Tissue triglycerides, insulin resistance, and insulin production: implications for hyperinsulinemia of obesity

1Gifford

KAZUNORI KOYAMA,1 GUOXUN CHEN,1 YOUNG LEE,1 AND ROGER H. UNGER1,2 Laboratories, Center for Diabetes Research, Department of Internal Medicine, University of Texas Southern Medical Center, Dallas 75235; and 2Department of Veterans Affairs Medical Center, Dallas, Texas 75216 obesity, such as -cell hyperplasia and increased insulin secretion at substimulatory glucose levels (16, 26). Third, it has been shown that islets of hyperinsulinemic obese rats have an extremely high TG content compared with normal littermates (20). There being no direct way to test the possibility that the coupling of insulin production to insulin need is mediated by tissue lipid content, we relied on correlations between -cell function and insulin effectiveness and tissue TG content of groups of rats with a widely varying tissue fat content. At one extreme of the spectrum of fat content were obese rats, in which tissue lipids are markedly increased (20). At the other extreme we exploited a novel syndrome of profound tissue lipid depletion that is the antithesis of obesity; this syndrome was produced by inducing chronic hyperleptinemia in normal rats by means of adenovirus-leptin gene transfer (3, 34). These animals undergo rapid and selective loss of all grossly visible fat (3); additionally the TG content in skeletal muscle, liver, and pancreas declines to 1/1,000 of that of obese rats and 1/10 of that of pair-fed controls (34). Between these two extremes of severe lipopenia and obesity, we studied free-feeding normal rats and normal rats whose food intake was restricted by pair feeding to the hyperleptinemic rats. The high correlations between indexes of insulin resistance and insulin production and the TG content in the target tissues of insulin and in the tissue of insulin production are consistent with the hypothesis that tissue fat content might provide the link between insulin resistance and hyperinsulinemia.
METHODS AND MATERIALS

Koyama, Kazunori, Guoxun Chen, Young Lee, and Roger H. Unger. Tissue triglycerides, insulin resistance, and insulin production: implications for hyperinsulinemia of obesity. Am. J. Physiol. 273 (Endocrinol. Metab. 36): E708 E713, 1997.Obesity is associated with both insulin resistance and hyperinsulinemia. Initially hyperinsulinemia compensates for the insulin resistance and thereby maintains normal glucose homeostasis. Obesity is also associated with increased tissue triglyceride (TG) content. To determine whether both insulin resistance and hyperinsulinemia might be secondary to increased tissue TG, we studied correlations between TG content of skeletal muscle, liver, and pancreas and plasma insulin, plasma [insulin] [glucose], and -cell function in four rat models with widely varying fat content: obese Zucker diabetic fatty rats, free-feeding lean Wistar rats, hyperleptinemic Wistar rats with profound tissue lipopenia, and rats pair fed to hyperleptinemics. Correlation coefficients 0.9 (P 0.05) were obtained among TG of skeletal muscle, liver, and pancreas and among plasma insulin, [insulin] [glucose] product, and -cell function as gauged by basal, glucose-stimulated, and arginine-stimulated insulin secretion by the isolated perfused pancreas. Although these correlations cannot prove cause and effect, they are consistent with the hypothesis that the TG content of tissues sets the level of both insulin resistance and insulin production. tissue fat; obese Zucker diabetic fatty rats; -cell function

OBESITY is the most prevalent health problem in the United States and is currently estimated to afflict 75,000,000 Americans (28). Although the risk of diabetes is high, glucose homeostasis remains relatively normal for long periods of time despite insulin resistance and excessive intake of food. This ability to maintain euglycemia despite diminished insulin effectiveness indicates that insulin production is somehow matched to insulin requirements. The mechanism by which -cells recognize the level of insulin resistance that must be overcome is unknown. Because hyperinsulinemia may be present in obesity even when glucose tolerance is normal, subtle glycemic elevations are not the signal for the enhanced insulin production at that stage of the disorder. This study was undertaken to identify a nonglycemic signal that might link insulin action to insulin production. Tissue triglyceride (TG) content seemed to be a plausible candidate for this dual role. Long-chain free fatty acids (FFA) have long been known to interfere with insulin-mediated glucose metabolism (2, 8, 13, 15, 18, 24, 25, 29) and to stimulate insulin secretion acutely (5, 11, 22, 26, 31). Moreover, high FFA levels in vitro have been shown to induce in normal islets the same changes described in the compensating -cells of

Animals. All animals were 79 wk of age at the start of experiments. Obese, prediabetic Zucker diabetic fatty (ZDF) rats (fa/fa) were bred in our laboratory from ZDF/Drt-fa (F10) stock purchased from R. Peterson (University of Indiana School of Medicine, Indianapolis, IN). Male rats exhibited the previously described phenotype (30). Male Wistar rats purchased from Charles River Breeding Laboratories (Wilmington, MA) were used as free-feeding lean controls and diet-restricted controls. Diet-restricted rats were Wistar rats pair fed to the leptin-overexpressing lipopenic rats described in the next paragraph; this resulted in a 42% reduction in their total caloric intake. The hyperleptinemic, lipopenic animals were normal Wistar rats that had received an infusion of a recombinant adenovirus containing the leptin cDNA (AdCMV-leptin). As reported previously (3), leptin mRNA appeared in the liver in association with a rise in plasma leptin levels and a reduction in food intake and body weight (see Fig. 1). Another group of Wistar rats was infused with adenovirus containing the cDNA of an

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irrelevant protein, bacterial -galactosidase (AdCMV-Gal), as a control for the viral infection. Gene transfer studies. To clone the rat leptin cDNA and measure its mRNA, total RNA was prepared from 1 g of epididymal adipose tissue of rats by extraction with TRIzol as recommended by the manufacturer. Oligo(dT) was used to prime rst-strand cDNA synthesis by use of a cDNA synthesis kit (Clontech, Palo Alto, CA). After treatment of the rststrand cDNA with deoxyribonuclease-free ribonuclease, the leptin gene product was amplied by polymerase chain reaction (PCR) with upstream sense primer 5-GGAGGAATCCCTGCTCCAGC-3 and downstream antisense primer 5CTTCTCCTGAGGATACCTGG-3 based on the rat leptin gene sequence (27). For both cDNA cloning and leptin mRNA measurement, amplication was performed using 1 cycle at 94C for 3 min, followed by 35 cycles at 92C for 45 s, at 53C for 45 s, and at 72C for 1 min, and then nal extension at 70C for 10 min. We also measured -actin expression with the same amplication conditions as for leptin and a previously described oligonucleotide pair (23). To prepare recombinant virus, a 640-bp PR fragment containing the entire leptin coding region was ligated to pCR TM 2.1 (Invitrogen, San Diego, CA) according to the manufacturers protocol. Sequence analysis conrmed that several clones contained the intact leptin cDNA. A BamH I- and Xba I-restricted leptin cDNA fragment that included 60 bp of 5 untranslated region and 76 bp of 3 untranslated region was ligated to similarly treated pACCMVpLpA (10). The resulting plasmid was cotransfected with pJM17 (23) into 293 cells by calcium phosphate/DNA coprecipitation to generate the new recombinant virus AdCMV-leptin by use of previously described methods (1). Virus DNA was isolated, and the presence of the leptin gene insert was conrmed by PCR by use of the primers described above and by Southern blotting with an oligonucleotide (5-GGATACCGACTGC GTGTGTGAAATGTCAT-3) complementary to the rat leptin cDNA. Stocks of AdCMV-leptin were amplied and puried as described (1) and stored at 70C in phosphate-buffered saline with 0.2% bovine serum albumin (BSA) and 10% glycerol at 13 1012 plaque-forming units (pfu)/ml. A virus containing the bacterial -galactosidase gene under control of the CMV promoter, AdCMV-Gal, was prepared and utilized as described previously (14). The virus was infused in male Wistar rats obtained from Charles River Laboratories (Wilmington, MA). Before adenovirus infusion studies, all rats received standard rat chow (Teklad F6 8664, Madison, WI) ad libitum and had free access to water. Polyethylene tubing (PE-50, Becton-Dickinson, Franklin Lakes, NJ) was anchored in the left carotid artery of 9-wk-old Wistar rats of 250300 g under pentobarbital sodium anesthesia (50 mg/kg, Abbott Laboratories, North Chicago, IL) and exteriorized via a subcutaneous tunnel. Tubing was lled with heparinized saline (1,000 U/dl) until the virus infusion was begun 3 days after surgery. Before infusion, adenovirus samples were suspended in saline and ltered through a 0.2-m lter. Two milliliters of AdCMVleptin or AdCMV--Gal containing a total of 1 1012 pfu were infused into anesthetized animals over a 1-h period. Animals were studied in individual metabolic cages (Nalgene, Rochester, NY), and food intake and body weight were measured daily. Plasma measurements. Beginning 1 day after adenovirus infusion, fasting blood samples (13 PM) were collected from the tail vein in capillary tubes coated with EDTA. Plasma was stored at 20C until the time of leptin assay. Plasma leptin was assayed using the Linco leptin assay kit (Linco Research, St. Charles, MO). Plasma insulin was assayed by standard

methods (37). Plasma glucose was measured by Glucose Analyzer II (Beckman, Brea, CA). Pancreas perfusion. Pancreata were isolated and perfused by the method of Grodsky and Fanska (12) as previously modied (17). The perfusate consisted of Krebs-Ringer bicarbonate buffer containing 4.5% Dextran T70, 5.6 mM glucose, 1% BSA, and 5 mM each of sodium pyruvate, sodium glutamate, and sodium fumarate. The ow rate was 3.0 ml/min. After a 20-min equilibration period, the pancreas was perfused for 10 min with 5.6 mM glucose. Then the glucose concentration was increased to 20 mM for 10 min. After a 10-min rest, during which the glucose level was returned to baseline, arginine was perfused at a concentration of 8 mM for a total of 10 min. Samples were collected at 1-min intervals for determination of insulin concentration. They were placed in chilled tubes containing 0.3 ml of 0.15 M NaCl, 0.05 M Na2EDTA, and 0.3 M benzamide and were frozen until the time of assay. TG content of liver, skeletal muscle, pancreas, and islets. Tissues were dissected and placed in liquid nitrogen. About 100 mg of tissues were placed in 4 ml of homogenizing buffer containing 18 mM of tris(hydroxymethyl)aminomethane HCl (pH 7.5), 300 mM of D-mannitol, and 5 mM of ethylene glycol-bis(-aminoethyl ether)-N, N, N, N-tetraacetic acid and were homogenized using a hand-held polytron (Kontes Glass, Vineland, NJ) for 10 s. Lipids were extracted by the method of Folch et al. (9). Total TG were assayed by the method of Danno et al. (6). Statistical analysis. All data are expressed as means SE. Statistical difference was analyzed by unpaired t-test. P 0.05 was considered statistically signicant. Regression line was calculated using the Stat View program (Abacus Concepts, Berkeley, CA).
RESULTS

Clinical and laboratory ndings. AdCMV-leptininfused rats developed hyperleptinemia averaging 13.7 2.1 ng/ml during the 14 days of the study (Fig. 1 A ). Compared with pair-fed normoleptinemic controls, these rats appeared hyperactive and disinterested in food but in otherwise good health. Their total feed intake over the period of 14 days averaged 58% of that of Gal-treated controls (Fig. 1B ). Body weight declined during the 1st wk and failed to increase during the 14-day period of study (Fig. 1C ), thus conrming our previous report (3). Visible body fat was absent or profoundly reduced in all sites after 1 wk of hyperleptinemia. Blood glucose levels fell below 2.6 mM and remained below normal throughout the 14 days (Fig. 2A ). Fasting plasma insulin levels averaged only 33.6 5.0 pM 14 days after the viral infusion, compared with 79.3 5.7 and 95.7 3.6 pM in pair-fed and free-feeding Galtreated controls, respectively (Fig. 2B ). Tissue lipid content in the various groups. Two weeks after the AdCMV-leptin infusion, the tissue content of TG was measured in liver, skeletal muscle, whole pancreas, and islets of age-matched hyperleptinemic rats, pair-fed and free-feeding control rats, and obese rats. The TG content is shown in Table 1, expressed both per gram of wet weight and as a percentage of the tissues of the three control groups. In the hyperleptinemic rats, the TG content of liver averaged 13.3% of that of free-feeding Gal-infused rats and was only

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A highly signicant relationship between the plasma [insulin] [glucose] and TG content of tissues was observed (r 0.93, P 0.0001). A similar correlation was observed between plasma insulin alone and tissue TG content (r 0.93, P 0.0001). The relationships between TG content of the three tissues and [insulin] [glucose] are shown in Fig. 3. Correlation with TG content of isolated islets is not shown because islets could not be weighed accurately and varied in size by a factor of 4. In the studies of perfused pancreata, insulin production was lowest in the hyperleptinemic group and increased progressively as the TG content of the pancreas increased. Thus, in the hyperleptinemic group, insulin secretion at 5.6 mM glucose was low but not signicantly different from that of pair-fed controls, whereas the responses to both glucose and arginine were completely absent (Fig. 4). At the other extreme of tissue TG, insulin production was greatest in the obese ZDF group and intermediate in the AdCMV-Galinfused and pair-fed control groups. Pancreata from obese ZDF rats were highest in fat content and exhibited the highest basal insulin secretion rate and the most brisk responses to glucose and arginine in absolute terms (Fig. 4). The coefficients of correlation in TG content among the three parameters of -cell function (basal, glucose-stimulated, and arginine-stimulated insulin secretion) ranged from 0.89 to 0.99.
DISCUSSION Fig. 1. Comparison of plasma leptin levels (A ), body weight (B ), and food intake (C ) in rats infused with a recombinant adenovirus containing leptin cDNA (AdCMV-leptin), normal rats pair fed to the former, free-feeding normal rats infused with an adenovirus containing cDNA of -galactosidase (AdCMV-gal), and obese Zucker diabetic fatty rats [ZDF (fa/fa )].

6.4% of that of obese rats; the fat content of their skeletal muscle was 8% of that of free-feeding Galinfused controls. In whole pancreas, TG content of the hyperleptinemics was 5.3% of that of AdCMV-Gal controls and 12.9% of that of pair-fed controls. In islets isolated from hyperleptinemic rats, the TG content per islet was below the sensitivity of the assay as employed. Islets of free-feeding AdCMV-Galinfused rats contained 28 ng/islet of TG, those of pair-fed rats contained 14 ng/islet, whereas islets of obese rats averaged 990 ng/islet. Correlations between TG content of pancreas and the product of plasma insulin and plasma glucose and insulin production by isolated pancreata. To determine the relationship between -cell function and islet fat content, we examined the correlations between pancreatic fat content and insulin production by isolated perfused pancreata from all groups. The perfusate consisted of either 5.6 mM glucose, 20 mM glucose, or 8 mM arginine plus 5.6 mM glucose. We also measured the correlation between pancreatic fat and plasma [insulin] plasma [glucose], which distinguished between hypoinsulinemia that results in increased blood glucose concentrations and hypoinsulinemia associated with increased insulin effectiveness.

The goal of this study was to determine whether the fat content in the target tissues of insulin and in the pancreas is correlated with both the effectiveness of insulin and the rate of its secretion. If so, it would be consistent with the notion that the responses of -cells are tailored to insulin need by a derivative of TG such as FFA. This would facilitate maintenance of euglycemia despite the changing body composition associated with obesity. It should, however, be stressed that corre-

Fig. 2. Comparison of plasma insulin (A ) and glucose (B ) in AdCMVleptin-infused rats, normal rats pair fed to the former, free-feeding AdCMV-gal-infused normal rats, and ZDF(fa/fa ) rats.

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Table 1. TG content in muscle, liver, and pancreas of AdCMV-leptin-infused rats compared with 3 control groups
Tissue AdCMV- Leptin Pair Fed AdCMV- Gal ZDF ( fa/fa)

mg/g wet wt Muscle Liver Pancreas 0.33 0.12 (6)* 0.64 0.19 (4)* 0.47 0.39 (4)* 1.31 0.26 (3) 4.59 1.17 (5) 3.63 2.00 (5) % of control groups Muscle Liver Pancreas 25.2 13.9 12.9 8.0 13.3 5.3 2.4 6.4 3.2 4.12 1.16 (5) 4.82 1.03 (4) 8.86 1.86 (3) 13.80 3.45 10.02 1.15 14.91 1.89

Values are means SE. Nos. of experiments are in parentheses. AdCMV-leptin and AdCMV-Gal, adenovirus containing leptin cDNA and -galactosidase, respectively; pair fed, normal rats pair fed to AdCMV-leptin-infused rats; ZDF ( fa/fa), obese Zucker diabetic fatty rats. Signicant (P 0.05) differences: * vs. pair fed; vs. AdCMV-Gal; vs. ZDF ( fa/fa).

lations cannot prove cause and effect and that alternative interpretations of these correlations are equally possible. For example, primary hyperinsulinemia could increase lipogenesis and cause increased tissue TG, which could in turn give rise to insulin resistance (24, 25). We employed both obese ZDF rats and lean Wistar rats. The availability of a unique fat-free animal, the hyperleptinemic rat, a novel model of extreme lipopenia that is the antithesis of obesity, made it possible to examine these correlations over the broadest possible spectrum of TG content, ranging from extreme lipopenia to severe obesity. In acute perfusion experiments, leptin had no effect on insulin production (data not shown), but in chronic studies of cultured islets, leptin abolished the insulin responses to both glucose- and arginine-stimulated insulin secretion while depleting islet TG; FFA restored the insulin response almost immediately (19). We observed that the production of the fasting plasma insulin and blood glucose levels, a crude index of insulin sensitivity, was signicantly correlated with TG content, as was the fasting plasma insulin level by itself. Insulin sensitivity was markedly enhanced (low

insulin-glucose product) in the lipopenic hyperleptinemic group. Fasting glucose levels in these groups were in a hypoglycemic range despite reduction in fasting plasma insulin levels; the insulin-glucose product of hyperleptinemic rats averaged 13% of normal controls and 4% of obese rats, evidence of exquisite insulin sensitivity. Clearly lipid content in target organs was correlated with this index of insulin sensitivity; r values ranged from 0.96 to 0.99 and were statistically signicant (P 0.05). However, because serum cortisol levels were not measured, a role of hypocortisolism in the insulin sensitivity of the hyperleptinemic rats cannot be excluded. Pancreatic TG content was correlated with -cell function as reected by the fasting insulin level (r 0.97; P 0.037). All phases of insulin secretion by the isolated perfused pancreas, basal and both glucose- and arginine-stimulated insulin secretion, also varied directly with the TG content of the pancreas.

Fig. 3. Correlations between triglyceride (TG) content of all tissues surveyed (skeletal muscle, liver, and pancreas) in AdCMV-leptininfused rats, rats pair fed to the former, free-feeding AdCMV-galinfused normal rats, and ZDF(fa/fa ) rats and the [insulin] [glucose] product. Correlation with plasma insulin alone gave an identical r value (not shown).

Fig. 4. Comparison of TG content of pancreas (A ) and insulin production by perfused pancreata (B ) isolated from hyperleptinemic rats, normal Wistar rats pair fed to the former, free-feeding AdCMVgal-infused rats, and ZDF(fa/fa ) rats.

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TISSUE TRIGLYCERIDES AND INSULIN PRODUCTION AND ACTION 10. Gomez-Foix, A. M., W. S. Coats, S. Baque, T. Alam, R. D. Gerard, and C. B. Newgard. Adenovirus-mediated transfer of the muscle glycogen phosphorylase gene into hepatocytes confers altered regulation of glycogen metabolism. J. Biol. Chem. 267: 2512925134, 1992. 11. Greenough, W. B., S. R. Crespin, and D. Steinberg. Hypoglycemia and hyperinsulinemia in response to raised free-fatty acid levels. Lancet 2: 13341336, 1967. 12. Grodsky, G. M., and R. E. Fanska. The in vitro perfused pancreas. Methods Enzymol. 39: 364372, 1975. 13. Groop, L. C., C. Saloranta, M. Shank, R. C. Bonadonna, E. Ferrannini, and R. A. DeFronzo. The role of free fatty acid metabolism in the pathogenesis of insulin resistance in obesity and noninsulin-dependent diabetes mellitus. J. Clin. Endocrinol. Metab. 72: 96107, 1991. 14. Herz, J., and R. D. Gerard. Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice. Proc. Natl. Acad. Sci. USA 90: 28122816, 1993. 15. Hettiarachchi, M., A. Watkinson, A. B. Jenkins, V. Theos, K. K. Ho, and E. W. Kraegen. Growth hormone-induced insulin resistance and its relationship to lipid availability in the rat. Diabetes 45: 415421, 1996. 16. Hirose, H., Y. H. Lee, L. R. Inman, Y. Nagasawa, J. H. Johnson, and R. H. Unger. Defective fatty acid-mediated -cell compensation in Zucker diabetic fatty rats. J. Biol. Chem. 271: 56335637, 1996. 17. Hisatomi, A., H. Maruyama, L. Orci, M. Vasko, and R. H. Unger. Adrenergically mediated intrapancreatic control of the glucagon responses to glucopenia in the isolated rat pancreas. J. Clin. Invest. 75: 420426, 1985. 18. Jenkins, A. B., L. H. Storlien, G. J. Cooney, G. S. Denyer, I. D. Caterson, and E. W. Kraegen. Effects of blockade of fatty acid oxidation on whole body and tissue-specic glucose metabolism in rats. Am. J. Physiol. 265 (Endocrinol. Metab. 28): E592E600, 1993. 19. Koyama, K., G. Chen, M.-Y. Wang, Y. Lee, M. Shimabukuro, C. B. Newgard, and R. H. Unger. -Cell function in normal rats made chronically hyperleptinemic by adenovirus-leptin gene therapy. Diabetes 46:12761280, 1997. 20. Lee, Y., H. Hirose, M. Ohneda, J. H. Johnson, J. D. McGarry, and R. H. Unger. -Cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: impairment in adipocyte--cell relationships. Proc. Natl. Acad. Sci. USA 91: 1087810882, 1994. 21. Lee, Y. H., H. Hirose, Y.-T. Zhou, V. Esser, J. D. McGarry, and R. H. Unger. Increased lipogenic capacity of the islets of obese rats: a role in the pathogenesis of NIDDM. Diabetes 46: 408413, 1997. 22. Madison, L. L., W. A. Seyffert, R. H. Unger, and B. Barker. Effect of plasma free fatty acids on plasma glucagon and serum insulin concentrations. Metabolism 17: 301304, 1968. 23. McCrory, W. J., D. S. Bautista, and F. L. Graham. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology 163: 614617, 1988. 24. McGarry, J. D. What if Minkowski had been ageusic? An alternative angle on diabetes. Science 258: 766774, 1992. 25. McGarry, J. D. Disordered metabolism in diabetes: have we underemphasized the fat cell component? J. Cell Biochem. 555: 2938, 1994. 26. Milburn, J. L., H. Hirose, Y. H. Lee, Y. Nagasawa, A. Ogawa, M. Ohneda, H. BeltrandelRio, C. Newgard, J. H. Johnson, and R. H. Unger. Pancreatic -cells in obesity: evidence for induction of functional, morphologic and metabolic abnormalities by increased long-chain fatty acids. J. Biol. Chem. 270: 12951299, 1995. 27. Murakami, T., and K. Shima. Cloning of rat obese cDNA and its expression in obese rats. Biochem. Biophys. Res. Commun. 209: 944952, 1995. 28. National Center for Health Statistics. An epidemic of obesity. Newsweek 1 Aug. 1994, p. 62.

The results are consistent with, but do not prove, the hypothesis that tissue TG content determines the level of insulin production and matches it to the level of insulin effectiveness, perhaps by providing an intracellular source of FFA retrieved from intracellular (TG) storage sites. Because the TG content of skeletal muscle and pancreas is also positively correlated, it is likely that it reects generalized changes in tissue fat. Teleologically, this postulated linkage between insulin requirement and insulin secretion would serve to reduce the risk of diabetes in obesity and to minimize the possibility of hypoglycemia in undernutrition. These observations follow more than three decades of research linking fatty acid metabolism to carbohydrate metabolism and diabetes (2, 4, 5, 7, 8, 11, 13, 15, 16, 18, 2022, 2426, 29, 3135, 38). Most attention has been directed at the effects of plasma lipids in reducing the insulin sensitivity of target tissues (3, 8, 13, 18, 23). However, the initial reports of stimulatory effects of FFA on islets (5, 11, 22) and, more recently, their inhibitory actions on -cell function (2, 7, 16, 38) have laid the groundwork for the present studies. The main difference is the emphasis on tissue TG content rather than perfusing levels of FFA and TG.
We thank Kay McCorkle, who provided technical support, and Sharryn Harris, who provided secretarial assistance. This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-0270037, the National Institutes of Health/Juvenile Diabetes Foundation Diabetes Interdisciplinary Research Program, and the Department of Veterans Affairs Institutional Research Support Grant SMI 821109. Address for reprint requests: R. H. Unger, Center for Diabetes Research, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 752358854. Received 14 March 1997; accepted in nal form 19 June 1997. REFERENCES 1. Becker, T. C., R. J. Noel, W. S. Coats, A. M. Gomez-Foix, T. Alam, R. D. Gerard, and C. B. Newgard. Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol. 43: 161189, 1994. 2. Capito, K., S. E. Hansen, C. J. Hedeskov, H. Islin, and P. Thams. Fat-induced changes in mouse pancreatic islet insulin secretion, insulin biosynthesis and glucose metabolism. Acta Diabetol. Lat. 28: 193198, 1992. 3. Chen, G., K. Koyama, X. Yuan, Y. Lee, Y. T. Zhou, R. ODoherty, C. B. Newgard, and R. H. Unger. Disappearance of body fat in normal rats induced by adenovirus-mediated leptin gene therapy. Proc. Natl. Acad. Sci. USA 93: 1479514799, 1996. 4. Corkey, B. E., M. C. Glennon, K. S. Chen, J. T. Denney, F. M. Matschinsky, and M. Prentki. A role for malonyl-CoA in glucose-stimulated insulin secretion from clonal pancreatic cells. J. Biol. Chem. 264: 2160821612, 1989. 5. Crespin, S. R., D. B. Greenough, and D. Steinberg. Stimulation of insulin secretion by long-chain free fatty acids. J. Clin. Invest. 48: 19341943, 1969. 6. Danno, H., Y. Jincho, S. Budiyanto, Y. Furukawa, and S. Kimura. A simple enzymatic quantitative analysis of triglycerides in tissues. J. Nutr. Sci. Vitaminol. 38: 517521, 1992. 7. Elks, M. L. Chronic perifusion of rat islets with palmitate suppresses glucose-stimulated insulin release. Endocrinology 133: 208214, 1993. 8. Ferrannini, E., E. J. Barrett, S. Bevilacqua, and R. A. DeFronzo. Effect of fatty acids on glucose production and utilization in man. J. Clin. Invest. 72: 17371747, 1983. 9. Folch, J., M. Lees, and G. H. S. Stanley. A simple method for the isolation and purication of total lipids from animal tissues. J. Biol. Chem. 226: 497509, 1957.

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