Reduction of hypoglycaemia, lifestyle modifications and psychological distress during lockdown following SARS-CoV-2 outbreak in type 1 diabetes.

AIMS: To assess changes in glucose metrics and their association with psychological distress and lifestyle changes in patients with type 1 diabetes (T1D) using flash glucose monitoring (FGM) during lockdown following severe acute respiratory syndrome coronavirus 2 outbreak. MATERIALS AND METHODS: Single-centre, observational, retrospective study enrolling T1D patients who attended a remote visit on April 2020 at the Endocrinology division of the University Hospital Policlinico Consorziale, Bari, Italy. Lockdown-related changes in physical activity level and dietary habits were assessed on a semi-quantitative basis. Changes in general well-being were assessed by the General Health Questionnaire-12 items with a binary scoring system. Glucose metrics were obtained from the Libreview platform for the first 2 weeks of February 2020 (T0) and the last 2 weeks before the phone visit (T1). RESULTS: Out of 84 patients assessed for eligibility, 48 had sufficient FGM data to be included in the analysis. FGM data analysis revealed significant reductions in coefficient of variation, number of hypoglycaemic events, and time below range, while no changes were found in time in range, time above range, mean sensor glucose, and glucose management indicator. Moreover, the frequency of sweets consumption was inversely related to the occurrence of hypoglycaemic events during lockdown. CONCLUSIONS: Lockdown-related lifestyle changes, albeit unhealthy, may lead to reduction in FGM-derived measures of hypoglycaemia and glycaemic variability in patients with T1D.

The p66(Shc) redox adaptor protein is induced by saturated fatty acids and mediates lipotoxicity-induced apoptosis in pancreatic beta cells.

AIMS/HYPOTHESIS: The role of the redox adaptor protein p66(Shc) as a potential mediator of saturated fatty acid (FA)-induced beta cell death was investigated. METHODS: The effects of the FA palmitate on p66(Shc) expression were evaluated in human and murine islets and in rat insulin-secreting INS-1E cells. p66(Shc) expression was also measured in islets from mice fed a high-fat diet (HFD) and from human donors with different BMIs. Cell apoptosis was quantified by two independent assays. The role of p66(Shc) was investigated using pancreatic islets from p66 (Shc-/-) mice and in INS-1E cells with knockdown of p66(Shc) or overexpression of wild-type and phosphorylation-defective p66(Shc). Production of reactive oxygen species (ROS) was evaluated by the dihydroethidium oxidation method. RESULTS: Palmitate induced a selective increase in p66(Shc) protein expression and phosphorylation on Ser(36) and augmented apoptosis in human and mouse islets and in INS-1E cells. Inhibiting the tumour suppressor protein p53 prevented both the palmitate-induced increase in p66(Shc) expression and beta cell apoptosis. Palmitate-induced apoptosis was abrogated in islets from p66 (Shc-/-) mice and following p66 (Shc) knockdown in INS-1E cells; by contrast, overexpression of p66(Shc), but not that of the phosphorylation-defective p66(Shc) mutant, enhanced palmitate-induced apoptosis. The pro-apoptotic effects of p66(Shc) were dependent upon its c-Jun N-terminal kinase-mediated phosphorylation on Ser(36) and associated with generation of ROS. p66(Shc) protein expression and function were also elevated in islets from HFD-fed mice and from obese/overweight cadaveric human donors. CONCLUSIONS/INTERPRETATION: p53-dependent augmentation of p66(Shc) expression and function represents a key signalling response contributing to beta cell apoptosis under conditions of lipotoxicity.

The p66Shc protein controls redox signaling and oxidation-dependent DNA damage in human liver cells.

The p66Shc protein mediates oxidative stress-related injury in multiple tissues. Steatohepatitis is characterized by enhanced oxidative stress-mediated cell damage. The role of p66Shc in redox signaling was investigated in human liver cells and alcoholic steatohepatitis. HepG2 cells with overexpression of wild-type or mutant p66Shc, with Ser36 replacement by Ala, were obtained through infection with recombinant adenoviruses. Reactive oxygen species and oxidation-dependent DNA damage were assessed by measuring dihydroethidium oxidation and 8-hydroxy-2'-deoxyguanosine accumulation into DNA, respectively. mRNA and protein levels of signaling intermediates were evaluated in HepG2 cells and liver biopsies from control and alcoholic steatohepatitis subjects. Exposure to H2O2 increased reactive oxygen species and phosphorylation of p66Shc on Ser36 in HepG2 cells. Overexpression of p66Shc promoted reactive oxygen species synthesis and oxidation-dependent DNA damage, which were further enhanced by H2O2. p66Shc activation also resulted in increased Erk-1/2, Akt, and FoxO3a phosphorylation. Blocking of Erk-1/2 activation inhibited p66Shc phosphorylation on Ser36. Increased p66Shc expression was associated with reduced mRNA levels of antioxidant molecules, such as NF-E2-related factor 2 and its target genes. In contrast, overexpression of the phosphorylation defective p66Shc Ala36 mutant inhibited p66Shc signaling, enhanced antioxidant genes, and suppressed reactive oxygen species and oxidation-dependent DNA damage. Increased p66Shc protein levels and Akt phosphorylation were observed in liver biopsies from alcoholic steatohepatitis compared with control subjects. In human alcoholic steatohepatitis, increased hepatocyte p66Shc protein levels may enhance susceptibility to DNA damage by oxidative stress by promoting reactive oxygen species synthesis and repressing antioxidant pathways.

Exendin-4 protects pancreatic beta cells from palmitate-induced apoptosis by interfering with GPR40 and the MKK4/7 stress kinase signalling pathway.

AIMS/HYPOTHESIS: The mechanisms of the protective effects of exendin-4 on NEFA-induced beta cell apoptosis were investigated. METHODS: The effects of exendin-4 and palmitate were evaluated in human and murine islets, rat insulin-secreting INS-1E cells and murine glucagon-secreting alpha-TC1-6 cells. mRNA and protein expression/phosphorylation were measured by real-time RT-PCR and immunoblotting or immunofluorescence, respectively. Small interfering (si)RNAs for Ib1 and Gpr40 were used. Cell apoptosis was quantified by two independent assays. Insulin release was assessed with an insulin ELISA. RESULTS: Exposure of human and murine primary islets and INS-1E cells, but not alpha-TC1-6 cells, to exendin-4 inhibited phosphorylation of the stress kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and prevented apoptosis in response to palmitate. Exendin-4 increased the protein content of islet-brain 1 (IB1), an endogenous JNK blocker; however, siRNA-mediated reduction of IB1 did not impair the ability of exendin-4 to inhibit JNK and prevent apoptosis. Exendin-4 reduced G-protein-coupled receptor 40 (GPR40) expression and inhibited palmitate-induced phosphorylation of mitogen-activated kinase kinase (MKK)4 and MKK7. The effects of exendin-4 were abrogated in the presence of the protein kinase A (PKA) inhibitors, H89 and KT5720. Knockdown of GPR40, as well as use of a specific GPR40 antagonist, resulted in diminished palmitate-induced JNK and p38 MAPK phosphorylation and apoptosis. Furthermore, inhibition of JNK and p38 MAPK activity prevented palmitate-induced apoptosis. CONCLUSIONS/INTERPRETATION: Exendin-4 counteracts the proapoptotic effects of palmitate in beta cells by reducing GPR40 expression and inhibiting MKK7- and MKK4-dependent phosphorylation of the stress kinases, JNK and p38 MAPK, in a PKA-dependent manner.

Evaluation of a digital tool supporting therapeutic decision making for the personalized management of patients with type 2 diabetes not treated with insulin: A pilot study.

AIMS: To investigate the benefits of using the Personalized Treatment Tool (PTT), a web-based clinical decision support tool assisting the diabetologist in the evaluation of patient's clinical characteristics and SMBG data, in the management of patients with non-insulin treated type 2 diabetes and inadequate glucose control. METHODS: We conducted a single-center, 16-week, cluster-randomized controlled trial. RESULTS: Eighty-two patients with 64.3 ± 9.4 years of age, disease duration 13.2 ± 9.1 years and HbA1c 7.8 ± 0.6%, 41 in the PTT group and 41 in the control group, completed the study. At follow-up, changes in indicators of glucose control and variability were not statistically different between the two groups. However, when considering the subgroup of patients on a single anti-diabetes drug at baseline (9 in the PTT group, 14 in the control group), changes in HbA1c and CGM-derived TIR 70-140 mg/dl, 24-hour MSG, GRADE, and HBGI were significantly improved in the PTT group compared to the control group. CONCLUSION: When performed in a structured manner and used to modify the diabetes therapy through an algorithm-driven digital tool, SMBG can lead to significant improvements of glycemic control and variability in patients with type 2 diabetes not treated with insulin.

Different gene expression in human heart tissue and progenitor cells from control and diabetic subjects: relevance to the pathogenesis of human diabetic cardiomyopathy.

The The aim of our study is to investigate the molecular mechanisms of diabetic cardiomyopathy through the identification of remarkable genes for the myocardial function that are expressed differently between diabetic and normal subjects. Moreover, we intend to characterize both in human myocardial tissue and in the related cardiac progenitor cells the pattern of gene expression and the levels of expression and protein activation of molecular effectors involved in the regulation of the myocardial function and differentiation to clarify whether in specific human pathological conditions (type 2 diabetes mellitus, cardiac failure, coronary artery disease) specific alterations of the aforementioned factors could take place. Thirty-five patients scheduled for coronary artery bypass grafting (CABG) or for aortic or mitral valve replacement were recruited into the study. There were 13 men and 22 women with a mean age of 64.8 +/- 13.4 years. A list of anamnestic, anthropometric, clinical, and instrumental data required for an optimal phenotypical characterization of the patients is reported. The small cardiac biopsy specimens were placed in the nourishing buffer, in a sterile tube provided the day of the procedure, to maintain the stability of the sample for several hours at room temperature. The cells were isolated by a dedicated protocol and then cultured in vitro. The sample was processed for total RNA extraction and levels of gene expression and protein activation of molecular effectors involved in the regulation of function and differentiation of human myocardium was analyzed. In particular, cardiac genes that modulate the oxidative stress response or the stress induced by pro-inflammatory cytokines (p66Shc, SOCS-1, SOCS-3) were analyzed. From a small sample of myocardium cardiac stem cells and cardiomyoblasts were also isolated and characterized. These cells showed a considerable proliferative capacity due to the fact that they demonstrate stability up to the eleventh passage. Analysis of gene expression in a subgroup of subjects showed the trend of a decrease in levels of expression of cardiac-specific transcription genes and oxidative stress-related proteins in tissues of diabetic patients compared with controls subjects. This trend is not confirmed in isolated cells. As for the coronary artery disease, diabetic cardiomyopathy could be associated with a reduction of the cardiac stem and progenitor cells pool. The expansion of the cardiac resident cells pool could be associated with a preservation of cardiac performance, suggesting that a preserved stamina compartment can counteract the impact of diabetes on the myocardium.

The Role of Oxidative Stress in Cardiac Disease: From Physiological Response to Injury Factor.

Reactive oxygen species (ROS) are highly reactive chemical species containing oxygen, controlled by both enzymatic and nonenzymatic antioxidant defense systems. In the heart, ROS play an important role in cell homeostasis, by modulating cell proliferation, differentiation, and excitation-contraction coupling. Oxidative stress occurs when ROS production exceeds the buffering capacity of the antioxidant defense systems, leading to cellular and molecular abnormalities, ultimately resulting in cardiac dysfunction. In this review, we will discuss the physiological sources of ROS in the heart, the mechanisms of oxidative stress-related myocardial injury, and the implications of experimental studies and clinical trials with antioxidant therapies in cardiovascular diseases.

Abnormalities of insulin-like growth factor-I signaling and impaired cell proliferation in osteoblasts from subjects with osteoporosis.

IGF-I regulates bone acquisition and maintenance, even though the cellular targets and signaling pathways responsible for its action in human bone cells are poorly understood. Whether abnormalities in IGF-I action and signaling occur in human osteoblasts under conditions of net bone loss has not been determined. Herein we carried out a comparative analysis of IGF-I signaling in primary cultures of human osteoblasts from osteoporotic and control donors. In comparison with control cells, osteoporotic osteoblasts showed increased tyrosine phosphorylation of the IGF-I receptor in the basal state and blunted stimulation of receptor phosphorylation by IGF-I. Augmentation of basal IGF-I receptor phosphorylation was associated with coordinate increases in basal tyrosine phosphorylation of insulin receptor substrate (IRS)-2 and activation of Erk, which were also minimally responsive to IGF-I stimulation. By contrast, phosphorylation levels of IRS-1, Akt, and glycogen synthase kinase-3 were similar in the basal state in control and osteoporotic osteoblasts and showed marked increases after IGF-I stimulation in both cell populations, even though these responses were significantly lower in the osteoporotic osteoblasts. The IGF-I signaling abnormalities in osteoporotic osteoblasts were associated with reduced DNA synthesis both under basal conditions and after stimulation with IGF-I. Interestingly, treatment of the osteoporotic osteoblasts with the MAPK kinase inhibitor PD098059 reduced the elevated levels of Erk phosphorylation and increased basal DNA synthesis. Collectively, our data show that altered osteoblast proliferation in human osteoporosis may result from dysregulation of IGF-I receptor signaling, including constitutive activation of the IRS-2/Erk signaling pathway, which becomes unresponsive to IGF-I, and defective induction of the IRS-1/Akt signaling pathway.

Insulin signaling in human visceral and subcutaneous adipose tissue in vivo.

In this study, we evaluated the activation of various insulin signaling molecules in human fat in vivo and compared signaling reactions in visceral and subcutaneous fat depots. Paired abdominal omental and subcutaneous fat biopsies were obtained from nonobese subjects with normal insulin sensitivity under basal conditions and 6 and 30 min following administration of intravenous insulin. Insulin receptor phosphorylation was more intense and rapid and insulin receptor protein content was greater in omental than in subcutaneous adipose tissue (P < 0.05). Insulin-induced phosphorylation of Akt also occurred to a greater extent and earlier in omental than in subcutaneous fat (P < 0.05) in the absence of significant changes in Akt protein content. Accordingly, phosphorylation of the Akt substrate glycogen synthase kinase-3 was more responsive to insulin stimulation in omental fat. Protein content of extracellular signal-regulated kinase (ERK)-1/2 was threefold higher in omental than in subcutaneous fat (P < 0.05), and ERK phosphorylation showed an early 6-min peak in omental fat, in contrast with a more gradual increase observed in subcutaneous fat. In conclusion, the adipocyte insulin signaling system of omental fat shows greater and earlier responses to insulin than that of subcutaneous fat. These findings may contribute to explain the biological diversity of the two fat depots.

GLP-1 Receptor Activation Inhibits Palmitate-Induced Apoptosis via Ceramide in Human Cardiac Progenitor Cells.

Context: Increased apoptosis of cardiomyocytes and cardiac progenitor cells (CPCs) in response to saturated fatty acids (SFAs) can lead to myocardial damage and dysfunction. Ceramides mediate lipotoxicity-induced apoptosis. Glucagonlike peptide-1 receptor (GLP1R) agonists exert beneficial effects on cardiac cells in experimental models. Objective: To investigate the protective effects of GLP1R activation on SFA-mediated apoptotic death of human CPCs. Design: Human CPCs were isolated from cardiac appendages of nondiabetic donors and then exposed to palmitate with or without pretreatment with the GLP1R agonist exendin-4. Ceramide accumulation was evaluated by immunofluorescence. Expression of key enzymes in de novo ceramide biosynthesis was studied by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Apoptosis was evaluated by measuring release of oligonucleosomes, caspase-3 cleavage, caspase activity, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. Results: Exposure of the CPCs to palmitate resulted in 2.3- and 1.9-fold higher expression of ceramide synthase 5 (CERS5) and ceramide desaturase-1, respectively (P < 0.05). This was associated with intracellular accumulation of ceramide and activation of c-Jun NH2-terminal protein kinase (JNK) signaling and apoptosis (P < 0.05). Both coincubation with fumonisin B1, a specific ceramide synthase inhibitor, and CERS5 knockdown prevented ceramide accumulation, JNK activation, and apoptosis in response to palmitate (P < 0.05). Exendin-4 also prevented the activation of the ceramide biosynthesis and JNK in response to palmitate, inhibiting apoptosis (P < 0.05). Conclusions: Excess palmitate results in activation of ceramide biosynthesis, JNK signaling, and apoptosis in human CPCs. GLP1R activation counteracts this lipotoxic damage via inhibition of ceramide generation, and this may represent a cardioprotective mechanism.

Long-Term Exposure of Pancreatic ß-Cells to Palmitate Results in SREBP-1C-Dependent Decreases in GLP-1 Receptor Signaling via CREB and AKT and Insulin Secretory Response.

The effects of prolonged exposure of pancreatic ß-cells to high saturated fatty acids on glucagon-like peptide-1 (GLP-1) action were investigated. Murine islets, human pancreatic 1.1B4 cells, and rat INS-1E cells were exposed to palmitate for 24 hours. mRNA and protein expression/phosphorylation were measured by real-time RT-PCR and immunoblotting, respectively. Specific short interfering RNAs were used to knockdown expression of the GLP-1 receptor (Glp1r) and Srebf1. Insulin release was assessed with a specific ELISA. Exposure of murine islets, as well as of human and INS-1E ß-cells, to palmitate reduced the ability of exendin-4 to augment insulin mRNA levels, protein content, and release. In addition, palmitate blocked exendin-4-stimulated cAMP-response element-binding protein and v-akt murine thymoma viral oncogene homolog phosphorylation, whereas phosphorylation of MAPK-ERK kinase-1/2 and ERK-1/2 was not altered. Similarly, RNA interference-mediated suppression of Glp1r expression prevented exendin-4-induced cAMP-response element-binding protein and v-akt murine thymoma viral oncogene homolog phosphorylation, but did not impair exendin-4 stimulation of MAPK-ERK kinase-1/2 and ERK-1/2. Both islets from mice fed a high fat diet and human and INS-1E ß-cells exposed to palmitate showed reduced GLP-1 receptor and pancreatic duodenal homeobox-1 (PDX-1) and increased sterol regulatory element-binding protein (SREBP-1C) mRNA and protein levels. Furthermore, suppression of SREBP-1C protein expression prevented the reduction of PDX-1 and GLP-1 receptor levels and restored exendin-4 signaling and action. Finally, treatment of INS-1E cells with metformin for 24 h resulted in inhibition of SREBP-1C expression, increased PDX-1 and GLP-1 receptor levels, consequently, enhancement of exendin-4-induced insulin release. Palmitate impairs exendin-4 effects on ß-cells by reducing PDX-1 and GLP-1 receptor expression and signaling in a SREBP-1C-dependent manner. Metformin counteracts the impairment of GLP-1 receptor signaling induced by palmitate.

Intrauterine growth restriction in humans is associated with abnormalities in placental insulin-like growth factor signaling.

The IGFs promote the growth and development of the feto-placental unit during gestation, and impairment of their placental actions may result in altered intrauterine growth of the fetus. In this study, proteins involved in IGF signaling were investigated in human placentas from pregnancies complicated by intrauterine growth restriction (IUGR) compared with those from normal pregnancies. IUGR placentas exhibited 33% reduction in the protein content of IGF-I receptors, but no changes in insulin receptor protein levels. In addition, insulin receptor substrate-2 (IRS-2) protein levels were reduced in IUGR placentas, with no changes in IRS-1 or Shc protein content, and this was associated with a parallel decrease in IRS-2-associated phosphatidyl inositol 3-kinase. Akt protein expression was also reduced in IUGR, whereas phosphorylation of Akt and its substrate glycogen synthase kinase-3 was unchanged. Finally, in IUGR placentas there was impaired activation of multiple members of the MAPK family, because phosphorylation of p38 and c-Jun N-terminal kinase was reduced 70%. In conclusion, human placentas from pregnancies complicated by IUGR are characterized by decreased IGF-I receptor content, selective impairment of the IRS-2/ phosphatidyl inositol 3-kinase pathway, and reduced p38 and c-Jun N-terminal kinase activation. The observed abnormalities in IGF-I signaling may contribute to altered fetal growth and development in human IUGR.

Pathophysiology of type 2 diabetes: rationale for different oral antidiabetic treatment strategies.

Type 2 diabetes mellitus is a chronic metabolic disorder that results from defects in both insulin secretion and insulin action. Both insulin resistance and beta-cell failure are genetically determined to some extent; however, environmental factors contribute to exacerbate both abnormalities. Type 2 diabetic individuals are also characterised by reduced beta-cell mass likely due to increased cellular apoptosis. The early use of insulin therapy in type 2 diabetes may prove beneficial to prevent further beta-cell loss and need for exogenous insulin. Treatment options with oral agents are quite diverse, including insulin sensitizers, alpha-glucosidase inhibitors, and beta-cell secretagogues. Although in recent years the emphasis on initial therapy has been shifting from insulin secretagogues to insulin sensitizers such as metformin and thiazolidinediones, questions remain as to genetic and/or phenotypic factors may dictate a different choice of the first antidiabetic drug to be used. It is not totally clear whether monotherapy should be pursued until the maximally effective dose of a given drug or combination therapy should be used to target distinct pathogenic defects in a single patient. Individual phenotypic and genetic characterisation of the patients may help to solve this conundrum, eventually providing tailored treatment algorithms.

Abnormalities of the cardiac stem and progenitor cell compartment in experimental and human diabetes.

Diabetic cardiomyopathy consists of a series of structural and functional changes. Accumulating evidence supports the concept that a "cardiac stem cell compartment disease" plays an important role in the pathophysiology of diabetic cardiomyopathy. In diabetic hearts, human cardiac stem/progenitor cells (CSPC) are reduced and manifest defective proliferative capacity. Hyperglycaemia, hyperlipidemia, inflammation, and the consequent oxidative stress are enhanced in diabetes: these conditions can induce defects in both growth and survival of these cells with an imbalance between cell death and cell replacement, thus favouring the onset of diabetic cardiomyopathy and its progression towards heart failure. The preservation of CSPC compartment can contribute to counteract the negative impact of diabetes on the myocardium. The recent studies summarized in this review have improved our understanding of the development and stem cell biology within the cardiovascular system. However, several issues remain unsolved before cell therapy can become a clinical therapeutically relevant strategy.

Cardiovascular disease and glycemic control in type 2 diabetes: now that the dust is settling from large clinical trials.

The relationship between glucose control and cardiovascular outcomes in type 2 diabetes has been a matter of controversy over the years. Although epidemiological evidence exists in favor of an adverse role of poor glucose control on cardiovascular events, intervention trials have been less conclusive. The Action to Control Cardiovascular Risk in Diabetes (ACCORD) study, the Action in Diabetes and Vascular Disease (ADVANCE) study, and the Veterans Affairs Diabetes Trial (VADT) have shown no beneficial effect of intensive glucose control on primary cardiovascular endpoints in type 2 diabetes. However, subgroup analysis has provided evidence suggesting that the potential beneficial effect largely depends on patients' characteristics, including age, diabetes duration, previous glucose control, presence of cardiovascular disease, and risk of hypoglycemia. The benefit of strict glucose control on cardiovascular outcomes and mortality may be indeed hampered by the extent and frequency of hypoglycemic events and could be enhanced if glucose-lowering medications, capable of exerting favorable effects on the cardiovascular system, were used. This review examines the relationship between intensive glucose control and cardiovascular outcomes in type 2 diabetes, addressing the need for individualization of glucose targets and careful consideration of the benefit/risk profile of antidiabetes medications.

TNFα signals via p66(Shc) to induce E-Selectin, promote leukocyte transmigration and enhance permeability in human endothelial cells.

Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and ß-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66(Shc) acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66(Shc) in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66(Shc) on Ser(36) and the stress kinase c-Jun NH2-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66(Shc) in HUVEC resulted in enhanced p66(Shc) phosphorylation on Ser(36), increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66(Shc) protein, in which Ser(36) was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66(Shc) resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66(Shc) acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66(Shc) on Ser(36).

Exendin-4 prevents c-Jun N-terminal protein kinase activation by tumor necrosis factor-alpha (TNFalpha) and inhibits TNFalpha-induced apoptosis in insulin-secreting cells.

Glucagon-like peptide-1 and its analogs may preserve pancreatic beta-cell mass by promoting resistance to cytokine-mediated apoptosis. The mechanisms of TNFalpha-induced apoptosis and of its inhibition by exendin-4 were investigated in insulin-secreting cells. INS-1 and MIN6 insulinoma cells were exposed to 20 ng/ml TNFalpha, with or without pretreatment with 10 nm exendin-4. Treatment with TNFalpha increased c-Jun N-terminal protein kinase (JNK) phosphorylation 2-fold, reduced inhibitor-kappaBalpha (IkappaBalpha) protein content by 50%, induced opposite changes in caspase-3 and Bcl-2 protein content, and increased cellular apoptosis. Moreover, exposure to TNFalpha resulted in increased serine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-2 and reduced basal and insulin-induced Akt phosphorylation. However, in the presence of a JNK inhibitor, TNFalpha-induced apoptosis was diminished and serine phosphorylation of IRS proteins was prevented. When cells were pretreated with exendin-4, TNFalpha-induced JNK and IRS-1/2 serine phosphorylation was markedly reduced, Akt phosphorylation was increased, caspase-3 and Bcl-2 protein levels were restored to normal, and TNFalpha-induced apoptosis was inhibited by 50%. This was associated with a 2-fold increase in IRS-2 expression levels. A similar ability of exendin-4 to prevent TNFalpha-induced JNK phosphorylation was found in isolated pancreatic human islets. The inhibitory effect of exendin-4 on TNFalpha-induced JNK phosphorylation was abrogated in the presence of the protein kinase A inhibitor H89. In conclusion, JNK activation mediates TNFalpha-induced apoptosis and impairment of the IRS/Akt signaling pathway in insulin-secreting cells. By inhibiting JNK phosphorylation in a PKA-dependent manner, exendin-4 counteracts TNFalpha-mediated apoptosis and reverses the inhibitory events in the IRS/Akt pathway, resulting in promotion of cell survival.

Cross-Talk between PPARgamma and Insulin Signaling and Modulation of Insulin Sensitivity.

PPARgamma activation in type 2 diabetic patients results in a marked improvement in insulin and glucose parameters, resulting from an improvement of whole-body insulin sensitivity. Adipose tissue is the major mediator of PPARgamma action on insulin sensitivity. PPARgamma activation in mature adipocytes induces the expression of a number of genes involved in the insulin signaling cascade, thereby improving insulin sensitivity. PPARgamma is the master regulator of adipogenesis, thereby stimulating the production of small insulin-sensitive adipocytes. In addition to its importance in adipogenesis, PPARgamma plays an important role in regulating lipid, metabolism in mature adipocytes by increasing fatty acid trapping. Finally, adipose tissue produces several cytokines that regulate energy homeostasis, lipid and glucose metabolism. Disturbances in the production of these factors may contribute to metabolic abnormalities, and PPARgamma activation is also associated with beneficial effects on expression and secretion of a whole range of cytokines.

Involvement of the p66Shc protein in glucose transport regulation in skeletal muscle myoblasts.

The p66(Shc) protein isoform regulates MAP kinase activity and the actin cytoskeleton turnover, which are both required for normal glucose transport responses. To investigate the role of p66(Shc) in glucose transport regulation in skeletal muscle cells, L6 myoblasts with antisense-mediated reduction (L6/p66(Shc)as) or adenovirus-mediated overexpression (L6/p66(Shc)adv) of the p66(Shc) protein were examined. L6/(Shc)as myoblasts showed constitutive activation of ERK-1/2 and disruption of the actin network, associated with an 11-fold increase in basal glucose transport. GLUT1 and GLUT3 transporter proteins were sevenfold and fourfold more abundant, respectively, and were localized throughout the cytoplasm. Conversely, in L6 myoblasts overexpressing p66(Shc), basal glucose uptake rates were reduced by 30% in parallel with a approximately 50% reduction in total GLUT1 and GLUT3 transporter levels. Inhibition of the increased ERK-1/2 activity with PD98059 in L6/(Shc)as cells had a minimal effect on increased GLUT1 and GLUT3 protein levels, but restored the actin cytoskeleton, and reduced the abnormally high basal glucose uptake by 70%. In conclusion, p66(Shc) appears to regulate the glucose transport system in skeletal muscle myoblasts by controlling, via MAP kinase, the integrity of the actin cytoskeleton and by modulating cellular expression of GLUT1 and GLUT3 transporter proteins via ERK-independent pathways.