.

\\lb

RICAN

JOURNAL

OF

PHYSIOLOGY

\ 01. 222, No. 4, April

1972.

Printed in U.S.A.

Glucose-independent lipogenesis by insulin

stimulation

of

V. K. MURTHY AND G STEINER Departments of Medicine and Physiology,

University

of Toronto,

Toronto,

Ontario,

Canada

MURTHY, V. K., AND G. STEINER. Glucose-independent stimulation II/ Qogenesis by insulin. Am. J. Physiol. 222(4) : 983-987. 1972.lkown adipose tissue slices, taken from alloxan-diabetic and from nondiabetic rats, were incubated in medium containing acetateZ-W but no glucose. In tissue slices from diabetic rats, 14C re((Ivery in fatty acids was decreased. Insulin added in vitro to the glucose-free medium partly reversed the inhibition of lipogenesis. insulin did not alter the concentration of glycogen or free fatty xid in these tissues. The tissue slices obtained from diabetic rats osidized more palmitate-1-14C to 14C02, but insulin added in 1 itro had no effect on this. Isolated cells from brown adipose time of nondiabetic rats were prepared in the presence of soy1wan trypsin inhibitor and incubated in glucose-free medium. Insulin increased the amounts of acetate incorporated into tatty acid by such cells only if the trypsin inhibitor is used in their pleparation. Mitochondria-free homogenates of brown adipose tissue from alloxan-diabetic rats incorporated less acetate into fatty acid than did homogenates of tissue from nondiabetic rats. lhis could be reversed by preincubating tissue slices from diabetic r*ats in glucose-free, but insulin-containing, medium before preparing the homogenates. It is concluded that insulin increases lipogenesis from acetate in brown adipose tissue through an effect of the hormone which is independent of any action on glucose transport or metabolism.

chondria, in supply of reductive hydrogen, or in the lipogenie reactions occurring in the extramitochondrial compartment of the cell. The present studies have been undertaken in order to examine whether such glucose-independent stimulation of lipogenesis occurred in another form of adipose tissue, interscapular brown adipose tissue and whether it occurred beyond the pyruvate(BAT), dehydrogenase step. We observed that BAT slices obtained from diabetic rats incorporated less acetate carbon in fatty acid compared to nondiabetic controls. In vitro addition of insulin to BAT slices of diabetic rats increased the recovery of acetate carbon in fatty acid in the complete absence of glucose transport or metabolism. The results of these studies have been communicated in preliminary form (25).
MATERIALS

alloxan-diabetes; XUP; fatty acid

brown

adipose

tissue;

isolated

fat

cells;

cyclic

BEEN DEMONSTRATED that liver preparations (3, 5, I;] and epididymal adipose tissue (14) obtained from diabetic rats incorporate less glucose or acetate (5) into fatty acids than do the similar tissues taken from normal rats. The low level of lipogenesis in diabetes is reversed by adlninistering insulin to the whole animal (6, 18, 28). Furthermore, insulin added in vitro to epididymal fat pads from diabetic rats increases lipogenesis from acetate, proAded that glucose is present in the incubation medium (30). The latter results are thought to be the consequence of alterations in glucose utilization which result from insulins ability to increase glucose transport into the cell (19). Recently, it has been shown that the conversion of pyruvate to fatty acid by rat epididymal adipose tissue slices is increased significantly in the presence of insulin and that the increase in lipogenesis is independent of insulins action on glucose transport (13). This could result either from an alteration in pyruvate dehydrogenase activity (15), in transport of two carbon units out of mito1'~ HAS

Radioactive biochemicals were obtained from New England Nuclear Corporation, alloxan was from British Drug Houses, Ltd., crystalline zinc insulin (free of glucagon), CoA, GSH, soybean trypsin inhibitor, glucose 6phosphate, and glucose-6-phosphate dehydrogenase were from Sigma Chemical Co., NADP and ATP were from Pabst Laboratories, and crude collagenase was obtained from Worthington Biochemical Corporation. Insulin was dissolved in 0.25 % bovine serum albumin solution before being added to incubation mixture. Palmitic acid was converted to the potassium salt and bound on to KrebsRinger bicarbonate buffer containing 4 % dialyzed bovine serum albumin, pH 7.4 (KRBA), as described by BorgStrom and Olivecrona (2). Male Wistar rats (High Oak Breeding Farms), weighing about 200 g and fed ad libitum on Purina laboratory chow, were used throughout the course of this investigation.
METHODS

Diabetes was induced by injecting alloxan (65 mg/kg iv) 5 days before each experiment. All animals had a blood glucose concentration over 250 mg/lOO ml. The rats were sacrificed by decapitation. Their interscapular BAT was dissected with care to avoid contamination by muscle or white adipose tissue. The BAT was sliced into pieces weighing about 5 mg each. Sixty milligrams of tissue slices were incubated under 95 % 02, 5 % CO:! at 37 C in 2 ml Krebs-Ringer bicarbonate buffer with 4 %

984 dialyzed serum albumin (KRBA) and either 2.5 mM sodium acetate-2-14C (1 &flask) or 0.5 mM palmitate-l14C (1 &flask). Each animal donated tissue to two flasks, one with insulin (12 mu/ml) added and one without. All incubations were carried out, in KRBA, for 90 min according to techniques already described (22). At the end of the incubation, the tissues were washed in saline, and tissue lipids were extracted and washed according to the method of Folch et al. (12). Tissue free fatty acids (FFA) were measured by the procedure described by Dole and Meinertz (9) as modified by Trout et al. (27). The methods used to measure 14C incorporation into tissue fatty acids (22, 23) and into 14COa (8) and to measure tissue glycogen content (23) have been previously described. Isolated cells from BAT slices were prepared by digestion with collagenase as described by Rodbell (20) modified as follows: no glucose was present in the medium either during digestion of tissue or during subsequent washing of the cells. BAT slices obtained from nondiabetic rats were incubated in polyethylene bottles; each bottle had 6 ml KRBA containing 2.5 mM acetate, 30 mg collagenase, and 10 mg soybean trypsin inhibitor. Seven hundred and fifty milligrams of BAT slices were added to each flask. Incubations were carried out for 90 min under atmosphere of 95 % 02, 5 % CO 2. The flasks were shaken at 130 cycles/min. At the end of incubation, the suspension was gently stirred with siliconized glass rod and filtered through nylon mesh into a polyethylene test tube. The contents of the tube were centrifuged at room temperature at 400 X g for 1 min and the floating fat, the sedimented debris, and the excess medium were removed by aspiration using siliconized Pasteur pipette. The cells were then washed 4 times, each time using 6 ml KRBA containing 2.5 mM acetate. The washing medium was maintained at 37 C. The cells were then suspended in KRBA containing 2.5 mM acetate and dispensed with a polyethylene pipette into siliconized incubation flasks containing KRBA and sodium acetate-2-14C (see Table 4). At the end of the incubation period, 20 ml of chloroform: methanol (2: 1 v/ v ) were added to the flasks, and the lipids were extracted and washed as described by Folch et al. (12) and counted in a Packard scintillation counter. In order to have a better understanding of the mechanism of action of insulin, it was decided to extend our studies to cell-free systems obtained from BAT. No information was available on the ability of cell-free extracts of BAT of diabetic rats to convert acetate to fatty acid compared to that of nondiabetic controls. Hence, the first series of experiments examined this. BAT of nondiabetic and alloxan-diabetic rats were homogenized in 0.25 M sucrose, and mitochondria-free supernatants were prepared and incubated as described by Steiner and Cahill (24). LMitochondria-free homogenates of BAT of diabetic rats showed significantly decreased ability to synthesize fatty acid. Therefore, a second series of experiments was done to determine whether preincubating these BAT slices in glucose-free, insulin-containing, medium before homogenization could improve their ability to synthesize fatty acid. (Conditions for preincubation are given under Table 5.) Each rat supplied tissue to two preincubation
TABLE

V.

R.

MURTHY

AND

G.

STEINER

1. EJect of in vitro insuhn addition lipogenesis from acetate by BAT slices

on

Acetate Recovered in Tissue Fatty Acids per Gram Lipid-Free Dry Weight, pmoles Source of BAT Control + Insulin Insul~ntMo~ PA

Nondiabetic rats Diabetic P1

59.8&16.3(8) rats 7.5&1.2(24) <O.OOl

51.7&10.4(8) 11.1&1.9(24)

-8.1&8.2 +3.6&1.5

>O.l =0.02

Conditions of the experiment are described in METHODS. Data given as mean & SE. Significance: PA was calculated by test of paired samples; P, by the Student t test. The numbers in parentheses represent numbers of animals used.

flasks, one with insulin and one without. After the preincubation, the slices were washed free of acetate with plain KRBA (37 C). They were then homogenized in 0.25 M sucrose; mitochondria-free supernatants were prepared, and fatty acid synthesis was studied in the system described above.
RESULTS

We have previously observed that BAT slices obtained from nondiabetic rats incorporated label from acetate-20 14C into tissue lipids (17.4 zt 3.6 pmoles/g lipid-free dry weight) and that label could be completely accounted for in the fatty acid fraction (20.8 =I= 7.1 pmoles/g lipidfree dry weight) (unpublished observations). It can be concluded from these results that BAT slices did not incorporate acetate J4C into glyceride-glycerol. Effect of addition of insulin on lipogenesis by diabetic BAT slices. The in vitro incorporation of acetateJ4C into tissue fatty acids was less in BAT slices obtained from diabetic rats than in tissue from nondiabetic rats (Table 1). Insulin, added to the glucose-free medium, significantly increased the recovery of label in lipids of BAT of diabetic rats, but had no effect when added to BAT slices of nondiabetic rats (Table 1). Tissue glycogen concentrations. Tissue glycogen concentrations were measured in order to determine whether they were altered under the experimentai conditions and, if so, whether this could account for insulins effect. These measurements were made in tissues before and at the end of a 90-min incubation in medium containing acetate but the glycogen content of the BAT no glucose. Although from diabetic rats was lower than that from normal rats, insulin, added in vitro, did not affect it (Table 2). Tissue FFA concentrations. Insulin, added in vitro to glucose-free medium, did not change the FFA levels in diabetic BAT (Table 2). Thus, it is improbable that alterations in tissue FFA account for insulins effect on lipogenesis under the present conditions. Oxidation of palmitate by BAT slices. The recovery of acetate label in tissue fatty acid is the net result of the formation and the degradation of fatty acid. Hence, it was important to assess the effect of insulin on oxidation of palmitate1-14C by diabetic BAT slices. The results, shown in Table 3, demonstrate that BAT slices obtained from

LHKECT

STIMULATION

OF

LIPOGENESIS

BY

INSULIN

985

PJUX 2. E$ect of insulin -. .

Determination (;lycogen*

on BAT

glycogen and FFA

Insulin Incubation Conditions 0 Insulin Minus Control PA

Source of BAT

12 mu/ml (7) (7) (ll)$ 27.0 27.3 rt rt 4.9 5.3 (3) (3)

Nondiabetic

rats

Unincubated Incubated with no substrate acetate

128.9 : 23.1 23.7

=t

17.6

+ 3.8 =t 4.2 P1 > 0.2

Diabetic

rats

Unincubated Incubated with no substrate acetate rats Incubated acetate Incubated acetate with with

24.7 : 8.8 10.8 50.0 55.2

=t 6.9 rt 0.4 =t 3.9 * 3.3

(6)s (3) (14)s (5) (5) 44.6 rt 4.9 (5) 10.6 it 4.5 >O.l 8.7 11.1 =t 2.6 zt 3.9 (3) (14) -0.1 0.3 rt: 0.03 * 0.3 >0.2 >0.2

Nondiabetic Diabetic rats

=t 5.7

-_ -_

-per Data gram are expressed lipid-free dry per gram weight. lipid-free dry weight. $ P > 0.2 compared * Micromoles to no-substrate glucose per controls.

Experimental conditions are as described in qrmn lipid-free dry weight. t Microequivalents $ Y < 0.001 compared to nondiabetic control.
TILE

METHODS.

_ -.

3. Oxidation

of palmitate-1-W

to CO2 in BAT

TABLE

4. Promotion of fatty acid synthesis by insulin in isolated brown adibocvtes

Acetate Recovered in Fatty Acid per Gram Lipid, pmoles Condition During Collagenase Digestion -IT--- XT-

Palmitate Recovered in CO2 per Gram Lipid-Free Dry Weight, pmoles

>ource of

RLiT

PA 0

12 mu/ml 34.Ort2.9(3) +5.Ort4.6

I
1 2 3 1 2 3

I No insulin

+ insulin,

--1____

.
>0.2 Trypsin absent inhibitor 0.20 0.23 0.86 0.44 1.60 1.07

12mU/ml 0.21 0.23 0.85 0.67 3.03 1.86

A due to

insulin

y0 change due to insulin

Nondiabe tic rats l>i:ibetic rats

29.0&2.8(3)

+0.01 0.00 -0.01 +0.23 +1.43 +0.79

+5.0 0 -1.2 +52.3 +89.4 +73.8

1,495.0&145.8(6)

1,52O.O=t247.0(6)

+25.0&10.(

1 >0.05

Trypsin present

inhibitor

Experimental

conditions

are

as described

in

METHODS.

diabetic rats oxidized palmitate to CO2 to a greater extent than did tissue from normal rats. Insulin, however, did not have any significant effect on palmitate oxidation by RXlY obtained from normal or from diabetic animals. EJect of insulin on fatty acid synthesis by isolated cells of BA T. It was possible that insulin augmented fatty acid synthesis c\-en in glucose-free medium as a result of increased transport of glucose from the interstitial space of BAT slices irr to the cell. This could account for the insulin effect hing observed in BAT slices from diabetic, but not in tissue slices from nondiabetic, rats. In order to check this l~~~ssibility, the effect of insulin on brown adipocytes isolated in absence of glucose was examined. Because of ready xailability of tissue, isolated cells were first prepared from BAT of nondiabetic rats (see METHODS). Our initial expcriments showed that insulin did not significantly influence the recovery of acetate label in fatty acid when isolated cells were prepared by collagenase digestion in hsence of trypsin inhibitor (Table 4). Fain and Loken (10) prepared isolated cells by digestion of rat BAT by collagerlase in presence of trypsin. They observed that insulin kreased the recovery of glucose carbon in CO2 and total lipid in brown adipocytes prepared in absence, but not

Incubation was carried out in siliconized 25-ml Erlenmeyer flasks maintained at 37 C. The flasks had 2 ml of KRBA containing 2.5 mM acetate (1 PC acetate-2 J4C per flask) and were gassed with a mixture of 95% 02, 57, COZ. The reaction was started by adding 0.5 ml of cell suspension (see METHODS) in KRBA containing 2.5 mM acetate; gassing was continued for 5 more min, and the flasks were incubated at 37 C for 90 min with shaking at 60 cycles/min. Insulin concentration was 12 mu/ml of incubation medium.

in presence, of trypsin. Since it has been reported that crude collagenase preparations have contaminating proteolytic activities (2 l), we decided to repeat our experiments using isolated cells prepared from BAT by digestion with collagenase in presence of soybean trypsin inhibitor. The cells were prepared, washed, and the effect of insulin on the recovery of acetate carbon in fatty acid was studied. Insulin now significantly increased fatty acid biosynthesis by isolated cells prepared in presence of trypsin inhibitor (Table 4). Fatty acid synthesis in mitochondria-free homogenates of BAT. Results presented thus far showed that insulin probably has a direct effect on fatty acid-synthesizing system and that this effect is independent of insulins action on glucose transport. In an attempt to explore further the mode of action of insulin, a series of experiments was carried out

986
TABLE

V.

K.

MURTHY

AND

G.

STEINER

5. Efect of preincubating BA T slices with insulin on fatty acid synthesis by mitochondria-free supernatant
Status of Rats Experimental Condition

Nondiabetic Diabetic Diabetic Diabetic

No

preincubation slices No preincubation

of BAT

189.1 50.5 68.8 133.5

rt rf rt rt

17.9 3.6 8.8 19.9

(3) (3)* (3) (3)T

Preincubation of slices but no insulin Preincubation with insulin

P values compared to corresponding controls. Conditions for preincubation : BAT slices from each rat were divided into two portions and added to two flasks, each containing 12 ml of KRBA and 2.5 mM acetate. The flasks were maintained at 37 C, were gassed with a mixture of 95% 02 and 5% COZ, and shaken at 60 cycles/ min. One flask contained insulin (12 mu/ml), and the other had no insulin. At the end of 90 min the slices were washed several times with plain KRBA (37 C). They were then homogenized at 2 C in 0.25 M sucrose. Mitochondria-free homogenates were prepared and incubated according to Steiner and Cahill (24). The incubation system contained: 40 mM potassium phosphate buffer, 3.5 mM NADP; 5 mM ATP; 0.1 mM CoA; pH 7.0; 50 mM MgC12; 50 mM potassium citrate; 10 mM KHC03; 50 mM GSH; 5 mM sodium acetate-2-14C (1 &flask); 15 mM glucose 6-phosphate; 2 EU glu* P < 0.001. $ P < 0.05. cose-6-phosphate dehydrogenase.

in which BAT slices were homogenized and in vitro fatty acid synthesis from acetate-2-14C by mitochondria-free homogenates of tissue was examined. Results shown in Table 5 demonstrated that mitochondria-free homogenates of BAT of nondiabetic rats synthesized more fatty acid from acetate compared to the corresponding extracts from alloxan-diabetic rats. The effect of preincubating BAT slices from diabetic rats with insulin was examined. BAT slices from diabetic rats were incubated in glucose-free medium with or without insulin. After preincubation for 90 min, they were washed with KRBA and mitochondria-free homogenates were prepared. Fatty acid synthesis from acetate was then studied. Preincubation with insulin significantly increased the recovery of acetate carbon in fatty acid synthesized in vitro (Table 5).
DISCUSSION

Insulin (12 mu/ml) augmented the recovery of acetate carbon in fatty acid of slices of BAT from diabetic rats. This occurred despite the absence of glucose from the incubation medium. Foa et al. (11) had similar observations in chick embryo heart. The increase in lipogenesis in BAT from diabetic rats was observed to be independent of any change in tissue concentration of glycogen. It is also interesting to note that there was no change in the glycogen content whether the slices were incubated in the presence or absence of acetate. Williamson (29) and Bethencourt et al. (1) have shown that in rat hearts perfused with acetate there was inhibition of oxidation of

glucose to CO 2. Williamson (29) also observed a marked increase in citrate concentration in these hearts and suggested that such an increase in citrate concentration inhibited glycolysis by inhibiting phosphofructokinase activity (16, 17). The reason for acetates failure to influence the residual glycogen concentration in BAT slices is not known. Under the conditions of present studies, incubation of BAT slices with insulin in the absence of glucose did not result in any change in tissue fatty acid concentration or in palmitate oxidation. Thus, it was also unlikely that the effect of insulin on acetate, 14C recovery in fatty acid could be explained by a decreased dilution of the acetyl CoA pool by nonradioactive products of tissue glycogen or fatty acid. The present studies suggest that insulin can enhance lipogenesis in BAT without affecting glucose utilization. This conclusion is strengthened by the observation of the insulin effect on isolated brown adipocytes prepared in absence of glucose. Halperin and Robinson (13) have demonstrated that in epididymal adipose tissue, insulin (10 mu/ml) enhances lipogenesis from pyruvate by a mechanism independent of an effect of glucose transport. They suggest that insulin in some way increased the binding affinity for pyruvate or one of the other intermediates at the rate-limiting step between pyruvate and fatty acid. Jungas (15) has demonstrated that insulin (1 mu/ml) augments the conversion of pyruvate to fatty acid in rat epididymal adipose tissue and that homogenates of tissues exposed to insulin showed greater pyruvate dehydrogenase activity compared to controls. Our studies demonstrate that insulin enhanced fatty acid synthesis from acetate. We also observed a glucose-independent insulin enhancement of lipogenesis in mitochondria-free homogenates of BAT. Insulin had not affected tissue concentrations of FFA, and all homogenates were incubated in the presence of optimal concentrations of cofactors (24). It is realized that these studies have not excluded the possibility of an alteration of FFA concentration in one specific compartment of the tissue. However, it would appear more likely that insulin may have an additional effect, namely to increase by an unknown mechanism the overall synthesis of fatty acid through the extramitochondrial pathway. One possible way whereby insulin may produce this effect on lipogenesis is suggested by preliminary work in our laboratory. The addition of 1 mM cyclic adenosine 3, 5-monophosphate (cyclic AMP) to mitochondriafree homogenates of BAT in presence of 0.5 mM theophylline produced about 50 % inhibition of incorporation of acetate into fatty acids (159.3 rfi 9.8 pmoles acetate recovered in fatty acid per gram N of supernatant for control ; 7 1.8 =t 20.3 pmoles in the presence of cyclic AMP). Such an inhibition occurred without any increase in FFA levels in incubation mixtures. Since it is known that insulin reduces cyclic AIM:P levels in brown and white adipose tissues (4, 26), it is possible that insulin promoted lipogenesis in brown adipocyte by lowering cyclic AMP levels.
Received for publication 14 June 1971.

DIRECT REFERENCES

STIMULATION

OF

LIPOGENESIS

BY

INSULIN

987

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fructokinase
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and
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Res. Commun.

17. PASSONNEAU, J., AND 0. H. LOWRY. Phosphofructokinase and the control of citric acid cycle. Biochem. Biophys. Res. Commun. 13: 372-379, 1963. 18. RENOLD, A. E., A. BAIRD HASTINGS, F. B. NESBETT, AND J. ASHMORE. Studies on carbohydrate metabolism in rat liver slices. IV. Biochemical sequence of events after insulin administration. J. BioZ. Chem. 213: 135-145, 1955. 19. RIESER, P. Insulin, Membranes and Metabolism. Baltimore: Williams & Wilkins, 1967, p. 6 l-86. M. Metabolism of isolated fat cells. I. Effect of hormones 20. RODBELL, on glucose metabolism and lipolysis. J. BioZ. Chem. 239: 375-380, 1964. 21. SCHREIBMAN, P. H., D. E. WILSON, AND R. A. ARKY. Degradation of (1311) insulin and inhibition of postheparin lipolytic activity by collagenase. Life Sci. 7 : 1295-130 1, 1968. G., AND G. F. CAHILL, JR. Brown and white adipose 22. STEINER, tissue metabolism in cold-exposed rats. Am. J. Physiol. 207: 840844, 1964. 23. STEINER, G., AND G. F. CAHILL, JR. Brown and white adipose tissue metabolism response to norepinephrine and insulin. Am. J. Physiol. 2 11: 1325-l 328, 1966. 24. STEINER, G., AND G. F. CAHILL, JR. Fatty acid synthesis and control in brown adipose tissue homogenates. Can. J. Biochem. 44: 1587-1596, 1966. 25. STEINER, G., AND V. K. MURTHY. Increased lipogenesis by insulin independent of its action on glucose transport. Diabetes 20: 379, 1971. 26. SUTHERLAND, E. W., AND G. ALAN ROBINSON. The role of cyclic AMP in the control of carbohydrate metabolism. Diabetes 18: 797-819, 1969. Titration 27. TROUT, D. L., E. H. E~TES, JR., AND S. I. FREIDBERG. of free fatty acids of plasma. A study of current methods and a new modification. J. Lipid Res. 1: 199-202, 1960. W. R., R. HILL, AND I. L. CHAIKOFF. Portal venous 28. WILLIAMS, injection of insulin in the diabetic rat-time of induction of changes cholesterogenesis and glycogenesis. J. in hepatic lipogenesis, Lz$id Res. 1: 236-240, 1960. 29. WILLIAMSON, J. R. Glycolytic control mechanisms. I. Inhibition of glycolysis by acetate and pyruvate in the isolated, perfused heart. J. BioZ. Chem. 240: 2308-232 1, 1965. A. I., AND A. E. RENOLD. Studies in rat adipose tissue 30. WINEGRAD, in vitro. I. Effects of insulin on the metabolism of glucose, pyruvate and acetate. J. Biol. Chem. 233: 267-272, 1958.