insulin effect on lipogenesis

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. \ \ lb RICAN JOURNAL OF PHYSIOLO GY\ 01. 222, No. 4, Apri l 1972. Printed in U.S.A.

Glucose-independent stimulation oflipogenesis by insulin

V. K . MURTHY AND G STEINERDepartmen ts o f Medicine and Physiology, University of Toronto, Toronto, O ntar io, Canada

MURTHY, V. K., AN D G. STEINER. Glucose-independent stimulationII/ Qogenesis by insulin. Am. J. Physiol. 222(4) : 983-987. 1972.-lkown adipose tissue slices, taken from alloxan-diabetic and fromnondiabetic rats, were incubated in mediu m containing acetate-Z-W but no glucose. In tissue slices from diabetic rats, 14C re-((Ivery in fatty acids was decreased. Insulin added in vitro to theglucose-free mediu m partly reversed the inhibi tion of lipogenesis.insulin did not alter the concentration of glycogen or free fattyxid in these tissues. The tissue slices obtained from diabetic ratsosidized more palmitate-1-14C to 14C02, but insulin added in1 tro had no effect on this. Isolated cells from brown adiposetime of nondiabetic rats were prepared in the presence of soy-1wan trypsin inhibito r and incubated in glucose-free medium .Insulin increased the amounts of acetate incorporated intotatty acid by such cells only if the trypsin inhibitor is used in theirpleparation. Mitochondria-free homogenates of brown adiposetissue from alloxan-diabetic rats incorporated less acetate intofatty acid than did homogenates of tissue from nondiabetic rats.lhis could be reversed by preincubating tissue slices from diabeticr*a s in glucose-free, but insulin-containing, mediu m before pre-paring the homogenates. It is concluded that insulin increaseslipogenesis from acetate in brown adipose tissue through an effectof the hormone which is independent of any action on glucosetransport or metabolism .

alloxan-diabetes; brown adipose tissue; isolated fat cells; cyclicXUP; fatty acid

1 '~ HAS BE EN DEMONSTRATED that liver preparations (3, 5,I;] and epididymal adipose t issue (14) obtained from dia-betic rats incorporate less glucose or acetate (5) into fa ttyacids than do the similar t issues taken from normal rats.The low level of l ipogenesis in diabetes is reversed by ad-lninistering insulin to the whole animal (6, 18, 28). Fur-thermore, insulin added in vitro to epididymal fat padsfrom diabetic rats increases lipogenesis from acetate, pro-Aded that glucose is present in the incubation medium(30). The latter results are thought to be the consequenceof alterations in glucose util ization which result from in-sulins abil ity to increase glucose transport into the cell(19). Recen tly, it has been shown that the conversion ofpyruvate to fatty acid by rat epididymal adipose t issueslices is increased signif icantly in the presence of insulinand that the increase in l ipogenesis is independent of in-sulins action on glucose transport (13). This could resulteither from an alteration in pyruvate dehydrogenase activ-ity (15), in transport of two carbon units out of mito-

chondria, in supply of reductive hydroge n, or in the lipo-genie reactions occurr ing in the extramitochondrial com-partment of the cell. The present studies have been under-taken in order to examine whether such glucose-inde-pendent stimulation of l ipogenesis occurred in anotherform of adipose t issue, interscapular brown adipose t issue(BAT), and whether it occurred beyond the pyruvate-dehydrogenase step. We observed that BAT slices ob-tained from diabetic rats incorporated less acetate carbonin fatty acid compared to nondiabetic controls. In vitroaddit ion of insulin to BAT slices of diabetic rats increasedthe recovery of acetate carbon in fatty acid in the com-plete absence of glucose transport or metabolism. Theresults of these studies have been commu nicated in pre-liminary form (25).MATERIALS

Radioactive biochemicals were obtained from NewEngland Nuclear Corporation, alloxan was from Brit ishDrug Ho uses, Ltd., crystall ine zinc insulin ( free of gluca-gon), CoA, GSH, soybean trypsin inhibitor, glucose 6-phosphate, and glucose-6-phosphate dehydrogenase werefrom Sigma Chemical Co., NADP and ATP were fromPabst Laborator ies, and crude collagenase was obtainedfrom Worthington Biochemical Corporation. Insulin wasdissolved in 0.25 % bovine serum albumin solution beforebeing added to incubation mixture. Palmit ic acid was con-verted to the potassium salt and bound on to Krebs-Ringer bicarbonate buffer containing 4 % dialyzed bovineserum album in, pH 7.4 (KRBA), as described by Borg-Strom and Olivecrona (2).Male Wistar rats (High Oak Breeding Farms ), weighingabout 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/kgiv) 5 days before each experiment. All animals had ablood glucose concentration over 250 mg/lOO ml. Therats were sacrif iced by decapitation. Their interscapularBAT was dissected with care to avoid contaminationby muscle or white adipose t issue. The BAT was slicedinto pieces weighing about 5 mg each. Sixty milligrams oft issue s lices were incubated under 95 % 02, 5 % CO :!at 37 C in 2 ml Krebs-Ringer bicarbonate buffer with 4 %


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98 4 V. R. MURTHY AND G. STEINERdialyzed serum albumin (KRBA) and either 2.5 mMsodium acetate-2-14C (1 &flask) or 0.5 mM palmitate-l-14C (1 &flask). Each animal donated t issue to two f lasks ,one with insulin (12 mu/ml) added and one without.All incubations were carr ied out, in KRBA, for 90 minaccording to techniques already described (22). At theend of the incubation, the t issues were washed in saline,and t issue lipids were extracted and washed accordingto the method of Folch et al. (12). T issue free fatty acids(FFA) were measured by the procedure described byDole and Meinertz (9) as modif ied by Trout et al. (27).The methods used to measure 14C incorporation intotissue fatty acids (22, 23) and into 14COa (8) and to meas-ure t issue glycogen content (23) have been previouslydescribed.

TABLE 1. EJec t of in vitro insuhn addition onlipogenesis from acetate by BAT slices

Acetate Recovered in Tissue Fatty Acids per GramLipid-Free Dry Weight, pmolesSource of BAT

Control + Insul in Insul~ntMo~ PA

NondiabeticratsDiabetic ratsP1

59.8&16.3(8) 51.7&10.4(8) -8.1&8.2 >O.l7.5&1.2(24) 11.1&1.9(24) +3.6&1.5 =0.02


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LHKECT STIMULATION OF LIPOGENESIS BY INSULIN 98 5PJUX 2. E$ect o f insulin on BAT glycogen and FFA-. .

Insul inIncubation Condit ions Insulin Minus Controlource of BATetermination

-(;lycogen*

PA

>0.2>0.2

>O.l

12 mu/ml128.9 =t 17.6 (7)

23.1 + 3.8 (7)23.7 =t 4.2 (ll)$

P1 > 0.224.7 =t 6.9 (6)s

8.8 rt 0.4 (3)10.8 =t 3.9 (14)s50.0 * 3.3 (5)55.2 =t 5.7 (5)

Nondiabetic rats UnincubatedIncubated with :no substrateacetate

27.0 rt 4.9 (3)27.3 rt 5.3 (3)

Diabetic rats UnincubatedIncubated with :

no substrateacetate

8.7 =t 2.6 (3)11.1 zt 3.9 (14)

44.6 rt 4.9 (5)

-0.1 rt: 0.030.3 * 0.3

Nondiabetic ratsDiabetic rats

Incubated withacetateIncubated withacetate

10.6 it 4.5-_ -_ - -

Experimental conditions are as described in METHODS. Data are expressed per gram lipid-free dry weight. * Micromoles glucose perqrmn lipid-free dry weight. t Microequivalents per gram lipid-free dry weight. $ P > 0.2 compared to no-substrate controls.$ Y < 0.001 compared to nondiabetic control.

TILE 3. Oxidation of palmitate-1-W to CO2 in BAT TABLE 4. Promotion of fatty acid synthesis by insulin_ -. in isolated brown adibocvtesPalmitate Recovered in CO2 per GramLipid-Free Dry Weight, pmoles Acetate Recovered in Fatty Acidper Gram Lipid, pmoles

-IT--- XT->ource ofRLiT

--1____Nondia-

be ticrats

l>i:ibeticrats

P A Condit ion DuringCollagenase Digestion INo insul in y0 changedue toinsul in+ insul in, A due to12mU/ml insulin1 0.20 0.21 +0.01 +5.02 0.23 0.23 0.00 03 0.86 0.85 -0.01 -1.21 0.44 0.67 +0.23 +52.32 1.60 3.03 +1.43 +89.43 1.07 1.86 +0.79 +73.8

12 mu/ml .29.0&2.8(3)

1,495.0&145.8(6)

34.Ort2.9(3)

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

+5.Ort4.6

+25.0&10.(

>0.2 Trypsin inhibitorabsent

>0.05 Trypsin inhibitorpresent

1

Experimental conditions are as described in METHODS.Incubation was carried out in siliconized 25-ml Erlenmeyer

flasks maintai ned at 37 C. The flasks had 2 ml of KRB A containing2.5 mM acetate (1 PC acetate-2 J4C per flask) and were gassed witha mixture of 95% 02, 57, COZ. The reaction was started by adding0.5 ml of cell suspension (see METHODS) in KRBA containing 2.5mM acetate; gassing was continued for 5 more min, a nd the flaskswere incubated at 37 C for 90 min w ith shaking at 60 cycles/min.Insulin concentration was 12 mu/m l of incubation medium .

diabetic rats oxidized palmitate to CO2 to a greater extentthan did t issue from normal rats. Insulin, however, didnot have any signif icant effect on palmitate oxidation byRXlY obtained from normal or from diabetic animals.EJec t of insulin on fatty acid synthesis b y isolated cells of BA T.

It was possible that insulin augmented fatty acid synthesisc\-en in glucose-free medium as a result of increased trans-port of glucose from the interstit ial space of BAT slicesirr to the cell. This could account for the insulin effecthing observed in BAT slices from diabetic, but not intissue slices from nondiabetic, rats. In order to check thisl~~~ssibil ity, the effect of insulin on brown adipocytes iso-lated in absence of glucose was examined. Because of readyxailabil ity of t issue, isolated cells were f irst prepared fromBAT of nondiabetic rats (see METHODS). Our initial ex-pcr iments show ed that insulin did not signif icantly in-f luence the recovery of acetate label in fatty acid whenisolated cells were prepared by collagenase digestion inhsenc e of trypsin inhibitor (Table 4). Fain and Loken (10)prepared isolated cells by digestion of rat BAT by collag-er lase in presence of trypsin. They observed that insulinkreased the recovery of glucose carbon in CO2 and totall ipid in brown adipocytes prepared in absence, but not

in presence, of trypsin. Since it has been reported thatcrude collagenase preparations have contaminating pro-teolytic activit ies (2 l) , we decided to repeat our experi-men ts using isolated cells prepared from BAT by digestionwith collagenase in presence of soybean trypsin inhibitor.The cells were prepared, washed, and the effect of insulinon the recovery of acetate carbon in fatty acid was studied.Insulin now signif icantly increased fatty acid biosynthe-sis by isolated cells prepared in presence of trypsin in-hibitor (Table 4).

Fatty acid synthesis in mitochondria-free homogenates of BAT.Results presented thus far showed that insulin probablyhas a direct effe ct on fatty acid-synthesizing sys tem andthat this effect is independent of insulins action on glucosetransport. In an attempt to explore further the mode ofaction of insulin, a series of experiments was carr ied out


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98 6TABLE 5. Efect of preincubating BA T slices with insulinon fatty acid synthesis by mitochondria-free supernatant

Status of Rats

NondiabeticDiabeticDiabeticDiabetic

Exper imental Condi t ion

No preincubation of BATslicesNo preincubation

Preincubation of slicesbut no insulin

Preincubation with insu-li n

189.1 rt 17.9 (3)50.5 rf 3.6 (3)*68.8 rt 8.8 (3)

133.5 rt 19.9 (3)T

P values compared to corresponding controls. Conditions forpreincubation : BAT slices from each rat were divided into twoportions and added to two flasks, each containing 12 ml of KRB Aand 2.5 mM acetate. The flasks were maintain ed at 37 C, were gassedwith a mixture of 95% 02 and 5% COZ, and shaken at 60 cycles/min. One flask contained insulin (12 mu/ml), and the other hadno insulin. At the end of 90 min the slices were washed severaltimes with plain KRBA (37 C). They were then homogenized at2 C in 0.25 M sucrose. Mitochondria-free homogenates were pre-pared and incubated according to Steiner and Cahil l (24). Theincubation system contained: 40 mM potassium phosphate buffer,pH 7.0; 50 mM MgC12; 3.5 mM NADP; 5 mM ATP; 0.1 mM CoA;50 mM potassium citrate; 10 mM KHC03; 50 mM GSH; 5 mM sodiumacetate-2-14C (1 &flask); 15 mM glucose 6-phosphate; 2 EU glu-cose-6-phosphate dehydrogenase. * P < 0.001. $ P < 0.05.

in which BAT slices were homogenized and in vitro fattyacid synthesis from acetate-2-14C by mitochondria-freehomogenates of t issue was examined. Results sh own inTable 5 demonstrated that mitochondria-free homogenatesof BAT of nondiabetic rats synthesized more fatty acidfrom acetate compared to the corresponding extracts fromalloxan-diabetic rats.

The effect of preincubating BAT slices from diabeticrats with insulin was examined. BAT slices from diabeticrats were incubated in glucose-free medium with or with-out insulin. After preincubation for 90 min, they werewashed with KRBA and mitochondria-free homogenateswere prepared. Fatty acid synthesis from acetate was thenstudied. Preincubation with insulin signif icantly increasedthe recovery of acetate carbon in fatty acid synthesizedin vitro (Table 5).DISCUSSION

Insulin (12 mu/ml) augmented the recovery of acetatecarbon in fatty acid of slices of BAT from diabetic rats.This occurred despite the absence of glucose from theincubation medium. Foa et al. (11) had similar observa-tions in chick em bryo heart. The increase in l ipogenesisin BAT from diabetic rats was observed to be independentof any change in t issue concentration of glycogen. It isalso interesting to note that there w as no change in theglycogen content whether the slices were incubated inthe presence or absence of acetate. W ill iamson (29) andBethencourt et al. (1) have shown that in rat hearts per-fused with acetate there was inhibit ion of oxidation of

V. K. MURTHY AND G. STEINER

glucose to CO 2. Will iamson (29) also observed a markedincrease in citrate concentration in these hearts and sug-gested that such an increase in citrate concentration in-hibited glycolysis by inhibit ing phosphofructokinase activ-ity (16, 17). The reason for aceta tes failure to influencethe residual glycogen concentration in BAT slices is notknown. Under the condit ions of present studies, incubationof BAT slices with insulin in the absence of glucose did notresult in any change in t issue fatty acid concentration orin palmitate oxidation. Thus, it was also unlikely that theeffect of insulin on acetate, 14C recovery in fatty acid couldbe explained by a decreased dilution of the acetyl CoApool by nonradioactive products of t issue glycogen orfatty acid.

The present studies sugg est that insulin can enhancelipogenesis in BAT without affecting glucose util ization.This conclusion is strengthened by the observation of theinsulin eff ect on isolated b rown a dipoc ytes prepared inabsence of glucose. Halperin and Robinson (13) havedemonstrated that in epididymal adipose t issue, insulin(10 mu/ml) enhances lipogenesis from pyruvate by amechanism independent of an effect of glucose transport.They suggest that insulin in some way increased the bindingaff inity for pyruvate or one of the other intermediates atthe rate-limit ing step between pyruvate and fatty acid.

Jungas (15) has demonstrated that insulin (1 mu/ml)augments the conversion of pyruvate to fatty acid in ratepididymal adipose t issue and that homogenates of t issuesexposed to insulin showed greater pyruvate dehydrogenaseactivity compared to controls. Our studies demonstratethat insulin enhanced fatty acid synthesis from acetate.We also observed a glucose-independent insulin enhance-ment of l ipogenesis in mitochondria-free homogenates ofBAT. Insulin had not affected t issue concentrations ofFFA, and all homogenates were incubated in the presenceof optimal concentrations of cofactors (24). It is realizedthat these studies have not excluded the possibil ity of analteration of FFA concentration in one specif ic compart-ment of the t issue. Howev er, it would appear more likelythat insulin may have an addit ional effect, namely toincrease by an unknown mechanism the overall sy nthesisof fatty acid through the extramitochondrial pathway.One possible way whereby insulin may produce thiseffect on lipogenesis is suggested by preliminary work inour laboratory. The addit ion of 1 mM cyclic adenosine3, 5-monophosphate (cyclic AMP) to mitochondria-free homogenates of BAT in presence of 0.5 mM theophyl-line produced about 50 % inhibition of incorporation ofacetate into fatty acids (159.3 rf i 9.8 pmoles acetaterecovered in fatty acid per gram N of supernatant forcontrol ; 7 1.8 =t 20.3 pmoles in the presence of cyclicAMP). Such an inhibit ion occurred without any increasein FFA levels in incubation mixtures. Since it is knownthat insulin reduces cyclic AIM:P levels in brown and whiteadipose t issues (4, 26), it is possible that insulin promotedlipogenesis in brown ad ipocyte by lowering cyclic AMPlevels.Received for publication 14 June 1971.


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DIRECT STIMULATION OF LIPOGEN ESIS BY INSULIN 987REFERENCES

1. BETHENCOURT, A. V., 0. E. MATOS, AND J. C. SHIPP . Acetate-14Cmetabolism and the effect of acetate on glucose and palmitat emetabolism in the perfused rat heart. Metabol i sm 15 : 847-855,1966.

BORGSTROM, B., AND T. OLIVECRONA. The metabolism of palmiticacid-1-14C in functionally hepactectomized rats. J. L$id Res.2: 263-267, 1961.BRADY , R. O., AND S. GURIN. Biosynthesis of labelle d fatty acidand cholesterol in experimental diabetes. J. B io l . Chem. 187:589-596, 1950.BUTCHER, R. W., AND C. E. BAIRD. The regulation of cyclic AMPand lipolysis in adipose tissue by hormones and other agents.In: Drugs Af fect ing L ip id Metabol i sm, edited by W. L. Holmes, L. A.Carlson, and R. Paoletti. New York: Plenum , 1969, p. 5-22.CHERNICK, S. S., AND I. L. CHAIKOFF. Two blocks in carbohydrateutilization in the liver of the diabetic rat. J. B ioZ. Chem. 188:389-396, 1951.CHERNICK , S. S., AND I. L. CHAIKOFF. Insulin and hepatic utiliza-tion of glucose for lipogenesis. J. B io l . Chem. 186 : 535-542, 1950.CHERNICK, S. S., I. L. CHAIKOFF, E. J. MASORO, AND L. E. ISAEFF.Lipogenesis and glucose oxidation in the liver of the alloxan-diabetic rat. J. B io l . Chem. 186: 527-534, 1950.CUPPY , D., AND L. E. CREV ASSE . An assembly for 14C02 collectionin metabolic studies for liquid scintillation counting. Anal . B iochem.5 : 462-463, 1963.DOLE, V. P., AND H. MEINERTZ. Microdetermination of longchain fatty acids in plasma and tissues. J. B ioZ. Chem. 235: 2595-2599, 1960.FAIN, J. N., AND S. C. LOKEN. Response of trypsin-treated brownand white fat cells to hormones. J. B ioZ. Chem. 244: 3500-3506,1969.FOA, P. P., M. MELLI, C. K. BERGER, D. BILLINGER, AND G. G.GUIDOTTI. Action of insulin on chick embryo heart. Federat ionProc. 24 : 1046-1052, 1965.FOLCH, J., M. LEES, AND G. H. SLOANE STANLEY. A simplemethod for the isolation and purification of total lipids from ani-mal tissues. J. B ioZ. Chem. 226 : 497-509, 1957.HALPER IN, M. L., AND B. H. ROBINSON. Mechanism of insulinaction on control of fatty acid synthesis independent of glucosetransport. Metabol i sm 20 : 78-86, 197 1.HAUSBE RGER, F. X., S. W. MILSTEIN, AND R. J. RUTMAN. Theinfluence of insulin on glucose utilization in adipose and hepatictissues in vi t ro. J. B ioZ. C hem. 208 : 43 l-438, 1954.JUNGAS, R. L. Hormonal regulation of pyruvate dehydrogenase.Metabol i sm 20: 43-53, 1971.PARMEGG IANI, A., AND R. H. BOWMAN. Regulation of phospho-

17.

18.

19.20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

fructokinase activity by citrate in normal and diabetic muscle.Biochem. Biophys. Res. Commun . 12 : 268-273, 1963.PASSO NNEAU , J., AND 0. H. LOWRY. Phosphofructokinase andthe control of citric acid cycle. Biochem. Biophys. Res. Commun .13: 372-379, 1963.

RENOLD, A. E., A. BAIRD HASTINGS, F. B. NESBETT, AND J. ASH-MORE. Studies on carbohydrate metabolism in rat liver slices. IV.Biochemical sequence of events after insulin administration. J.BioZ. Chem. 213: 135-145, 1955.RIESER, P. Insul in , Membranes and Metabol i sm. Baltimore: Will iams& Wilkins, 1967, p. 6 l-86.RODBELL, M. Metabolism of isolated fat cells. I. Effect of hormoneson glucose m etabolism and lipolysis. J. B ioZ. Chem. 239: 375-380,1964.SCHREIBMAN, P. H., D. E. WILSON, AND R. A. ARKY. Degradationof (1311) insulin and inhibit ion of postheparin lipolytic activity bycollagenase. Li fe Sci . 7 : 1295-130 1, 1968.STEINER, G., AND G. F. CAHILL, JR. Brown and white adiposetissue metabolism in cold-exposed rats. Am. J. Physiol . 207: 840-844, 1964.STEINER , G., AND G. F. CAHILL, JR. Brown and white adiposetissue metabolism response to norepinephrine and insulin. Am. J.Physiol . 2 11: 1325-l 328, 1966.STEINER, G., AND G. F. CAHILL, JR. Fatty acid synthesis andcontrol in brown adipose tissue homogenates. Can. J. B iochem.44: 1587-1596, 1966.STEINER , G., AND V. K. MURTHY. Increased lipogenesis by insulinindependent of its action on glucose transport. Diabetes 20: 379,1971.SUTHERLAN D, E. W., AND G. ALAN ROBINSON. The role of cyclicAMP in the control of carbohydrate metabolism. Diabetes 18:797-819, 1969.TROUT, D. L., E. H. E~TES, JR., AND S. I. FREIDBERG. Titrationof free fatty acids of plasma. A study of current methods and anew modification. J. Lipid Res. 1: 199-202, 1960.WILLIAMS, W. R., R. HILL, AND I. L. CHAIKOFF. Portal venousinjection of insulin in the diabetic rat-time of induction of changesin hepatic lipogenesis, cholesterogenesis and glycogenesis. J.Lz$id Res. 1: 236-240, 1960.WILLIAMSON, J. R. Glycolytic control mechanisms. I. Inhibitio nof glycolysis by acetate and pyruvate in the isolated, perfusedheart. J. B ioZ. Chem. 240: 2308-232 1, 1965.WINEGRAD, A. I., AND A. E. RENOLD. Studies in rat adipose tissuein vi t ro. I. Effects of insulin on the metabolism of glucose, pyruvateand acetate. J. B io l . Chem. 233: 267-272, 1958.

insulin effect on lipogenesis

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