Ernesto Bernal-Mizrachi

Type 2 diabetes mellitus is a complex disease characterized by β-cell failure in the setting of insulin resistance. In early stages of the disease, pancreatic β-cells adapt to insulin resistance by increasing mass and function. As nutrient excess persists, hyperglycemia and elevated free fatty acids negatively impact β-cell function. This happens by numerous mechanisms, including the generation of reactive oxygen species, alterations in metabolic pathways, increases in intracellular calcium and the activation of endoplasmic reticulum stress. These processes adversely affect β-cells by impairing insulin secretion, decreasing insulin gene expression and ultimately causing apoptosis. In this review, we will first discuss the regulation of β-cell mass during normal conditions. Then, we will discuss the mechanisms of β-cell failure, including glucotoxicity, lipotoxicity and endoplasmic reticulum stress. Further research into mechanisms will reveal the key modulators of β-cell failure and thus identify possible novel therapeutic targets. Type 2 diabetes mellitus is a multifactorial disease that has greatly risen in prevalence in part due to the obesity and inactivity that characterize the modern Western lifestyle. Pancreatic β-cells possess the potential to greatly expand their function and mass in both physiologic and pathologic states of nutrient excess and increased insulin demand. β-cell response to nutrient excess occurs by several mechanisms, including hypertrophy and proliferation of existing β-cells, increased insulin production and secretion, and formation of new β-cells from progenitor cells [1, 2]. Failure of pancreatic β-cells to adequately expand in settings of increased insulin demand results in hyperglycemia and diabetes. In this review, we will first discuss the factors involved in β-cell growth and then discuss the mechanisms by which β-cell expansion fails and leads to β-cell failure and diabetes (Fig. 1). The mechanisms by which -cell failure and apoptosis occur are complex, not completely unraveled and involve the interplay of numerous factors and conditions. These factors are summarized in this figure. Glucotoxicity and lipotoxicity lead to the production of ROS, which activate JNK. JNK activity leads to a decrease in IRS signaling and may directly be involved in decreased Pdx-1 activity by translocation from the nucleus to the cytoplasm [183]. In addition, glucose and FA have both been found to induce ER stress. Chronic glucose elevation inhibits FA oxidation and favors the generation of ceramide and lipid partitioning, which ultimately results in β-cell dysfunction and apoptosis. AMPK activation promotes fatty acid oxidation by phosphorylation and inhibition of acetyl-CoA carboxylase or via down regulation of the transcription factor sterol-regulatory-element-binding-protein-1c (SREBP1c) and subsequent decreases in acetyl-CoA carboxylase. Glucose and FA also activate the UPR response and induce ER stress. The ER stress response and its effectors are activated in response to misfolded proteins in order to protect β-cells from apoptosis; however, activation of these processes under conditions of long-term elevation of FFA and glucose can lead to β-cell dysfunction and ultimately apoptosis. Activation of ER stress leads to inhibition of insulin mRNA and protein expression and may also be pro-apoptotic. The mechanisms for induction of apoptosis by ER stress are not completely known, but induction of CHOP is an important component. In addition, induction of ATF3 and SREBP can downregulate IRS signaling by repressing IRS2 transcription. One interesting finding is that inhibition of IRS signaling seems to be a common pathway induced by the majority of the mechanisms described for β-cell failure. An additional event is the increase in mTOR signaling by nutrient excess (glucose). This results in negative feedback inhibition on IRS1 and possibly IRS2 by activation of S6K signaling. The decrease in IRS signaling induces GSK3β and Foxo1 function. Activation of these molecules ultimately reduces Pdx1 levels and increases the levels of the cell cycle inhibitor p27

The serine‐threonine kinase Akt regulates multiple biological processes. An important strategy to study Akt signaling in different tissues is targeted activation of this pathway in vivo. The current studies describe the generation of a mouse model that combines a double reporter system with activation of a constitutively active form of Akt1 (caAkt) in a Cre‐dependent manner. Before Cre recombination, these mice express LacZ during development as well as in most adult tissues. After Cre‐mediated excision of the LacZ reporter, functionality of the transgene was demonstrated by expression of the caAkt mutant along with the second reporter, EGFP in different pancreatic compartments and in the nervous system. This animal model provides a critical reagent for assessing the effects of Akt activation in specific tissues. The lineage‐tracing properties provide a useful tool to study the role of Akt signaling in regulation of differentiation programs during development and plasticity of mature tissues. genesis 46:256–264, 2008.

The insulin and IGF signaling pathways are critical for development and maintenance of pancreatic β cell mass and function. The serine-threonine kinase Akt is one of several mediators regulated by these pathways. We have studied the role of Akt in pancreatic β cell physiology by generating transgenic mice expressing a kinase-dead mutant of this enzyme in β cells. Reduction of Akt activity in transgenic animals resulted in impaired glucose tolerance due to defective insulin secretion. The mechanisms involved in dysregulation of secretion in these mice lie at the level of insulin exocytosis and are not the result of abnormalities in glucose signaling or function of voltage-gated Ca²⁺ channels. Therefore, transgenic mice showed increased susceptibility to developing glucose intolerance and diabetes following fat feeding. These observations suggest that Akt plays a novel and important role in the regulation of distal components of the secretory pathway and that this enzyme represents a therapeutic target for improvement of β cell function in diabetes.

The purpose of the current study was to determine whether nuclear factor-κB (NF-κB) activation is a component of the depolarization/Ca2+-dependent signaling in β-cells. MIN6 cells were transfected with a plasmid containing five tandem repeats of NF-κB binding sites linked to a luciferase reporter. The results of these experiments showed that KCl induced depolarization-activated NF-κB-dependent transcription (3.8-fold at 45 mmol/l, P < 0.01) in a concentration-dependent manner. Tumor necrosis factor-α (TNF-α), a known inducer of NF-κB signaling, activated this construct by 3.4-fold (P < 0.01). The response of NF-κB to depolarization was inhibited by the Ca2+-channel blocker verapamil and by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 (70 and 62%, respectively). TNF-α, glucose, and KCl treatment resulted in inhibitory κBα degradation by Western blot analysis. TNF-α treatment and depolarization activation of NF-κB differed significantly in that TNF-α activation was not blocked by PD98059. Transfection with PKA, MEK, and MEK kinase induced NF-κB-dependent transcription by 20-, 90-, and 300-fold, respectively, suggesting that these pathways contribute to the activation in the depolarization response. These findings demonstrate that depolarization/Ca2+ influx, as well as TNF-α treatment, can activate NF-κB-dependent transcription in pancreatic β-cells, but by different signaling pathways. The current studies show that Ca2+ signals in pancreatic β-cells can activate transcription factors involved in the regulation of cell cycle and apoptosis. These findings now add NF-κB to the list of depolarization-induced transcription factors in pancreatic β-cells.

Elk-1, a member of the ternary complex factor family of Ets domain proteins that bind serum response elements, is activated by phosphorylation in a cell-specific manner in response to growth factors and other agents. The purpose of the current study was to determine whether Elk-1 activation contributes to glucose-/depolarization-induced Ca²⁺-dependent induction of immediate early response genes in pancreatic islet β-cells. The results of experiments in insulinoma (MIN6) cells demonstrated that Elk-1-binding sites (Ets elements) in the Egr-1 gene promoter contribute to transcriptional activation of the gene. Treatment with either epidermal growth factor (EGF), a known inducer of β-cell hyperplasia, glucose, or KCl-induced depolarization resulted in Ser³⁸³phosphorylation and transcriptional activation of Elk-1 (4 ± 0.3-, P = 0.003, 2.3 ± 0.19-, P = 0.002, and 2.2 ± 0.1- fold, P= 0.001 respectively). The depolarization response was inhibited by the Ca²⁺channel blocker verapamil and by the MEK inhibitor PD98059 (53 ± 6 and 55 ± 0.5%, respectively). EGF-induced activation of Elk-1 was also inhibited by PD98059 (60 ± 5%). A dominant negative Ras produced partial inhibition (42%) of the depolarization-induced Elk-1 transcriptional activation. Transfection with a constitutively active Ca²⁺/calmodulin kinase IV plasmid also resulted in Elk-1 transcriptional activation. Experiments with p38, phosphatidylinositol 3-kinase, and protein kinase A inhibitors indicated that these pathways are not involved. We conclude that Elk-1 activation contributes to glucose-/depolarization-induced Ca²⁺-dependent induction of immediate early growth response genes in pancreatic islet β-cells. Furthermore, the results demonstrated a convergence of nutrient- and growth factor-mediated signaling pathways on Elk-1 activation through induction of Ras/mitogen-activated protein kinase ERK-1 and -2. The role of these pathways in the glucose-induced proliferation of islet β-cells can now be assessed.

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.