Genetic Deficiency of Glycogen Synthase Kinase-3β Corrects Diabetes in Mouse Models of Insulin Resistance

Despite treatment with agents that enhance β-cell function and insulin action, reduction in β-cell mass is relentless in patients with insulin resistance and type 2 diabetes mellitus. Insulin resistance is characterized by impaired signaling through the insulin/insulin receptor/insulin receptor substrate/PI-3K/Akt pathway, leading to elevation of negatively regulated substrates such as glycogen synthase kinase-3β (Gsk-3β). When elevated, this enzyme has antiproliferative and proapoptotic properties. In these studies, we designed experiments to determine the contribution of Gsk-3β to regulation of β-cell mass in two mouse models of insulin resistance. Mice lacking one allele of the insulin receptor (Ir+/−) exhibit insulin resistance and a doubling of β-cell mass. Crossing these mice with those having haploinsufficiency for Gsk-3β (Gsk-3β+/−) reduced insulin resistance by augmenting whole-body glucose disposal, and significantly reduced β-cell mass. In the second model, mice missing two alleles of the insulin receptor substrate 2 (Irs2−/−), like the Ir+/− mice, are insulin resistant, but develop profound β-cell loss, resulting in early diabetes. We found that islets from these mice had a 4-fold elevation of Gsk-3β activity associated with a marked reduction of β-cell proliferation and increased apoptosis. Irs2−/− mice crossed with Gsk-3β+/− mice preserved β-cell mass by reversing the negative effects on proliferation and apoptosis, preventing onset of diabetes. Previous studies had shown that islets of Irs2−/− mice had increased cyclin-dependent kinase inhibitor p27kip1 that was limiting for β-cell replication, and reduced Pdx1 levels associated with increased cell death. Preservation of β-cell mass in Gsk-3β+/−Irs2−/− mice was accompanied by suppressed p27kip1 levels and increased Pdx1 levels. To separate peripheral versus β-cell–specific effects of reduction of Gsk3β activity on preservation of β-cell mass, mice homozygous for a floxed Gsk-3β allele (Gsk-3F/F) were then crossed with rat insulin promoter-Cre (RIP-Cre) mice to produce β-cell–specific knockout of Gsk-3β (βGsk-3β−/−). Like Gsk-3β+/− mice, βGsk-3β−/− mice also prevented the diabetes of the Irs2−/− mice. The results of these studies now define a new, negatively regulated substrate of the insulin signaling pathway specifically within β-cells that when elevated, can impair replication and increase apoptosis, resulting in loss of β-cells and diabetes. These results thus form the rationale for developing agents to inhibit this enzyme in obese insulin-resistant individuals to preserve β-cells and prevent diabetes onset

Glucose and Fatty Acids Synergize to Promote B-Cell Apoptosis through Activation of Glycogen Synthase Kinase 3β Independent of JNK Activation

The combination of elevated glucose and free-fatty acids (FFA), prevalent in diabetes, has been suggested to be a major contributor to pancreatic β-cell death. This study examines the synergistic effects of glucose and FFA on β-cell apoptosis and the molecular mechanisms involved. Mouse insulinoma cells and primary islets were treated with palmitate at increasing glucose and effects on apoptosis, endoplasmic reticulum (ER) stress and insulin receptor substrate (IRS) signaling were examined.Increasing glucose (5-25 mM) with palmitate (400 µM) had synergistic effects on apoptosis. Jun NH2-terminal kinase (JNK) activation peaked at the lowest glucose concentration, in contrast to a progressive reduction in IRS2 protein and impairment of insulin receptor substrate signaling. A synergistic effect was observed on activation of ER stress markers, along with recruitment of SREBP1 to the nucleus. These findings were confirmed in primary islets. The above effects associated with an increase in glycogen synthase kinase 3β (Gsk3β) activity and were reversed along with apoptosis by an adenovirus expressing a kinase dead Gsk3β.Glucose in the presence of FFA results in synergistic effects on ER stress, impaired insulin receptor substrate signaling and Gsk3β activation. The data support the importance of controlling both hyperglycemia and hyperlipidemia in the management of Type 2 diabetes, and identify pancreatic islet β-cell Gsk3β as a potential therapeutic target

Islet β cell expression of constitutively active Akt1/PKBα induces striking hypertrophy, hyperplasia, and hyperinsulinemia

The phosphoinositide 3-kinase–Akt/PKB pathway mediates the mitogenic effects various nutrients and growth factors in cultured cells. To study its effects in vivo in pancreatic islet β cells, we created transgenic mice that expressed a constitutively active Akt1/PKBα linked to an Insulin gene promoter. Transgenic mice exhibited a grossly visible increase in islet mass, largely due to proliferation of insulin-containing β cells. Morphometric analysis verified a six-fold increase in β cell mass/pancreas, a two-fold increase in 5-bromo-2′-deoxyuridine incorporation, a four-fold increase in the number of β cells per pancreas area, and a two-fold increase in cell size in transgenic compared with wild-type mice at 5 weeks. At least part of the increase in β cell number may be accounted for by neogenesis, defined by criteria that include β cells proliferating from ductular epithelium, and by a six-fold increase in the number of single and doublet β cells scattered throughout the exocrine pancreas of the transgenic mice. Glucose tolerance was improved, and fasting as well as fed insulin was greater compared with wild-type mice. Glucose-stimulated insulin secretion was maintained in transgenic mice, which were resistant to streptozotocin–induced diabetes. We conclude that activation of the Akt1/PKBα pathway affects islet β cell mass by alteration of size and number

Glucose and Fatty Acids Synergize to Promote B-Cell Apoptosis through Activation of Glycogen Synthase Kinase 3β Independent of JNK Activation

Background: The combination of elevated glucose and free-fatty acids (FFA), prevalent in diabetes, has been suggested to be a major contributor to pancreatic β-cell death. This study examines the synergistic effects of glucose and FFA on β-cell apoptosis and the molecular mechanisms involved. Mouse insulinoma cells and primary islets were treated with palmitate at increasing glucose and effects on apoptosis, endoplasmic reticulum (ER) stress and insulin receptor substrate (IRS) signaling were examined.Principal Findings: Increasing glucose (5–25 mM) with palmitate (400 µM) had synergistic effects on apoptosis. Jun NH2-terminal kinase (JNK) activation peaked at the lowest glucose concentration, in contrast to a progressive reduction in IRS2 protein and impairment of insulin receptor substrate signaling. A synergistic effect was observed on activation of ER stress markers, along with recruitment of SREBP1 to the nucleus. These findings were confirmed in primary islets. The above effects associated with an increase in glycogen synthase kinase 3β (Gsk3β) activity and were reversed along with apoptosis by an adenovirus expressing a kinase dead Gsk3β.Conclusions/Significance: Glucose in the presence of FFA results in synergistic effects on ER stress, impaired insulin receptor substrate signaling and Gsk3β activation. The data support the importance of controlling both hyperglycemia and hyperlipidemia in the management of Type 2 diabetes, and identify pancreatic islet β-cell Gsk3β as a potential therapeutic target.</p

The synergistic effects of glucose and palmitate on ER stress results in concomitant effects on activation of SREBP1.

<p>(<b>A</b>) MIN6 cells were treated with either control 0.5% BSA or 400 µM palmitate+0.5% BSA at a concentration of 5, 10, 15, 25 mM glucose for 18-h. Nuclear fractions were extracted from the cells and were subjected to Western blot analyses using anti-SREBP1 and anti-Lamin antibodies. 25 µg of nuclear protein was loaded in each lane. The upper band normalized over Lamin was used to do the quantification (the lower band is nonspecific). The relative ratio of nuclear SREBP1 over Lamin calculated by densitometries was summarized as means ± S.E.M. in the graph respectively. The representative results of three experiments are shown, and graphically illustrated, * p<0.05. (<b>B</b>) MIN6 cells were treated with either control 0.5% BSA or 400 µM palmitate+0.5% BSA at a concentration of either 5 or 25 mM glucose for 24-h. Total cell lysates were subjected to Western blot analysis using anti-acetyl CoA carboxylase (ACC) and anti-α-Tubulin antibodies. The representative results of two individual experiments are shown. (<b>C</b>) Cells were treated with either 500 µg/ml NaCl or 500 µg/ml TUDCA 15-h prior to beginning of palmitate treatment. Cells were co-treated with either 0.5% BSA or 400 µM palmitate+0.5% BSA with 25 mM glucose and NaCl or TUDCA for 18-h. Nuclear fractions were extracted from the cells and were subjected to Western blot analyses using anti-SREBP1 and anti-Lamin antibodies. The upper band normalized over Lamin was used to do the quantification (the lower band is nonspecific). 25 µg of nuclear extracts were loaded in each lane. The representative results of three individual experiments are shown. The relative ratio of nuclear SREBP1 over Lamin calculated by densitometries was summarized as means ± S.E.M. in the graph respectively **p<0.01.</p

The synergistic effects of glucose and palmitate on ER stress and reduction of insulin signaling is attenuated by addition of a chemical chaperon.

<p>(<b>A</b>) MIN6 cells were treated with either control 0.5% BSA or 400 µM palmitate+0.5% BSA at a concentration of 5, 10, 25 mM glucose for 8 hours. Total cell lysates were extracted at indicated time points and were subjected to Western blot analysis using anti-phospho-PERK (980Thr), anti-phopsho-eIF2α (51Ser), and (<b>B</b>) MIN6 cells were treated with either control 0.5% BSA or 400 µM palmitate+0.5% BSA at a concentration of 5, 10, 25 mM glucose for 18 hours and blotted with anti-ATF3 and anti-CHOP antibodies. β-Actin was detected for loading control. Tunicamycin treatment was control for ER stress. (<b>C</b>) Cells were treated with either 500 µg/ml NaCl (ionic control) or 500 µg/ml TUDCA 15-h prior to beginning of palmitate treatment and then were co-treated with either 0.5% BSA or 400 µM palmitate+0.5% BSA with either 5 mM or 25 mM glucose and NaCl or TUDCA for 24 h. Total cell lysates were subjected to Western blot analysis with antibodies to the indicated proteins. Densitometry of total CHOP and cleaved Caspase3 and Pdx1 were measured and normalized over α-Tubulin, respectively. Densitometry of phospho-cJun was measured and normalized over total JNK. The representative results of three individual experiments are shown. The effects on CHOP, cleaved Caspase3 and phospho-cJun and Pdx1 protein are graphically illustrated. *p<0.05. (<b>D</b>) Cells were treated with either 500 µg/ml NaCl (ionic control) or 500 µg/ml TUDCA 15-h prior to beginning of palmitate treatment and then were co-treated with either 0.5% BSA or 400 µM palmitate+0.5% BSA with either 5 mM, 10 mM or 25 mM glucose and NaCl or TUDCA for 24-h. Total cell lysates were subjected to Western blot analysis with antibodies to the indicated proteins. Densitometry of total IRS2 was measured and normalized over α-Tubulin and densitometry of phospho-Akt was measured and normalized over total Akt. The representative results of three individual experiments are shown. The effects on IRS2 protein are graphically illustrated, *p<0.05, **p<0.01.</p

Working diagram illustrating some of the key steps involved in “glucolipotoxicity” of β-cells.

<p>High glucose and FFA together result in a vicious negative cycle that ultimately promotes β-cell death. As suggested by our findings, high glucose addition to FFA treated β-cells results in much more activation of SREBP1 than glucose alone. SREBP1 enhances ACC expression with generation of malonyl-CoA which impairs FFA oxidation. This in turn leads to augmented ER stress with further activation of ER-localized SREBP1 as a result of degradation of the anchoring protein Insig1. The excess non-metabolized FFA due to more impairment of FFA oxidation would partition in ER membranes compounding ER stress. In addition to SREBP1, ER stress activates ATF3. Both nuclear SREBP1 and ATF3 result in inhibition of IRS2, with concomitant impairment of insulin signaling, activation of Gsk3β and reduction of Pdx1 leading to apoptosis.</p

Synergistic effects of glucose and palmitate on cell death but not JNK activation in MIN6 cells.

<p>MIN6 cells were treated with either control 0.5% BSA or 400 µM palmitate+0.5% BSA at a concentration of 5, 10, 15, 25 mM glucose for 24-h. (<b>A</b>) The percentage of cell death was then assessed by adding propidium iodide for the last hour of incubation as described under Methods. The bar graph depicts the averages of the data obtained from five individual experiments, and data are expressed as means ±S.E.M. ** p<0.01, *** p<0.001; (<b>B</b>) The cell lysates were subjected to Western blot analysis using anti-cleaved Caspase3, anti-phospho-JNK, anti-total JNK and anti-α-Tubulin antibodies. Protein level of phospho-JNK was normalized over total JNK. Cleaved Caspase3 levels were normalized over α-Tubulin. The representative result of three individual experiments is shown. The data obtained from three individual experiments are expressed as means ± S.E.M. * p<0.05, ** p<0.01.</p

Inhibition of Gsk3β protects against glucose and palmitate-induced apoptosis in MIN6 cells.

<p>MIN6 cells were infected with 100, 200, or 400 MOI of adenovirus expressing a catalytically inactive mutant of the human Gsk3β (Adv-Gsk3βKM) or adenovirus expressing GFP (Adv-GFP) 24 hours prior to palmitate treatment. Cells were treated with 25 mM glucose and with either 0.5% BSA or 400 µM PA+0.5% BSA for 24 hours. A GFP control was placed on either end of the blot to facilitate comparison of control and Adv-Gsk3βKM. (<b>A</b>) Western blots using the indicated antibodies, with relative expression of Pdx1 normalized over α-Tubulin, and expression levels of cleaved Caspase3 normalized over α-tubulin. (<b>B</b>) Percentage of Propidium Iodide incorporation (n = 3, means ± S.E.M., *p<0.05, *** p<0.001).</p