We have proposed the "glucolipotoxicity" hypothesis in which elevated free fatty acids (FFAs) together with hyperglycemia are synergistic in causing islet beta-cell damage because high glucose inhibits fat oxidation and consequently lipid detoxification. The effects of 1-2 d culture of both rat INS 832/13 cells and human islet beta-cells were investigated in medium containing glucose (5, 11, 20 mM) in the presence or absence of various FFAs. A marked synergistic effect of elevated concentrations of glucose and saturated FFA (palmitate and stearate) on inducing beta-cell death by apoptosis was found in both INS 832/13 and human islet beta-cells. In comparison, linoleate (polyunsaturated) synergized only modestly with high glucose, whereas oleate (monounsaturated) was not toxic. Treating cells with the acyl-coenzyme A synthase inhibitor triacsin C, or the AMP kinase activators metformin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside that redirect lipid partitioning to oxidation, curtailed glucolipotoxicity. In contrast, the fat oxidation inhibitor etomoxir, like glucose, markedly enhanced palmitate-induced cell death. The data indicate that FFAs must be metabolized to long chain fatty acyl-CoA to exert toxicity, the effect of which can be reduced by activating fatty acid oxidation. The results support the glucolipotoxicity hypothesis of beta-cell failure proposing that elevated FFAs are particularly toxic in the context of hyperglycemia.

␤-Cells possess inherent mechanisms to adapt to overnutrition and the prevailing concentrations of glucose, fatty acids, and other fuels to maintain glucose homeostasis. However, this is balanced by potentially harmful actions of the same nutrients. Both glucose and fatty acids may cause good/adaptive or evil/toxic actions on the ␤-cell, depending on their concentrations and the time during which they are elevated. Chronic high glucose dramatically influences ␤-cell lipid metabolism via substrate availability, changes in the activity and expression of enzymes of glucose and lipid metabolism, and modifications in the expression level of key transcription factors. We discuss here the emerging view that ␤-cell "glucotoxicity" is in part indirectly caused by "lipotoxicity," and that ␤-cell abnormalities will become particularly apparent when both glucose and circulating fatty acids are high. We support the concept that elevated glucose and fatty acids synergize in causing toxicity in islets and other organs, a process that may be instrumental in the pleiotropic defects associated with the metabolic syndrome and type 1 and type 2 diabetes. The mechanisms by which hyperglycemia and hyperlipidemia alter insulin secretion are discussed and a model of ␤-cell "glucolipotoxicity" that implicates alterations in ␤-cell malonyl-CoA concentrations; peroxisome proliferator؊activated receptor-␣ and -␥ and sterol regulatory element binding protein-1c expression; and lipid partitioning is proposed. Diabetes 51 (Suppl. 3):S405-S413, 2002

Glucagon-like peptide-1-(7±36)amide (GLP-1) is secreted by the enteroendocrine L-cells in response to fat meals and carbohydrates [1,2]. It is a potent glucoincretin hormone which acts as a competence factor in determining the ability of the beta-cell to respond to glucose by secretion of insulin [3], K/ATP channels closure [4] and the induction of immediate early response genes (IEG) [5]. It is a potentially important drug in the treatment of diabetes particularly Diabetologia (1999) AbstractAims/hypothesis. Glucagon-like peptide-1 is a potent glucoincretin hormone and a potentially important drug in the treatment of Type II (non-insulin-dependent) diabetes mellitus. We have investigated whether it acts as a growth factor in beta (INS-1)-cells and have studied the signalling pathways and transcription factors implicated in this process. Methods. Cell proliferation was assessed by tritiated thymidine incorporation measurements. We have examined the action of glucagon-like peptide-1 on the enzymatic activity of phosphatidylinositol 3-kinase. The DNA binding activity of transcription factors was investigated by electrophoretic mobility shift assay. Measurements of mRNA were done using the northern technique. Results. Glucagon-like peptide-1 caused an increase in tritiated thymidine incorporation in beta (INS-1)-cells and phosphatidylinositol 3-kinase activity in a dose-dependent manner non-additively with glucose. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294 002 blocked the effects of glucagon-like peptide-1 on DNA synthesis. Transcription factor pancreatic and duodenal homebox gene 1 (PDX-1) DNA binding activity was increased by glucagon-like peptide-1 at 3 or 11 mmol/l glucose and the phosphatidylinositol 3-kinase inhibitor LY294 002 suppressed the action of glucagon-like peptide-1 on PDX-1 DNA binding activity. Glucagon-like peptide-1 and glucose alone did not change activating protein-1 DNA binding activity. They synergised, however, to increase the activity of activating protein-1. Glucagon-like peptide-1 also increased the expression of PDX-1, glucose transporter 2, glucokinase and insulin mRNAs. Finally, glucagon-like peptide-1 increased the incorporation of tritiated thymidine in isolated rat islets. Conclusion/interpretation. The results suggest that glucagon-like peptide-1 may act as a growth factor for the beta cell by a phosphatidylinositol 3-kinase mediated event. Glucagon-like peptide-1 could also regulate the expression of the insulin gene and genes encoding enzymes implicated in glucose transport and metabolism through the phosphatidylinositol 3-kinase/PDX-1 transduction signalling pathway. [Diabetologia (1999) 42: 856±864]

The malonyl-CoA/long-chain acyl-CoA (LC-CoA) model of glucose-induced insulin secretion (GIIS) predicts that malonyl-CoA derived from glucose metabolism inhibits fatty acid oxidation, thereby increasing the availability of LC-CoA for lipid signaling to cellular processes involved in exocytosis. For directly testing the model, INSr3 cell clones overexpressing malonylCoA decarboxylase in the cytosol (MCDc) in a tetracycline regulatable manner were generated, and INS(832/ 13) and rat islets were infected with MCDc-expressing adenoviruses. MCD activity was increased more than fivefold, and the malonyl-CoA content was markedly diminished. This was associated with enhanced fat oxidation at high glucose, a suppression of the glucoseinduced increase in cellular free fatty acid (FFA) content, and reduced partitioning at elevated glucose of exogenous palmitate into lipid esterification products. MCDc overexpression, in the presence of exogenous FFAs but not in their absence, reduced GIIS in all ␤-cell lines and in rat islets. It also markedly curtailed the stimulation of insulin secretion by other fuel and nonfuel secretagogues. In the absence of MCDc overexpression, the secretory responses to all types of secretagogues were amplified by the provision of exogenous fatty acids. In the presence of exogenous FFAs, the fatty acyl-CoA synthetase inhibitor triacsin C reduced secretion in response to glucose and nonfuel stimuli. The data show the existence of important links between the metabolic coupling factor malonyl-CoA, the partitioning of fatty acids, and the stimulation of insulin secretion to both fuel and nonfuel stimuli. Diabetes 53:

Macrophage migration inhibitory factor (MIF), originally identified as a cytokine secreted by T lymphocytes, was found recently to be both a pituitary hormone and a mediator released by immune cells in response to glucocorticoid stimulation. We report here that the insulinsecreting ␤ cell of the islets of Langerhans expresses MIF and that its production is regulated by glucose in a time-and concentration-dependent manner. MIF and insulin colocalize by immunocytochemistry within the secretory granules of the pancreatic islet ␤ cells, and once released, MIF appears to regulate insulin release in an autocrine fashion. In perifusion studies performed with isolated rat islets, immunoneutralization of MIF reduced the first and second phase of the glucose-induced insulin secretion response by 39% and 31%, respectively. Conversely, exogenously added recombinant MIF was found to potentiate insulin release. Constitutive expression of MIF antisense RNA in the insulin-secreting INS-1 cell line inhibited MIF protein synthesis and decreased significantly glucose-induced insulin release. MIF is therefore a glucose-dependent, islet cell product that regulates insulin secretion in a positive manner and may play an important role in carbohydrate metabolism.

The role of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I, in regulating the oxidation of fatty acids in rat skeletal (1, 2) and cardiac (3, 4) muscle has been intensively investigated. Recent studies have demonstrated that its concentration in rat muscle is governed, at least in part, by changes in the activity of the muscle isoform of acetyl-CoA carboxylase (ACC ␤ ) 1 (5), the enzyme that catalyzes malonylCoA synthesis. Thus, in keeping with their observed effects on malonyl-CoA concentration and fatty acid oxidation, insulin and glucose appear to activate ACC ␤ in muscle by increasing the cytosolic concentration of citrate, an allosteric activator of ACC ␤ and a precursor of its substrate, cytosolic acetyl-CoA. Conversely, decreases in malonyl-CoA concentration and increases in fatty acid oxidation in muscle during exercise (contraction) have been linked to decreases in ACC ␤ activity, attributable to its phosphorylation and inhibition by the ␣ 2 isoform of AMP-activated protein kinase (AMPK) (5). AMPK can also be activated and the concentration of malonyl-CoA decreased by exposing resting muscle to 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which is taken into the muscle and phosphorylated to form the 5Ј-AMP analogue ZMP (6).Whether a change in malonyl-CoA turnover contributes to the alterations in its concentration in muscle during exercise and other conditions is not known. In a lipogenic tissue such as liver, the de novo synthesis of fatty acids is thought to be the major mechanism by which malonyl-CoA is utilized. In contrast, in skeletal muscle fatty acid synthesis occurs at a very low rate, if at all (7), and attention has been focused on malonyl-CoA decarboxylase (MCD) for removal of malonyl-CoA (1). Evidence has been presented that MCD is present in both cardiac (8, 9) and skeletal (1, 10, 11) muscle. In skeletal muscle, its activity is similar to that of ACC (1). In heart, in which MCD activity is substantially greater than in skeletal muscle, a decrease in the K m of MCD for malonyl-CoA has been reported following an increase in its work load (9). On the other hand, no change in activity has been observed following ischemia-reperfusion of the heart, a situation in which AMPK is activated (8). The question of whether MCD is acutely regulated in skeletal muscle and, if so, how has not been studied previously.In this study, we describe the characteristics of purified MCD from rat skeletal muscle and contraction-induced changes in its maximal activity and affinity for malonyl-CoA. In addition, the effects of the AMPK activator 5-aminoimida-

Pancreatic islet transplantation may successfully restore normoglycemia in type 1 diabetic patients. However, successful grafting requires transplantation of a sufficient number of islets, usually requiring two or more donors. During the isolation process and following clinical transplantation, islets are subjected to severe adverse conditions that impair survival and ultimately contribute to graft failure. Here, we have mapped the major intracellular stress-signaling pathways that may mediate human islet loss during isolation and following cytokine attack. We found that the isolation procedure potently recruits two pathways consisting of ͦmitogen-activated protein kinase kinase (MKK)7 3 Jun NH 2 -terminal kinase (JNK)/p38 3 c-fosͦ and the ͦnuclear factor-B (NF-B) 3 iNOSͦ module. Cytokines activate the ͦNF-B 3 iNOSͦ and ͦMKK4/MKK3/6 3 JNK/p38ͦ pathways without recruitment of c-fos. Culturing the islets for 48 h after isolation allows for the activated pathways to return to background levels, with expression of MKK7 becoming undetectable. These data indicate that isolation and cytokines recruit different death pathways. Therefore, strategies might be rationally developed to avoid possible synergistic activation of these pathways in mediating islet loss during isolation and following grafting.

Endogenous lipid stores are thought to be involved in the mechanism whereby the ␤-cell adapts its secretory capacity in obesity and diabetes. In addition, hormonesensitive lipase (HSL) is expressed in ␤-cells and may provide fatty acids necessary for the generation of coupling factors linking glucose metabolism to insulin release. We have recently created HSL-deficient mice that were used to directly assess the role of HSL in insulin secretion and action. HSL ؊/؊ mice were normoglycemic and normoinsulinemic under basal conditions, but showed an ϳ30% reduction of circulating free fatty acids (FFAs) with respect to control and heterozygous animals after an overnight fast. An intraperitoneal glucose tolerance test revealed that HSL-null mice were glucose-intolerant and displayed a lack of a rise in plasma insulin after a glucose challenge. Examination of plasma glucose during an insulin tolerance test suggested that HSL-null mice were insulin-resistant, because plasma glucose was barely lowered after the injection of insulin. Freshly isolated islets from HSL-deficient mice displayed elevated secretion at low (3 mmol/l) glucose, failed to release insulin in response to high (20 mmol/l) glucose, but had a normal secretion when challenged with elevated KCl. The phenotype of heterozygous mice with respect to the measured parameters in vitro was similar to that of wild type. Finally, the islet triglyceride content of HSL ؊/؊ mice was 2-2.5 fold that in HSL ؊/؉ and HSL ؉/؉ animals. The results demonstrate an important role of HSL and endogenous ␤-cell lipolysis in the coupling mechanism of glucose-stimulated insulin secretion. The data also provide direct support for the concept that some lipid molecule(s), such as FFAs, fatty acyl-CoA or their derivatives, are implicated in ␤-cell glucose signaling.