Insulin resistance is often associated with obesity and can precipitate type 2 diabetes. To date, most known approaches that improve insulin resistance must be preceded by the amelioration of obesity and hepatosteatosis. Here, we show that this provision is not mandatory; insulin resistance and hyperglycemia are improved by the modification of hepatic fatty acid composition, even in the presence of persistent obesity and hepatosteatosis. Mice deficient for Elovl6, the gene encoding the elongase that catalyzes the conversion of palmitate to stearate, were generated and shown to become obese and develop hepatosteatosis when fed a high-fat diet or mated to leptin-deficient ob/ob mice. However, they showed marked protection from hyperinsulinemia, hyperglycemia and hyperleptinemia. Amelioration of insulin resistance was associated with restoration of hepatic insulin receptor substrate-2 and suppression of hepatic protein kinase C epsilon activity resulting in restoration of Akt phosphorylation. Collectively, these data show that hepatic fatty acid composition is a new determinant for insulin sensitivity that acts independently of cellular energy balance and stress. Inhibition of this elongase could be a new therapeutic approach for ameliorating insulin resistance, diabetes and cardiovascular risks, even in the presence of a continuing state of obesity.

To determine the role of cholesterol synthesis in pancreatic b-cells, a transgenic model of in vivo activation of sterol-regulatory element binding protein 2 (SREBP-2) specifically in b-cells (TgRIP-SREBP-2) was developed and analyzed. Expression of nuclear human SREBP-2 in b-cells resulted in severe diabetes as evidenced by greater than 5-fold elevations in glycohemoglobin compared with C57BL/6 controls. Diabetes in TgRIP-SREBP-2 mice was primarily due to defects in glucose-and potassium-stimulated insulin secretion as determined by glucose tolerance test. Isolated islets of TgSREBP-2 mice were fewer in number, smaller, deformed, and had decreased insulin content. SREBP-2-expressing islets also contained increased esterified cholesterol and unchanged triglycerides with reduced ATP levels. Consistently, these islets exhibited elevated expression of HMG-CoA synthase and reductase and LDL receptor, with suppression of endogenous SREBPs. Genes involved in b-cell differentiation, such as PDX1 and BETA2, were suppressed, explaining loss of b-cell mass, whereas IRS2 expression was not affected. These phenotypes were dependent on the transgene expression. Taken Sterol-regulatory element binding protein 1c (SREBP-1c) is a membrane-bound transcription factor of the basic HLH (bHLH) leucine zipper family and has been established as a nutritional regulator of lipogenic enzymes in the liver (3, 4). Expression of SREBP-1c is highly upregulated by dietary intake of carbohydrates, sugars, and saturated FAs, whereas PUFAs, such as eicosapentaenoic acid, have been shown to inhibit hepatic SREBP-1c through multiple mechanisms (5, 6). These nutritional regulations of SREBP-1c are also observed in a cultured b-cell line and in isolated islets of mice (7,8). SREBP-1c also plays a role in insulin signaling by inhibiting insulin receptor substrate 2 (IRS-2), the major insulin-signaling mediator in the liver and in b-cells (9, 10).As a model for lipotoxicity by endogenous FAs in pancreatic b-cells, we previously developed transgenic mice overexpressing the active form of SREBP-1c under the insulin promoter expression (10). These mice exhibited impaired glucose tolerance in vivo due to both decreased b-cell mass and impaired insulin secretion estimated in isolated islets, which was enhanced by feeding the mice a high-fat, high-sucrose diet. The SREBP-1c-overexpressing islets had ATP depletion caused by enhanced lipogenesis and increased uncoupling protein 2 (UCP-2). Explaining the loss of b-cell mass, these islets had decreased expression of IRS-2 and PDX1. In addition to inhibition of GSIS,

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OBJECTIVE-Chronic exposure to fatty acids causes ␤-cell failure, often referred to as lipotoxicity. We investigated its mechanisms, focusing on contribution of SREBP-1c, a key transcription factor for lipogenesis. RESEARCH DESIGN AND METHODS-We studied in vitro and in vivo effects of saturated and polyunsaturated acids on insulin secretion, insulin signaling, and expression of genes involved in ␤-cell functions. Pancreatic islets isolated from C57BL/6 control and SREBP-1-null mice and adenoviral gene delivery or knockdown systems of related genes were used.RESULTS-Incubation of C57BL/6 islets with palmitate caused inhibition of both glucose-and potassium-stimulated insulin secretion, but addition of eicosapentaenoate (EPA) restored both inhibitions. Concomitantly, palmitate activated and EPA abolished both mRNA and nuclear protein of SREBP-1c, accompanied by reciprocal changes of SREBP-1c target genes such as insulin receptor substrate-2 (IRS-2) and granuphilin. These palmitate-EPA effects on insulin secretion were abolished in SREBP-1-null islets. Suppression of IRS-2/Akt pathway could be a part of the downstream mechanism for the SREBP-1c-mediated insulin secretion defect because adenoviral constitutively active Akt compensated it. Uncoupling protein-2 (UCP-2) also plays a crucial role in the palmitate inhibition of insulin secretion, as confirmed by knockdown experiments, but SREBP-1c contribution to UCP-2 regulation was partial. The palmitate-EPA regulation of insulin secretion was similarly observed in islets from C57BL/6 mice pretreated with dietary manipulations. Furthermore, administration of EPA to diabetic KK-Ay mice ameliorated impairment of insulin secretion in their islets.CONCLUSIONS-SREBP-1c plays a dominant role in palmitatemediated insulin secretion defect, and EPA prevents it through SREBP-1c inhibition, implicating a therapeutic potential for treating diabetes related to lipotoxicity. Diabetes 57:2382-2392, 2008

Granuphilin is a crucial component of the docking machinery of insulin-containing vesicles to the plasma membrane. Here, we show that the granuphilin promoter is a target of SREBP-1c, a transcription factor that controls fatty acid synthesis, and MafA, a beta cell differentiation factor. Potassium-stimulated insulin secretion (KSIS) was suppressed in islets with adenoviral-mediated overexpression of granuphilin and enhanced in islets with knockdown of granuphilin (in which granuphilin had been knocked down). SREBP-1c and granuphilin were activated in islets from beta cell-specific SREBP-1c transgenic mice, as well as in several diabetic mouse models and normal islets treated with palmitate, accompanied by a corresponding reduction in insulin secretion. Knockdown- or knockout-mediated ablation of granuphilin or SREBP-1c restored KSIS in these islets. Collectively, our data provide evidence that activation of the SREBP-1c/granuphilin pathway is a potential mechanism for impaired insulin secretion in diabetes, contributing to beta cell lipotoxicity.

Restriction endonucleases are widely used in laboratory applications from recombinant DNA technology to diagnostics, but engineering of restriction enzymes by structure-guided design and in vivo directed evolution is at an early stage. Here, we report the use of an in vitro compartmentalization system for completely in vitro selection of restriction enzymes. Compartmentalization of a single gene in a rabbit reticulocyte in vitro transcription/translation system serves to isolate individually synthesized enzymes from each other. In each compartment, an active enzyme cleaves only its own encoding gene, whereas genes encoding inactive enzymes remain intact. Affinity selection of the cleaved DNA encoding active restriction endonucleases was accomplished by the use of streptavidin-immobilized beads and dUTP-biotin, which was efficiently incorporated into the cohesive end of the cleaved DNA using a DNA polymerase. We confirmed that genes encoding active restriction endonuclease FokI could be selected from a randomized library. This method overcomes the limitations of current in vivo technologies and should prove useful for rapid screening and evolution of novel restriction enzymes from diverse mutant libraries, as well as for studies of catalytic and evolutionary mechanisms of restriction enzymes.

F-box and WD repeat domain-containing 7 (Fbw7) is the component of an evolutionarily conserved complex of the Skp1-Cul1-F-box protein ubiquitin ligase and is involved in substrate recognition of the complex (1, 2). Fbw7 targets several proto-oncogenes that function in cell growth and division pathways, including c-Myc (encoded by Myc), cyclin E, Notch, and c-Jun (encoded by Jun) (3-7). Fbw7 is perturbed in many human malignancies and is an established tumor suppressor (8 -11). Mouse Fbw7 exists in three different isoforms as follows: ␣, ␤, and ␥. The ␣ isoform is expressed ubiquitously, whereas the ␤ and ␥ isoforms are expressed restrictedly in the brain, heart, testis, and skeletal muscle (12). Intriguing characteristics of Fbw7␣ (encoded by the isoform1 of Fbxw7) have recently been described by Ericsson et al. (13) who demonstrated that this cell growth regulator also regulated the degradation of the nuclear forms of the sterol regulatory elementbinding protein (SREBP) 2 family (14). SREBPs, belonging to the bHLH-Zip transcription factor family, are established regulators of lipid synthesis. The unique features of SREBPs are their rough-surfaced endoplasmic reticulum membrane-bound transcription factors. These factors need to undergo proteolytic cleavage for nuclear transport to activate the expression of genes involved in lipid synthesis. This represents the crucial step for sterol and fatty acid synthetic gene regulation (15)(16)(17). The SREBP family includes three isoforms as follows: . SREBP-2 governs cellular sterol regulation, whereas hepatic SREBP-1c (encoded by the isoformb of Srebf1) controls fatty acid and triglyceride synthesis depending on the nutritional state of the liver. SREBP-1a is highly expressed in growing cells and contributes to the synthesis of cholesterol, triglyceride (TG), and phospholipid for the supply of membrane lipids during cell growth (21, 22). Nuclear SREBP-1a regulates the cell cycle and growth by itself, indicating its strong association with cell growth (23,24).Without a proteasome inhibitor such as calpain inhibitor I in cell cultures, nuclear SREBPs are rapidly degraded by the ubiquitin-proteasome pathway after cleavage. Recently, Fbw7 was reported to be the key factor for this degradation of SREBPs in cultured cells (14). SREBP-1a is phosphorylated at several sites * This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental