ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y₁ receptor agonist, induced a large signal while UTPyS (P2Y₂ agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y₁. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.     

Atg5 deficiency-mediated mitophagy aggravates cardiac inflammation and injury in response to angiotensin II

ObjectiveHypertension induces end-organ damage through inflammation, and autophagy plays a crucial role in the regulation of cellular homeostasis. In the present study, we aimed to define the role of autophagy in the development of inflammation and cardiac injury induced by angiotensin II (Ang II).Methods and ResultsAutophagy protein 5 (Atg5) haplodeficiency (Atg5^+/−) and age-matched wild-type (WT) C57BL/6 J mice were infused with Ang II (1500 ng/kg/min) or saline for 7 days. Heart sections were stained with hematoxylin and eosin (H&E), Masson's trichrome, and immunohistochemical stains. Cytokine and LC3 levels were measured using real-time PCR or western blot analysis. After Ang II infusion, the WT mice exhibited marked macrophage accumulation, cytokine expression, and reactive oxygen species (ROS) production compared with saline-infused controls. However, these effects induced by Ang II infusion were aggravated in Atg5^+/− mice. These effects were associated with Atg5-mediated impaired autophagy, accompanied by increased production of ROS and activation of nuclear factor-κB (NF-κB) in macrophages. Finally, increased cardiac inflammation in Atg5 haplodeficient mice was associated with increased cardiac fibrosis.ConclusionAtg5 deficiency-mediated autophagy increases ROS production and NF-κB activity in macrophages, thereby contributing to cardiac inflammation and injury. Thus, improving autophagy may be a novel therapeutic strategy to ameliorate hypertension-induced inflammation and organ damage.     

Regulation of Ca2+ homeostasis by glucose metabolism in rat brain

In a previous communication we reported that glucose deprivation from KHRB medium resulted in a marked stimulation of Ca²⁺ uptake by brain tissue, suggesting a relationship between glucose and Ca²⁺ homeostasis in brain tissue [17]. Experiments were carried out to investigate the significance of glucose in Ca²⁺ transport in brain cells. The replacement of glucose with either D-methylglucoside or 2-deoxyglucose, non-metabolizable analogues of glucose, resulted in stimulation of Ca²⁺ uptake just as by glucose deprivation. These data show that glucose metabolism rather than glucose transfer was necessary to stimulate Ca²⁺ uptake in brain tissue. Inhibition of glucose metabolism with either NaF, NaCN, or iodoacetate resulted in stimulation of Ca²⁺ uptake similar to that produced by glucose deprivation. These results lend further support for the concept that glucose metabolism is essential for Ca²⁺ homeostasis in brain. Anoxia promotes glucose metabolism through glycolytic pathway to keep up with the demand for ATP by cellular processes (the Pasteur effect). Incubation of brain slices under nitrogen gas did not alter Ca²⁺ uptake by brain tissue, as did glucose deprivation and the inhibitors of glucose metabolism. We conclude that glucose metabolism resulting in the synthesis of ATP is essential for Ca²⁺ homeostasis in brain. Verapamil and nifedipine which block voltage-gated Ca²⁺ channels, did not alter Ca²⁺ uptake stimulated by glucose deprivation, indicating that glucose deprivation-enhanced Ca²⁺ uptake was not mediated by Ca²⁺ channels. Tetrodotoxin which specifically blocks Na⁺ channels, abolished Ca²⁺ uptake enhanced by glucose deprivation, but had no effect on Ca²⁺ uptake in presence of glucose (controls). These results suggest that stimulation of Ca²⁺ uptake by glucose deprivation may be related to Na⁺ transfer via Na-Ca exchange in brain.     

Pancreatic mitochondrial complex I exhibits aberrant hyperactivity in diabetes

It is well established that NADH/NAD⁺ redox balance is heavily perturbed in diabetes, and the NADH/NAD⁺ redox imbalance is a major source of oxidative stress in diabetic tissues. In mitochondria, complex I is the only site for NADH oxidation and NAD⁺ regeneration and is also a major site for production of mitochondrial reactive oxygen species (ROS). Yet how complex I responds to the NADH/NAD⁺ redox imbalance and any potential consequences of such response in diabetic pancreas have not been investigated. We report here that pancreatic mitochondrial complex I showed aberrant hyperactivity in either type 1 or type 2 diabetes. Further studies focusing on streptozotocin (STZ)-induced diabetes indicate that complex I hyperactivity could be attenuated by metformin. Moreover, complex I hyperactivity was accompanied by increased activities of complexes II to IV, but not complex V, suggesting that overflow of NADH via complex I in diabetes could be diverted to ROS production. Indeed in diabetic pancreas, ROS production and oxidative stress increased and mitochondrial ATP production decreased, which can be attributed to impaired pancreatic mitochondrial membrane potential that is responsible for increased cell death. Additionally, cellular defense systems such as glucose 6-phosphate dehydrogenase, sirtuin 3, and NQO1 were found to be compromised in diabetic pancreas. Our findings point to the direction that complex I aberrant hyperactivity in pancreas could be a major source of oxidative stress and β cell failure in diabetes. Therefore, inhibiting pancreatic complex I hyperactivity and attenuating its ROS production by various means in diabetes might serve as a promising approach for anti-diabetic therapies.     

Ca mishandling and cardiac dysfunction in obesity and insulin resistance: Role of oxidative stress

Obesity and insulin resistance (IR) are strongly connected to the development of subclinical cardiac dysfunction and eventually can lead to heart failure, which is the main cause of morbidity and death in patients having these metabolic diseases. It has been considered that excessive fat tissue may play a critical role in producing systemic IR and enhancing reactive oxygen species (ROS) generation. This oxidative stress (OS) may elicit or exacerbate IR. On the other hand, evidence suggests that some of the cellular mechanisms involved in the pathophysiology of obesity and IR-related cardiomyopathy are excessive myocardial ROS production and abnormal Ca²⁺ homeostasis. In addition, emerging evidence suggests that augmented ROS production may contribute to Ca²⁺ mishandling by affecting the redox state of key proteins implicated in this process. In this review, we focus on the role of Ca²⁺ mishandling in the development of cardiac dysfunction in obesity and IR and address the evidence suggesting that OS might also contribute to cardiac dysfunction by affecting Ca²⁺ handling.     

MitoQ对高糖诱导的心肌细胞线粒体功能影响

目的:探讨MitoQ对高糖诱导的心肌细胞线粒体功能影响。方法:常规获取与纯化SD大鼠新生仔鼠心肌细胞,分为对照组、高糖组、实验组。对照组用含10%血清的DMEM培养基(5.5 mmol/L葡萄糖)培养;高糖组用含血清的高糖DMEM培养基(33mmol/L葡萄糖)培养;实验组用含血清的高糖DMEM培养基(33 mmol/L葡萄糖)和MitoQ。MTT法检测心肌细胞存活率,氯离子荧光探针检测细胞内氯离子浓度,流式细胞术检测各组心肌细胞凋亡率,超氧化物阴离子荧光染色检测心肌细胞活性氧(reactive oxygen,ROS)含量,利用ATP检测试剂盒检测心肌细胞中的ATP水平,Western blot法检测心肌细胞胱天蛋白酶3(caspase-3)蛋白水平。结果:与对照组相比,高糖组的心肌细胞增凋亡率、ROS产生、氯离子相对浓度均明显增加,ATP显著降低(P0.05),细胞内caspase-3蛋白表达显著上升(P0.05);与高糖组相比,实验组凋亡率降低,ROS产生、细胞内caspase-3蛋白表达均显著降低(P0.05)。结论:高糖会引起心肌细胞线粒体障碍,造成心肌细胞凋亡,MitoQ可降低细胞内ROS和caspase-3水平,抑制心肌细胞凋亡,改善心肌细胞线粒体功能。     

The role of Ca signaling in the coordination of mitochondrial ATP production with cardiac work

The heart is capable of balancing the rate of mitochondrial ATP production with utilization continuously over a wide range of activity. This results in a constant phosphorylation potential despite a large change in metabolite turnover. The molecular mechanisms responsible for generating this energy homeostasis are poorly understood. The best candidate for a cytosolic signaling molecule reflecting ATP hydrolysis is Ca²⁺. Since Ca²⁺ initiates and powers muscle contraction as well as serves as the primary substrate for SERCA, Ca²⁺ is an ideal feed-forward signal for priming ATP production. With the sarcoplasmic reticulum to cytosolic Ca²⁺ gradient near equilibrium with the free energy of ATP, cytosolic Ca²⁺ release is exquisitely sensitive to the cellular energy state providing a feedback signal. Thus, Ca²⁺ can serve as a feed-forward and feedback regulator of ATP production. Consistent with this notion is the correlation of cytosolic and mitochondrial Ca²⁺ with work in numerous preparations as well as the localization of mitochondria near Ca²⁺ release sites. How cytosolic Ca²⁺ signaling might regulate oxidative phosphorylation is a focus of this review. The relevant Ca²⁺ sensitive sites include several dehydrogenases and substrate transporters together with a post-translational modification of F1-FO-ATPase and cytochrome oxidase. Thus, Ca²⁺ apparently activates both the generation of the mitochondrial membrane potential as well as utilization to produce ATP. This balanced activation extends the energy homeostasis observed in the cytosol into the mitochondria matrix in the never resting heart.     

Effect of starvation of gastric somatostatin release

The present study is an investigation of the effects of 16 and 48 hours starvation on gastric somatostatin release using the isolated perfused rat stomach. Before sacrifice the body weights and blood glucose levels of fasted rats were significantly lower than fed rats. In the presence of 4.4 mM glucose, basal somatostatin concentrations in the stomach perfusate of fasted rats were also significantly lower. Gastric somatostatin release was stimulated in all three groups similarly by 5 × 10^?8 M glucagon when the decrease in basal levels is considered. These results suggest that gastric somatostatin as well as pancreatic somatostatin contributes to nutrient homeostasis and that nutrient homeostasis influences somatostatin levels in turn.     

R-type Ca(2+)-channel-evoked CICR regulates glucose-induced somatostatin secretion

Pancreatic islets have a central role in blood glucose homeostasis. In addition to insulin-producing beta-cells and glucagon-secreting alpha-cells, the islets contain somatostatin-releasing delta-cells. Somatostatin is a powerful inhibitor of insulin and glucagon secretion. It is normally secreted in response to glucose and there is evidence suggesting its release becomes perturbed in diabetes. Little is known about the control of somatostatin release. Closure of ATP-regulated K(+)-channels (K(ATP)-channels) and a depolarization-evoked increase in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) have been proposed to be essential. Here, we report that somatostatin release evoked by high glucose (>or=10 mM) is unaffected by the K(ATP)-channel activator diazoxide and proceeds normally in K(ATP)-channel-deficient islets. Glucose-induced somatostatin secretion is instead primarily dependent on Ca(2+)-induced Ca(2+)-release (CICR). This constitutes a novel mechanism for K(ATP)-channel-independent metabolic control of pancreatic hormone secretion.     

Calcium signalling in diabetes

Molecular cascades responsible for Ca²⁺ homeostasis and Ca²⁺ signalling could be assembled in highly plastic toolkits that define physiological adaptation of cells to the environment and which are intimately involved in all types of cellular pathology. Control over Ca²⁺ concentration in different cellular compartments is intimately linked to cell metabolism, because (i) ATP production requires low Ca²⁺, (ii) Ca²⁺ homeostatic systems consume ATP and (iii) Ca²⁺ signals in mitochondria stimulate ATP synthesis being an essential part of excitation–metabolic coupling. The communication between the ER and mitochondria plays an important role in this metabolic fine tuning. In the insulin resistance state and diabetes this communication has been impaired leading to different disorders, for instance, diminished insulin production by pancreatic β cells, reduced heart and skeletal muscle contractility, reduced NO production by endothelial cells, increased glucose production by liver, increased lipolysis by adipose cells, reduced immune responses, reduced cognitive functions, among others. All these processes eventually trigger degenerative events resulting in overt diabetes due to reduction of pancreatic β cell mass, and different complications of diabetes, such as retinopathy, nephropathy, neuropathy, and different cardiovascular diseases.     

Mitochondrial localization of NCXs: Balancing calcium and energy homeostasis

It is well established that mitochondria are the main source of ATP production within cells. However, mitochondria have other remarkable functions, serving as important modulators of cellular Ca²⁺ signaling, and it is now generally recognized that control over Ca²⁺ homeostasis is intrinsically interwoven with mitochondrial abilities to adjust and tune ATP production. In this review, we describe the mechanisms that mitochondria use to balance Ca²⁺ homeostasis maintenance and cell energy metabolism. In recent years, the knowledge on the molecular machinery mediating Ca²⁺ influx/efflux has been improved and, albeit still open to further investigations, several lines of evidence converge on the hypothesis that plasma membrane Na⁺/Ca²⁺ exchanger (NCX) isoforms are also expressed at the mitochondrial level, where they contribute to the Ca²⁺ and Na⁺ homeostasis maintenance. In particular, the connection between mitochondrial NCX activity and metabolic substrates utilization is further discussed here. We also briefly focus on the alterations of both mitochondrial Ca²⁺ handling and cellular bioenergetics in neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease.     

Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria

Although plant cell bioenergetics is strongly affected by abiotic stresses, mitochondrial metabolism under stress is still largely unknown. Interestingly, plant mitochondria may control reactive oxygen species (ROS) generation by means of energy-dissipating systems. Therefore, mitochondria may play a central role in cell adaptation to abiotic stresses, which are known to induce oxidative stress at cellular level. With this in mind, in recent years, studies have been focused on mitochondria from durum wheat, a species well adapted to drought stress. Durum wheat mitochondria possess three energy-dissipating systems: the ATP-sensitive plant mitochondrial potassium channel (PmitoK(ATP)); the plant uncoupling protein (PUCP); and the alternative oxidase (AOX). It has been shown that these systems are able to dampen mitochondrial ROS production; surprisingly, PmitoK(ATP) and PUCP (but not AOX) are activated by ROS. This was found to occur in mitochondria from both control and hyperosmotic-stressed seedlings. Therefore, the hypothesis of a 'feed-back' mechanism operating under hyperosmotic/oxidative stress conditions was validated: stress conditions induce an increase in mitochondrial ROS production; ROS activate PmitoK(ATP) and PUCP that, in turn, dissipate the mitochondrial membrane potential, thus inhibiting further large-scale ROS production. Another important aspect is the chloroplast/cytosol/mitochondrion co-operation in green tissues under stress conditions aimed at modulating cell redox homeostasis. Durum wheat mitochondria may act against chloroplast/cytosol over-reduction: the malate/oxaloacetate antiporter and the rotenone-insensitive external NAD(P)H dehydrogenases allow cytosolic NAD(P)H oxidation; under stress this may occur without high ROS production due to co-operation with AOX, which is activated by intermediates of the photorespiratory cycle.     

Autoregulation of Free Radicals via Uncoupling Protein Control in Pancreatic β-Cell Mitochondria

Pancreatic β-cells sense the ambient blood-glucose concentration and secrete insulin to signal other tissues to take up glucose. Mitochondria play a key role in this response as they metabolize nutrients to produce ATP and reactive oxygen species (ROS), both of which are involved in insulin secretion signaling. Based on data available in the literature and previously developed mathematical models, we present a model of glucose-stimulated mitochondrial respiration, ATP synthesis, and ROS production and control in β-cells. The model is consistent with a number of experimental observations reported in the literature. Most notably, it captures the nonlinear rise in the proton leak rate at high membrane potential and the increase in this leak due to uncoupling protein (UCP) activation by ROS. The functional forms used to model ROS production and UCP regulation yield insight into these mechanisms, as many details have not yet been unraveled in the experimental literature. We examine short- and long-term effects of UCP activation inhibition and changes in the mitochondrial density on mitochondrial responses to glucose. Results suggest increasing mitochondrial density while decreasing UCP activity may be an effective way to increase glucose-stimulated insulin secretion while decreasing oxidative stress.     

Nutritional Energy Stimulates NAD+ Production to Promote Tankyrase-Mediated PARsylation in Insulinoma Cells

The poly-ADP-ribosylation (PARsylation) activity of tankyrase (TNKS) regulates diverse physiological processes including energy metabolism and wnt/β-catenin signaling. This TNKS activity uses NAD⁺ as a co-substrate to post-translationally modify various acceptor proteins including TNKS itself. PARsylation by TNKS often tags the acceptors for ubiquitination and proteasomal degradation. Whether this TNKS activity is regulated by physiological changes in NAD⁺ levels or, more broadly, in cellular energy charge has not been investigated. Because the NAD⁺ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) in vitro is robustly potentiated by ATP, we hypothesized that nutritional energy might stimulate cellular NAMPT to produce NAD⁺ and thereby augment TNKS catalysis. Using insulin-secreting cells as a model, we showed that glucose indeed stimulates the autoPARsylation of TNKS and consequently its turnover by the ubiquitin-proteasomal system. This glucose effect on TNKS is mediated primarily by NAD⁺ since it is mirrored by the NAD⁺ precursor nicotinamide mononucleotide (NMN), and is blunted by the NAMPT inhibitor FK866. The TNKS-destabilizing effect of glucose is shared by other metabolic fuels including pyruvate and amino acids. NAD⁺ flux analysis showed that glucose and nutrients, by increasing ATP, stimulate NAMPT-mediated NAD⁺ production to expand NAD⁺ stores. Collectively our data uncover a metabolic pathway whereby nutritional energy augments NAD⁺ production to drive the PARsylating activity of TNKS, leading to autoPARsylation-dependent degradation of the TNKS protein. The modulation of TNKS catalytic activity and protein abundance by cellular energy charge could potentially impose a nutritional control on the many processes that TNKS regulates through PARsylation. More broadly, the stimulation of NAD⁺ production by ATP suggests that nutritional energy may enhance the functions of other NAD⁺-driven enzymes including sirtuins.     

Monoclonal antibody to six transmembrane epithelial antigen of prostate-4 influences insulin sensitivity by attenuating phosphorylation of P13K (P85) and Akt: Possible mitochondrial mechanism

We examined the effects of anti-six-transmembrane epithelial antigen of the prostate-4 (STEAP4) antibodies on glucose transport in mature adipocytes and determined the mechanism of insulin resistance in obesity. Western blotting was performed to determine STEAP4 expression, to assess translocation of insulin-sensitive glucose transporter 4 (GLUT4), and to measure phosphorylation and total protein content of insulin-signaling proteins. Confocal laser microscopy and flow cytometry were used to detect intracellular reactive oxygen species (ROS) and fluctuations in mitochondrial membrane potential (ΔΨ). ATP production was measured by using a luciferase-based luminescence assay kit. After the application of anti-STEAP4 antibodies at 0.002?mg/mL, adipocytes exhibited reduced insulin-stimulated glucose transport by attenuating the phosphorylation of IRS-1, PI3K (p85), and Akt. The antibodies also potentially increase the level of ROS and decrease cellular ATP production and ΔΨ. In conclusion, (i) STEAP4 regulates the function of IRS-1, PI3K, and Akt and decreases insulin-induced GLUT4 translocation and glucose uptake; (ii) ROS-related mitochondrial dysfunction may be related to a reduced IRS-1 correlation with the PI3K signaling pathway, leading to insulin resistance. These observations highlight the potential role of STEAP4 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity and may provide new insights into the mechanisms of insulin resistance in obesity.     

Vitamin K3 suppressed inflammatory and immune responses in a redox-dependent manner

Abstract

Recent investigations suggest that cellular redox status may play a key role in the regulation of several immune functions. Treatment of lymphocytes with vitamin K3 (menadione) resulted in a significant decrease in cellular GSH/GSSG ratio and concomitant increase in the ROS levels. It also suppressed Concanavalin A (Con A)-induced proliferation and cytokine production in lymphocytes and CD4 + T cells in vitro. Immunosuppressive effects of menadione were abrogated only by thiol containing antioxidants. Mass spectrometric analysis showed that menadione directly interacted with thiol antioxidant GSH. Menadione completely suppressed Con A-induced activation of ERK, JNK and NF-κB in lymphocytes. It also significantly decreased the homeostasis driven proliferation of syngeneic CD4 + T cells. Further, menadione significantly delayed graft-vs-host disease morbidity and mortality in mice. Menadione suppressed phytohemagglutinin-induced cytokine production in human peripheral blood mononuclear cells. These results reveal that cellular redox perturbation by menadione is responsible for significant suppression of lymphocyte responses.     

ATP-driven and AMPK-independent autophagy in an early branching eukaryotic parasite

Autophagy is a catabolic cellular process required to maintain protein synthesis, energy production and other essential activities in starved cells. While the exact nutrient sensor(s) is yet to be identified, deprivation of amino acids, glucose, growth factor and other nutrients can serve as metabolic stimuli to initiate autophagy in higher eukaryotes. In the early-branching unicellular parasite Trypanosoma brucei, which can proliferate as procyclic form (PCF) in the tsetse fly or as bloodstream form (BSF) in animal hosts, autophagy is robustly triggered by amino acid deficiency but not by glucose depletion. Taking advantage of the clearly defined adenosine triphosphate (ATP) production pathways in T. brucei, we have shown that autophagic activity depends on the levels of cellular ATP production, using either glucose or proline as a carbon source. While autophagosome formation positively correlates with cellular ATP levels; perturbation of ATP production by removing carbon sources or genetic silencing of enzymes involved in ATP generation pathways, also inhibited autophagy. This obligate energy dependence and the lack of glucose starvation-induced autophagy in T. brucei may reflect an adaptation to its specialized, parasitic life style.     

Neuroprotection — rationale for pharmacological modulation of Na+-channels

Summary The primary factor detrimental to neurons in neurological disorders associated with deficient oxygen supply or mitochondrial dysfunction is insufficient ATP production relative to their requirement. As a large part of the energy consumed by brain cells is used for maintenance of the Na⁺ gradient across the cellular membrane, reduction of energy demand by down-modulation of voltage-gated Na⁺-channels is a rational strategy for neuroprotection. In addition, preservation of the inward Na⁺ gradient may be beneficial because it is an essential driving force for vital ion exchanges and transport mechanisms such as Ca²⁺ homeostasis and neurotransmitter uptake.     

GRP75 mediates endoplasmic reticulum–mitochondria coupling during palmitate-induced pancreatic β-cell apoptosis

The endoplasmic reticulum (ER) and mitochondria are structurally connected with each other at specific sites termed mitochondria-associated membranes (MAMs). These physical links are composed of several tethering proteins and are important during varied cellular processes, such as calcium homeostasis, lipid metabolism and transport, membrane biogenesis, and organelle remodeling. However, the attributes of specific tethering proteins in these cellular functions remain debatable. Here, we present data to show that one such tether protein, glucose regulated protein 75 (GRP75), is essential in increasing ER–mitochondria contact during palmitate-induced apoptosis in pancreatic insulinoma cells. We demonstrate that palmitate increased GRP75 levels in mouse and rat pancreatic insulinoma cells as well as in mouse primary islet cells. This was associated with increased mitochondrial Ca²⁺ transfer, impaired mitochondrial membrane potential, increased ROS production, and enhanced physical coupling between the ER and mitochondria. Interestingly, GRP75 inhibition prevented these palmitate-induced cellular aberrations. Additionally, GRP75 overexpression alone was sufficient to impair mitochondrial membrane potential, increase mitochondrial Ca²⁺ levels and ROS generation, augment ER–mitochondria contact, and induce apoptosis in these cells. In vivo injection of palmitate induced hyperglycemia and hypertriglyceridemia, as well as impaired glucose and insulin tolerance in mice. These animals also exhibited elevated GRP75 levels accompanied by enhanced apoptosis within the pancreatic islets. Our findings suggest that GRP75 is critical in mediating palmitate-induced ER–mitochondrial interaction leading to apoptosis in pancreatic islet cells.