Human IGF binding protein-3 overexpression impairs glucose regulation in mice via an inhibition of insulin secretion.

Human IGF binding protein-3 (hIGFBP-3) overexpression in mice causes hyperglycemia, but its effect on ß-cell function is unknown. We compared wild-type mice with mice overexpressing hIGFBP-3 [phoshoglycerate kinase (PGK)BP3] and mutant (Gly56/Gly8°/Gly8¹)hIGFBP-3 devoid of IGF binding affinity (PGKmBP3). Intraperitoneal glucose and insulin tolerance tests were performed, and glucose, IGFBP-3, IGF-I, and insulin were determined. Pancreatic sections were used for islet histomorphometry and stained with antibodies against insulin, glucagon, and hIGFBP-3. Pancreatic islets were isolated to determine the expression of IGFBP-3, and glucose-stimulated insulin secretion was measured using both islet batch incubation and perifusion. IGFBP-3 was expressed in ß-cells but not in other islet cell types. Fasting glucose concentration was elevated in PGKBP3 mice (6.27 ± 0.31 mm) compared with PGKmBP3 mice (3.98 ± 0.36 mm) and wild-type mice (4.84 ± 0.07 mm). During glucose tolerance test, glucose declined more slowly in PGKBP3 and PGKmBP3 mice than in wild-type mice, and insulin secretion was impaired in PGKBP3 mice. During insulin tolerance test, insulin declined more slowly in both transgenic mice compared with wild-type mice. Insulin secretion in islets incubated with 3.3 mm glucose was similar among groups, but islet insulin response to 16.7 mm glucose alone, or with carbachol and cAMP enhancers, was reduced in PGKBP3 and PGKmBP3 mice compared with wild-type controls. ATP content, Akt phosphorylation, and phosphoglucose isomerase activity were reduced in islets from both transgenic mice. Thus, overexpression of hIGFBP-3 in mice delays in vivo insulin clearance and reduces glucose-stimulated insulin secretion in pancreatic islets by both IGF-dependent and IGF-independent mechanisms.

Interaction between O-GlcNAc modification and tyrosine phosphorylation of prohibitin: implication for a novel binary switch.

Prohibitin (PHB or PHB1) is an evolutionarily conserved, multifunctional protein which is present in various cellular compartments including the plasma membrane. However, mechanisms involved in various functions of PHB are not fully explored yet. Here we report for the first time that PHB interacts with O-linked beta-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and is O-GlcNAc modified; and also undergoes tyrosine phosphorylation in response to insulin. Tyrosine 114 (Tyr114) and tyrosine 259 (Tyr259) in PHB are in the close proximity of potential O-GlcNAc sites serine 121 (Ser121) and threonine 258 (Thr258) respectively. Substitution of Tyr114 and Tyr259 residues in PHB with phenylalanine by site-directed mutagenesis results in reduced tyrosine phosphorylation as well as reduced O-GlcNAc modification of PHB. Surprisingly, this also resulted in enhanced tyrosine phosphorylation and activity of OGT. This is attributed to the presence of similar tyrosine motifs in PHB and OGT. Substitution of Ser121 and Thr258 with alanine and isoleucine respectively resulted in attenuation of O-GlcNAc modification and increased tyrosine phosphorylation of PHB suggesting an association between these two dynamic modifications. Sequence analysis of O-GlcNAc modified proteins having known O-GlcNAc modification site(s) or known tyrosine phosphorylation site(s) revealed a strong potential association between these two posttranslational modifications in various proteins. We speculate that O-GlcNAc modification and tyrosine phosphorylation of PHB play an important role in tyrosine kinase signaling pathways including insulin, growth factors and immune receptors signaling. In addition, we propose that O-GlcNAc modification and tyrosine phosphorylation is a novel previously unidentified binary switch which may provide new mechanistic insights into cell signaling pathways and is open for direct experimental examination.

Prohibitin binds to C3 and enhances complement activation.

Prohibitin (PHB1) is a multifunction protein that is released in lipid droplets from adipocytes and possibly other cells and is detectable in the circulation. We used crosslinking, immunoprecipitation and proteomic analysis to investigate binding partners for circulating PHB1. Crosslinking of PHB1 to serum resulted in two complexes of approximately 150 and 100 kDa, which contained both PHB1 and fragments of C3. The binding of PHB1 to C3 was confirmed using a solid phase assay where the dissociation constant was approximately 90 fmol/l. PHB1, but not the closely related PHB2, was able to enhance complement activation and induce lysis of sensitized sheep erythrocytes when added with normal serum but not with C3-deficient serum. The ability of PHB1 to bind to, and activate C3 suggests that PHB1 may have a previously unrecognized role in innate immunity.

Prohibitin attenuates insulin-stimulated glucose and fatty acid oxidation in adipose tissue by inhibition of pyruvate carboxylase.

Prohibitin (PHB-1) is a highly conserved protein involved in mitochondrial biogenesis and function. It is secreted in lipid droplets from adipocytes and is present in the circulation. In adipose tissue it functions as a membrane receptor and can target binding partners to the mitochondria. Here we report that PHB-1 has a hitherto undescribed role as an inhibitor of pyruvate carboxylase (PC). As a consequence, it can modulate insulin-stimulated glucose and fatty acid oxidation. It had no effect on insulin-stimulated 2-deoxglucose uptake by isolated adipocytes but inhibited insulin-stimulated oxidation of [14C]glucose with a half-maximal concentration of approximately 4 nM. It also inhibited oleic acid oxidation in glucose-depleted adipocytes via depletion of oxaloacetate. In vitro experiments using broken-cell assays confirmed that PHB-1 inhibited PC. MALDI-TOF analysis of proteins identified by cross-linking of PHB-1 to adipocyte membranes indicated that PHB-1 is closely associated with PC and EH domain 2 (EHD2). On the basis of these data, we propose that PHB-1 is recycled between the extracellular space and the mitochondria by a mechanism involving lipid rafts and EHD2 and can modulate mitochondrial fuel metabolism by inhibition of PC.