Kaempferol alleviates palmitic acid-induced lipid stores,endoplasmic reticulum stress and pancreatic β-cell dysfunction through AMPK/mTOR-mediated lipophagy

Kaempferol, a natural flavonoid, has the beneficial effects of preserving pancreatic β-cell mass and function, but its action on β-cell lipid metabolism still remains elusive. Recently, autophagy has been reported to play a major role in lipid metabolism in various cell types, but its role in pancreatic β-cell's lipid metabolism is rarely reported. Here, we investigated the role of kaempferol-induced autophagy in inhibition of lipid stores, ER stress and β-cell dysfunction in palmitic acid-challenged RIN-5F cells and isolated pancreatic islets. The lipid-lowering effect of kaempferol was determined by Oil Red O staining, triglyceride assay, BODIPY labeling, RT-PCR and immunoblot analysis of PLIN2 (the lipid droplet coat protein) expression. Further, the involvement of AMPK/mTOR-mediated lipophagy was established by pharmacological and genetic inhibitors of autophagy and AMPK. The co-localization studies of lipid droplets with autophagosomes/lysosomes by BODIPY-MDC-LysoTracker co-staining, LC3/BODIPY labeling and LC3/PLIN2 double immunolabeling further strengthened the findings. Kaempferol treatment exhibited decreased lipid stores and increased co-localization of lipid droplets with autophagosomes and lysosomes in palmitic acid–challenged β-cells. Moreover, inhibition of autophagy led to decreased co-localization and increased lipid droplets accumulation. Kaempferol-induced alleviation of ER stress and β-cell dysfunctions was established by immunoblot analysis of CHOP-10 (a key mediator of cell death in response to ER stress) and insulin content/secretion analysis respectively. Together, these findings suggest that kaempferol prevents ectopic lipid accumulation and ER stress, thus restoring β-cell function through AMPK-mediated lipophagy. The current data implies that kaempferol may be a potential therapeutic candidate to prevent obesity-linked diabetic complications.     

PDI reductase acts on Akita mutant proinsulin to initiate retrotranslocation along the Hrd1/Sel1L-p97 axis

In mutant INS gene–induced diabetes of youth (MIDY), characterized by insulin deficiency, MIDY proinsulin mutants misfold and fail to exit the endoplasmic reticulum (ER). Moreover, these mutants bind and block ER exit of wild-type (WT) proinsulin, inhibiting insulin production. The ultimate fate of ER-entrapped MIDY mutants is unclear, but previous studies implicated ER-associated degradation (ERAD), a pathway that retrotranslocates misfolded ER proteins to the cytosol for proteasomal degradation. Here we establish key ERAD machinery components used to triage the Akita proinsulin mutant, including the Hrd1-Sel1L membrane complex, which conducts Akita proinsulin from the ER lumen to the cytosol, and the p97 ATPase, which couples the cytosolic arrival of proinsulin with its proteasomal degradation. Surprisingly, we find that protein disulfide isomerase (PDI), the major protein oxidase of the ER lumen, engages Akita proinsulin in a novel way, reducing proinsulin disulfide bonds and priming the Akita protein for ERAD. Efficient PDI engagement of Akita proinsulin appears linked to the availability of Hrd1, suggesting that retrotranslocation is coordinated on the lumenal side of the ER membrane. We believe that, in principle, this form of diabetes could be alleviated by enhancing the targeting of MIDY mutants for ERAD to restore WT insulin production.     

UCHL1 deficiency exacerbates human islet amyloid polypeptide toxicity in β-cells

The islet in type 2 diabetes mellitus (T2DM) is characterized by a deficit in β-cells and increased β-cell apoptosis attributable at least in part to intracellular toxic oligomers of IAPP (islet amyloid polypeptide). β-cells of individuals with T2DM are also characterized by accumulation of polyubiquitinated proteins and deficiency in the deubiquitinating enzyme UCHL1 (ubiquitin carboxyl-terminal esterase L1 [ubiquitin thiolesterase]), accounting for a dysfunctional ubiquitin/proteasome system. In the present study, we used mouse genetics to elucidate in vivo whether a partial deficit in UCHL1 enhances the vulnerability of β-cells to human-IAPP (hIAPP) toxicity, and thus accelerates diabetes onset. We further investigated whether a genetically induced deficit in UCHL1 function in β-cells exacerbates hIAPP-induced alteration of the autophagy pathway in vivo. We report that a deficit in UCHL1 accelerated the onset of diabetes in hIAPP transgenic mice, due to a decrease in β-cell mass caused by increased β-cell apoptosis. We report that UCHL1 dysfunction aggravated the hIAPP-induced defect in the autophagy/lysosomal pathway, illustrated by the marked accumulation of autophagosomes and cytoplasmic inclusions positive for SQSTM1/p62 and polyubiquitinated proteins with lysine 63-specific ubiquitin chains. Collectively, this study shows that defective UCHL1 function may be an early contributor to vulnerability of pancreatic β-cells for protein misfolding and proteotoxicity, hallmark defects in islets of T2DM. Also, given that deficiency in UCHL1 exacerbated the defective autophagy/lysosomal degradation characteristic of hIAPP proteotoxicity, we demonstrate a previously unrecognized role of UCHL1 in the function of the autophagy/lysosomal pathway in β-cells.     

The role of autophagy in endoplasmic reticulum stress-induced pancreatic β cell death

In pancreatic β-cells, the endoplasmic reticulum (ER) is the crucial site for insulin biosynthesis, as this is where the protein-folding machinery for secretory proteins is localized. Perturbations to ER function of the β-cell, such as those caused by high levels of free fatty acid and insulin resistance, can lead to an imbalance in protein homeostasis and ER stress, which has been recognized as an important mechanism for type 2 diabetes. Macroautophagy (hereafter referred to as autophagy) is activated as a novel signaling pathway in response to ER stress. In this review, we outline the mechanism of ER stress-mediated β-cell death and focus on the role of autophagy in ameliorating ER stress. The development of drugs to take advantage of the potential protective effect of autophagy in ER stress, such as glucagon like peptide-1, will be a promising avenue of investigation.     

Modulation of palmitate-induced endoplasmic reticulum stress and apoptosis in pancreatic β-cells by stearoyl-CoA desaturase and Elovl6

Induction of endoplasmic reticulum (ER) stress and apoptosis by elevated exogenous saturated fatty acids (FAs) plays a role in the pathogenesis of β-cell dysfunction and loss of islet mass in type 2 diabetes. Regulation of monounsaturated FA (MUFA) synthesis through FA desaturases and elongases may alter the susceptibility of β-cells to saturated FA-induced ER stress and apoptosis. Herein, stearoyl-CoA desaturase (SCD)1 and SCD2 mRNA expression were shown to be induced in islets from prediabetic hyperinsulinemic Zucker diabetic fatty (ZDF) rats, whereas SCD1, SCD2, and fatty acid elongase 6 (Elovl6) mRNA levels were markedly reduced in diabetic ZDF rat islets. Knockdown of SCD in INS-1 β-cells decreased desaturation of palmitate to MUFA, lowered FA partitioning into complex neutral lipids, and increased palmitate-induced ER stress and apoptosis. Overexpression of SCD2 increased desaturation of palmitate to MUFA and attenuated palmitate-induced ER stress and apoptosis. Knockdown of Elovl6 limited palmitate elongation to stearate, increasing palmitoleate production and attenuating palmitate-induced ER stress and apoptosis, whereas overexpression of Elovl6 increased palmitate elongation to stearate and palmitate-induced ER stress and apoptosis. Overall, these data support the hypothesis that enhanced MUFA synthesis via upregulation of SCD2 activity can protect β-cells from elevated saturated FAs, as occurs in prediabetic states. Overt type 2 diabetes is associated with diminished islet expression of SCD and Elovl6, and this can disrupt desaturation of saturated FAs to MUFAs, rendering β-cells more susceptible to saturated FA-induced ER stress and apoptosis.     

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.     

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.     

Endoplasmic reticulum stress response in an INS-1 pancreatic β-cell line with inducible expression of a folding-deficient proinsulin

Background  

Cells respond to endoplasmic reticulum stress (ER) stress by activating the unfolded protein response. To study the ER stress response in pancreatic β-cells we developed a model system that allows for pathophysiological ER stress based on the Akita mouse. This mouse strain expresses a mutant insulin 2 gene (C96Y), which prevents normal proinsulin folding causing ER stress and eventual β-cell apoptosis. A double-stable pancreatic β-cell line (pTet-ON INS-1) with inducible expression of insulin 2 (C96Y) fused to EGFP was generated to study the ER stress response.     

Endoplasmic Reticulum Oxidoreductin-1α (Ero1α) Improves Folding and Secretion of Mutant Proinsulin and Limits Mutant Proinsulin-induced Endoplasmic Reticulum Stress

Upon chronic up-regulation of proinsulin synthesis, misfolded proinsulin can accumulate in the endoplasmic reticulum (ER) of pancreatic β-cells, promoting ER stress and type 2 diabetes mellitus. In Mutant Ins-gene-induced Diabetes of Youth (MIDY), misfolded mutant proinsulin impairs ER exit of co-expressed wild-type proinsulin, limiting insulin production and leading to eventual β-cell death. In this study we have investigated the hypothesis that increased expression of ER oxidoreductin-1α (Ero1α), despite its established role in the generation of H₂O₂, might nevertheless be beneficial in limiting proinsulin misfolding and its adverse downstream consequences. Increased Ero1α expression is effective in promoting wild-type proinsulin export from cells co-expressing misfolded mutant proinsulin. In addition, we find that upon increased Ero1α expression, some of the MIDY mutants themselves are directly rescued from ER retention. Secretory rescue of proinsulin-G(B23)V is correlated with improved oxidative folding of mutant proinsulin. Indeed, using three different variants of Ero1α, we find that expression of either wild-type or an Ero1α variant lacking regulatory disulfides can rescue mutant proinsulin-G(B23)V, in parallel with its ability to provide an oxidizing environment in the ER lumen, whereas beneficial effects were less apparent for a redox-inactive form of Ero1. Increased expression of protein disulfide isomerase antagonizes the rescue provided by oxidatively active Ero1. Importantly, ER stress induced by misfolded proinsulin was limited by increased expression of Ero1α, suggesting that enhancing the oxidative folding of proinsulin may be a viable therapeutic strategy in the treatment of type 2 diabetes.     

A novel therapeutic combination of sitagliptin and melatonin regenerates pancreatic β-cells in mouse and human islets

Autoimmune-led challenge resulting in β-cell loss is responsible for the development of type 1 diabetes (T1D). Melatonin, a pineal hormone or sitagliptin, a dipeptidyl peptidase IV (DPP-IV) inhibitor, has increased β-cell mass in various diabetic models and has immunoregulatory property. Both β-cell regenerative capacity and melatonin secretion decrease with ageing. Thus, we aimed to investigate the therapeutic potential of melatonin combined with sitagliptin on β-cell regeneration under glucotoxic stress, in the streptozotocin-induced young and old diabetic mouse models, and euglycemic humanized islet transplant mouse model. Our results suggest that combination therapy of sitagliptin and melatonin show an additive effect in inducing mouse β-cell regeneration under glucotoxic stress, and in the human islet transplant mouse model. Further, in the young diabetic mouse model, the monotherapies induce β-cell transdifferentiation and reduce β-cell apoptosis whereas, in the old diabetic mouse model, melatonin and sitagliptin induce β-cell proliferation and β-cell transdifferentiation, and it also reduces β-cell apoptosis. Further, in both the models, combination therapy reduces fasting blood glucose levels, increases plasma insulin levels and glucose tolerance and promotes β-cell proliferation, β-cell transdifferentiation, and reduces β-cell apoptosis. It can be concluded that combination therapy is superior to monotherapies in ameliorating diabetic manifestations, and it can be used as a future therapy for β-cell regeneration in diabetes patients.     

Lentinan protects against pancreatic β-cell failure in chronic ethanol consumption-induced diabetic mice via enhancing β-cell antioxidant capacity

Chronic ethanol consumption is a well-established independent risk factor for type 2 diabetes mellitus (T2DM). Recently, increasing studies have confirmed that excessive heavy ethanol exerts direct harmful effect on pancreatic β-cell mass and function, which may be a mechanism of pancreatic β-cell failure in T2DM. In this study, we evaluated the effect of Lentinan (LNT), an active ingredient purified from the bodies of Lentinus edodes, on pancreatic β-cell apoptosis and dysfunction caused by ethanol and the possible mechanisms implicated. Functional studies reveal that LNT attenuates chronic ethanol consumption-induced impaired glucose metabolism in vivo. In addition, LNT ameliorates chronic ethanol consumption-induced β-cell dysfunction, which is characterized by reduced insulin synthesis, defected insulin secretion and increased cell apoptosis. Furthermore, mechanistic assays suggest that LNT enhances β-cell antioxidant capacity and ameliorates ethanol-induced oxidative stress by activating Nrf-2 antioxidant pathway. Our results demonstrated that LNT prevents ethanol-induced pancreatic β-cell dysfunction and apoptosis, and therefore may be a potential pharmacological agent for preventing pancreatic β-cell failure associated with T2DM and stress-induced diabetes.     

Dilation of the endoplasmic reticulum in beta cells due to molecular overcrowding?: Kinetic simulations of extension limits and consequences on proinsulin synthesis

Insulin regulates the energy homeostasis of the human body. This is synthesized in the endoplasmic reticulum (ER) of pancreatic beta cells from proinsulin. Chronic hyperglycemia increases considerably the proinsulin secretion, overcrowding the ER. Recent experimental evidence demonstrates that such states favor the proinsulin denaturation. The biophysical mechanism of this cellular dysfunction remains largely unknown. We use basic molecular principles and numerical simulations of time-dependent crowding conditions in the ER to show that crowding effects enhance the propensity of proinsulin molecules to (mis)fold in compressed, nonnative structures. Present results suggest: i) misfolding events and toxic accumulations increase dramatically if the proinsulin load exceeds 50% of the available space and ii) insufficient lag time for the relaxation of the ER between consecutive proinsulin uploads can cause irreversible alterations of folding capabilities. Present study may prove useful in generating new testable statements on circumstances leading to the development of diabetes.     

The glial glutamate transporter 1 (GLT1) is expressed by pancreatic beta-cells and prevents glutamate-induced beta-cell death

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained at physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans where it is secreted by the α-cells and acts as a signaling molecule to modulate hormone secretion. Whether glutamate plays a role in islet cell viability is presently unknown. We demonstrate that chronic exposure to glutamate exerts a cytotoxic effect in clonal β-cell lines and human islet β-cells but not in α-cells. In human islets, glutamate-induced β-cell cytotoxicity was associated with increased oxidative stress and led to apoptosis and autophagy. We also provide evidence that the key regulator of extracellular islet glutamate concentration is the glial glutamate transporter 1 (GLT1). GLT1 localizes to the plasma membrane of β-cells, modulates hormone secretion, and prevents glutamate-induced cytotoxicity as shown by the fact that its down-regulation induced β-cell death, whereas GLT1 up-regulation promoted β-cell survival. In conclusion, the present study identifies GLT1 as a new player in glutamate homeostasis and signaling in the islet of Langerhans and demonstrates that β-cells critically depend on its activity to control extracellular glutamate levels and cellular integrity.     

Targeting Nrf2 by dihydro-CDDO-trifluoroethyl amide enhances autophagic clearance and viability of β-cells in a setting of oxidative stress

Nrf2 appears to be a critical regulator of diabetes in rodents. However, the underlying mechanisms as well as the clinical relevance of the Nrf2 signaling in human diabetes remain to be fully understood. Herein, we report that islet expression of Nrf2 is upregulated at an earlier stage of diabetes in both human and mice. Activation of Nrf2 suppresses oxidative stress and oxidative stress-induced β-cell apoptosis while enhancing autophagic clearance in isolated rat islets. Additionally, oxidative stress per se activated autophagy in β-cells. Thus, these results reveal that Nrf2 drives a novel antioxidant independent autophagic clearance for β-cell protection in the setting of diabetes.     

Cellular Inhibitor of Apoptosis Protein-1 (cIAP1) Plays a Critical Role in β-Cell Survival under Endoplasmic Reticulum Stress: PROMOTING UBIQUITINATION AND DEGRADATION OF C/EBP HOMOLOGOUS PROTEIN (CHOP)

Lipotoxicity in pancreatic β-cells, arising from excess free fatty acid-induced endoplasmic reticulum (ER) stress response, has been recognized as a key pathogenic factor causing loss of β-cell mass and contributing to type 2 diabetes. However, how the adaptive ER stress response causes cell death remains enigmatic. We report herein a critical role of cellular inhibitor of apoptosis protein-1 (cIAP1) in controlling β-cell survival under ER stress. While both palmitate and palmitoleate induced an overt ER stress response, lipotoxicity was only observed in β-cells exposed to palmitate but not palmitoleate. Interestingly, cells treated with palmitoleate exerted a sustainable level of cIAP1, whereas the protein quickly degraded following palmitate treatment. Enforced overexpression of cIAP1 prevented palmitate-induced cell death. In contrast, siRNA-mediated knockdown of cIAP1 in β-cells or knock-out of cIap1 in mouse embryonic fibroblasts not only increased palmitate-induced apoptosis, but also committed cells to death in response to the nontoxic palmitoleate treatment. Of importance, we found that cIAP1 functions as an E3 ubiquitin ligase promoting ubiquitination and degradation of C/EBP homologous protein (CHOP), a key mediator of ER stress-induced cell death. These findings define a novel mechanism for β-cell survival under ER stress and help to identify targets for therapeutic intervention against lipotoxicity in β-cells.     

Autophagy fosters myofibroblast differentiation through MTORC2 activation and downstream upregulation of CTGF

Recent evidence suggests that autophagy may favor fibrosis through enhanced differentiation of fibroblasts in myofibroblasts. Here, we sought to characterize the mediators and signaling pathways implicated in autophagy-induced myofibroblast differentiation. Fibroblasts, serum starved for up to 4 d, showed increased LC3-II/-I ratios and decreased SQSTM1/p62 levels. Autophagy was associated with acquisition of markers of myofibroblast differentiation including increased protein levels of ACTA2/αSMA (actin, α 2, smooth muscle, aorta), enhanced gene and protein levels of COL1A1 (collagen, type I, α 1) and COL3A1, and the formation of stress fibers. Inhibiting autophagy with 3 different class I phosphoinositide 3-kinase and class III phosphatidylinositol 3-kinase (PtdIns3K) inhibitors or through ATG7 silencing prevented myofibroblast differentiation. Autophagic fibroblasts showed increased expression and secretion of CTGF (connective tissue growth factor), and CTGF silencing prevented myofibroblast differentiation. Phosphorylation of the MTORC1 target RPS6KB1/p70S6K kinase was abolished in starved fibroblasts. Phosphorylation of AKT at Ser473, a MTORC2 target, was reduced after initiation of starvation but was followed by spontaneous rephosphorylation after 2 d of starvation, suggesting the reactivation of MTORC2 with sustained autophagy. Inhibiting MTORC2 activation with long-term exposure to rapamycin or by silencing RICTOR, a central component of the MTORC2 complex abolished AKT rephosphorylation. Both RICTOR silencing and rapamycin treatment prevented CTGF and ACTA2 upregulation, demonstrating the central role of MTORC2 activation in CTGF induction and myofibroblast differentiation. Finally, inhibition of autophagy with PtdIns3K inhibitors or ATG7 silencing blocked AKT rephosphorylation. Collectively, these results identify autophagy as a novel activator of MTORC2 signaling leading to CTGF induction and myofibroblast differentiation.     

Endoplasmic reticulum stress-induced apoptosis in the development of diabetes: is there a role for adipose tissue and liver?

Diabetes mellitus (DM) is a multifactorial chronic metabolic disease characterized by hyperglycaemia. Several different mechanisms have been implicated in the development of the disease, including endoplasmic reticulum (ER) stress. ER stress is increasingly acknowledged as an important mechanism in the development of DM, not only for β-cell loss but also for insulin resistance. Accumulating evidence suggests that ER stress-induced apoptosis may be an important mode of β-cell loss and therefore important in the development of diabetes. Recent data also suggest a role of ER stress-induced apoptosis in liver and adipose tissue in relation to diabetes, but more extensive studies on human adipocyte and hepatocyte (patho)physiology and ER stress are needed to identify the exact interactions between environmental signals, ER stress and apoptosis in these organs.     

Inhibition of GPR40 protects MIN6 β cells from palmitate-induced ER stress and apoptosis

Chronic exposure to elevated concentration of free fatty acids (FFA) has been verified to induce endoplasmic reticulum (ER) stress, which leads to pancreatic β-cell apoptosis. As one of the medium and long chain FFA receptors, GPR40 is highly expressed in pancreatic β cells, mediates both acute and chronic effects of FFA on β-cell function, but the role of GPR40 in FFA-induced β-cell apoptosis remains unclear. In this study, we investigated the possible effects of GPR40 in palmitate-induced MIN6 β-cell apoptosis, and found that DC260126, a novel small molecular antagonist of GPR40, could protect MIN6 β cells from palmitate-induced ER stress and apoptosis. Similar results were observed in GPR40-deficient MIN6 cells, indicating that palmitate-induced β-cell apoptosis is at least partially dependent on ER stress pathway via GRP40.