Downregulation of mTORC1 and Mcl-1 by lipid-oversupply contributes to islet β-cell apoptosis and dysfunction

Pancreatic β-cell apoptosis is a key feature of diabetes and can be induced by chronic exposure to saturated fatty acids (FAs). However, the underlying mechanisms remain poorly understood. We presently evaluated the role of Mcl-1 and mTOR in mice fed with high-fat-diet (HFD) and β-cells exposed to the overloaded palmitic acid (PA). Compared with normal-chow-diet (NCD)-fed mice, HFD group showed impaired glucose tolerance after two months. Along with the diabetes progression, pancreatic islets first became hypertrophic and then atrophic, the ratio of β-cell:α-cell increased in the islets of four months HFD-fed mice while decreased after six months. This process was accompanied by significantly increased β-cell apoptosis and AMPK activity, and decreased Mcl-1 expression and mTOR activity. Consistently, glucose-induced insulin secretion dropped. In terms of mechanism, PA with lipotoxic dose could activate AMPK, which in turn inhibited ERK-stimulated Mcl-1^Thr163 phosphorylation. Meanwhile, AMPK blocked Akt activity to release Akt inhibition on GSK3β, followed by GSK3β-initiated Mcl-1^Ser159 phosphorylation. The context of Mcl-1 phosphorylation finally led to its degradation by ubiquitination. Also, AMPK inhibited the activity of mTORC1, resulting in a lower level of Mcl-1. Suppression of mTORC1 activity and Mcl-1 expression positively related to β-cell failure. Alteration of Mcl-1 or mTOR expression rendered different tolerance of β-cell to different dose of PA. In conclusion, lipid oversupply-induced dual modulation of mTORC1 and Mcl-1 finally led to β-cell apoptosis and impaired insulin secretion. The study may help further understand the pathogenesis of β-cell dysfunction in case of dyslipidemia, and provide promising therapeutic targets for diabetes.     

Upregulation of PTEN by peroxynitrite contributes to cytokine-induced apoptosis in pancreatic β-cells

Phosphatase and tensin homolog (PTEN), a tumor suppressor gene, by negatively regulating the PI3K-Akt signaling pathway, participates in multiple biological processes such as cell proliferation, apoptosis, differentiation, and migration. Recent studies show that selective deletion of PTEN in pancreatic β-cells leads to resistance to streptozotocin (STZ)-induced diabetes, but the mechanism is unclear. One major mechanism underlying STZ toxicity is cytokine-mediated β-cell destruction in which oxidative stress plays a key role. The present study investigated the role of PTEN in cytokine-induced β-cell apoptosis, and further explored whether oxidative stress, particularly peroxynitrite formation, could regulate PTEN-Akt pathway. Incubation of βTC-6 cells with cytokine mixture (IL-1β, TNF-α, and IFN-γ) or exogenous peroxynitrite significantly increased apoptotic cell percentage, elevated PTEN and p-PTEN levels, and inhibited Akt activation. Transfection with PTEN-specific siRNA protected βTC-6 cells from cytokine or peroxynitrite-mediated cell apoptosis and partially reversed Akt inhibition. Furthermore, nitrotyrosine formation, an indicator of peroxynitrite production, was significantly elevated after cytokine treatment. Preventing peroxynitrite formation by administrating NAC/l-NMMA, or scavenging peroxynitrite directly by UA, attenuated cytokine-induced PTEN upregulation, Akt inhibition, and β-cell apoptosis. These findings suggest that peroxynitrite-mediated PTEN upregulation plays an important role in cytokine-induced pancreatic β-cell apoptosis.     

The role of autophagy in pancreatic β-cell and diabetes

Pancreatic β-cells play a key role in glucose homeostasis in mammals. Although large-scale protein synthesis and degradation occur in pancreatic β-cells, the mechanism underlying dynamic protein turnover in β-cells remains largely unknown. We found low-level constitutive autophagy in β-cells of C57BL/6 mice fed a standard diet; however, autophagy was markedly upregulated in mice fed a high-fat diet. β-cells of diabetic db/db mice contained large numbers of autophagosomes, compared with non-diabetic db/misty controls. The functional importance of autophagy was analyzed using β-cell-specific Atg7 knockout mice. Autophagy-deficient mice showed degeneration of β-cells and impaired glucose tolerance with reduced insulin secretion. While a high-fat diet stimulated β-cell autophagy in control mice, it induced a profound deterioration of glucose intolerance in β-cell autophagy-deficient mutants, partly because of the lack of a compensatory increase in β-cell mass. These results suggest that the degradation of unnecessary cellular components by autophagy is essential for maintenance of the architecture and function of β-cells. Autophagy also serves as a crucial element of stress responses to protect β-cells under insulin resistant states. Impairment of autophagic machinery could thus predispose individuals to type 2 diabetes.     

The regulation of the UCH-L1 gene by transcription factor NF-κB in podocytes

In kidney, the ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) is involved in podocyte injury and proteinuria but details of the mechanism underlying its regulation are not known. Activation of NF-κB is thought to be the predominant risk factor for kidney disease; therefore, it is postulated that UCH-L1 may be one of the NF-κB target genes. In this study, we investigated the involvement of NF-κB activation in the regulation of UCH-L1 expression and the function of murine podocytes. Stimulation of podocytes with the cytokines TNF-α and IL-1β up-regulated UCH-L1 expression rapidly at the mRNA and protein levels and the NF-κB-specific inhibitor pyrrolidine dithiocarbamate resulted in down-regulation. NF-κB up-regulates UCH-L1 via binding the ? 300 bp and ? 109 bp sites of its promoter, which was confirmed by the electrophoretic mobility shift assay of DNA–nuclear protein binding. In the renal biopsy from lupus nephritis patients, the expressions of NF-κB and UCH-L1 increased in immunohistochestry staining and were positively correlated. Activation of NF-κB up-regulates UCH-L1 expression following changing of other podocytes molecules, such as nephrin and snail. These results suggest that activation of the NF-κB signaling pathway could be the major pathogenesis to up-regulate UCH-L1 in podocyte injury, followed by the turnover of other molecules, which might result in morphological changes and dysfunction of podocytes. This work help us to understand the effect of NF-κB on specific target molecules of podocytes, and suggest that targeting the NF-κB–UCH-L1 interaction could be a novel therapeutic strategy for the treatment of podocyte lesions and proteinuria.     

Loss of βarrestin1 and βarrestin2 contributes to pulmonary hypoplasia and neonatal lethality in mice

Less is known about the connection between the malfunction of βarrestins and developmental defects as the mice with either of two βarrestin isoforms knockout appear normal. In order to address the biological function of βarrestins during developmental process, we generate βarrestin1/2 double knockout mice. We found that βarrestin1/2 dual-null mice developed respiratory distress and atelectasis that subsequently caused neonatal death. Morphological examination revealed type II pneumocyte immaturity. Our results indicate that not only βarrestin1/2 double knockout lung tissue show disturbances in cell proliferation but βarrestin1 and βarrestin2 contribute to pulmonary surfactant complex generation during pulmonary maturation. Intra-amniotic delivery of recombinant adenovirus expressing βarrestin1 or βarrestin2 enhances surfactant-associated proteins synthesis in vivo. Our mRNA microarray data further reveal that βarrestin1/2 double knockout results in downregulation of a significant proportion of genes involved in several lung morphogenesis processes. Together, our study demonstrates that βarrestin1 and βarrestin2 collaborate in embryonic development processes for epithelial pneumocyte differentiation and lung maturation.     

The pancreatic islet β-cell-enriched transcription factor Pdx-1 regulates Slc30a8 gene transcription through an intronic enhancer

PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumour progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small-molecule trypsin inhibitors have low affinity and little selectivity, whereas protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. In the present study, we use mutagenesis of a mesotrypsin substrate, APPI (amyloid precursor protein Kunitz protease inhibitor domain), and of a poor mesotrypsin inhibitor, BPTI (bovine pancreatic trypsin inhibitor), to dissect mesotrypsin specificity at the key P(2)' position. We find that bulky and charged residues strongly disfavour binding, whereas acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexes with BPTI variants provide structural insights into mesotrypsin specificity and inhibition. Through optimization of the P(1) and P(2)' residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant K(i) of 5.9 nM, a >2000-fold improvement in affinity over native BPTI. Using this engineered inhibitor, we demonstrate the efficacy of pharmacological inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. Although further improvements in inhibitor selectivity will be important before clinical potential can be realized, the results of the present study support the feasibility of engineering protein protease inhibitors of mesotrypsin and highlight their therapeutic potential.     

mTORC1 signaling and regulation of pancreatic β-cell mass

The capacity of β cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of β cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for β-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of β-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in β cells. This review will discuss recent advances in the understanding of how this pathway regulates β-cell mass and present data on the role of TSC1 in modulation of β-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic β cells results in improved glucose tolerance, hyperinsulinemia and expansion of β-cell mass that persists with aging.     

mTORC1 signaling and regulation of pancreatic β-cell mass

The capacity of β cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of β cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for β-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of β-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in β cells. This review will discuss recent advances in the understanding of how this pathway regulates β-cell mass and present data on the role of TSC1 in modulation of β-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic β cells results in improved glucose tolerance, hyperinsulinemia and expansion of β-cell mass that persists with aging.     

In vivo activating transcription factor 3 silencing ameliorates the AMPK compensatory effects for ER stress-mediated β-cell dysfunction during the progression of type-2 diabetes

In obese Zucker diabetic fatty (ZDF) rats, ER stress is associated with insulin resistance and pancreatic β-cell dysfunction; however the exact mechanisms by which ER stress drives type-2 diabetes remain uncertain. Here, we investigated the role of ATF3 on the preventive regulation of AMPK against ER stress-mediated β-cell dysfunction during the end-stage progression of hyperglycemia in ZDF rats. The impaired glucose metabolism and β-cell dysfunction were significantly increased in late-diabetic phase 19-week-old ZDF rats. Although AMPK phosphorylation reduced in 6- and 12-week-old ZDF rats was remarkably increased at 19 weeks, the increases of lipogenice genes, ATF3, and ER stress or ROS-mediated β-cell dysfunction were still remained, which were attenuated by in vivo-injection of chemical chaperon tauroursodeoxycholate (TUDCA), chronic AICAR, or antioxidants. ATF3 did not directly affect AMPK phosphorylation, but counteracts the preventive effects of AMPK for high glucose-induced β-cell dysfunction. Moreover, knockdown of ATF3 by delivery of in vivo-jetPEI ATF3 siRNA attenuated ER stress-mediated β-cell dysfunction and enhanced the beneficial effect of AICAR. Our data suggest that ATF3 may play as a counteracting regulator of AMPK and thus promote β-cell dysfunction and the development of type-2 diabetes and could be a potential therapeutic target in treating type-2 diabetes.     

Regulation of pancreatic β-cell function and mass dynamics by prostaglandin signaling

Prostaglandins (PGs) are signaling lipids derived from arachidonic acid (AA), which is metabolized by cyclooxygenase (COX)-1 or 2 and class-specific synthases to generate PGD₂, PGE₂, PGF_2α, PGI₂ (prostacyclin), and thromboxane A₂. PGs signal through G-protein coupled receptors (GPCRs) and are important modulators of an array of physiological functions, including systemic inflammation and insulin secretion from pancreatic islets. The role of PGs in β-cell function has been an active area of interest, beginning in the 1970s. Early studies demonstrated that PGE₂ inhibits glucose-stimulated insulin secretion (GSIS), although more recent studies have questioned this inhibitory action of PGE₂. The PGE₂ receptor EP3 and one of the G-proteins that couples to EP3, Gα_Z, have been identified as negative regulators of β-cell proliferation and survival. Conversely, PGI₂ and its receptor, IP, play a positive role in the β-cell by enhancing GSIS and preserving β-cell mass in response to the β-cell toxin streptozotocin (STZ). In comparison to PGE₂ and PGI₂, little is known about the function of the remaining PGs within islets. In this review, we discuss the roles of PGs, particularly PGE₂ and PGI₂, PG receptors, and downstream signaling events that alter β-cell function and regulation of β-cell mass.     

NADPH oxidase 2 plays a critical role in dysfunction and apoptosis of pancreatic β-cells induced by very low-density lipoprotein

In type 2 diabetes, pancreatic β-cells cannot secret enough insulin compensate for insulin resistance, which are often accompanied by abnormality in lipid metabolism such as hypertriglyceridemia. It is reported that oxidative stress is involved in pancreatic β-cell dysfunction. However, molecular mechanisms linking between excessive generations of reactive oxygen species (ROS) and β-cell dysfunction and apoptosis induced by high levels of very low-density lipoprotein (VLDL) are poorly understood. In this study, we test the hypothesis that NADPH oxidase 2 (NOX2)-derived ROS may play a key role in dysfunction and apoptosis of pancreatic β-cell induced by VLDL. Our results show that the ApoCIII transgenic mice displayed increased serum TG levels, enhanced generation of ROS and impaired insulin content in pancreatic β-cells. In vitro, the treatment of pancreatic NIT-1 cells with 1?mg/ml VLDL for 12?h stimulated NOX2-derived ROS generation, decreased expression and secretion of insulin. Furthermore, we found that VLDL induced dysfunction and apoptosis of pancreatic β-cells through JNK and p53 pathways, which were rescued by siRNA-mediated NOX2 reduction. In conclusion, our data demonstrate a critical role of NOX2-derived ROS in dysfunction and apoptosis through JNK and p53 pathways in pancreatic β-cells induced by VLDL.