on post-prandial and heart of alloxan-diabetic rats (22). The stimulatory effect of glycerol 3-phosphate specimens of non-diabetic subjects (Table V and Figure 1). It is reasonable to assume in this on fatty acid synthesis was reported (23, 24). case, that the skin might have been already This product of the glycolytic pathway is stimulated to the maximum in vivo, after in- needed for the synthesis of glycerides and phospholipids. The mechanism of its enhancement gestion of food prior to excision. An alternative interpretation of the decreased of fatty acid synthesis was proposed as to faincorporation of 'C into lipids from acetate-i- cilitate the transfer of acyl groups from acyl 140 may be that an increased endogenous acetate carrier protein so that more free acyl carrier pooi existed in skin of the diabetic and fasting protein becomes available for fatty acid synindividuals. A larger acetate pool would result thesis (24). in greater dilution of the acetate-1-14C and hence
Recently Wakil et at. (25) reported that
less amounts of 140 incorporated. The actual size of acetate pooi in skin under the various conditions is not known, but studies on liver of diabetic rats by Elwood and Van Bruggen (4) indicated a decreased rather than an increased acetate pool. If the pool size of acetate were to increase in acidosis and during fasting, the ob-
phosphorylated sugars, particularly fructose 1, 6-diphosphate can stimulate fatty acid syn-
served decrease of 14C incorporation must be in-
terpreted with caution as to indicate decrease of lipogenesis. On the other hand, the increased incorporation of 14C in the presence of added glucose and insulin (Tables IV, VI and Figure 1) undoubtedly indicated increase of lipogenesis.
thesis using enzyme preparations of pigeon liver
and E. coli. Considerations were given to the cofactors, ATP and TPNH which are needed in lipogenesis
and can be furnished by glucose utilization. The controversies concerning the supply of TPNH as a regulatory mechanism were discussed by Masoro (26). The role of co-enzymes and 02 in the control of lipogenesis in adipose
tissue was discussed by Flatt and Ball (27). From these studies with animal and microbial
The findings of this study thus corroborate systems, it appears that lipogenesis may be previous findings, suggesting that lipogenesis regulated by carbohydrate metabolism at multimay be in some way regulated by carbohydrate ple points. Whether any or all of these mechautilization. The exact mechanism by which this nisms, or still other mechanisms operate in huhomeostatic regulation is accomplished has been man skin can only be clarified by further studies.
448
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY SUMMARY
Skin biopsy specimens from 10 non-diabetic subjects and 6 patients in diabetic acidosis were compared with regard to their ability to incorporate 14C into lipids from acetate-1-'4C. Specimens from the diabetics were found to incorporate significantly lower amounts of 14C. After insulin therapy, skin specimens taken from 4 of these patients were again studied, and increased incorporation was noted in each case. Specimens of non-diabetic subjects in fasting incorporated less amounts of '4C into lipids than post-prandial specimens. Stimulatory effects on
lipogenesis by the addition of glucose and insulin in vitro were demonstrated in skin specimens of diabetics and fasting non-diabetics. The controlling mechanisms of lipogenesis in
animal and microbial systems were briefly reviewed.
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