SirT3 Regulates the Mitochondrial Unfolded Protein Response

The mitochondria of cancer cells are characterized by elevated oxidative stress caused by reactive oxygen species (ROS). Such an elevation in ROS levels contributes to mitochondrial reprogramming and malignant transformation. However, high levels of ROS can cause irreversible damage to proteins, leading to their misfolding, mitochondrial stress, and ultimately cell death. Therefore, mechanisms to overcome mitochondrial stress are needed. The unfolded protein response (UPR) triggered by accumulation of misfolded proteins in the mitochondria (UPR^mt) has been reported recently. So far, the UPR^mt has been reported to involve the activation of CHOP and estrogen receptor alpha (ERα). The current study describes a novel role of the mitochondrial deacetylase SirT3 in the UPR^mt. Our data reveal that SirT3 acts to orchestrate two pathways, the antioxidant machinery and mitophagy. Inhibition of SirT3 in cells undergoing proteotoxic stress severely impairs the mitochondrial network and results in cellular death. These observations suggest that SirT3 acts to sort moderately stressed from irreversibly damaged organelles. Since SirT3 is reported to act as a tumor suppressor during transformation, our findings reveal a dual role of SirT3. This novel role of SirT3 in established tumors represents an essential mechanism of adaptation of cancer cells to proteotoxic and mitochondrial stress.     

Mitochondrial longevity pathways

Production of reactive oxygen species (ROS) is a tightly regulated process, and increased levels of ROS within mitochondria are the principal trigger not only for mitochondrial dysfunctions but, more in general, for the diseases associated with aging, thus representing a powerful signaling molecules. One of the key regulators of ROS production, mitochondrial dysfunction, and aging is the 66-kDa isoform of the growth factor adapter shc (p66^shc) that is activated by stress and generates ROS within the mitochondria, driving cells to apoptosis. Accordingly, p66^shc knockout animals are one of the best characterized genetic model of longevity.On the other hand, caloric restriction is the only non-genetic mechanism that is shown to increase life span. Several studies have revealed a complex network of signaling pathways modulated by nutrients, such as IGF-1, TOR, sirtuins, AMP kinase, and PGC-1α that are connected and converge to inhibit oxidative stresses within the mitochondria. Animal models in which components of these signaling pathways are induced or silenced present a general phenotype characterized by the deceleration of the aging process. This review will summarize the main findings in the process that link mitochondria to longevity and the connections between the different signaling molecules involved in this intriguing relationship.     

Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress‐mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells—the major effectors of host adaptive immunity against infection and malignancy—is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (¹O₂) only at telomeres or mitochondria in Jurkat T cells. We found that targeted ¹O₂ production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted ¹O₂ formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X‐ray repair cross‐complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet ¹O₂ formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging‐associated diseases.     

Mitochondrial signaling, TOR, and life span

Growing evidence supports the concept that mitochondrial metabolism and reactive oxygen species (ROS) play a major role in aging and determination of an organism's life span. Cellular signaling pathways regulating mitochondrial activity, and hence the generation of ROS and retrograde signaling events originating in mitochondria, have recently moved into the spotlight in aging research. Involvement of the energy-sensing TOR pathway in both mitochondrial signaling and determination of life span has been shown in several studies. This brief review summarizes the recent progress on how mitochondrial signaling might contribute to the aging process with a particular emphasis on TOR signaling from invertebrates to humans.     

In quiescence of fission yeast,autophagy and the proteasome collaborate for mitochondrial maintenance and longevity

Regulation of proliferation and quiescence in response to intra- or extracellular environmental signals are important for medicine and basic biology. Quiescence is relevant to tumorigenesis and tissue regeneration, and the maintenance of post-mitotic cells is important with regard to a number of senescence-related diseases such as neurodegeneration. We employ fission yeast, Schizosaccharomyces pombe, as a model to study quiescence and longevity as this lower eukaryote has a long chronological life span (over months) in quiescence that is induced by nitrogen starvation. We recently reported that autophagy and the proteasome cooperate in proper mitochondrial maintenance in the quiescent phase. Such cooperativity is not found in proliferating cells. In quiescence, the proteasome is required for normal mitochondrial functions; inactivation of the proteasome results in a large accumulation of reactive oxygen species (ROS), diminished mitochondrial function, and the elevation of proteins and compounds having anti-oxidant activities. Autophagy contributes to preventing the lethal accumulation of ROS by degrading mitochondria, the primary source of ROS. Our results indicate that the degradation of mitochondria by autophagy during proteasome dysfunction is a defense mechanism of quiescenct cells against the accumulation of ROS.     

Mitochondrial ROS generation for regulation of autophagic pathways in cancer

Mitochondria, the main source of reactive oxygen species (ROS), are required for cell survival; yet also orchestrate programmed cell death (PCD), referring to apoptosis and autophagy. Autophagy is an evolutionarily conserved lysosomal degradation process implicated in a wide range of pathological processes, most notably cancer. Accumulating evidence has recently revealed that mitochondria may generate massive ROS that play the essential role for autophagy regulation, and thus sealing the fate of cancer cell. In this review, we summarize mitochondrial function and ROS generation, and also highlight ROS-modulated core autophagic pathways involved in ATG4–ATG8/LC3, Beclin-1, p53, PTEN, PI3K–Akt–mTOR and MAPK signaling in cancer. Therefore, a better understanding of the intricate relationships between mitochondrial ROS and autophagy may ultimately allow cancer biologists to harness mitochondrial ROS-mediated autophagic pathways for cancer drug discovery.     

Targeting proteomics to investigate metastasis-associated mitochondrial proteins

Mitochondria are essential organelles in eukaryotic cells and are responsible for regulating energy metabolism, ROS production, and cell survival. Recently, various cellular pathogeneses, including tumorigenesis and metastasis, have been reported to be associated with mitochondrial homeostasis. Consequently, exploiting the correlation between dysfunctional mitochondria and tumor progression has been implicated in the understanding of tumorigenesis, tumor metastasis, and chemoresistance, along with novel strategies to develop cancer therapeutics. To comprehensively understand the role of the mitochondria in cancer metastasis, it is necessary to resolve thousands of mitochondrial proteins and their post-translational modifications with high-throughput global assessments. We introduce mitochondrial proteomic strategies in this review and a discussion on their recent findings related to cancer metastasis. Additionally, the mitochondrial respiratory chain is believed to be a major site for ROS production, and elevated ROS is likely a key source to trigger dysfunctional mitochondria and impaired mitochondrial metabolism that subsequently contribute to the development of cancer progression. Equipment-based metabolomic analysis now allows the monitoring of disease progression and diagnosis. These newly emerging techniques, including proteomics, redox-proteomics, and metabolomics, are described in this review.     

Redox-optimized ROS balance: A unifying hypothesis

While it is generally accepted that mitochondrial reactive oxygen species (ROS) balance depends on the both rate of single electron reduction of O₂ to superoxide (O₂^?) by the electron transport chain and the rate of scavenging by intracellular antioxidant pathways, considerable controversy exists regarding the conditions leading to oxidative stress in intact cells versus isolated mitochondria. Here, we postulate that mitochondria have been evolutionarily optimized to maximize energy output while keeping ROS overflow to a minimum by operating in an intermediate redox state. We show that at the extremes of reduction or oxidation of the redox couples involved in electron transport (NADH/NAD⁺) or ROS scavenging (NADPH/NADP⁺, GSH/GSSG), respectively, ROS balance is lost. This results in a net overflow of ROS that increases as one moves farther away from the optimal redox potential. At more reduced mitochondrial redox potentials, ROS production exceeds scavenging, while under more oxidizing conditions (e.g., at higher workloads) antioxidant defenses can be compromised and eventually overwhelmed. Experimental support for this hypothesis is provided in both cardiomyocytes and in isolated mitochondria from guinea pig hearts. The model reconciles, within a single framework, observations that isolated mitochondria tend to display increased oxidative stress at high reduction potentials (and high mitochondrial membrane potential, ?Ψ_m), whereas intact cardiac cells can display oxidative stress either when mitochondria become more uncoupled (i.e., low ?Ψ_m) or when mitochondria are maximally reduced (as in ischemia or hypoxia). The continuum described by the model has the potential to account for many disparate experimental observations and also provides a rationale for graded physiological ROS signaling at redox potentials near the minimum.     

Mitochondria from rat uterine smooth muscle possess ATP-sensitive potassium channel

The objective of this study was to detect ATP-sensitive K⁺ uptake in rat uterine smooth muscle mitochondria and to determine possible effects of its activation on mitochondrial physiology. By means of fluorescent technique with usage of K⁺-sensitive fluorescent probe PBFI (potassium-binding benzofuran isophthalate) we showed that accumulation of K ions in isolated mitochondria from rat myometrium is sensitive to effectors of K_ATP-channel (ATP-sensitive K⁺-channel) – ATP, diazoxide, glibenclamide and 5HD (5-hydroxydecanoate). Our data demonstrates that K⁺ uptake in isolated myometrium mitochondria results in a slight decrease in membrane potential, enhancement of generation of ROS (reactive oxygen species) and mitochondrial swelling. Particularly, the addition of ATP into incubation medium led to a decrease in mitochondrial swelling and ROS production, and an increase in membrane potential. These effects were eliminated by diazoxide. If blockers of K_ATP-channel were added along with diazoxide, the effects of diazoxide were removed. So, we postulate the existence of K_ATP-channels in rat uterus mitochondria and assume that their functioning may regulate physiological conditions of mitochondria, such as matrix volume, ROS generation and polarization of mitochondrial membrane.     

ROS-induced mitochondrial depolarization initiates PARK2/PARKIN-dependent mitochondrial degradation by autophagy

Reactive oxygen species (ROS) have been implicated as a signal for general autophagy. Both mitochondrial-produced and exogenous ROS induce autophagosome formation. However, it is unclear whether ROS are required for the selective autophagic degradation of mitochondria, a process called mitophagy. Recent work using carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial-uncoupling reagent, has been shown to induce mitophagy. However, CCCP treatment may not be biologically relevant since it causes the depolarization of the entire mitochondrial network. Since mitochondria are the main ROS production sites in mammalian cells, we propose that short bursts of ROS produced within mitochondria may be involved in the signaling for mitophagy. To test this hypothesis, we induced an acute burst of ROS within mitochondria using a mitochondrial-targeted photosensitizer, mitochondrial KillerRed (mtKR). Using mtKR, we increased ROS levels in the mitochondrial matrix, which resulted in the loss of membrane potential and the subsequent activation of PARK2-dependent mitophagy. Importantly, we showed that overexpression of the mitochondrial antioxidant protein, superoxide dismutase-2, can squelch mtKR-induced mitophagy, demonstrating that mitochondrial ROS are responsible for mitophagy activation. Using this assay, we examined the impact of mitochondrial morphology on mitophagy. It was shown recently that elongated mitochondria are more resistant to mitophagy through unknown mechanisms. Here, we show that elongated mitochondria are more resistant to ROS-induced damage and mitophagy compared with fragmented mitochondria, suggesting that mitochondrial morphology has an important role in regulating ROS and mitophagy. Together, our results suggest that ROS-induced mitochondrial damage may be an important upstream activator of mitophagy.     

Oxidative modification sensitizes mitochondrial apoptosis-inducing factor to calpain-mediated processing

Although processing of mitochondrial apoptosis-inducing factor (AIF) is essential for its function during apoptosis in most cell types, the detailed mechanisms of AIF cleavage remain elusive. Recent findings indicate that the proteolytic process is Ca²⁺-dependent and that it is mediated by a calpain located in the mitochondrial intermembrane space. We can now report that, in addition to a sustained intracellular Ca²⁺ elevation, enhanced formation of reactive oxygen species (ROS) is a prerequisite step for AIF to be cleaved and released from mitochondria in staurosporine-treated cells. These events occurred independent of the redox state of the mitochondria and were not influenced by binding of pyridine nucleotides to AIF. Chelation of cytosolic Ca²⁺ by BAPTA/AM suppressed the elevation of both Ca²⁺ and ROS, suggesting that the Ca²⁺ rise was the most upstream signal required for AIF processing. We could further show that the stimulated ROS production leads to oxidative modification (carbonylation) of AIF, which markedly increases its rate of cleavage by calpain. Accordingly, pretreatment of the cells with antioxidants blocked AIF carbonylation, as well as its subsequent cleavage and release from the mitochondria. Combined, our data provide evidence that ROS-mediated, posttranslational modification of AIF is critical for its cleavage by calpain and thus for AIF-mediated cell death.     

Mechanisms of Rapid Reactive Oxygen Species Generation in Response to Cytosolic Ca2+ or Zn2+ Loads in Cortical Neurons

Excessive “excitotoxic” accumulation of Ca²⁺ and Zn²⁺ within neurons contributes to neurodegeneration in pathological conditions including ischemia. Putative early targets of these ions, both of which are linked to increased reactive oxygen species (ROS) generation, are mitochondria and the cytosolic enzyme, NADPH oxidase (NOX). The present study uses primary cortical neuronal cultures to examine respective contributions of mitochondria and NOX to ROS generation in response to Ca²⁺ or Zn²⁺ loading. Induction of rapid cytosolic accumulation of either Ca²⁺ (via NMDA exposure) or Zn²⁺ (via Zn²⁺/Pyrithione exposure in 0 Ca²⁺) caused sharp cytosolic rises in these ions, as well as a strong and rapid increase in ROS generation. Inhibition of NOX activation significantly reduced the Ca²⁺-induced ROS production with little effect on the Zn²⁺- triggered ROS generation. Conversely, dissipation of the mitochondrial electrochemical gradient increased the cytosolic Ca²⁺ or Zn²⁺ rises caused by these exposures, consistent with inhibition of mitochondrial uptake of these ions. However, such disruption of mitochondrial function markedly suppressed the Zn²⁺-triggered ROS, while partially attenuating the Ca²⁺-triggered ROS. Furthermore, block of the mitochondrial Ca²⁺ uniporter (MCU), through which Zn²⁺ as well as Ca²⁺ can enter the mitochondrial matrix, substantially diminished Zn²⁺ triggered ROS production, suggesting that the ROS generation occurs specifically in response to Zn²⁺ entry into mitochondria. Finally, in the presence of the sulfhydryl-oxidizing agent 2,2''-dithiodipyridine, which impairs Zn²⁺ binding to cytosolic metalloproteins, far lower Zn²⁺ exposures were able to induce mitochondrial Zn²⁺ uptake and consequent ROS generation. Thus, whereas rapid acute accumulation of Zn²⁺ and Ca²⁺ each can trigger injurious ROS generation, Zn²⁺ entry into mitochondria via the MCU may do so with particular potency. This may be of particular relevance to conditions like ischemia in which cytosolic Zn²⁺ buffering is impaired due to acidosis and oxidative stress.     

Diet-Sensitive Sources of Reactive Oxygen Species in Liver Mitochondria: Role of Very Long Chain Acyl-CoA Dehydrogenases

High fat diets and accompanying hepatic steatosis are highly prevalent conditions. Previous work has shown that steatosis is accompanied by enhanced generation of reactive oxygen species (ROS), which may mediate further liver damage. Here we investigated mechanisms leading to enhanced ROS generation following high fat diets (HFD). We found that mitochondria from HFD livers present no differences in maximal respiratory rates and coupling, but generate more ROS specifically when fatty acids are used as substrates. Indeed, many acyl-CoA dehydrogenase isoforms were found to be more highly expressed in HFD livers, although only the very long chain acyl-CoA dehydrogenase (VLCAD) was more functionally active. Studies conducted with permeabilized mitochondria and different chain length acyl-CoA derivatives suggest that VLCAD is also a source of ROS production in mitochondria of HFD animals. This production is stimulated by the lack of NAD⁺. Overall, our studies uncover VLCAD as a novel, diet-sensitive, source of mitochondrial ROS.     

Mitochondrial calcium uniporter blocker effectively prevents brain mitochondrial dysfunction caused by iron overload

AimsAlthough iron overload induces oxidative stress and brain mitochondrial dysfunction, and is associated with neurodegenerative diseases, brain mitochondrial iron uptake has not been investigated. We determined the role of mitochondrial calcium uniporter (MCU) in brain mitochondria as a major route for iron entry. We hypothesized that iron overload causes brain mitochondrial dysfunction, and that the MCU blocker prevents iron entry into mitochondria, thus attenuating mitochondrial dysfunction.Main methodsIsolated brain mitochondria from male Wistar rats were used. Iron (Fe^2 + and Fe^3 +) at 0–286 μM were applied onto mitochondria at various incubation times (5–30 min), and the mitochondrial function was determined. Effects of MCU blocker (Ru-360) and iron chelator were studied.Key findingsBoth Fe^2 + and Fe^3 + entered brain mitochondria and caused mitochondrial swelling in a dose- and time-dependent manner, and caused mitochondrial depolarization and increased ROS production. However, Fe^2 + caused more severe mitochondrial dysfunction than Fe^3 +. Although all drugs attenuated mitochondrial dysfunction caused by iron overload, only an MCU blocker could completely prevent ROS production and mitochondrial depolarization.SignificanceOur findings indicated that iron overload caused brain mitochondrial dysfunction, and that an MCU blocker effectively prevented this impairment, suggesting that MCU could be the major portal for brain mitochondrial iron uptake.     

MAPK15 protects from oxidative stress‐dependent cellular senescence by inducing the mitophagic process

Mitochondria are the major source of reactive oxygen species (ROS), whose aberrant production by dysfunctional mitochondria leads to oxidative stress, thus contributing to aging as well as neurodegenerative disorders and cancer. Cells efficiently eliminate damaged mitochondria through a selective type of autophagy, named mitophagy. Here, we demonstrate the involvement of the atypical MAP kinase family member MAPK15 in cellular senescence, by preserving mitochondrial quality, thanks to its ability to control mitophagy and, therefore, prevent oxidative stress. We indeed demonstrate that reduced MAPK15 expression strongly decreases mitochondrial respiration and ATP production, while increasing mitochondrial ROS levels. We show that MAPK15 controls the mitophagic process by stimulating ULK1‐dependent PRKN Ser¹⁰⁸ phosphorylation and inducing the recruitment of damaged mitochondria to autophagosomal and lysosomal compartments, thus leading to a reduction of their mass, but also by participating in the reorganization of the mitochondrial network that usually anticipates their disposal. Consequently, MAPK15‐dependent mitophagy protects cells from accumulating nuclear DNA damage due to mitochondrial ROS and, consequently, from senescence deriving from this chronic DNA insult. Indeed, we ultimately demonstrate that MAPK15 protects primary human airway epithelial cells from senescence, establishing a new specific role for MAPK15 in controlling mitochondrial fitness by efficient disposal of old and damaged organelles and suggesting this kinase as a new potential therapeutic target in diverse age‐associated human diseases.     

Mitochondrial Remodeling in Endothelial Cells under Cyclic Stretch is Independent of Drp1 Activation

Mitochondria in endothelial cells remodel morphologically when supraphysiological cyclic stretch is exerted on the cells. During remodeling, mitochondria become shorter, but how they do so remains elusive. Drp1 is a regulator of mitochondrial morphologies. It shortens mitochondria by shifting the balance from mitochondrial fusion to fission. In this study, we hypothesized that Drp1 activation is involved in mitochondrial remodeling under supraphysiological cyclic stretch. To verify the involvement of Drp1, its activation was first quantified with Western blotting, but Drp1 was not significantly activated in endothelial cells under supraphysiological cyclic stretch. Next, Drp1 activation was inhibited with Mdivi-1, but this did not inhibit mitochondrial remodeling. Intracellular Ca²⁺ increase activates Drp1 through calcineurin. First, we inhibited the intracellular Ca²⁺ increase with Gd³⁺ and thapsigargin, but this did not inhibit mitochondrial remodeling. Next, we inhibited calcineurin with cyclosporin A, but this also did not inhibit mitochondrial remodeling. These results indicate that mitochondrial remodeling under supraphysiological cyclic stretch is independent of Drp1 activation. In endothelial cells under supraphysiological cyclic stretch, reactive oxygen species (ROS) are generated. Mitochondrial morphologies are remodeled by ROS generation. When ROS was eliminated with N-acetyl-L-cysteine, mitochondrial remodeling was inhibited. Furthermore, when the polymerization of the actin cytoskeleton was inhibited with cytochalasin D, mitochondrial remodeling was also inhibited. These results suggest that ROS and actin cytoskeleton are rather involved in mitochondrial remodeling. In conclusion, the present results suggest that mitochondrial remodeling in endothelial cells under supraphysiological cyclic stretch is induced by ROS in association with actin cytoskeleton rather than through Drp1 activation.     

Mitochondrial metabolism of reactive oxygen species

For a long time mitochondria have mainly been considered for their role in the aerobic energy production in eukaryotic cells, being the sites of the oxidative phosphorylation, which couples the electron transfer from respiratory substrates to oxygen with the ATP synthesis. Subsequently, it was showed that electron transfer along mitochondrial respiratory chain also leads to the formation of radicals and other reactive oxygen species, commonly indicated as ROS. The finding that such species are able to damage cellular components, suggested mitochondrial involvement in degenerative processes underlying several diseases and aging.More recently, a new role for mitochondria, as a system able to supply protection against cellular oxidative damage, is emerging. Experimental evidence indicates that the systems, evolved to protect mitochondria against endogenously produced ROS, can also scavenge ROS produced by other cellular sources. It is possible that this action, particularly relevant in physio-pathological conditions leading to increased cellular ROS production, is more effective in tissues provided with abundant mitochondrial population. Moreover, the mitochondrial dysfunction, resulting from ROS-induced inactivation of important mitochondrial components, can be attenuated by the cell purification from old ROS-overproducing mitochondria, which are characterized by high susceptibility to oxidative damage. Such an elimination is likely due to two sequential processes, named mitoptosis and mitophagy, which are usually believed to be induced by enhanced mitochondrial ROS generation. However, they could also be elicited by great amounts of ROS produced by other cellular sources and diffusing into mitochondria, leading to the elimination of the old dysfunctional mitochondrial subpopulation.     

Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism

The recent knowledge on mitochondria as the substantial source of reactive oxygen species, namely superoxide and hydrogen peroxide efflux from mitochondria, is reviewed, as well as nitric oxide and subsequent peroxynitrite generation in mitochondria and their effects. The reactive oxygen species formation in extramitochondrial locations, in peroxisomes, by cytochrome P450, and NADPH oxidase reaction, is also briefly discussed. Conditions are pointed out under which mitochondria represent the major ROS source for the cell: higher percentage of non-phosphorylating and coupled mitochondria, in vivo oxygen levels leading to increased intensity of the reverse electron transport in the respiratory chain, and nitric oxide effects on the redox state of cytochromes. We formulate hypotheses on the crucial role of ROS generated in mitochondria for the whole cell and organism, in concert with extramitochondrial ROS and antioxidant defense. We hypothesize that a sudden decline of mitochondrial ROS production converts cells or their microenvironment into a “ROS sink” represented by the instantly released excessive capacity of ROS-detoxification mechanisms. A partial but immediate decline of mitochondrial ROS production may be triggered by activation of mitochondrial uncoupling, specifically by activation of recruited or constitutively present uncoupling proteins such as UCP2, which may counterbalance the mild oxidative stress.     

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.