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Genetic Manipulations of Fatty Acid Metabolism in -Cells Are Associated with Dysregulated Insulin Secretion

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Genetic Manipulations of Fatty Acid Metabolism in -Cells Are Associated with Dysregulated Insulin Secretion Genetic Manipulations of Fatty Acid Metabolism in ␤-Cells Are Associated With Dysregulated Insulin Secretion Kazuhiro Eto,1 Tokuyuki Yamashita,1 Junji Matsui,1 Yasuo Terauchi,1 Mitsuhiko Noda,2 and Takashi Kadowaki1 Triacylglyceride (TG) accumulation in pancreatic ␤-cells is associated with impaired insulin secretion, which is called lipotoxicity. To gain a better under- t has been widely known that the accumulation of standing of the pathophysiology of lipotoxicity, we gen- triacylglycerides (TGs) in pancreatic ␤-cells is asso- erated three models of dysregulated fatty acid ciated with dysfunction of the cells, including im- metabolism in ␤-cells. The overexpression of sterol Ipaired insulin secretory response to glucose and regulatory element binding protein-1c induced lipogenic lipoapoptosis (1–3). The mechanisms by which TG accu- genes and TG accumulation. Under these conditions, we mulation leads to these dysfunctions, however, have re- observed a decrease in glucose oxidation and upregula- mained largely elusive. In the present study, we tion of uncoupling protein-2, which might be causally established three models of dysregulated metabolism of related to the decreased glucose-stimulated insulin se- fatty acids in ␤-cells and studied their effects on cell cretion. The overexpression of AMP-activated protein functions, especially insulin secretion, as follows: 1) over- kinase was accompanied by decreased lipogenesis, in- expression of sterol regulatory element binding protein creased fatty acid oxidation, and decreased glucose (SREBP)-1c (a master transcription factor for lipogenesis oxidation; insulin secretions to glucose and depolariza- [4–7]) in ␤-cells to reconstitute lipotoxicity with a geneti- tion stimuli were decreased, probably because of the cally determined cause; 2) overexpression of AMP-acti- decrease in glucose oxidation and cellular insulin con- vated protein kinase (AMPK; a metabolic master switch tent. It was notable that the secretory response to for energy utilization [8,9]) in ␤-cells to dissect its role in palmitate was blunted, which would suggest a role of the insulin secretion in relation to glucose and lipid metabo- fatty acid synthesis pathway, but not its oxidative path- ␤ way in palmitate-stimulated insulin secretion. Finally, lism; and 3) -cells with a moderately decreased activity of ␥ ؉ ؊ Ϫ we studied islets of PPAR-␥ / mice that had increased peroxisome proliferator activated receptor (PPAR)- ␥ϩ/Ϫ insulin sensitivity and low TG content in white adipose from PPAR- mice to better understand the role of tissue, skeletal muscle, and liver. On a high-fat diet, PPAR-␥ in the regulation of lipid distribution to multiple glucose-stimulated insulin secretion was decreased in organs and the influence on ␤-cells (10–12). association with increased TG content in the islets, which might be mediated through the elevated serum free fatty acid levels and their passive transport into ROLE OF SREBPs IN LIPID METABOLISM ␤-cells. These results revealed some aspects about the SREBPs are comprised of three forms, SREBP-1a, -1c, and mechanisms by which alterations of fatty acid metabo- -2, which belong to the basic helix-loop-helix leucine lism affect ␤-cell functions. Diabetes 51 (Suppl. 3): zipper transcription factors (5). SREBP-1a and -1c are S414–S420, 2002 encoded by a single gene through the use of alternative transcription start sites and differ in the first exon. SREBP-2 arises from a separate gene. Although all three forms of SREBP contribute to lipid biosynthesis, From the 1Department of Metabolic Diseases, Graduate School of Medicine, SREBP-1a and -1c are preferentially important for fatty University of Tokyo, Tokyo, Japan; and the 2Institute for Diabetes Care and Research, Asahi Life Foundation, Tokyo, Japan. acid and TG synthesis, whereas SREBP-2 is important for Address correspondence and reprint requests to Takashi Kadowaki, Depart- cholesterol synthesis (13). SREBP-1a has a longer NH2- ment of Metabolic Diseases, Graduate School of Medicine, University of terminal acidic activation domain than SREBP-1c, render- Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: kadowaki- [email protected]. ing the 1a isoform much more active in stimulating Received for publication 23 March 2002 and accepted in revised form 3 April transcription (14). In addition to their roles in transcrip- 2002. tion, SREBPs have been implicated in enhancing adipo- ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide-1- ␤-D-ribofuranoside; AMPK, AMP-activated protein kinase; AMPK/CA, consti- cyte differentiation (7). In one study, homozygous tutive active form of AMPK; CPT-1, carnitine palmitoyltransferase-1; HNF, knockout of the SREBP-1 gene in mice resulted in 50–85% hepatocyte nuclear factor; KATP channel, ATP-sensitive potassium channel; L-PK, liver-type pyruvate kinase; PPAR, peroxisome proliferator–activated of embryonic lethality, but the surviving mice appeared receptor; RXR, retinoid X receptor; SRE, sterol regulatory element; SREBP, normal with unchanged amounts of white adipose tissue sterol regulatory element binding protein; TG, triacylglyceride; TZD, thiazo- and mRNA levels for lipogenic enzymes, lipoprotein lipase, lidinedione; UCP, uncoupling protein. The symposium and the publication of this article have been made possible and leptin (15). These results suggest that SREBP-2 can by an unrestricted educational grant from Servier, Paris. compensate, at least in part, for SREBP-1 in the regulation S414 DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002 K. ETO AND ASSOCIATES FIG. 1. A: Increased insulin secretion at 1 mmol/l glucose and decreased insulin secretion at 10 mmol/l glucose in INS-1 cells overexpressing SREBP-1c. B: Preserved potentiation of glucose-stimulated insulin secretion by palmitate in INS-1 cells overexpressing SREBP-1c. Increase by .P < 0.01* .(6 ؍ mmol/l palmitate in insulin secretion at 10 mmol/l glucose is indicated. Values are expressed as means ؎ SE (n 1 of lipid synthesis. The transgenic overexpression of glucose was increased by 50%, whereas the enhancement SREBP-1a in liver, driven by the phosphoenolpyruvate in insulin secretion in response to 10 mmol/l glucose was carboxykinase promoter, developed into progressive and decreased by 70% as compared to controls (Fig. 1A). The massive enlargement of the liver owing to the engorge- insulin response to depolarizing stimuli such as 50 mmol/l ment of the hepatocytes with TGs and cholesterol, accom- KCl and 5 ␮mol/l glibenclamide was decreased by 35 and panied by markedly elevated mRNA levels for acetyl-CoA 13%, respectively. The effect of palmitate on initiating carboxylase, fatty acid synthase, and stearoyl-CoA desatu- insulin secretion in the presence of 1 mmol/l glucose and rase-1 (16). In ␤-cells, although all three forms of SREBPs potentiating insulin secretion in response to 10 mmol/l were expressed, the amounts were small and barely de- glucose was relatively preserved in cells overexpressing tectable by RT-PCR in islets of Zucker lean wild-type rats SREBP-1c as compared to controls (Fig. 1B). Consistent (17). with the reduced secretory response to glucose, [6-14C]glu- cose oxidation was decreased by 20% in cells overexpress- DEFECTIVE GLUCOSE-STIMULATED INSULIN ing SREBP-1c. We then measured cellular contents of ATP SECRETION IN ␤-CELLS OVEREXPRESSING SREBP-1c and ADP. The ATP/ADP ratio in the presence of 10 mmol/l To generate a ␤-cellϪspecific primary lipotoxicity model, glucose was significantly decreased as compared to con- we overexpressed a constitutively active form of trols, a finding that supports the reduced catabolism of SREBP-1c (1-403 aa) in the insulin-secreting cell line INS-1 glucose. Thus, these impairments in glucose utilization via an adenoviral vector (7,18). The overexpression may have affected the ATP-sensitive potassium (KATP) evoked a marked increase in the transcriptional activity of channelϪdependent pathway of the glucose-stimulated a reporter gene driven by the sterol regulatory element insulin secretion in this model. (SRE) cognitive sequences and resulted in a 60% increase Interestingly, the expression of uncoupling protein in TG content at 48 h after the adenoviral infection at (UCP)-2, which dissipates mitochondrial electrochemical 10 m.o.i. Indeed, the expression of lipogenic enzymes, potential to heat, thereby costing ATP generation (19), was such as ATP-citrate lyase, acetyl-CoA carboxylase, acyl- increased twofold at both mRNA and protein levels. In CoA synthase, fatty acid synthase, and glycerol-phosphate agreement with this observation, SRE has been found in acyltransferase, were markedly increased; on the other the promoter region of the UCP-2 gene (20). It has also hand, the expression of enzymes for fatty acid oxidation, been reported that oleic acid and linolenic acid activate such as acetyl-CoA oxidase and acetyl-CoA dehydroge- the transcription of the UCP-2 gene (21,22). Thus, the nase, was almost unchanged as compared to control cells upregulation of UCP-2 directly through the SRE by infected with an adenoviral vector harboring the LacZ SREBP-1c, and/or indirectly through generation of specific cDNA. Under these conditions, cellular insulin content fatty acids, is another plausible mechanism by which was slightly decreased, but transcriptional activity of the lipotoxicity causes the reduced responsiveness of insulin pre-proinsulin gene, as measured with a luciferase re- secretion to glucose. This hypothesis would be verifiable porter gene assay, was not affected. under
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