HSPB1 influences mitochondrial respiration in ER-stressed beta cells

Beta-cell death and dysfunction are involved in the development of type 1 and 2 diabetes. ER-stress impairs beta-cells function resulting in pro-apoptotic stimuli that promote cell death. Hence, the identification of protective mechanisms in response to ER-stress could lead to novel therapeutic targets and insight in the pathology of these diseases. Here, we report the identification of proteins involved in dysregulated pathways upon thapsigargin treatment of MIN6 cells. Utilizing quantitative proteomics we identified upregulation of proteins involved in protein folding, unfolded protein response, redox homeostasis, proteasome processes associated with endoplasmic reticulum and downregulation of TCA cycle, cellular respiration, lipid metabolism and ribosome assembly processes associated to mitochondria and eukaryotic initiation translation factor components. Subsequently, pro-inflammatory cytokine treatment was performed to mimic pathological changes observed in beta-cells during diabetes. Cytokines induced ER stress and impaired mitochondrial function in beta-cells corroborating the results obtained with the proteomic approach. HSPB1 levels are increased by prolactin on pancreatic beta-cells and this protein is a key factor for cytoprotection although its role has not been fully elucidated. Here we show that while up-regulation of HSPB1 was able to restore the mitochondrial dysfunction induced by beta-cells' exposure to inflammatory cytokines, silencing of this chaperone abrogated the beneficial effects promoted by PRL. Taken together, our results outline the importance of HSPB1 to mitigate beta-cell dysfunction. Further studies are needed to elucidate its role in diabetes.     

Unraveling the pathogenesis of type 1 diabetes with proteomics: present and future directions

Type 1 diabetes (T1D) is the result of selective destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. T1D is due to a complex interplay between the beta-cell, the immune system, and the environment in genetically susceptible individuals. The initiating mechanism(s) behind the development of T1D are largely unknown, and no genes or proteins are specific for most T1D cases. Different pro-apoptotic cytokines, IL-1 beta in particular, are present in the islets during beta-cell destruction and are able to modulate beta-cell function and induce beta-cell death. In beta-cells exposed to IL-1 beta, a race between destructive and protective events are initiated and in susceptible individuals the deleterious events prevail. Proteins are involved in most cellular processes, and it is thus expected that their cumulative expression profile reflects the specific activity of cells. Proteomics may be useful in describing the protein expression profile and thus the diabetic phenotype. Relatively few studies using proteomics technologies to investigate the T1D pathogenesis have been published to date despite the defined target organ, the beta-cell. Proteomics has been applied in studies of differentiating beta-cells, cytokine exposed islets, dietary manipulated islets, and in transplanted islets. Although that the studies have revealed a complex and detailed picture of the protein expression profiles many functional implications remain to be answered. In conclusion, a rather detailed picture of protein expression in beta-cell lines, islets, and transplanted islets both in vitro and in vivo have been described. The data indicate that the beta-cell is an active participant in its own destruction during diabetes development. No single protein alone seems to be responsible for the development of diabetes. Rather the cumulative pattern of changes seems to be what favors a transition from dynamic stability in the unperturbed beta-cell to dynamic instability and eventually to beta-cell destruction.     

Baculovirus p35 increases pancreatic beta-cell resistance to apoptosis

beta-cells die by apoptosis in type 1 diabetes as a result of autoimmune attack mediated by cytokines, and in type 2 diabetes by various perpetrators including human islet amyloid polypeptide (hIAPP). The cascade of apoptotic events induced by cytokines and hIAPP is mediated through caspases and reactive oxygen species. The baculovirus p35 protein is a potent anti-apoptotic agent shown to be effective in a variety of species and able to inhibit a number of apoptotic pathways. Here, we aimed at determining the protective potential of p35 in beta-cells exposed to cytokines and hIAPP, as well as the effects of p35 on beta-cell function. The p35 gene was introduced into betaTC-tet cells, a differentiated murine beta-cell line capable of undergoing inducible growth-arrest. Both proliferating and growth-arrested cells expressing p35 manifested increased resistance to cytokines and hIAPP, compared with control cells, as judged by cell viability, DNA fragmentation, and caspase-3 activity assays. p35 was significantly more protective in growth-arrested, compared with proliferating, cells. No significant differences were observed in proliferation and insulin content between cells expressing p35 and control cells. In contrast, p35 manifested a perturbing effect on glucose-induced insulin secretion. These findings suggest that p35 could be incorporated as part of a multi-pronged approach of immunoprotective strategies to provide protection from recurring autoimmunity for transplanted beta-cells, as well as in preventive gene therapy in type 1 diabetes. p35 may also be protective from beta-cell damage caused by hIAPP in type 2 diabetes.     

Oxidative stress and the JNK pathway are involved in the development of type 1 and type 2 diabetes

Failure of pancreatic beta-cells is the common characteristic of type 1 and type 2 diabetes. Type 1 diabetes mellitus is induced by destruction of pancreatic beta-cells which is mediated by an autoimmune mechanism and consequent inflammatory process. Various inflammatory cytokines and oxidative stress are produced during this process, which has been proposed to play an important role in mediating beta-cell destruction. The JNK pathway is also activated by such cytokines and oxidative stress, and is involved in beta-cell destruction. Type 2 diabetes is the most prevalent and serious metabolic disease, and beta-cell dysfunction and insulin resistance are the hallmark of type 2 diabetes. Under diabetic conditions, chronic hyperglycemia gradually deteriorates beta-cell function and aggravates insulin resistance. This process is called "glucose toxicity". Under such conditions, oxidative stress is provoked and the JNK pathway is activated, which is likely involved in pancreatic beta-cells dysfunction and insulin resistance. In addition, oxidative stress and activation of the JNK pathway are also involved in the progression of atherosclerosis which is often observed under diabetic conditions. Taken together, it is likely that oxidative stress and subsequent activation of the JNK pathway are involved in the pathogenesis of type 1 and type 2 diabetes.     

Beta-cell ABCA1 influences insulin secretion, glucose homeostasis and response to thiazolidinedione treatment

Type 2 diabetes is characterized by both peripheral insulin resistance and reduced insulin secretion by beta-cells. The reasons for beta-cell dysfunction in this disease are incompletely understood but may include the accumulation of toxic lipids within this cell type. We examined the role of Abca1, a cellular cholesterol transporter, in cholesterol homeostasis and insulin secretion in beta-cells. Mice with specific inactivation of Abca1 in beta-cells had markedly impaired glucose tolerance and defective insulin secretion but normal insulin sensitivity. Islets isolated from these mice showed altered cholesterol homeostasis and impaired insulin secretion in vitro. We found that rosiglitazone, an activator of the peroxisome proliferator-activated receptor-gamma, which upregulates Abca1 in beta-cells, requires beta-cell Abca1 for its beneficial effects on glucose tolerance. These experiments establish a new role for Abca1 in beta-cell cholesterol homeostasis and insulin secretion, and suggest that cholesterol accumulation may contribute to beta-cell dysfunction in type 2 diabetes.     

Cell surface trafficking of Fas in NIT-1 cells and dissection of surface and total Fas expression.

The appearance of Fas receptor at the surface of pancreatic beta-cells affected by progressive insulitis strongly suggests that Fas-mediated beta-cell apoptosis plays an important role in the pathogenesis of type 1 diabetes. In support of this concept, the present study has shown that islet cells from NOD mice and the beta-cell line NIT-1 respond to the proinflammatory cytokines IL-1beta and IFN-gamma with Fas surface expression in a dose- and time-dependent manner. Moreover, the prevention of cytokine-induced surface Fas expression by actinomycin D, cycloheximide, and brefeldin A demonstrated that trafficking of Fas to the beta-cell surface requires RNA and protein synthesis and, in addition is critically dependent on intracellular protein transport. Compared with total cellular Fas protein, the amount of Fas at the cell surface was relatively small and indicated that Fas is preferentially expressed in cytoplasmic compartments of NIT-1 cells. It is concluded that inflammatory insults specifically induce translocation of Fas to the beta-cell surface and that interference with cell surface Fas expression is a new strategy to improve beta-cell survival in inflamed islets.     

Activation of the Reg family genes by pancreatic-specific IGF-I gene deficiency and after streptozotocin-induced diabetes in mouse pancreas

We have recently reported that Pdx1-Cre-mediated whole pancreas inactivation of IGF-I gene [in pancreatic-specific IGF-I gene-deficient (PID) mice] results in increased beta-cell mass and significant protection against both type 1 and type 2 diabetes. Because the phenotype is unlikely a direct consequence of IGF-I deficiency, the present study was designed to explore possible activation of proislet factors in PID mice by using a whole genome DNA microarray. As a result, multiple members of the Reg family genes (Reg2, -3alpha, and -3beta, previously not known to promote islet cell growth) were significantly upregulated in the pancreas. This finding was subsequently confirmed by Northern blot and/or real-time PCR, which exhibited 2- to 8-fold increases in the levels of these mRNAs. Interestingly, these Reg family genes were also activated after streptozotocin-induced beta-cell damage and diabetes (wild-type T1D mice) when islet cells were undergoing regeneration. Immunohistochemistry revealed increased Reg proteins in exocrine as well as endocrine pancreas and suggested their potential role in beta-cell neogenesis in PID or T1D mice. Previously, other Reg proteins (Reg1 and islet neogenesis-associated protein) have been shown to promote islet cell replication and neogenesis. These uncharacterized Reg proteins may play a similar but more potent role, not only in normal islet cell growth in PID mice, but also in islet cell regeneration after T1D.     

Mitochondrial proteomic analysis of a cell line model of familial amyotrophic lateral sclerosis

Mutations in copper-zinc superoxide dismutase (SOD1) have been linked to a subset of familial amytrophic lateral sclerosis (fALS), a fatal neurodegenerative disease characterized by progressive motor neuron death. An increasing amount of evidence supports that mitochondrial dysfunction and apoptosis activation play a critical role in the fALS etiology, but little is known about the mechanisms by which SOD1 mutants cause the mitochondrial dysfunction and apoptosis. In this study, we use proteomic approaches to identify the mitochondrial proteins that are altered in the presence of a fALS-causing mutant G93A-SOD1. A comprehensive characterization of mitochondrial proteins from NSC34 cells, a motor neuron-like cell line, was achieved by two independent proteomic approaches. Four hundred seventy unique proteins were identified in the mitochondrial fraction collectively, 75 of which are newly discovered proteins that previously had only been reported at the cDNA level. Two-dimensional gel electrophoresis was subsequently used to analyze the differences between the mitochondrial proteomes of NSC34 cells expressing wild-type and G93A-SOD1. Nine and 36 protein spots displayed elevated and suppressed abundance respectively in G93A-SOD1-expressing cells. The 45 spots were identified by MS, and they include proteins involved in mitochondrial membrane transport, apoptosis, the respiratory chain, and molecular chaperones. In particular, alterations in the post-translational modifications of voltage-dependent anion channel 2 (VDAC2) were found, and its relevance to regulating mitochondrial membrane permeability and activation of apoptotic pathways is discussed. The potential role of other proteins in the mutant SOD1-mediated fALS is also discussed. This study has produced a short list of mitochondrial proteins that may hold the key to the mechanisms by which SOD1 mutants cause mitochondrial dysfunction and neuronal death. It has laid the foundation for further detailed functional studies to elucidate the role of particular mitochondrial proteins, such as VDAC2, in the pathogenesis of familial ALS.     

The central role of calcium in the effects of cytokines on beta-cell function: implications for type 1 and type 2 diabetes

The appropriate regulation of intracellular calcium is a requirement for proper cell function and survival. This review focuses on the effects of proinflammatory cytokines on calcium regulation in the insulin-producing pancreatic beta-cell and how normal stimulus-secretion coupling, organelle function, and overall beta-cell viability are impacted. Proinflammatory cytokines are increasingly thought to contribute to beta-cell dysfunction not only in type 1 diabetes (T1D), but also in the progression of type 2 diabetes (T2D). Cytokine-induced disruptions in calcium handling result in reduced insulin release in response to glucose stimulation. Cytokines can alter intracellular calcium levels by depleting calcium from the endoplasmic reticulum (ER) and by increasing calcium influx from the extracellular space. Depleting ER calcium leads to protein misfolding and activation of the ER stress response. Disrupting intracellular calcium may also affect organelles, including the mitochondria and the nucleus. As a chronic condition, cytokine-induced calcium disruptions may lead to beta-cell death in T1D and T2D, although possible protective effects are also discussed. Calcium is thus central to both normal and pathological cell processes. Because the tight regulation of intracellular calcium is crucial to homeostasis, measuring the dynamics of calcium may serve as a good indicator of overall beta-cell function.     

Beta-cells in type 2 diabetes: a loss of function and mass

Type 2 diabetes mellitus manifests itself in individuals who lose the ability to produce sufficient amounts of insulin to maintain normoglycaemia in the face of insulin resistance. The ability to secrete adequate amounts of insulin depends on beta-cell function and mass. Chronic hyperglycaemia is detrimental to pancreatic beta-cells, causing impaired insulin secretion and playing an essential role in the regulation of beta-cell turnover. This paper will address the effect of chronically elevated glucose levels on beta-cell turnover and function. In previous studies we have shown that elevated glucose concentrations induce apoptosis in human beta-cells due to an interaction between constitutively expressed Fas ligand and upregulated Fas. Human beta-cells produce interleukin (IL)-1beta in response to high glucose concentrations, independently of an immune-mediated process. This was antagonized by the IL-1 receptor antagonist (IL-1Ra), a naturally occurring anti-inflammatory cytokine also found in the beta-cell. Therefore the balance of IL-1beta and IL-1Ra may play a crucial role in the pathogenesis of diabetes. Inhibition of glucotoxicity represents a promising therapeutic stratagem in diabetes therapy to preserve functional beta-cell mass.     

Caspase-3-dependent beta-cell apoptosis in the initiation of autoimmune diabetes mellitus

beta-Cell apoptosis is a key event contributing to the pathogenesis of type 1 diabetes mellitus. In addition to apoptosis being the main mechanism by which beta cells are destroyed, beta-cell apoptosis has been implicated in the initiation of type 1 diabetes mellitus through antigen cross-presentation mechanisms that lead to beta-cell-specific T-cell activation. Caspase-3 is the major effector caspase involved in apoptotic pathways. Despite evidence supporting the importance of beta-cell apoptosis in the pathogenesis of type 1 diabetes, the specific role of caspase-3 in this process is unknown. Here, we show that Caspase-3 knockout (Casp3(-/-) mice were protected from developing diabetes in a multiple-low-dose streptozotocin autoimmune diabetes model. Lymphocyte infiltration of the pancreatic islets was completely absent in Casp3(-/-) mice. To determine the role of caspase-3-dependent apoptosis in disease initiation, a defined antigen-T-cell receptor transgenic system, RIP-GP/P14 double-transgenic mice with Casp3 null mutation, was examined. beta-cell antigen-specific T-cell activation and proliferation were observed only in the pancreatic draining lymph node of RIP-GP/P14/Casp3(+/-) mice, but not in mice lacking caspase-3. Together, our findings demonstrate that caspase-3-mediated beta-cell apoptosis is a requisite step for T-cell priming, a key initiating event in type 1 diabetes.     

Two-dimensional electrophoretic profiling of atopic dermatitis in primary cultured keratinocytes from patients

Recently, we reported altered protein expression in primary cultured fibroblasts from atopic dermatitis (AD) patients. As a sequential study, we conducted proteomic analysis of primary keratinocytes derived from AD patients to further identify AD-related proteins. Three pH ranges, 4-7, 6-9, and 7-11, were used to profile the altered protein expression in AD. We obtained 46 candidate spots from the 2-D gel image analysis: 18 proteins were up-regulated and 27 proteins were down-regulated. Among the several important candidate proteins, NCC27 showed the same profile of a defect in PTM in both AD-derived keratinocytes and fibroblasts. On the basis of current and previous reports, real-time PCR was performed on select candidate genes to compare RNA and protein expression levels in AD-derived keratinocytes and fibroblasts. Our results provide new clues to aid in understanding the mechanism of atopic alterations in keratinocytes and suggest new AD-associated proteins that are important in AD pathogenesis.     

Pancreatic beta-cell growth and survival--a role in obesity-linked type 2 diabetes?

Obesity-linked type 2 diabetes is a disease of insulin resistance combined with pancreatic beta-cell dysfunction. Although a role for beta-cell mass in the pathogenesis of obesity-linked type 2 diabetes has recently gained prominence, the idea is still being developed. It is proposed that in early obesity an increase in beta-cell mass and function might compensate for peripheral insulin resistance. However, as time and/or the severity of the obesity continue, there is decay in such adaptation and the beta-cell mass becomes inadequate. This, together with beta-cell dysfunction, leads to the onset of type 2 diabetes. It is becoming evident that elements in insulin and insulin growth factor (IGF)-1 signal-transduction pathways are key to regulating beta-cell growth. Current evidence indicates that interference of insulin signaling in obesity contributes to peripheral insulin resistance. This article examines whether a similar interference of IGF-1 signaling in the beta-cell could hinder upregulation of beta-cell mass and/or function, resulting in a failure to compensate for insulin resistance.     

Increased cytokine-induced cytotoxicity of pancreatic islet cells from transgenic mice expressing the Src-like tyrosine kinase GTK

BACKGROUND: The loss of beta cells in type 1 diabetes may involve protein kinases because they control cell growth, differentiation, and survival. Previous studies have revealed that GTK, a Src-like protein tyrosine kinase expressed in beta cells (also named Bsk/Iyk), regulates multiple responses including growth and survival of rat insulinoma cells (RINm5F) and differentiation of neuronal PC12 cells. In the present study, we have generated a transgenic mouse expressing a kinase active GTK mutant (GTK-Y504F) under the control of the rat insulin I promoter to establish a role of GTK in beta cells. MATERIALS AND METHODS: Control and GTK-transgenic CBA mice were used for determination of in vivo glucose tolerance and the relative insulin-positive area. Isolated islets from both groups were cultured in the absence and presence of cytokines and insulin secretion, viability and protein expression were assessed. RESULTS: The beta-cell mass of GTK-transgenic mice was increased as a consequence of a larger pancreas and an increased relative beta-cell area. Islets isolated from the transgenic animals exhibited an enhanced glucose-induced insulin release and reduced viability in response to cytokines that could not be explained by higher levels of nitric oxide (NO) compared with control islets. Extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), and Akt were all activated by cytokines, but GTK-transgenic islets contained higher basal levels of phosphorylated ERK1/2 and lower basal levels of phosphorylated p38 compared with the control islets. The total amount of activated MAPKs was, however, higher in the cytokine-stimulated transgenic islets compared with the control islets due to increased levels of phospho-ERK1/2. Moreover, the proline-rich tyrosine kinase (PYK) 2 (also named RAFTK/CAK beta/CADTK) levels were elevated in response to a 24-hr exposure to cytokines in control islets but not in the GTK-transgenic islets. CONCLUSIONS: These results suggest that although GTK increases the beta-cell mass, it also enhances islet cell death in response to cytokines and may thus be involved in the beta-cell damage in type 1 diabetes.     

The hexosamine biosynthesis pathway regulates insulin secretion via protein glycosylation in mouse islets

The hexosamine biosynthesis pathway plays a role in the modification of cellular proteins via the provision of substrate for addition of O-linked N-acetylglucosamine (GlcNAc). The relative importance of the GlcNAc modification of proteins to insulin secretion from pancreatic beta-cells has not been investigated and so remains unclear. In the present study, we show that inhibition of the hexosamine biosynthesis pathway decreases insulin secretion from mouse islets in response to a number of secretagogues, including glucose. This impairment in beta-cell function could not be attributed to reduced islet insulin content, altered ATP levels, or cell death and was restored with the addition of N-acetylglucosamine, a substrate that enters the pathway below the point of inhibition. Western blot analysis revealed that decreased islet protein glycosylation paralleled the decrease in insulin secretion following inhibition of the pathway. In conclusion, the data suggest a role for the hexosamine biosynthesis pathway in regulating the secretion of insulin by altering protein glycosylation. This finding may have implications for the development of type 2 diabetes, as chronic increase in flux through the hexosamine biosynthesis pathway may lead to the deterioration of beta-cell function via abnormal protein glycosylation.