Accumulation of depolarized mitochondria within b-cells has been associated with oxidative damage and development of diabetes. To determine the source and fate of depolarized mitochondria, individual mitochondria were photolabeled and tracked through fusion and fission. Mitochondria were found to go through frequent cycles of fusion and fission in a 'kiss and run' pattern. Fission events often generated uneven daughter units: one daughter exhibited increased membrane potential (Dw m ) and a high probability of subsequent fusion, while the other had decreased membrane potential and a reduced probability for a fusion event. Together, this pattern generated a subpopulation of nonfusing mitochondria that were found to have reduced Dw m and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1 K38A or FIS1 RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion. Pulse chase and arrest of autophagy at the pre-proteolysis stage reveal that before autophagy mitochondria lose Dw m and OPA1, and that overexpression of OPA1 decreases mitochondrial autophagy. Together, these findings suggest that fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy.

OBJECTIVEPrevious studies have reported that β-cell mitochondria exist as discrete organelles that exhibit heterogeneous bioenergetic capacity. To date, networking activity, and its role in mediating β-cell mitochondrial morphology and function, remains unclear. In this article, we investigate β-cell mitochondrial fusion and fission in detail and report alterations in response to various combinations of nutrients.RESEARCH DESIGN AND METHODSUsing matrix-targeted photoactivatable green fluorescent protein, mitochondria were tagged and tracked in β-cells within intact islets, as isolated cells and as cell lines, revealing frequent fusion and fission events. Manipulations of key mitochondrial dynamics proteins OPA1, DRP1, and Fis1 were tested for their role in β-cell mitochondrial morphology. The combined effects of free fatty acid and glucose on β-cell survival, function, and mitochondrial morphology were explored with relation to alterations in fusion and fission capacity.RESULTSβ-Cell mitochondria are constantly involved in fusion and fission activity that underlies the overall morphology of the organelle. We find that networking activity among mitochondria is capable of distributing a localized green fluorescent protein signal throughout an isolated β-cell, a β-cell within an islet, and an INS1 cell. Under noxious conditions, we find that β-cell mitochondria become fragmented and lose their ability to undergo fusion. Interestingly, manipulations that shift the dynamic balance to favor fusion are able to prevent mitochondrial fragmentation, maintain mitochondrial dynamics, and prevent apoptosis.CONCLUSIONSThese data suggest that alterations in mitochondrial fusion and fission play a critical role in nutrient-induced β-cell apoptosis and may be involved in the pathophysiology of type 2 diabetes.

Adrenergic stimulation of brown adipocytes (BA) induces mitochondrial uncoupling, thereby increasing energy expenditure by shifting nutrient oxidation towards thermogenesis. Here we describe that mitochondrial dynamics is a physiological regulator of adrenergically-induced changes in energy expenditure. The sympathetic neurotransmitter Norepinephrine (NE) induced complete and rapid mitochondrial fragmentation in BA, characterized by Drp1 phosphorylation and Opa1 cleavage. Mechanistically, NE-mediated Drp1 phosphorylation was dependent on Protein Kinase-A (PKA) activity, whereas Opa1 cleavage required mitochondrial depolarization mediated by FFAs released as a result of lipolysis. This change in mitochondrial architecture was observed both in primary cultures and brown adipose tissue from cold-exposed mice. Mitochondrial uncoupling induced by NE in brown adipocytes was reduced by inhibition of mitochondrial fission through transient Drp1 DN overexpression. Furthermore, forced mitochondrial fragmentation in BA through Mfn2 knock down increased the capacity of exogenous FFAs to increase energy expenditure. These results suggest that, in addition to its ability to stimulate lipolysis, NE induces energy expenditure in BA by promoting mitochondrial fragmentation. Together these data reveal that adrenergically-induced changes to mitochondrial dynamics are required for BA thermogenic activation and for the control of energy expenditure.

Recent studies have shown that autophagy is essential for proper ␤-cell function and survival. However, it is yet unclear under what pathogenic conditions autophagy is inhibited in ␤-cells. Here, we report that long term exposure to fatty acids and glucose block autophagic flux in ␤-cells, contributing to their toxic effect. INS1 cells expressing GFP-LC3 (an autophagosome marker) were treated with 0.4 mM palmitate, 0.4 mM oleate, and various concentrations of glucose for 22 h. Kinetics of the effect of fatty acids on autophagy showed a biphasic response. During the second phase of autophagy, the size of autophagosomes and the content of autophagosome substrates (GFP-LC3, p62) and endogenous LC3 was increased. During the same phase, fatty acids suppressed autophagic degradation of long lived protein in both INS1 cells and islets. In INS1 cells, palmitate induced a 3-fold decrease in the number and the acidity of Acidic Vesicular Organelles. This decrease was associated with a suppression of hydrolase activity, suppression of endocytosis, and suppression of oxidative phosphorylation. The combination of fatty acids with glucose synergistically suppressed autophagic turnover, concomitantly suppressing insulin secretion. Rapamycin treatment resulted in partial reversal of the inhibition of autophagic flux, the inhibition of insulin secretion, and the increase in cell death. Our results indicate that excess nutrient could impair autophagy in the long term, hence contributing to nutrient-induced ␤-cell dysfunction. This may provide a novel mechanism that connects diet-induced obesity and diabetes.Macroautophagy (hereafter named autophagy) is the main mechanism the cell uses to degrade damaged and redundant organelles. It involves the formation of a double-membrane structure called the phagophore, which evolves into the autophagosome (AP), 2 an organelle that sequesters cytoplasmic material such as mitochondria, peroxisomes, endoplasmic reticulum, protein aggregates, and lipids. Upon acidification (1), the AP fuses with the lysosome to form the autolysosome, which degrades its content (2).The main approach to study autophagy is by tracking APs using LC3 (microtubule-associated protein 1 light chain 3), a cytosolic protein that upon stimulation of autophagy is lipidated and recruited to the AP membrane. LC3 remains bound to the AP until released to the cytosol or degraded by lysosomal enzymes (3).Stimulators of autophagy are known to increase the number of APs. However, the quantification of APs to assess autophagy can be misleading, APs being but one component in the chain constituting autophagic degradation (3). Thus, for example, in the case of various neuronal diseases, the increase in the number of APs was originally falsely interpreted as an increase in autophagic turnover, although it is now known to be the result of a decrease in autophagic turnover downstream to AP formation (4, 5).Type 2 diabetes is a disease in which glucose homeostasis is impaired due to peripheral insulin resistance accompanied by a decrease...

Lipotoxicity is frequently associated with alkalinization of lysosomes and impaired autophagic flux but the relationship between the two is unclear. Trudeau et al. show that autophagic flux can be restored in β islet cells chronically exposed to fatty acids by re-acidification of lysosomes with photoactivatable nanoparticles.

AL amyloidosis is the consequence of clonal production of amyloidogenic immunoglobulin light chain (LC) proteins, often resulting in a rapidly progressive and fatal amyloid cardiomyopathy. Recent work has found that amyloidogenic LC directly initiate a cardio-toxic response underlying the pathogenesis of the cardiomyopathy; however, the mechanisms that contribute to this proteotoxicity remain unknown. Using human amyloidogenic LC isolated from patients with amyloid cardiomyopathy, we reveal that dysregulation of autophagic flux is critical for mediating amyloidogenic LC proteotoxicity. Restoration of autophagic flux by pharmacological intervention using rapamycin protected against amyloidogenic light chain protein-induced pathologies including contractile dysfunction and cell death at the cellular and organ level and also prolonged survival in an in vivo zebrafish model of amyloid cardiotoxicity. Mechanistically, we identify impaired lysosomal function to be the major cause of defective autophagy and amyloidogenic LC-induced proteotoxicity. Collectively, these findings detail the downstream molecular mechanisms underlying AL amyloid cardiomyopathy and highlight potential targeting of autophagy and lysosomal dysfunction in patients with amyloid cardiomyopathy.

Studies in various types of cells find that, on average, each mitochondrion becomes involved in a fusion event every 15 min, depending on the cell type. As most contact events do not result in mitochondrial fusion, it is expected that properties of the individual mitochondrion determine the likelihood of a fusion event. However, apart from membrane potential, the properties that influence the likelihood of entering a fusion event are not known. Here, we tag and track individual mitochondria in H9c2, INS1, and primary beta-cells and determine the biophysical properties that increase the likelihood of a fusion event. We found that the probability for fusion is independent of contact duration and organelle dimensions, but it is influenced by organelle motility. Furthermore, the history of a previous fusion event of the individual mitochondrion influenced both the likelihood for a subsequent fusion event, as well as the site on the mitochondrion at which the fusion occurred. These observations unravel the specific properties that distinguish mitochondria that will enter fusion events from the ones that will not. Altogether, these properties may help to elucidate the molecular mechanisms that regulate fusion at the level of the single mitochondrion.

Autophagy, a ubiquitous catabolic pathway involved in both cell survival and cell death, has been implicated in many age-associated diseases. Recent findings have shown autophagy to be crucial for proper insulin secretion and β-cell viability. Transgenic mice lacking autophagy in their β-cells showed decreased β-cell mass and suppressed glucose-stimulated insulin secretion. Several studies showed that stress can stimulate autophagy in β-cells: the number of autophagosomes is increased in different in vivo models for diabetes, such as db/db mice, mice fed high-fat diet, pdx-1 knockout mice, as well as in in vitro models of glucotoxicity and lipotoxicity. Pharmacological and molecular inhibition of autophagy increases the susceptibility to cell stress, suggesting that autophagy protects against diabetes-relevant stresses. Recent findings, however, question these conclusions. Pancreases of diabetics and β-cells exposed to fatty acids show accumulation of abnormal autophagosome morphology and suppression of lysosomal gene expression suggesting impairment in autophagic turnover. In this review we attempt to give an overview of the data generated by others and by us in view of the possible role of autophagy in diabetes, a role which depending on the conditions, could be beneficial or detrimental in coping with stress.