Aims/hypothesis Activation of the G protein-coupled receptor (GPR)40 by long-chain fatty acids potentiates glucosestimulated insulin secretion (GSIS) from pancreatic beta cells, and GPR40 agonists are in clinical development for type 2 diabetes therapy. GPR40 couples to the G protein subunit Gα q/11 but the signalling cascade activated downstream is unknown. This study aimed to determine the mechanisms of GPR40-dependent potentiation of GSIS by fatty acids. Methods Insulin secretion in response to glucose, oleate or diacylglycerol (DAG) was assessed in dynamic perifusions and static incubations in islets from wild-type (WT) and Gpr40 −/− mice. Depolymerisation of filamentous actin (F-actin) was visualised by phalloidin staining and epifluorescence. Pharmacological and molecular approaches were used to ascertain the roles of protein kinase D (PKD) and protein kinase C delta in GPR40-mediated potentiation of GSIS.Results Oleate potentiates the second phase of GSIS, and this effect is largely dependent upon GPR40. Accordingly, oleate induces rapid F-actin remodelling in WT but not in Gpr40islets. Exogenous DAG potentiates GSIS in both WT and Gpr40 −/− islets. Oleate induces PKD phosphorylation atElectronic supplementary material The online version of this article

There is growing concern over confounding artifacts associated with β-cell–specific Cre-recombinase transgenic models, raising questions about their general usefulness in research. The inducible β-cell–specific transgenic (MIP-CreERT1Lphi) mouse was designed to circumvent many of these issues, and we investigated whether this tool effectively addressed concerns of ectopic expression and disruption of glucose metabolism. Recombinase activity was absent from the central nervous system using a reporter line and high-resolution microscopy. Despite increased pancreatic insulin content, MIP-CreERT mice on a chow diet exhibited normal ambient glycemia, glucose tolerance and insulin sensitivity, and appropriate insulin secretion in response to glucose in vivo and in vitro. However, MIP-CreERT mice on different genetic backgrounds were protected from high-fat/ streptozotocin (STZ)-induced hyperglycemia that was accompanied by increased insulin content and islet density. Ectopic human growth hormone (hGH) was highly expressed in MIP-CreERT islets independent of tamoxifen administration. Circulating insulin levels remained similar to wild-type controls, whereas STZ-associated increases in α-cell number and serum glucagon were significantly blunted in MIP-CreERT1Lphi mice, possibly due to paracrine effects of hGH-induced serotonin expression. These studies reveal important new insight into the strengths and limitations of the MIP-CreERT mouse line for β-cell research.

Ββ-Cell adaptation to insulin resistance is necessary to maintain glucose homeostasis in obesity. Failure of this mechanism is a hallmark of type 2 diabetes (T2D). Hence, factors controlling functional β-cell compensation are potentially important targets for the treatment of T2D. Protein kinase D1 (PKD1) integrates diverse signals in the β-cell and plays a critical role in the control of insulin secretion. However, the role of β-cell PKD1 in glucose homeostasis in vivo is essentially unknown. Using β-cell-specific, inducible PKD1 knockout mice (βPKD1KO), we examined the role of β-cell PKD1 under basal conditions and during high-fat feeding. βPKD1KO mice under a chow diet presented no significant difference in glucose tolerance or insulin secretion compared with mice expressing the Cre transgene alone; however, when compared with wild-type mice, both groups developed glucose intolerance. Under a high-fat diet, deletion of PKD1 in β-cells worsened hyperglycemia, hyperinsulinemia, and glucose intolerance. This was accompanied by impaired glucose-induced insulin secretion both in vivo in hyperglycemic clamps and ex vivo in isolated islets from high-fat diet-fed βPKD1KO mice without changes in islet mass. This study demonstrates an essential role for PKD1 in the β-cell adaptive secretory response to high-fat feeding in mice.

Along with insulin, β‐cells co‐secrete the neurotransmitter ATP which acts as a positive autocrine signal via P2Y1 receptors to activate phospholipase C and increase the production of diacylglycerol (DAG). However, the downstream signaling that couples P2Y1 activation to insulin secretion remains to be fully elucidated. Since DAG activates protein kinase D1 (PKD1) to potentiate glucose‐stimulated insulin release, we hypothesized that autocrine ATP signaling activates downstream PKD1 to regulate insulin secretion. Indeed, we find that the P2Y1 receptor agonists, MRS2365 and ATP induce, PKD1 phosphorylation at serine 916 in mouse islets. Similarly, direct depolarization of islets by KCl caused PKD1 activation, which is reduced upon P2Y1 antagonism. Potentiation of insulin secretion by P2Y1 activation was lost from PKD1−/− mouse islets, and knockdown of PKD1 reduced the ability of P2Y1 activation to facilitate exocytosis in single mouse β‐cells. Finally, qPCR analysis confirmed PKD1 transcript (PRKD1) expression in human islets, and insulin secretion assays showed that inhibition of either P2Y1 or PKD1 signaling impaired glucose‐stimulated insulin secretion. Human islets showed donor‐to‐donor variation in their responses to both P2Y1 and PKD1 inhibition, however, and we find that the P2Y1‐PKD1 pathway contributes a substantially greater proportion of insulin secretion from islets of overweight and obese donors. Thus, PKD1 promotes increased insulin secretion, likely mediating an autocrine ATP effect via P2Y1 receptor activation which may be more important in islets of donors who are overweight or obese.

Free fatty acid receptor 1 (FFA1/GPR40) plays a key role in the potentiation of glucose-stimulated insulin secretion by fatty acids in pancreatic β cells. We previously demonstrated that GPR40 signaling leads to cortical actin remodeling and potentiates the second phase of insulin secretion. In this study, we examined the role of p21 activated kinase 4 (PAK4), a known regulator of cytoskeletal dynamics, in GPR40-dependent potentiation of insulin secretion. The fatty acid oleate induced PAK4 phosphorylation in human islets, in isolated mouse islets and in the insulin secreting cell line INS832/13. However, oleate-induced PAK4 phosphorylation was not observed in GPR40-null mouse islets. siRNA-mediated knockdown of PAK4 in INS832/13 cells abrogated the potentiation of insulin secretion by oleate, whereas PAK7 knockdown had no effect. Our results indicate that PAK4 plays an important role in the potentiation of insulin secretion by fatty acids downstream of GPR40.

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Schools for Growth: Radical Alternatives to Current Educational Models. Lois Holzman. Mahwah, NJ: Lawrence Erlbaum Associates, 1997. xi + 152 pp.

processes and that at high glucose islets convert a significant portion of the sugar into glycerol and FFA, which are exported outside the b-cell. The identification of fuel surfeit detoxification mechanisms by a targeted metabolomic approach should reveal additional pathways of b-cell fuel detoxification. Targeting fuel detoxification processes may provide a novel approach to preserve b-cell mass and function.