Fibroblast growth factor-21 (FGF-21) is a recently discovered metabolic regulator. Here, we investigated the effects of FGF-21 in the pancreatic ␤-cell. In rat islets and INS-1E cells, FGF-21 activated extracellular signal-regulated kinase 1/2 and Akt signaling pathways. In islets isolated from healthy rats, FGF-21 increased insulin mRNA and protein levels but did not potentiate glucose-induced insulin secretion. Islets and INS-1E cells treated with FGF-21 were partially protected from glucolipotoxicity and cytokineinduced apoptosis. In islets isolated from diabetic rodents, FGF-21 treatment increased islet insulin content and glucose-induced insulin secretion. Short-term treatment of normal or db/db mice with FGF-21 lowered plasma levels of insulin and improved glucose clearance compared with vehicle after oral glucose tolerance testing. Constant infusion of FGF-21 for 8 weeks in db/db mice nearly normalized fed blood glucose levels and increased plasma insulin levels. Immunohistochemistry of pancreata from db/db mice showed a substantial increase in the intensity of insulin staining in islets from FGF-21-treated animals as well as a higher number of islets per pancreas section and of insulin-positive cells per islet compared with control. No effect of FGF-21 was observed on islet cell proliferation. In conclusion, preservation of ␤-cell function and survival by FGF-21 may contribute to the beneficial effects of this protein on glucose homeostasis observed in diabetic animals. Diabetes 55:2470 -2478, 2006 P ancreatic ␤-cell dysfunction is a central component of the pathogenesis of all forms of diabetes. Type 1 diabetes manifests from the autoimmune destruction of ␤-cells, whereas type 2 diabetes is characterized by reduced ␤-cell mass and marked functional defects, including impaired first-phase insulin secretion, increased proinsulin-to-insulin ratio, and elevated rate of ␤-cell apoptosis (1-3). The glucose-sensing and insulin-signaling pathways have been shown to play important roles in insulin secretion as well as ␤-cell growth and survival. For example, mice lacking insulin receptors, insulin receptor substrate-2, or Akt (protein kinase B) display marked defects in glucose sensing, insulin secretion, and ␤-cell mass (4 -6). The amount of secreted insulin is determined by the secretory activity of the ␤-cell and the total number of ␤-cells in the pancreas. Glucose plays an essential role in the control of secretory activity of ␤-cells. Metabolism of glucose leads to an increase in the ATP-to-ADP ratio, membrane depolarization, Ca 2ϩ influx, and stimulation of insulin secretion (7). ␤-Cell mass is governed by the balance between ␤-cell growth and ␤-cell death (apoptosis). Type 2 diabetic patients display a progressive loss of ␤-cells caused by an increased rate of ␤-cell apoptosis (8). However, the cause and mechanism(s) responsible for the increased apoptosis rate in type 2 diabetes are not well understood (9). Preventing ␤-cell death and increasing survival of the ␤-cell can be a valuable therapeutic approa...

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By using the yeast two-hybrid system we identified a novel protein from the human brain interacting with the C terminus of somatostatin receptor subtype 2. This protein termed somatostatin receptor interacting protein is characterized by a novel domain structure, consisting of six N-terminal ankyrin repeats followed by SH3 and PDZ domains, several proline-rich regions, and a C-terminal sterile ␣ motif. It consists of 2185 amino acid residues encoded by a 9-kilobase pair mRNA; several splice variants have been detected in human and rat cDNA libraries. Sequence comparison suggests that the novel multidomain protein, together with cortactinbinding protein, forms a family of cytoskeletal anchoring proteins. Fractionation of rat brain membranes indicated that somatostatin receptor interacting protein is enriched in the postsynaptic density fraction. The interaction of somatostatin receptor subtype 2 with its interacting protein was verified by overlay assays and coimmunoprecipitation experiments from transfected human embryonic kidney cells. Somatostatin receptor subtype 2 and the interacting protein display a striking overlap of their expression patterns in the rat brain. Interestingly, in the hippocampus the mRNA for somatostatin receptor interacting protein was not confined to the cell bodies but was also observed in the molecular layer, suggesting a dendritic localization of this mRNA.Targeting of neurotransmitter receptors to postsynaptic or presynaptic sites is an area that has been widely studied in recent years; a large body of evidence has accumulated showing that many receptors are anchored at their specifc site of action by specialized anchoring proteins, which may link receptors to components of the synaptic structure or the cytoskeleton (1, 2). This is true for inhibitory as well as excitatory receptors of the family of ligand-gated ion channels. For the second large family of neurotransmitter receptors, the seven transmembrane domain G-protein-coupled receptors, only very recently have some proteins been identified that may be involved in anchoring or linkage to the cytoskeleton. These include the homer proteins, which are tightly associated with metabotropic glutamate receptors via a PDZ 1 domain in homer and the C terminus of the mGluRs (3). However, for the large majority of G-protein-coupled receptors, no intracellular associated proteins have been identified so far beyond those proteins which are necessary for signal transduction and functional regulation of the receptors, i.e. the G-proteins and proteins of the arrestin family (4).We have begun to address this issue for members of the somatostatin receptor family (SSTRs). SSTRs are widely expressed in neuronal tissue and modulate synaptic responses by interacting with inhibitory G-proteins in presynaptic as well as postsynaptic compartments of neurons (e.g. Refs. 5-8). Recently we have used the yeast two-hybrid system to screen for proteins intracellularly associated with SSTR2, one of the major SSTR subtypes in the mammalian brain. Here we show t...

Subtypes of the calcium-independent receptors for ␣؊latrotoxin (CIRL1-3) define a distinct subgroup within the large family of the seven-transmembrane region cell surface receptors. The physiological function of CIRLs is unknown because neither extracellular ligands nor intracellular coupling proteins (G-proteins) have been identified. Using yeast two-hybrid screening, we identified a novel interaction between the C termini of CIRL1 and -2 and the PSD-95/discs large/ZO-1 (PDZ) domain of a recently discovered multidomain protein family (ProSAP/SSTRIP/Shank) present in human and rat brain. In vitro, CIRL1 and CIRL2 interacted strongly with the PDZ domain of ProSAP1. The specificity of this interaction has been verified by in vivo experiments using solubilized rat brain membrane fractions and Pro-SAP1 antibodies; only CIRL1, but not CIRL2, was coimmunoprecipitated with ProSAP1. In situ hybridization revealed that ProSAP1 and CIRL1 are co-expressed in the cortex, hippocampus, and cerebellum. Colocalization was also observed at the subcellular level, as both CIRL1 and ProSAP1 are enriched in the postsynaptic density fraction from rat brain. Expression of all three CIRL isoforms is highly regulated during postnatal brain development, with CIRL3 exhibiting its highest expression levels immediately after birth, followed by CIRL2 and finally CIRL1 in aged rats.Most neuronal cell surface receptors are attached to intracellular proteins via specific protein/protein interactions; this has been demonstrated in a number of cases involving ligand gated ion channels and G-protein-coupled receptors, as well as cell adhesion molecules (e.g. Refs. 1-3). A protein module frequently involved in this type of interaction is the PDZ 1 domain,

Acute-phase serum amyloid A (A-SAA) was shown recently to correlate with obesity and insulin resistance in humans. However, the mechanisms linking obesity-associated inflammation and elevated plasma A-SAA to insulin resistance are poorly understood. Using high-fat diet- (HFD-) fed mice, we found that plasma A-SAA was increased early upon HFD feeding and was tightly associated with systemic insulin resistance. Plasma A-SAA elevation was due to induction of Saa1 and Saa2 expression in liver but not in adipose tissue. In adipose tissue Saa3 was the predominant isoform and the earliest inflammatory marker induced, suggesting it is important for initiation of adipose tissue inflammation. To assess the potential impact of A-SAA on adipose tissue insulin resistance, we treated 3T3-L1 adipocytes with recombinant A-SAA. Intriguingly, physiological levels of A-SAA caused alterations in gene expression closely resembling those observed in HFD-fed mice. Proinflammatory genes (Ccl2, Saa3) were induced while genes critical for insulin sensitivity (Irs1, Adipoq, Glut4) were down-regulated. Our data identify HFD-fed mice as a suitable model to study A-SAA as a biomarker and a novel possible mediator of insulin resistance.

We report here an interaction between the C terminus of the rat somatostatin receptor subtype 2 (SSTR2) and a protein that has recently been identified as cortactinbinding protein 1 (CortBP1). Interaction is mediated by the PDZ (PSD-95/discs large/ZO-1) domain of CortBP1. As shown by in situ hybridization, SSTR2 and cortactinbinding protein are coexpressed in the rat brain. The association between SSTR2 and the PDZ-domain of CortBP1 was verified by overlay assays and by coprecipitation after transfection in human embryonic kidney (HEK) cells. Analysis by confocal microscopy indicates that CortBP1 is distributed diffusely throughout the cytosol in transfected cells and that it becomes concentrated at the plasma membrane when SSTR2 is present. This process is largely increased when the receptor is stimulated by somatostatin; as CortBP1 interacts with the C terminus of SSTR2, our data suggest that the binding of agonist to the receptor increase the accessibility of the receptor C terminus to the PDZ domain of CortBP1. Our data for the first time establish a link between a G-protein coupled receptor and constituents of the cytoskeleton.Neuronal receptors for chemical transmitters are not uniformly distributed throughout the cell but are concentrated at specific cellular sites; this is particularly true for receptors belonging to the group of ligand-gated ion channels, which are clustered at postsynaptic sites through interaction with specific proteins such as rapsyn (1), gephyrin (2), or the PSD/SAP proteins (3). In contrast, very little is known about proteins that are involved in the targeting of G-protein-coupled receptors (GPCR) 1 to their sites of action. Proteins that are known to interact intracellularly with this group of receptors are either involved in the function (G-proteins) or in the regulation (arrestins and the receptor kinases) of GPCRs (4), but not in the subcellular localization. Only recently several reports have suggested that additional proteins may interact with certain GPCRs (5-8). However, for the large majority of GPCRs it remains unclear if specific proteins exist that are responsible for anchoring or subcellular targeting of the receptors. We have addressed this issue by searching for proteins that interact with the intracellular C terminus of receptors for the neuropeptide somatostatin (SST). Somatostatin acts through five different G-protein-coupled receptors (SSTR1-5), which are widely expressed in the mammalian central nervous system; frequently more than one receptor subtype is detectable in one cell type (9), and it may be difficult to assign the effects of somatostatin and its analogues to any specific receptor subtype. One possible reason for this multiplicity of subtypes may be the differential subcellular localization that has recently been observed using subtype-specific antibodies (10 -13).Here we show that the C terminus of the rat SSTR2 interacts specifically with the PDZ domain of a cortactin-binding protein 1 (CortBP1). As the C-terminal sequences of SSTRs are largely di...

Liver X receptors (LXRs) form functional heterodimers with the retinoid X receptors (RXRs) and regulate cholesterol, lipid, and glucose metabolism. We demonstrated previously that activation of LXR modulates insulin secretion in MIN6 cells and pancreatic islets. In this study we investigated the effects of the LXR agonist T0901317 and the RXR agonist 9-cis-retinoic acid (9cRA) on cell proliferation and apoptosis in MIN6 cells. Whereas T0901317 showed no effect on proliferation of MIN6 cells, combination of T0901317 with 9cRA inhibited cell proliferation. Flow cytometry analysis of cell cycle demonstrated that activation of LXR/RXR prevented MIN6 cells from G1 to G2 phase progression. Combination of T0901317 and 9cRA increased apoptosis rate and caspase-3/7 activity in MIN6 cells. Moreover, T0901317 or its combination with 9cRA significantly increased the cell susceptibility to free fatty acid- and cytokine-induced apoptosis. Treatment of MIN6 cells with LXR and RXR agonists produced a strong increase in expression of mothers against decapentaplegic homolog 3, a protein known to inhibit cell cycle G1/S phase progression and induce apoptosis. In isolated rat islets, the effect of palmitic acid on caspase-3/7 activity was increased with T0901317 alone and even more with the combination of T0901317 and 9cRA. Thus, activation of LXR/RXR signaling inhibits cell proliferation and induces apoptosis in pancreatic beta-cells.

The human vitamin D receptor (hVDR) is a ligand-regulated transcription factor that mediates the actions of the 1,25-dihydroxyvitamin D3 hormone to effect bone mineral homeostasis. Employing mutational analysis, we characterized Arg-18/Arg-22, hVDR residues immediately N-terminal of the first DNA binding zinc finger, as vital for contact with human basal transcription factor IIB (TFIIB). Alteration of either of these basic amino acids to alanine also compromised hVDR transcriptional activity. In contrast, an artificial hVDR truncation devoid of the first 12 residues displayed both enhanced interaction with TFIIB and transactivation. Similarly, a natural polymorphic variant of hVDR, termed F/M4 (missing a FokI restriction site), which lacks only the first three amino acids (including Glu-2), interacted more efficiently with TFIIB and also possessed elevated transcriptional activity compared with the full-length (f/M1) receptor. It is concluded that the functioning of positively charged Arg-18/Arg-22 as part of an hVDR docking site for TFIIB is influenced by the composition of the adjacent polymorphic N terminus. Increased transactivation by the F/M4 neomorphic hVDR is hypothesized to result from its demonstrated enhanced association with TFIIB. This proposal is supported by the observed conversion of f/M1 hVDR activity to that of F/M4 hVDR, either by overexpression of TFIIB or neutralization of the acidic Glu-2 by replacement with alanine in f/M1 hVDR. Because the f VDR genotype has been associated with lower bone mineral density in diverse populations, one factor contributing to a genetic predisposition to osteoporosis may be the F/f polymorphism that dictates VDR isoforms with differential TFIIB interaction.