IV- The mitochondria and cellular stress

1. The mitochondria

Mitochondria provide most of the energy for cells through oxidative phosphorylation and are thus critical to cell function. Mitochondria are highly dynamic organelles whose morphology is regulated by cycles of fusion and fission, collectively termed mitochondrial dynamics. In beta-cells these mechanisms may function to negate the detrimental effects of long-term exposure to high levels of nutrients; indeed combination of high fat and glucose gradually led to the arrest of fusion activity and complete fragmentation of the mitochondrial architecture. Inhibiting mitochondrial fission preserved mitochondrial morphology and dynamics and prevented beta-cell apoptosis (Shirihai, Lecture).22 In neurons, mitochondria have the ability to fuse, divide, and migrate to provide energy throughout the extended neuronal processes. Mitochondrial dysfunction is an early event in virtually all common neurodegenerative diseases, including Huntington’s disease, Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, stroke, and epilepsy. It was
speculated that increased mitochondrial fission also plays an important role in the pathology of diabetic neuropathies (Bossy-Wetzel, Lecture).23

FOCUS – The VHL gene and beta cell function

Defective insulin secretion in response to glucose is an important component of the beta-cell dysfunction seen in type 2 diabetes. As mitochondrial oxidative phosphorylation plays a key role in GSIS, oxygen-sensing pathways may modulate insulin release. The von Hippel–Lindau (VHL) protein controls the degradation of hypoxia-inducible factor (HIF) to coordinate cellular and organismal responses to altered oxygenation: at low oxygen concentrations and in the absence of VHL, HIF-alpha is stabilized and active. Experiments on rodents evidenced that mouse lacking Vhl in pancreatic beta-cells (betaVhlKO mice) and in the pancreas (PVhlKO mice) developed glucose intolerance with impaired insulin secretion, impaired glucose uptake and altered expression of genes involved in beta-cell function. This phenotype was completely reversed by additional deletion of floxed Hif1alpha. Consistent with this, expression of activated Hif-1alpha in a mouse beta-cell line was shown to impair GSIS by switching glucose metabolism from aerobic oxidative phosphorylation to anaerobic glycolysis.24 Since HIF upregulates expression of the high-affinity glucose transporter GLUT1 and glycolytic enzymes and decreases mitochondrial oxygen consumption, activation of the HIF pathway has the potential to provide a major input modulating GSIS. This could potentially be important in a wide range of disease states in which oxygen delivery is altered, or when islet oxygenation is directly compromised, such as in islet transplantation (Withers, Lecture).

2. Oxidative and ER stress

Cellular redox biology is inseparable from redox compartmentalization in eukaryotic cells. Mitochondria are the most redox-active compartment, accounting for more than 90% of electron transfer to O2 as the terminal electron acceptor. The predominant electron transfer occurs through a central redox circuit which uses the potential energy available from oxidation of various metabolic substrates (eg, pyruvate, fatty acids) to generate ATP. The molecular machinery required for oxidative phosphorylation is highly dependent upon critical cysteine residues of proteins for enzymatic and transport activities, and the mitochondrial genome is susceptible to oxidative damage. These processes are directly relevant to mitochondrial oxidative stress-related diseases such diabetes, and improved understanding
can be expected to aid the development of improved therapies for these diseases (Jones, Lecture).25

1. Reactive oxygen species (ROS) and ER stress

Glycolytic and oxidative events leading to accelerated ATP generation are key transduction phenomena in beta-cell signaling. However, they are also coupled with the generation of reactive oxygen species (ROS) mostly during oxidative phosphorylation within mitochondria. Superoxide anion radical (O2.-) is a very reactive molecule, which can be converted to less reactive H2O2 by superoxide dismutase (SOD) isoenzymes, and then to oxygen and water mainly by catalase (CAT), glutathione peroxidases (GPxs) and peroxiredoxin. beta-cells are equipped with moderate, but physiologically sufficient, catalytic capacities for conversion of O2.- into H2O2 in cytoplasm and mitochondria.

However, levels of the H2O2-inactivating enzymes GPxs and CAT are extremely low potentially making beta-cells vulnerable to H2O2 accumulation. In many instances, H2O2 generation is not a useless or harmful process but, rather, an essential element for certain biological responses. Indeed, Pi et al (2007)26 evidenced that low concentration of H2O2 derived from glucose metabolism is one of the metabolic signals that stimulates insulin secretion. However, chronic oxidative stress (as accumulation of H2O2 that is caused, for instance, by gluco- and lipotoxicity) has been increasingly implicated in the impaired state of beta-cells observed in metabolic disease, since it may induce endogenous antioxidant enzymes to blunt H2O2 signaling and GSIS (Collins, Lecture) (Figure 7).27

The molecular pathways that couple ER stress and oxidative stress are poorly understood.

The ER provides a unique oxidizing environment for protein folding and disulfide bond formation before transit to the Golgi compartment. Malhotra et al (2008)28 evidenced that accumulation of unfolded protein in the ER lumen is sufficient to produce ROS and both ROS and unfolded protein are required in concert to activate the UPR and apoptosis. They suggested several possible mechanisms to explain how protein misfolding in the ER may generate ROS (Figure 8): first, misfolded proteins bind protein chaperones, such as GRP78/BiP, that consume ATP that may stimulate mitochondrial oxidative phosphorylation to produce ROS as a byproduct. Second, ROS may be produced as a consequence of disulfide bond formation in the ER during the transfer of electrons from thiol groups in folding substrates through protein disulfide isomerase (PDI) and ER oxidoreductase 1 (ERO1) to molecular oxygen to produce hydrogen peroxide. Interestingly, islets explanted from homozygous ERO1- beta (the inducible isoform of ERO1) mutant mice (and ERO1-beta –deficient Min6 cells) have a conspicuous kinetic defect (delay) in oxidative folding, proinsulin maturation and exhibit glucose intolerance (Ron, Lecture).29 Third, glutathione (GSH) may be consumed during reduction of unstable and/or improper disulfide bonds in misfolded proteins. Finally, protein misfolding in the ER lumen can cause Ca2+ leak from the ER and uptake into the mitochondria to disrupt the electron transport chain (Kaufman Lecture).28

Figure 7. Model of ROS as a signal in GSIS and relationship to oxidative stress. Adapted from ref 27. © American Diabetes Association, 2007

Figure 7. Model of ROS as a signal in GSIS and relationship to oxidative stress. Adapted from ref 27. © American Diabetes Association, 2007

2. UCP2, oxidative stress and beta-cell function

Uncoupling protein 2 (UCP2) is a widely expressed mitochondrial inner membrane carrier protein that was discovered through its sequence homology to the brown fat UCP1. UCP1 dissipates caloric energy as heat by uncoupling mitochondrial respiration from ATP production. However, UCP2 is not a physiologically relevant “uncoupling protein” in the manner of UCP1 and does not contribute to adaptive thermogenesis. An ever-increasing number of studies highlight the significance of UCP2 in the regulation of glucose sensing in brain and pancreas and classify it as a critical
link between obesity, beta-cell dysfunction, and type 2 diabetes.30,31 UCP2 is an endogenous suppressor of insulin secretion, as demonstrated by the inhibition of GSIS in isolated beta-cells with UCP2 overexpression. Furthermore, Ucp2-/- mouse exhibited increased glucose tolerance and increased GSIS, and acute in vivo knockdown of UCP2 in diabetic animals caused a significant improvement in insulin secretion and enhanced whole-body sensitivity to insulin (Brand, Lecture). However, recent work from the group of Collins revealed confounding effects of genetic background and suggested that the chronic absence of UCP2 resulted in persistent oxidative stress and impairment of beta-cell function (Collins, Lecture).27 Nevertheless it is currently admitted that UCP2 effects can be mediating by lowering the coupling efficiency of oxidative phosphorylation, which results in decreased ATP/ADP ratio, leading to decreased stimulation of KATP channels and lowered insulin secretion. This UCP2-induced blunting of insulin secretion could be a physiological response to protect against hypoglycaemia (for example during fasting). Additionally, oxidative stress has been shown to induce the transcription of the UCP2 gene whose product would attenuate mitochondrial production of ROS and thus decrease GSIS (Brand, Lecture).

Figure 8. Protein misfolding and oxidative stress create a vicious cycle leading to ER stress and cell death. Adapted from ref 29. © National Academy of Sciences, U.S.A., 2008.

Figure 8. Protein misfolding and oxidative stress create a vicious cycle leading to ER stress and cell death. Adapted from ref 29. © National Academy of Sciences, U.S.A., 2008.

“The Stressed Beta-Cell”
I- ER and the canonical unfolded protein response (UPR)
II- When UPR leads to cell death
III- Cellular stress in type 2 diabetes
IV- The mitochondria and cellular stress
V- Therapeutic targeting of ER dysfunction
VI- Conclusions