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Comparison of basal metabolic activities and hormonal sensitivities between mesenteric and epididymal adipocytes
Camp. Biochem. Physiol. Vol. 82A, No. 2, pp. 311-383, Printed in Great Britain
0300-9629/85 S3.00+ 0.00 0 1985Pergamon Press Ltd
1985
COMPARISON OF BASAL METABOLIC ACTIVITIES AND HORMONAL SENSITIVITIES BETWEEN MESENTERIC AND EPIDIDYMAL ADIPOCYTES MASAHIRO
Department
of Physiological
TSHUCHIMOTO*,
Chemistry,
YUIGKO
TOKUMITW
Faculty of Pharmaceutical
and MICHO UI Sciences, Hokkaido University,
Sapporo, 060, Japan (Received
23 January
1985)
Abstract-l. The mesenteric adipose tissue, which is present in a considerable amount in the rat but had not been used for the experiments, contained a larger number of adipocytes than the epididymal adipose tissue per wet weight of the tissue. 2. The basal metabolic activities and the responses to epinephrine and insulin in the mesenteric adipocytes were essentially equal to those in the epididymal adipocytes. 3. The mesenteric adipocytes are useful for elucidation of metabolism in white adipose tissue.
MATERIALS
INTRODUaION
Rodbell (1964) was the first to prepare free fat cells from rat epididymal adipose tissues by the collagenase method. Compared with the slice of the adipose tissue, isolated fat cells prepared from adipose tissue have the following advantages. The cells separated easily from other kinds of cells contained in the adipose tissue by centrifugation are composed purely of adipocytes. The adipocyte preparations exhibit generally higher hormone sensitivity than the tissue slices. Because of these advantages, the cell preparations have been widely used in investigations on the metabolic responses of glucose and lipids to various hormones such as epinephrine, ACTH, PGE,, somatostatin or growth hormone. In general, the adipocyte preparations are obtained from the epididymal adipose tissue. The adipocytes are also present in the mesenteric adipose tissue, which is a white adipose tissue. Much less attention has, however, been paid on the mesenteric adipose tissue, no matter how an amount of this tissue present in one rat is compared to that of the epididymal adipose tissue. Hence no data are available for the metabolic activities and the hormonal sensitivities in the adipocytes prepared from the mesenteric adipose tissue. Itaya and Ui (1964) found that the mesenteric adipose tissue slices showed higher metabolic responses to serotonin than the epididymal adipose tissue. Thus, the cell preparations from the mesenteric adipose tissue must be a useful system for exploring the mechanism involved in regulation of adipose tissue. In the present paper, we prepared the adipocytes from the mesenteric adipose tissue and compared their basal metabolic activities and hormonal sensitivities with the epididymal adipocytes which are known to present exquisite sensitivities to various hormones.
Preparation
of adipocytes
The adipocytes were prepared from the mesenteric and the epididymal adipose tissue of rats by the collagenase method of Rodbell (1964) with minor modifications. In brief, the minced adipose tissue was digested with collagenase. (2 mgjml) for 5&55 min (mesenteric adipose tissue) or 40-45 min (enididvmal adioose tissue) at 37°C in Ktebs-Ringer b&&&ate buffer (PH 7.4) ‘containing 2% bovine serum albumin (BSA) in an atmosphere of 95% 0?-5% CO,. Adipocytes obtained after the digestion were washed three times with the buffer. The cells suspended in the buffer were prcincubated for 1 hr at 37°C before incubation for measurement of metabolic activities. Incubation of adipose tissue slices and meawrements of the metabolic activities
adipocytes
for
A quarter of adipose tissue slices from a rat or one-tenth of adipocytes prepared from a rat was used for one incubation. To each flask was added 1.5ml of Krebs-Ringer bicarbonate buffer (pH 7.4) containing 2% BSA and 5 mM i4C(U)-glucose. Where indicated, pahnitate bound to albumin by the method of Spector and Hoak (1969) was added. Incubation was carried out with shaking at 37°C for 1 hr in an atmosphere of 95% 0,-5x CO,. The reaction was terminated by putting the flask into an ice-water bath to measure incorporation of “C(U)-glucose into glycogen, lactate production, FFA release and glycerol release. The incubation medium for measurement of glucose oxidation was acidified with HCIO, (final concentration was 0.3 N). ‘TO, liberated was trapped by hyamine in a well hung from a rubber cap. Radioactivity was determined in a liquid scintillation counter. “C(U>glucose incorporated into glycogen and the amounts of lactate, FFA and glycerol were determined by the methods of Yajima and I%-(1974), Barker and Summerson (1941), Tokumitsu et al. (1977) and Pinter et al. (1967), respectively.
The flasks containing 377
CBP82,2A~I
Male rats of the Wistarderived strain, weighing 130-190 g, were used and given free access to food and water.
Incubation of adipocytes 3’,5’-cyclic monophosphate
*Present address: Pharmacological Research Department, Teijin Institute for Bio-medical Research, Teijin Limited, Tokyo 191, Japan.
AND METHOD!?+
Animals
for measurement (CAMP)
of aaknosine
0.3 ml of the medium were incu-
378
MASAH~RO TSIRJCH~MOTO et al.
These effects are biphasic with increasing epinephrine concentration, which are consistent with the data reported with the epididymal adipocytes (Allen et al., 1969; Atlen and MacLaren, 1970; Allen and Beck, 1972; Kono and Barham, 1973; Luzio et al., 1974; Miller and Allen, 1973). The stimulating effects of epinephrine on the lipolytic activities of the epiMeasurement of DNA content in the adipose tissues DNA was extracted from the tissues by the method of didymal adipocytes are also higher than those of Denton et al. (1966) and determined by the method of mesenteric adipocytes. Figure 4 shows the effects of an c1-or a jl -adrenergic antagonist on lipolysis and Burton (1956). CAMP formation stimulated by epinephrine in both Reagenrs types of adipocytes. The concentration of epinephrine Reagents used for radioimmunoassay of CAMP were employed is 4pM at which lipolytic activities reach kindly supplied from Yamasa Shoyu Co. Sources of other the first peak of curves shown in Fig. 3. Propranolol reagents were: coilagenase (Type I), Worthington Biochemi(~-adrener~c antagonist) inhibits completely the cal-co.; insulin, Eh Lilly 8r Co.; epineph~ne, E. Merck, activities of glycerol release, FFA release and CAMP Darmstadt; phenoxybenxamine, Nakarai Chemical, Ltd; propranolol,- Otsuka Pharmaceutical Co.; i4C(U)-glucose formation stimulated by epinephrine in the mesenand 57COC12 used for FFA determination, New England teric adipocytes as well as in the epididymal adipo(a-adrenergic antagonist) Nuclear Corp.; DNA (Type I), BSA (Fraction V) and cytes. Phenoxybenzamine enzymes, co-factors and substrates for the determination of exhibits no inhibitory effect in both types of adipoglycerol, Sigma Chemical Co. cytes. These results indicate that the activation of lipolysis by epinephrine is mediated through /Iadrenergic receptor and the inhibition of the lipolytic RESULTS action by propranolol is due to a decrease in intraComparison of FFA uptake, glycerol release and cellular CAMP concentration. The dose-response bated for 4 min. Other conditions for incubation were the same as those for measurement of the metabolites described above. The incubation was terminated by the addition of HCI (final ~nc~tration was 0.1 N). CAMP was determined by the radioimm~~h~i~l method of Honma ei al. (1977).
factatepro~cfion cytes
in mesenteric and epididymu~ adipo-
Figure 1 shows the time course of FFA uptake, glycerol release and lactate production in the presence of palmitate in the mesenteric adipose tissue slices and in the epididymal adipose tissue slices. The figure shows that these activities of the mesenteric adipose tissue slices are higher than those of epididymal adipose tissue slices when the activities are expressed per wet weight. However, the cell number and DNA content per wet weight were different between both tissues. That is, the cell number per g wet weight is 14.5 f 1.03 x lo6 cells for the mesenteric adipose tissue and 8.9 + 0.54 x lo6 cells for the epididymal adipose tissue. The DNA content per g wet weight is 555 + 51.6 ,ug for the mesenteric adipose tissue and 124 1: 20.1 pg for the epididymal adipose tissue. In the experiments for Fig. 2, the time courses of FFA uptake, glycerol release and lactate production were observed with adipocytes prepared from both types of tissues. Figure 2 shows these activities per lo6 cells, indicating that the activities per cell in both types of adipocytes are almost equal to each other. Comparison of responses to epinephrine in mesenteric and epididymal adipocytes In the mesenteric adipocytes as well as the epididymal adipocytes, epinephrine stimulates glucose oxidation (Fig. 3A) and lactate production (Fig. 33) in a concentration-dependent manner. The effects of epinephrine on these activities in the epididymal adipocytes are higher than those in the mesenteric adipocytes. Epin~~~e enhances in~~oration of “C(U)glucose into glycogen at low concentrations (0.03-0.4pM), which is decreased with a further increase in epinephrine concentration (Fig. 3C). No appreciable difference is seen between the activities of both types of adipocytes. Epinephrine stimulates FFA release (Fig. 3D) and glycerol release (Fig. 3E).
Time (hours)
Fig. 1. Comparison of the basal metabolic activities between mesenteric and epididymal adipose tissue slices. (A) FFA release; (B) glycerol reiease; (C) lactate production. Tissue slices were incubated in the medium containing plamitate. See Materials and Methods for other details. A: Mesenteric adipose tissue; 0: epididymal adipose tissue. Each point represents the mean for metabolic activities per g tissue (number of observations, 5).
Metabolic study of mesenteric adipocytes
379
1973). The concentration-response curves for both types of adipocytes are identical to each other. DISCUSSION
-3.oJ 0.50-l
Time fhars)
Fig. 2. Comparison of the basal metabolic activities between mesenteric and epididymal adipocytes. (A) FFA release; (B) glycerol release; (C) lactate production. Adipocytes were incubated in the medium c&aining paImitate. -See Materials and Methods for other details. A: Mesenteric adipocytes; 0: epididymal adipocytes. Each point represents the mean for metabolic activities per lo6 cells (number of observations 3).
relationships for propranolol-induced inhibition of lipolysis and CAMP formation in the mesenteric adipocytes are similar to those observed in the epididymal adipocytes. Similar results were obtained with 275pM epinephrine (the second peak of the curve shown in Fig. 3) (data not shown). The effects of propranolol on epinephrina stimulated glucose oxidation and lactate production in the mesenteric adipocytes are shown in Fig. 5. Propranolol inhibits dose-dependently the stimulating effects of epineph~ne, while phenoxybenzamine does not in practice inhibt the epinephrinestimulated effects. Comparison of responses to insulin in mesenteric and epididymal adipocy tes
Figure 6 shows effects of insulin on FFA release and glycerol release stimulated by 1.4 FM epinephrine. Both the FFA and the glycerol release stimulated by epinephrine are inhibited by a low concentration of insulin {l-lo ,uU/ml). A further increase in insulin con~ntration leads to an activation of lipolysis. These results are consistent with those obtained with the epididymal adipocytes (Kono and Barham,
The present results show that the mesenteric adipose tissue slices exhibit higher metabolic activities than the epididymal adipose tissue slices, which is consistent with previous data in our laboratory (Itaya and Ui, 1964). However, these results do not imply that the activities of the mesenteric adipocytes per cell are higher than those of the epididymal adipocytes. That is, the cell number and the DNA content of the mesenteric adipose tissue per g wet weight were 1.6 times (1.45 x lo’/&9 x 106)and 4.5 times (555 pg/ 124pg) those of the epididymal adipose tissue, respectively. The reason for the higher activities of the mesenteric adipose tissue is because a larger number of adipocytes per wet weight are contained in this tissue. Thus the mesenteric adipocyte has the same basal metabolic activities as the epididymal adipocytes. In addition, the present results show that the mesenteric adipose tissue contains a larger number of cells besides adipocytes than the epididymal adipose tissue as indicated by the measurement of the DNA content. The mesenteric adipocytes have the responses to epinephrine on metabolic activities as the epididymal adipocytes. That is, the effects of epinephrine on “C(U)-glucose into glycogen is identical in both types of cells, but the effects of epinephrine on glucose oxidation, lactate production, FFA release and glycerol release of the mesenteric adipocytes are significantly lower than those of the epididymal adipocytes (Fig. 3). An a-adrenergic antagonist has practically no effect on FFA release, glycerol release and CAMP formation stimulated by epinephrine in both types of adipocytes. A j3-adrenergic antagonist suppresses the activities of both types of adipocytes to a similar extent. In general, the inhibitions of FFA release, glycerol release and CAMP fo~ation by B-adrenergic antagonist are considered to be brought about by changes in CAMP concentration mediated through a /?-adrenergic receptor. Some reports, however, emphasized that lipolytic responses in the epididymal adipocytes are more complex than are responses simply in proportion to intracellular CAMP concentration (Lang et al., 1976; Siddle and Hales, 1974; Schimmel et al., 1984). The present results support the hypothesis that intracellular CAMP may play an important role in, or is at least one of the factors controlling, lipolysis in adipocytes. Many reports have been published on the stimulating effects of catecholamines on glucose uptake in the rat epididymal adipocytes. Luzio et al. (1974) reported that an u-adrenergic antagonist, phenoxybenzamine, inhibited epineph~ne-stimulate glucose uptake in the rat epididymal adipocytes. In contrast, Kashiwagi et al. (1983) observed that a p-adrenergic agonist, L-isoproterenol stimulated the basal 3-0methylglucose transport in both the rat epididymal and the human subcutaneous adipocytes. Ludvigsen et al. (1980) also observed that a p-adrenergic antagonist, propranoiol, inhibited epinephrineor L-isoproterenol-stimulated glucose transport. On the
MASAHIRO TSHUCIUMOTO et al.
380
0
B) 0.6
160
K 1
(El
A Y
OAA
0
0
d- 0.1
0
1
O$
10 1001000 Epinephrine (@4)
Fig. 3. Comparison of the responses to epinephrine between mesenteric and epididymal adipocytes. (A) Glucose oxidation, expressed by the amount of the glucose oxidized to C02; (B) lactate production; (C) W(U)-glucose incorporation into glycogen, expressed by the amount of the glucose incorporated into glycogen; (D) FFA release; (E) glycerol release. gee Materials and Methods for experimental details. A: Mesenteric adipocytes; 0: epididymal adipocytes. Each point represents the mean from two observations.
other
hand,
Mills
et
al.
(1984)
suggested
that
forskolin, which stimulates CAMP accumulation, inhibited the glucose metabolism via a mechanism independent of CAMP in the rat epididymal adipocytes. The present results show that glucose oxidation and lactate production are inhibited by propranolol in the mesenteric adipocytes (Fig. 5). Corrected ZDm values of propranolol calculated according to equation (1) are shown in Table 1. All of the corrected
ID, values (0.05-0.26pM) from each other.
corrected IDso=
are not greatly different 14, 1 + [agonist].
(1)
EDu, These results suggest that lipolysis, glucose oxidation and lactate production are mediated by the same
381
Metabolic study of mesenteric adipocytes
and a /I-antagonist on lipolysis are the same in both types of adipocytes. In addition, the responses of the mesenteric and the epididymal adipocytes to insulin are identical in respect to its antagonism against lipolytic responses stimulated by epinephrine (Fig. 6). Thus the responses of the mesenteric adipocytes are essentially similar to those of the epididymal adipocytes. An amount of the mesenteric adipose tissue present in one rat is comparable to that of the epididymal adipose tissue, and the content of adipocytes in the mesenteric adipose tissue is higher than that in the epididymal adipose tissue. Thus the mesenteric adipocytes are useful for the elucidation of metabolism in a white adipose tissue.
150-
SUMMARY
Basal metabolic activities (free fatty acid uptake, glycerol release and lactate production) of the mesenteric adipose tissue slices were found to be higher
rE
0
I
0.1
I
1 Antagonist
I
10 (PM)
1
,
100
1000
Fig. 4. Effects of an CL-or a /?-adrenergic antagonist on epinephrine-stimulated lipolysis in mesenteric and epididymal adipocytes. (A) FFA release; (B) glycerol release;
(C) CAMP fonnation. The values show % of the control (epinephrine alone) calculated from the mean from two observations. Control values in the mesenteric and the epididymal adipocytes were 1.38 and 2.58 pEq/106 cells (FFA release), 1.98 and 3.81 pmol/106 cells (glycerol release), and 41.2 and 60.5 pmol/106 cells (CAMP formation), respectively. The epinephrine concentration employed was 5.5 PM, at which lipolysis reached the first peak (see Fig. 3). Closed symbols: an a-adrenergic antagonist, phenoxybenzamine; open symbols: a p-adrenergic antagonist, propranolol. AA: Mesenteric adipocytes; 00: epididymal adipocytes.
b-adrenergic receptor. This supports an idea proposed previously that glucose transport is mediated by B-adrenergic receptor. Recently, it was found that the stimulatory effect of epinephrine on glucose uptake was associated with the presence of adenosine released from adipocytes. That is, the effect of epinephrine which mimicked that of insulin-stimulated glucose transport was diminished and furthermore the insulin-stimulated transport itself was suppressed by epinephrine when adenosine was removed from the incubation medium by the addition of adenosine deaminase (Green, 1983). In addition, this suppressive effect of epinephrine was blocked by the /I-adrenergic antagonist, propranolol. These suppressive effects of epinephrine in the absence of adenosine were also observed in the present studies (data not shown). As described above, the effects of an a-antagonist
odt
I
1 10 100 Antagonist ( pM )
<
1000
Fig. 5. Effects of an a- and a /?-adrenergic antagonist on epinephrine-stimulated glucose oxidation and lactate production in mesenteric adipocytes. (A) Glucose oxidation, expressed by the amount of glucose oxidized to CO,; (B) lactate production. The values show “/, of the control (epinephrine alone) calculated from the-mean from two observations. Control value is 0.35 flmol/106 cells (glucose oxidation) and 0.34 ymol/106 cells (lactate product&) respectively. Epinephrine concentration employed was 100 phi. Closed symbols: an a-adrenergic antagonist, phenoxybenxamine, open symbols: a /I-adrenergic antagonist, propranolol.
MASAHIROTFHICH~MOTO et al.
382
adrenergic antagonists on epinephrine-stimulated activities (lipolysis, glucose oxidation and lactate production) or the inhibitory effects of insulin on epineph~n5stimuIated lipolysis were similar in both
types of adipocytes. Therefore, it can be considered that responses of the mesenteric adipocytes to epinephrine and insulin are essentially as high as those of the epididymal adipocytes. The total amount of mesenteric adipocytes present in one rat was larger than that of the epidid~al adipocytes. Hence the mesenteric adipocytes are useful for elucidation of metabolism in a white adipose tissue.
REFERENCES
OJ 6’
i
io 100 1000 Insulin (pU/ml)
,
I
10000
Fig. 6. Comparison of the responses to insulin between mesenteric and epididymai adipocytes. Antiiipoiytic effect of insulin against epinephrine-stimulated iipoiysis was evaiuated. (A) FFA release; (B) glycerol release. The values show % of the control (epinephrine alone) calculated from the mean from two observations. Control values in mesentet-ic and epidid~al adipocytes were 0.34 and 0.62 pEq/i06 cells (FFA release) and 0.68 and O.SSrmoi/io6 cells (glycerol release), respectively. The epinephrine concentration employed was 1.4 p M. A Mesenteric adipocytes; 0 epididymai adipocytes.
Table 1. Affinity of a fi-adrenergic antagonist, propranolol, receptor of mesenteric adipocytes Epinephrine concentration
ID,
&I
0.22
5.5 275 5.5 275 215
0.4 40 0.7 6.5 259
0.09 0.25 0.11 0.26 0.20
0.22
21.5
65
0.05
I.7
release
Glycerol release Glucose oxidation Lactate production
Corrected
WW
E&O FFA ____
for the
1.1
ED,,: The concentration of epinephrine required for obtaining 50% of the maximal effect. ID,: The concentration of propranolol required for a 50% inhibition of the effect of epinephrine. Corrected ID, was calculated with the equation (1).
than those of epididymal adipose tissue slices when the activities were expressed per g wet weight of tissue. These activities of the mesenteric adipocytes per cell were, however, equal to those of the epididymal adipocytes because the cell number in the former tissue was larger than that in the latter. The ratio of DNA content in the mesenteric adipose tissue to that in the epididymal adipose tissue per g wet weight was much higher (about three times) than the ratio of cell number in both types of adipocytes. This suggested that other kinds of cells were contained
much more in the mesenteric adipose tissue than in the epididymal adipose tissue. Responses to epinephrine on glucose oxidation, lactate production and lipofysis in the mesenteric adipocytes were lower than those in the epidid~al adipocytes. The effects of
Alien D. O., Hillman C. C. and Ashmore J. (1969) Studies on a biphasic lipolytic response to catechoiamine in
isolated fat ceils. Biochem. Pharmac. 18, 2233-2240. Alien D. 0. and MacLaren P. J. (1970) Effect of potassium ion, theophyiiine and propranoioi on the biphasic response to some catechoi~ines. Biochem. Pharmac. t9, 2569-2578. Alien D. 0. and Beck R. R. (1972) Alterations in lipolysis, adenyiate cyciase and adenosine 3’,5’-monophosphate levels in isolated fat ceils following adrenaiectomy. Endocrinology 91, 504-510. Barker S. B. and Su~e~on W. H. (1941) The ~lo~metric determination of lactic acid in biological material. J. biol. Chem. 138, 535-554. Burton K. (1956) A study of the conditions and mechanism of the diphenylamine reaction for the coiorimetric estimation of deoxyribonucleic acid. Biochem. J. 62,315323. Denton R. M., Yorke R. E. and Randle P. J. (1966) Measurement of concentrations of metaboiite in adipose tissue and effects of insulin, ailoxandiabetes and adrenaline. Biochem. J. 100, 407-419. Green A. (1983) Catechoiamines inhibit insulin-stimulated glucose transport in adipocytes, in the presence of adenosine deaminase. FEBS Lerr. 152, 261-264. Honma M., Satoh T., Takezawa J. and Ui M. (1977) An ultrasensitive method for the simultaneous determination of cyclic AMP and cyclic GMP in small-vohune samples from blood and tissue. Biochem. Med. 18, 257-213. Itaya K. and Wi M. (1964) The inhibitory action of serotonin on free fatty acid utilization by rat mesenteric adipose tissue. Biochim. biophys. Aeta 84, 604+506. Kashiwagi A., Huecksteadt T. P. and Foley J. E. (1983) Regulation of glucose transport by cAMPstimulators via three different mechanisms in rat and human adipocytes. J. biol. Chem. 288, 13685-13692. Kono T. and Barham F. W. (1973) Effects of insulin on the levels of adenosine 3’, ~-monophospha~ and Iipoiysis in isolated rat epididymai fat c&is. J. biol. Chem. 248, 1417-7426. Lang U., Fauchere J. L., Pelican G. M., Karlaganis G. and Schwyzer R. (1976) Hormone receptor interactions, adrenocorticotrophin (7-24) octadecapptide stimulates adipocyte membrane adenyiate cyclase without causing tipoiysis. FEBS Mt. 66, 246-249. Ludvigsen C., Jarett L. and McDonald J. M. (1980) The characterization of catechoiamine stimulation of gh~cose transport by rat adipocytes and isolated plasma membranes. Endocrinology 106, 786790. Luzio J. P., Jones R. C., Siddie K. and Hales C. N. (1974) Dissociation of the effect of adrenaline on glucose uptake from that on adenosine cyclic 3’, 5’-monophosphate ieveis and on lipolysis in rat-isolated fat cells. Biochim. biophys. Acta 362, 29-36. Miller E. A. and Allen D. 0. (1973) Hormone-stimulated iipoiysis in isolated fat ceils from “young” and “old” rats. J. Lipid Res. 14, 331-336.
Metabolic study of mesenteric adipocytes Mills I., Moreno F. J. and Fain J. N. (1984) Forskolin inhibition of glucose metabolism in rat adipocytes independent of adenosine 3’,5’-monophosphate accumulation and lipolysis. Endocrinology 115, 10661069. Pinter J. K., Hayashi J. A. and Watson J. A. (1967) Enzymatic assay of glycerol, dihydroxyacetone and glyceraldehyde. Archs B&hem. Biophys. 121, 404-414. Rodbell M. (1964) Metabolism of isolated fat cells. J. 6iol. Chem.
239,
37S380.
Schimmel R. J. (1984) Stimulation of CAMP accumulation and lipolysis in hamster adipocytes with forskolin. Am. J. Physiol. 246, C63-C68.
383
Siddle K. and Hales C. N. (1974) The action of local anesthetics on lipolysis and on adenosine 3’. S-cyclic monophosphate content in isolated rat fat cells. Biochem. J. 142, 345-351. Spector A. A. and Hoak J. C. (1969) An improved method for the addition of long-chain free fatty acid to protein solutions. Analyt. Biochem. 32, 297-302. Tokumitsu Y., Kondoh T. and Ui M. (1977) Radiochemical assay of free fatty acids in blood using s7Co as a tracer. Analyt. Biochem. 81, 488490. Yajima M. and Ui M. (1974) Gluconeogenesis in epinephtine-induced hyperglycemia. Am. J. Physiol. 227, l-8.
0300-9629/85 S3.00+ 0.00 0 1985Pergamon Press Ltd
1985
COMPARISON OF BASAL METABOLIC ACTIVITIES AND HORMONAL SENSITIVITIES BETWEEN MESENTERIC AND EPIDIDYMAL ADIPOCYTES MASAHIRO
Department
of Physiological
TSHUCHIMOTO*,
Chemistry,
YUIGKO
TOKUMITW
Faculty of Pharmaceutical
and MICHO UI Sciences, Hokkaido University,
Sapporo, 060, Japan (Received
23 January
1985)
Abstract-l. The mesenteric adipose tissue, which is present in a considerable amount in the rat but had not been used for the experiments, contained a larger number of adipocytes than the epididymal adipose tissue per wet weight of the tissue. 2. The basal metabolic activities and the responses to epinephrine and insulin in the mesenteric adipocytes were essentially equal to those in the epididymal adipocytes. 3. The mesenteric adipocytes are useful for elucidation of metabolism in white adipose tissue.
MATERIALS
INTRODUaION
Rodbell (1964) was the first to prepare free fat cells from rat epididymal adipose tissues by the collagenase method. Compared with the slice of the adipose tissue, isolated fat cells prepared from adipose tissue have the following advantages. The cells separated easily from other kinds of cells contained in the adipose tissue by centrifugation are composed purely of adipocytes. The adipocyte preparations exhibit generally higher hormone sensitivity than the tissue slices. Because of these advantages, the cell preparations have been widely used in investigations on the metabolic responses of glucose and lipids to various hormones such as epinephrine, ACTH, PGE,, somatostatin or growth hormone. In general, the adipocyte preparations are obtained from the epididymal adipose tissue. The adipocytes are also present in the mesenteric adipose tissue, which is a white adipose tissue. Much less attention has, however, been paid on the mesenteric adipose tissue, no matter how an amount of this tissue present in one rat is compared to that of the epididymal adipose tissue. Hence no data are available for the metabolic activities and the hormonal sensitivities in the adipocytes prepared from the mesenteric adipose tissue. Itaya and Ui (1964) found that the mesenteric adipose tissue slices showed higher metabolic responses to serotonin than the epididymal adipose tissue. Thus, the cell preparations from the mesenteric adipose tissue must be a useful system for exploring the mechanism involved in regulation of adipose tissue. In the present paper, we prepared the adipocytes from the mesenteric adipose tissue and compared their basal metabolic activities and hormonal sensitivities with the epididymal adipocytes which are known to present exquisite sensitivities to various hormones.
Preparation
of adipocytes
The adipocytes were prepared from the mesenteric and the epididymal adipose tissue of rats by the collagenase method of Rodbell (1964) with minor modifications. In brief, the minced adipose tissue was digested with collagenase. (2 mgjml) for 5&55 min (mesenteric adipose tissue) or 40-45 min (enididvmal adioose tissue) at 37°C in Ktebs-Ringer b&&&ate buffer (PH 7.4) ‘containing 2% bovine serum albumin (BSA) in an atmosphere of 95% 0?-5% CO,. Adipocytes obtained after the digestion were washed three times with the buffer. The cells suspended in the buffer were prcincubated for 1 hr at 37°C before incubation for measurement of metabolic activities. Incubation of adipose tissue slices and meawrements of the metabolic activities
adipocytes
for
A quarter of adipose tissue slices from a rat or one-tenth of adipocytes prepared from a rat was used for one incubation. To each flask was added 1.5ml of Krebs-Ringer bicarbonate buffer (pH 7.4) containing 2% BSA and 5 mM i4C(U)-glucose. Where indicated, pahnitate bound to albumin by the method of Spector and Hoak (1969) was added. Incubation was carried out with shaking at 37°C for 1 hr in an atmosphere of 95% 0,-5x CO,. The reaction was terminated by putting the flask into an ice-water bath to measure incorporation of “C(U)-glucose into glycogen, lactate production, FFA release and glycerol release. The incubation medium for measurement of glucose oxidation was acidified with HCIO, (final concentration was 0.3 N). ‘TO, liberated was trapped by hyamine in a well hung from a rubber cap. Radioactivity was determined in a liquid scintillation counter. “C(U>glucose incorporated into glycogen and the amounts of lactate, FFA and glycerol were determined by the methods of Yajima and I%-(1974), Barker and Summerson (1941), Tokumitsu et al. (1977) and Pinter et al. (1967), respectively.
The flasks containing 377
CBP82,2A~I
Male rats of the Wistarderived strain, weighing 130-190 g, were used and given free access to food and water.
Incubation of adipocytes 3’,5’-cyclic monophosphate
*Present address: Pharmacological Research Department, Teijin Institute for Bio-medical Research, Teijin Limited, Tokyo 191, Japan.
AND METHOD!?+
Animals
for measurement (CAMP)
of aaknosine
0.3 ml of the medium were incu-
378
MASAH~RO TSIRJCH~MOTO et al.
These effects are biphasic with increasing epinephrine concentration, which are consistent with the data reported with the epididymal adipocytes (Allen et al., 1969; Atlen and MacLaren, 1970; Allen and Beck, 1972; Kono and Barham, 1973; Luzio et al., 1974; Miller and Allen, 1973). The stimulating effects of epinephrine on the lipolytic activities of the epiMeasurement of DNA content in the adipose tissues DNA was extracted from the tissues by the method of didymal adipocytes are also higher than those of Denton et al. (1966) and determined by the method of mesenteric adipocytes. Figure 4 shows the effects of an c1-or a jl -adrenergic antagonist on lipolysis and Burton (1956). CAMP formation stimulated by epinephrine in both Reagenrs types of adipocytes. The concentration of epinephrine Reagents used for radioimmunoassay of CAMP were employed is 4pM at which lipolytic activities reach kindly supplied from Yamasa Shoyu Co. Sources of other the first peak of curves shown in Fig. 3. Propranolol reagents were: coilagenase (Type I), Worthington Biochemi(~-adrener~c antagonist) inhibits completely the cal-co.; insulin, Eh Lilly 8r Co.; epineph~ne, E. Merck, activities of glycerol release, FFA release and CAMP Darmstadt; phenoxybenxamine, Nakarai Chemical, Ltd; propranolol,- Otsuka Pharmaceutical Co.; i4C(U)-glucose formation stimulated by epinephrine in the mesenand 57COC12 used for FFA determination, New England teric adipocytes as well as in the epididymal adipo(a-adrenergic antagonist) Nuclear Corp.; DNA (Type I), BSA (Fraction V) and cytes. Phenoxybenzamine enzymes, co-factors and substrates for the determination of exhibits no inhibitory effect in both types of adipoglycerol, Sigma Chemical Co. cytes. These results indicate that the activation of lipolysis by epinephrine is mediated through /Iadrenergic receptor and the inhibition of the lipolytic RESULTS action by propranolol is due to a decrease in intraComparison of FFA uptake, glycerol release and cellular CAMP concentration. The dose-response bated for 4 min. Other conditions for incubation were the same as those for measurement of the metabolites described above. The incubation was terminated by the addition of HCI (final ~nc~tration was 0.1 N). CAMP was determined by the radioimm~~h~i~l method of Honma ei al. (1977).
factatepro~cfion cytes
in mesenteric and epididymu~ adipo-
Figure 1 shows the time course of FFA uptake, glycerol release and lactate production in the presence of palmitate in the mesenteric adipose tissue slices and in the epididymal adipose tissue slices. The figure shows that these activities of the mesenteric adipose tissue slices are higher than those of epididymal adipose tissue slices when the activities are expressed per wet weight. However, the cell number and DNA content per wet weight were different between both tissues. That is, the cell number per g wet weight is 14.5 f 1.03 x lo6 cells for the mesenteric adipose tissue and 8.9 + 0.54 x lo6 cells for the epididymal adipose tissue. The DNA content per g wet weight is 555 + 51.6 ,ug for the mesenteric adipose tissue and 124 1: 20.1 pg for the epididymal adipose tissue. In the experiments for Fig. 2, the time courses of FFA uptake, glycerol release and lactate production were observed with adipocytes prepared from both types of tissues. Figure 2 shows these activities per lo6 cells, indicating that the activities per cell in both types of adipocytes are almost equal to each other. Comparison of responses to epinephrine in mesenteric and epididymal adipocytes In the mesenteric adipocytes as well as the epididymal adipocytes, epinephrine stimulates glucose oxidation (Fig. 3A) and lactate production (Fig. 33) in a concentration-dependent manner. The effects of epinephrine on these activities in the epididymal adipocytes are higher than those in the mesenteric adipocytes. Epin~~~e enhances in~~oration of “C(U)glucose into glycogen at low concentrations (0.03-0.4pM), which is decreased with a further increase in epinephrine concentration (Fig. 3C). No appreciable difference is seen between the activities of both types of adipocytes. Epinephrine stimulates FFA release (Fig. 3D) and glycerol release (Fig. 3E).
Time (hours)
Fig. 1. Comparison of the basal metabolic activities between mesenteric and epididymal adipose tissue slices. (A) FFA release; (B) glycerol reiease; (C) lactate production. Tissue slices were incubated in the medium containing plamitate. See Materials and Methods for other details. A: Mesenteric adipose tissue; 0: epididymal adipose tissue. Each point represents the mean for metabolic activities per g tissue (number of observations, 5).
Metabolic study of mesenteric adipocytes
379
1973). The concentration-response curves for both types of adipocytes are identical to each other. DISCUSSION
-3.oJ 0.50-l
Time fhars)
Fig. 2. Comparison of the basal metabolic activities between mesenteric and epididymal adipocytes. (A) FFA release; (B) glycerol release; (C) lactate production. Adipocytes were incubated in the medium c&aining paImitate. -See Materials and Methods for other details. A: Mesenteric adipocytes; 0: epididymal adipocytes. Each point represents the mean for metabolic activities per lo6 cells (number of observations 3).
relationships for propranolol-induced inhibition of lipolysis and CAMP formation in the mesenteric adipocytes are similar to those observed in the epididymal adipocytes. Similar results were obtained with 275pM epinephrine (the second peak of the curve shown in Fig. 3) (data not shown). The effects of propranolol on epinephrina stimulated glucose oxidation and lactate production in the mesenteric adipocytes are shown in Fig. 5. Propranolol inhibits dose-dependently the stimulating effects of epineph~ne, while phenoxybenzamine does not in practice inhibt the epinephrinestimulated effects. Comparison of responses to insulin in mesenteric and epididymal adipocy tes
Figure 6 shows effects of insulin on FFA release and glycerol release stimulated by 1.4 FM epinephrine. Both the FFA and the glycerol release stimulated by epinephrine are inhibited by a low concentration of insulin {l-lo ,uU/ml). A further increase in insulin con~ntration leads to an activation of lipolysis. These results are consistent with those obtained with the epididymal adipocytes (Kono and Barham,
The present results show that the mesenteric adipose tissue slices exhibit higher metabolic activities than the epididymal adipose tissue slices, which is consistent with previous data in our laboratory (Itaya and Ui, 1964). However, these results do not imply that the activities of the mesenteric adipocytes per cell are higher than those of the epididymal adipocytes. That is, the cell number and the DNA content of the mesenteric adipose tissue per g wet weight were 1.6 times (1.45 x lo’/&9 x 106)and 4.5 times (555 pg/ 124pg) those of the epididymal adipose tissue, respectively. The reason for the higher activities of the mesenteric adipose tissue is because a larger number of adipocytes per wet weight are contained in this tissue. Thus the mesenteric adipocyte has the same basal metabolic activities as the epididymal adipocytes. In addition, the present results show that the mesenteric adipose tissue contains a larger number of cells besides adipocytes than the epididymal adipose tissue as indicated by the measurement of the DNA content. The mesenteric adipocytes have the responses to epinephrine on metabolic activities as the epididymal adipocytes. That is, the effects of epinephrine on “C(U)-glucose into glycogen is identical in both types of cells, but the effects of epinephrine on glucose oxidation, lactate production, FFA release and glycerol release of the mesenteric adipocytes are significantly lower than those of the epididymal adipocytes (Fig. 3). An a-adrenergic antagonist has practically no effect on FFA release, glycerol release and CAMP formation stimulated by epinephrine in both types of adipocytes. A j3-adrenergic antagonist suppresses the activities of both types of adipocytes to a similar extent. In general, the inhibitions of FFA release, glycerol release and CAMP fo~ation by B-adrenergic antagonist are considered to be brought about by changes in CAMP concentration mediated through a /?-adrenergic receptor. Some reports, however, emphasized that lipolytic responses in the epididymal adipocytes are more complex than are responses simply in proportion to intracellular CAMP concentration (Lang et al., 1976; Siddle and Hales, 1974; Schimmel et al., 1984). The present results support the hypothesis that intracellular CAMP may play an important role in, or is at least one of the factors controlling, lipolysis in adipocytes. Many reports have been published on the stimulating effects of catecholamines on glucose uptake in the rat epididymal adipocytes. Luzio et al. (1974) reported that an u-adrenergic antagonist, phenoxybenzamine, inhibited epineph~ne-stimulate glucose uptake in the rat epididymal adipocytes. In contrast, Kashiwagi et al. (1983) observed that a p-adrenergic agonist, L-isoproterenol stimulated the basal 3-0methylglucose transport in both the rat epididymal and the human subcutaneous adipocytes. Ludvigsen et al. (1980) also observed that a p-adrenergic antagonist, propranoiol, inhibited epinephrineor L-isoproterenol-stimulated glucose transport. On the
MASAHIRO TSHUCIUMOTO et al.
380
0
B) 0.6
160
K 1
(El
A Y
OAA
0
0
d- 0.1
0
1
O$
10 1001000 Epinephrine (@4)
Fig. 3. Comparison of the responses to epinephrine between mesenteric and epididymal adipocytes. (A) Glucose oxidation, expressed by the amount of the glucose oxidized to C02; (B) lactate production; (C) W(U)-glucose incorporation into glycogen, expressed by the amount of the glucose incorporated into glycogen; (D) FFA release; (E) glycerol release. gee Materials and Methods for experimental details. A: Mesenteric adipocytes; 0: epididymal adipocytes. Each point represents the mean from two observations.
other
hand,
Mills
et
al.
(1984)
suggested
that
forskolin, which stimulates CAMP accumulation, inhibited the glucose metabolism via a mechanism independent of CAMP in the rat epididymal adipocytes. The present results show that glucose oxidation and lactate production are inhibited by propranolol in the mesenteric adipocytes (Fig. 5). Corrected ZDm values of propranolol calculated according to equation (1) are shown in Table 1. All of the corrected
ID, values (0.05-0.26pM) from each other.
corrected IDso=
are not greatly different 14, 1 + [agonist].
(1)
EDu, These results suggest that lipolysis, glucose oxidation and lactate production are mediated by the same
381
Metabolic study of mesenteric adipocytes
and a /I-antagonist on lipolysis are the same in both types of adipocytes. In addition, the responses of the mesenteric and the epididymal adipocytes to insulin are identical in respect to its antagonism against lipolytic responses stimulated by epinephrine (Fig. 6). Thus the responses of the mesenteric adipocytes are essentially similar to those of the epididymal adipocytes. An amount of the mesenteric adipose tissue present in one rat is comparable to that of the epididymal adipose tissue, and the content of adipocytes in the mesenteric adipose tissue is higher than that in the epididymal adipose tissue. Thus the mesenteric adipocytes are useful for the elucidation of metabolism in a white adipose tissue.
150-
SUMMARY
Basal metabolic activities (free fatty acid uptake, glycerol release and lactate production) of the mesenteric adipose tissue slices were found to be higher
rE
0
I
0.1
I
1 Antagonist
I
10 (PM)
1
,
100
1000
Fig. 4. Effects of an CL-or a /?-adrenergic antagonist on epinephrine-stimulated lipolysis in mesenteric and epididymal adipocytes. (A) FFA release; (B) glycerol release;
(C) CAMP fonnation. The values show % of the control (epinephrine alone) calculated from the mean from two observations. Control values in the mesenteric and the epididymal adipocytes were 1.38 and 2.58 pEq/106 cells (FFA release), 1.98 and 3.81 pmol/106 cells (glycerol release), and 41.2 and 60.5 pmol/106 cells (CAMP formation), respectively. The epinephrine concentration employed was 5.5 PM, at which lipolysis reached the first peak (see Fig. 3). Closed symbols: an a-adrenergic antagonist, phenoxybenzamine; open symbols: a p-adrenergic antagonist, propranolol. AA: Mesenteric adipocytes; 00: epididymal adipocytes.
b-adrenergic receptor. This supports an idea proposed previously that glucose transport is mediated by B-adrenergic receptor. Recently, it was found that the stimulatory effect of epinephrine on glucose uptake was associated with the presence of adenosine released from adipocytes. That is, the effect of epinephrine which mimicked that of insulin-stimulated glucose transport was diminished and furthermore the insulin-stimulated transport itself was suppressed by epinephrine when adenosine was removed from the incubation medium by the addition of adenosine deaminase (Green, 1983). In addition, this suppressive effect of epinephrine was blocked by the /I-adrenergic antagonist, propranolol. These suppressive effects of epinephrine in the absence of adenosine were also observed in the present studies (data not shown). As described above, the effects of an a-antagonist
odt
I
1 10 100 Antagonist ( pM )
<
1000
Fig. 5. Effects of an a- and a /?-adrenergic antagonist on epinephrine-stimulated glucose oxidation and lactate production in mesenteric adipocytes. (A) Glucose oxidation, expressed by the amount of glucose oxidized to CO,; (B) lactate production. The values show “/, of the control (epinephrine alone) calculated from the-mean from two observations. Control value is 0.35 flmol/106 cells (glucose oxidation) and 0.34 ymol/106 cells (lactate product&) respectively. Epinephrine concentration employed was 100 phi. Closed symbols: an a-adrenergic antagonist, phenoxybenxamine, open symbols: a /I-adrenergic antagonist, propranolol.
MASAHIROTFHICH~MOTO et al.
382
adrenergic antagonists on epinephrine-stimulated activities (lipolysis, glucose oxidation and lactate production) or the inhibitory effects of insulin on epineph~n5stimuIated lipolysis were similar in both
types of adipocytes. Therefore, it can be considered that responses of the mesenteric adipocytes to epinephrine and insulin are essentially as high as those of the epididymal adipocytes. The total amount of mesenteric adipocytes present in one rat was larger than that of the epidid~al adipocytes. Hence the mesenteric adipocytes are useful for elucidation of metabolism in a white adipose tissue.
REFERENCES
OJ 6’
i
io 100 1000 Insulin (pU/ml)
,
I
10000
Fig. 6. Comparison of the responses to insulin between mesenteric and epididymai adipocytes. Antiiipoiytic effect of insulin against epinephrine-stimulated iipoiysis was evaiuated. (A) FFA release; (B) glycerol release. The values show % of the control (epinephrine alone) calculated from the mean from two observations. Control values in mesentet-ic and epidid~al adipocytes were 0.34 and 0.62 pEq/i06 cells (FFA release) and 0.68 and O.SSrmoi/io6 cells (glycerol release), respectively. The epinephrine concentration employed was 1.4 p M. A Mesenteric adipocytes; 0 epididymai adipocytes.
Table 1. Affinity of a fi-adrenergic antagonist, propranolol, receptor of mesenteric adipocytes Epinephrine concentration
ID,
&I
0.22
5.5 275 5.5 275 215
0.4 40 0.7 6.5 259
0.09 0.25 0.11 0.26 0.20
0.22
21.5
65
0.05
I.7
release
Glycerol release Glucose oxidation Lactate production
Corrected
WW
E&O FFA ____
for the
1.1
ED,,: The concentration of epinephrine required for obtaining 50% of the maximal effect. ID,: The concentration of propranolol required for a 50% inhibition of the effect of epinephrine. Corrected ID, was calculated with the equation (1).
than those of epididymal adipose tissue slices when the activities were expressed per g wet weight of tissue. These activities of the mesenteric adipocytes per cell were, however, equal to those of the epididymal adipocytes because the cell number in the former tissue was larger than that in the latter. The ratio of DNA content in the mesenteric adipose tissue to that in the epididymal adipose tissue per g wet weight was much higher (about three times) than the ratio of cell number in both types of adipocytes. This suggested that other kinds of cells were contained
much more in the mesenteric adipose tissue than in the epididymal adipose tissue. Responses to epinephrine on glucose oxidation, lactate production and lipofysis in the mesenteric adipocytes were lower than those in the epidid~al adipocytes. The effects of
Alien D. O., Hillman C. C. and Ashmore J. (1969) Studies on a biphasic lipolytic response to catechoiamine in
isolated fat ceils. Biochem. Pharmac. 18, 2233-2240. Alien D. 0. and MacLaren P. J. (1970) Effect of potassium ion, theophyiiine and propranoioi on the biphasic response to some catechoi~ines. Biochem. Pharmac. t9, 2569-2578. Alien D. 0. and Beck R. R. (1972) Alterations in lipolysis, adenyiate cyciase and adenosine 3’,5’-monophosphate levels in isolated fat ceils following adrenaiectomy. Endocrinology 91, 504-510. Barker S. B. and Su~e~on W. H. (1941) The ~lo~metric determination of lactic acid in biological material. J. biol. Chem. 138, 535-554. Burton K. (1956) A study of the conditions and mechanism of the diphenylamine reaction for the coiorimetric estimation of deoxyribonucleic acid. Biochem. J. 62,315323. Denton R. M., Yorke R. E. and Randle P. J. (1966) Measurement of concentrations of metaboiite in adipose tissue and effects of insulin, ailoxandiabetes and adrenaline. Biochem. J. 100, 407-419. Green A. (1983) Catechoiamines inhibit insulin-stimulated glucose transport in adipocytes, in the presence of adenosine deaminase. FEBS Lerr. 152, 261-264. Honma M., Satoh T., Takezawa J. and Ui M. (1977) An ultrasensitive method for the simultaneous determination of cyclic AMP and cyclic GMP in small-vohune samples from blood and tissue. Biochem. Med. 18, 257-213. Itaya K. and Wi M. (1964) The inhibitory action of serotonin on free fatty acid utilization by rat mesenteric adipose tissue. Biochim. biophys. Aeta 84, 604+506. Kashiwagi A., Huecksteadt T. P. and Foley J. E. (1983) Regulation of glucose transport by cAMPstimulators via three different mechanisms in rat and human adipocytes. J. biol. Chem. 288, 13685-13692. Kono T. and Barham F. W. (1973) Effects of insulin on the levels of adenosine 3’, ~-monophospha~ and Iipoiysis in isolated rat epididymai fat c&is. J. biol. Chem. 248, 1417-7426. Lang U., Fauchere J. L., Pelican G. M., Karlaganis G. and Schwyzer R. (1976) Hormone receptor interactions, adrenocorticotrophin (7-24) octadecapptide stimulates adipocyte membrane adenyiate cyclase without causing tipoiysis. FEBS Mt. 66, 246-249. Ludvigsen C., Jarett L. and McDonald J. M. (1980) The characterization of catechoiamine stimulation of gh~cose transport by rat adipocytes and isolated plasma membranes. Endocrinology 106, 786790. Luzio J. P., Jones R. C., Siddie K. and Hales C. N. (1974) Dissociation of the effect of adrenaline on glucose uptake from that on adenosine cyclic 3’, 5’-monophosphate ieveis and on lipolysis in rat-isolated fat cells. Biochim. biophys. Acta 362, 29-36. Miller E. A. and Allen D. 0. (1973) Hormone-stimulated iipoiysis in isolated fat ceils from “young” and “old” rats. J. Lipid Res. 14, 331-336.
Metabolic study of mesenteric adipocytes Mills I., Moreno F. J. and Fain J. N. (1984) Forskolin inhibition of glucose metabolism in rat adipocytes independent of adenosine 3’,5’-monophosphate accumulation and lipolysis. Endocrinology 115, 10661069. Pinter J. K., Hayashi J. A. and Watson J. A. (1967) Enzymatic assay of glycerol, dihydroxyacetone and glyceraldehyde. Archs B&hem. Biophys. 121, 404-414. Rodbell M. (1964) Metabolism of isolated fat cells. J. 6iol. Chem.
239,
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Schimmel R. J. (1984) Stimulation of CAMP accumulation and lipolysis in hamster adipocytes with forskolin. Am. J. Physiol. 246, C63-C68.
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Siddle K. and Hales C. N. (1974) The action of local anesthetics on lipolysis and on adenosine 3’. S-cyclic monophosphate content in isolated rat fat cells. Biochem. J. 142, 345-351. Spector A. A. and Hoak J. C. (1969) An improved method for the addition of long-chain free fatty acid to protein solutions. Analyt. Biochem. 32, 297-302. Tokumitsu Y., Kondoh T. and Ui M. (1977) Radiochemical assay of free fatty acids in blood using s7Co as a tracer. Analyt. Biochem. 81, 488490. Yajima M. and Ui M. (1974) Gluconeogenesis in epinephtine-induced hyperglycemia. Am. J. Physiol. 227, l-8.