Fatty acid and phorbol ester-mediated interference of mitogenic signaling via novel protein kinase C isoforms in pancreatic beta-cells (INS-1).

It is possible that activation of protein kinase C (PKC) isoforms by free fatty acids (FFA) plays a role in the failure of pancreatic beta-cell mass expansion to compensate for peripheral insulin resistance in the pathogenesis of type-2 diabetes. The effect of lipid moieties on activation of conventional (PKC-alpha and -beta1), novel (PKC-delta) and atypical (PKC-zeta) PKC isoforms was evaluated in an in vitro assay, using biotinylated neurogranin as a substrate. Oleoyl-Coenzyme A (CoA) and palmitoyl-CoA, but not unesterified FFA, significantly increased the activity of all PKC isoforms (P< or =0.05), particularly that for PKC-delta. It was found that FFA (0.4 mM oleate/complexed to 0.5% bovine serum albumin) inhibited IGF-I-induced activation of protein kinase B (PKB) in the pancreatic beta-cell line (INS-1), but this was alleviated in the presence of the general PKC inhibitor (Gö6850; 1 microM). To further investigate whether conventional or novel PKC isoforms adversely affect beta-cell proliferation, the effect of phorbol ester (phorbol 12-myristate 13-acetate; PMA)-mediated activation of these PKC isoforms on glucose/IGF-I-induced INS-1 cell mitogenesis, and insulin receptor substrate (IRS)-mediated signal transduction was investigated. PMA-mediated activation of PKC (100 nM; 4 h) reduced glucose/IGF-I mediated beta-cell mitogenesis (>50%; P< or =0.05), which was reversible by the general PKC inhibitor Gö6850 (1 microM), indicating an effect of PKC and not due to a non-specific PMA toxicity. PMA inhibited IGF-I-induced activation of PKB, correlating with inhibition of IGF-I-induced association of IRS-2 with the p85 regulatory subunit of phosphatidylinositol-3 kinase. However, in contrast, PMA activated the mitogen-activated protein kinases, Erk1/2. Titration inhibition analysis using PKC isoform inhibitors indicated that these PMA-induced effects were via novel PKC isoforms. Thus, FFA/PMA-induced activation of novel PKC isoforms can inhibit glucose/IGF-I-mediated beta-cell mitogenesis, in part by decreasing PKB activation, despite an upregulation of Erk1/2. Thus, activation of novel PKC isoforms by long-chain acyl-CoA may well contribute to decreasing beta-cell mass in the pathogenesis of type-2 diabetes, similar to their inhibition of insulin signal transduction which causes insulin resistance.

Cooperativity between the preproinsulin mRNA untranslated regions is necessary for glucose-stimulated translation.

Glucose regulates proinsulin biosynthesis via stimulation of the translation of the preproinsulin mRNA in pancreatic beta-cells. However, the mechanism by which this occurs has remained unclear. Using recombinant adenoviruses that express the preproinsulin mRNA with defined alterations, the untranslated regions (UTRs) of the preproinsulin mRNA were examined for elements that specifically control translation of the mRNA in rat pancreatic islets. These studies revealed that the preproinsulin 5'-UTR was necessary for glucose stimulation of preproinsulin mRNA translation, whereas the 3'-UTR appeared to suppress translation. However, together the 5'- and 3'-UTRs acted cooperatively to markedly increase glucose-induced proinsulin biosynthesis. In primary hepatocytes the presence of the preproinsulin 3'-UTR led to reduced mRNA levels compared with the presence of the SV40 3'-UTR, consistent with the presence of mRNA stability determinants in the 3'-UTR that stabilize the preproinsulin mRNA in a pancreatic beta-cell-specific manner. Translation of these mRNAs in primary hepatocytes was not stimulated by glucose, indicating that regulated translation of the preproinsulin mRNA occurs in a pancreatic beta-cell-specific manner. Thus, the untranslated regions of the preproinsulin mRNA play crucial roles in regulating insulin production and therefore glucose homeostasis by regulating the translation and the stability of the preproinsulin mRNA.

Differential activation of protein kinase B and p70(S6)K by glucose and insulin-like growth factor 1 in pancreatic beta-cells (INS-1).

It has been shown that IGF-1-induced pancreatic beta-cell proliferation is glucose-dependent; however, the mechanisms responsible for this glucose dependence are not known. Adenoviral mediated expression of constitutively active phosphatidylinositol 3-kinase (PI3K) in the pancreatic beta-cells, INS-1, suggested that PI3K was not necessary for glucose-induced beta-cell proliferation but was required for IGF-1-induced mitogenesis. Examination of the signaling components downstream of PI3K, 3-phosphoinositide-dependent kinase 1, protein kinase B (PKB), glycogen synthase kinase-3, and p70-kDa-S6-kinase (p70(S6K)), suggested that a major part of glucose-dependent beta-cell proliferation requires activation of mammalian target of rapamycin/p70(S6K), independent of phosphoinositide-dependent kinase 1/PKB activation. Adenoviral expression of the kinase-dead form of PKB in INS-1 cells decreased IGF-1-induced beta-cell proliferation. However, a surprisingly similar decrease was also observed in adenoviral wild type and constitutively active PKB-infected cells. Upon analysis of extracellular signal-regulated protein kinase 1 and 2 (ERK1/ERK2), an increase in ERK1/ERK2 phosphorylation activation by glucose and IGF-1 was observed in kinase-dead PKB-infected cells, but this phosphorylation activation was inhibited in the constitutively active PKB-infected cells. Hence, there is a requirement for the activation of both ERK1/ERK2 and mammalian target of rapamycin/p70(S6K) signal transduction pathways for a full commitment to glucose-induced pancreatic beta-cell mitogenesis. However, for IGF-1-induced activation, these pathways must be carefully balanced, because chronic activation of one (PI3K/PKB) can lead to dampening of the other (ERK1/2), reducing the mitogenic response.

Effects of dichloroacetate (DCA) on serum insulin levels and insulin-controlled signaling proteins in livers of male B6C3F1 mice.

DCA is hepatocarcinogenic in rodents. At carcinogenic doses, DCA causes a large accumulation of liver glycogen. Thus, we studied the effects of DCA treatment on insulin levels and expression of insulin-controlled signaling proteins in the liver. DCA treatment (0.2-2.0 g/l in drinking water for 2 weeks) reduced serum insulin levels. The decrease persisted for at least 8 weeks. In livers of mice treated with DCA for 2-, 10-, and 52-week periods, insulin receptor (IR) protein levels were significantly depressed. Additionally, protein kinase B (PKBalpha) expression decreased significantly with DCA treatment. In normal liver, glycogen levels were increased as early as at 1 week, and this effect preceded changes in insulin and IR and PKBalpha. In contrast to normal liver, IR protein was elevated in DCA-induced liver tumors relative to that in liver tissue of untreated animals and to an even greater extent when compared to adjacent normal liver in the treated animal. Mitogen-activated protein kinase (MAP kinase) phosphorylation was also increased in tumor tissue relative to normal liver tissue and tissue from untreated controls. These data suggest that normal hepatocytes down-regulate insulin-signaling proteins in response to the accumulation of liver glycogen caused by DCA. Furthermore, these results suggest that the initiated cell population, which does not accumulate glycogen and is promoted by DCA treatment, responds differently from normal hepatocytes to the insulin-like effects of this chemical. The differential sensitivity of the 2 cell populations may contribute to the tumorigenic effects of DCA in the liver.

Effects of dichloroacetate on glycogen metabolism in B6C3F1 mice.

Dichloroacetate (DCA) is a by-product of drinking water chlorination. Administration of DCA in drinking water results in accumulation of glycogen in the liver of B6C3F1 mice. To investigate the processes affecting liver glycogen accumulation, male B6C3F1 mice were administered DCA in drinking water at levels varying from 0.1 to 3 g/l for up to 8 weeks. Liver glycogen synthase (GS) and glycogen phosphorylase (GP) activities, liver glycogen content, serum glucose and insulin levels were analyzed. To determine whether effects were primary or attributable to increased glycogen synthesis, some mice were fasted and administered a glucose challenge (20 min before sacrifice). DCA treatments in drinking water caused glycogen accumulation in a dose-dependent manner. The DCA treatment in drinking water suppressed the activity ratio of GS measured in mice sacrificed at 9:00 AM, but not at 3:00 AM. However, net glycogen synthesis after glucose challenge was increased with DCA treatments for 1-2 weeks duration, but the effect was no longer observed at 8 weeks. Degradation of glycogen by fasting decreased progressively as the treatment period was increased, and no longer occurred at 8 weeks. A shift of the liver glycogen-iodine spectrum from DCA-treated mice was observed relative to that of control mice, suggesting a change in the physical form of glycogen. These data suggest that DCA-induced glycogen accumulation at high doses is related to decreases in the degradation rate. When DCA was administered by single intraperitoneal (i.p.) injection to naïve mice at doses of 2-200 mg/kg at the time of glucose challenge, a biphasic response was observed. Doses of 10-25 mg/kg increased both plasma glucose and insulin concentrations. In contrast, very high i.p. doses of DCA (> 75 mg/kg) produced progressive decreases in serum glucose and glycogen deposition in the liver. Since the blood levels of DCA produced by these higher i.p. doses were significantly higher than observed with drinking water treatment, we conclude that apparent differences with data of previous investigations is related to substantial differences in systemic dose and/or dose-time relations.