Guy Las

Fission and selective fusion govern mitochondrial segregation and elimination by autophagy

Accumulation of depolarized mitochondria within β‐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 (Δψm) 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 non‐fusing mitochondria that were found to have reduced Δψm and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1K38A 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 Δψm 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.

Mitochondrial Networking Protects β-Cells From Nutrient-Induced Apoptosis

OBJECTIVE Previous 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 METHODS Using 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. RESULTS β-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. CONCLUSIONS These 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.

Fatty Acids Suppress Autophagic Turnover in β-Cells

Recent studies have shown that autophagy is essential for proper β-cell function and survival. However, it is yet unclear under what pathogenic conditions autophagy is inhibited in β-cells. Here, we report that long term exposure to fatty acids and glucose block autophagic flux in β-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 β-cell dysfunction. This may provide a novel mechanism that connects diet-induced obesity and diabetes.

Hormone‐induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure

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.

Biophysical properties of mitochondrial fusion events in pancreatic β-cells and cardiac cells unravel potential control mechanisms of its selectivity

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.

Lysosomal dysfunction and impaired autophagy underlie the pathogenesis of amyloidogenic light chain‐mediated cardiotoxicity

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.

Miro1: New wheels for transferring mitochondria

Mesenchymal stem cells (MSC) are capable of protecting cells harboring mitochondrial damage. This protection is associated with the transfer of mitochondria through tunneling nanotubes (TNT) from MSC to the injured cells. In this issue of The EMBO Journal, the group of Anurag Agrawal shows that mitochondrial transfer is dependent on the levels of Miro1, a mitochondrial Rho‐GTPase that regulates intercellular mitochondrial movement. Miro1 is the first protein shown to accelerate mitochondrial transfer. Amplifying the mitochondrial transfer phenomenon may allow for the study of the mechanisms that regulate it and contribute to our understanding of its role in disease and aging.

Modulation of Insulin Secretion by Fatty Acyl Analogs

The secretagogue, the incretin-like, and the suppressive activities of long-chain fatty acids (LCFAs) in modulating insulin secretion in vivo and in cultured islets were simulated here by β,β′-tetramethyl-hexadecanedioic acid (M16) and α,α′-tetrachloro-tetradecanedioic acid (Cl-DICA). M16, but not Cl-DICA, serves as a substrate for ATP-dependent CoA thioesterification but is not further metabolized. M16, but not Cl-DICA, acted as a potent insulin secretagogue in islets cultured in basal but not high glucose. Short-term exposure to M16 or Cl-DICA resulted in activation of glucose- but not arginine-stimulated insulin secretion. Long-term exposure to M16, but not to Cl-DICA, resulted in suppression of glucose-, arginine-, and K+-stimulated insulin secretion; inhibition of glucose-induced proinsulin biosynthesis; and depletion of islets insulin. β-Cell mass and islet ATP content remained unaffected. Hence, nonmetabolizable LCFA analogs may highlight discrete LCFA metabolites and pathways involved in modulating insulin secretion, which could be overlooked due to the rapid turnover of natural LCFA.

Autophagy in the Homeostasis of Pancreatic β-Cells

Autophagy is increasingly recognized as a key process for the homeostasis in β-cells. In vivo evidence in pancreata of diabetic patients suggests that autophagy is impaired, while selective arrest of autophagy in β-cells of mice alters insulin secretion, followed by hyperglycemia. Several cellular pathways have been implicated to be tightly coupled to autophagy. Autophagy is critical for mitochondrial homeostasis in β-cells and selectively removes mitochondria at a sustained lower energetic level. As mitochondria are critical for insulin secretion, arrest of their removal leads to accumulation of mitochondrial dysfunction and results in impaired insulin secretion. In addition, various lysosomal enzymes that are associated with various stages of autophagy are co-localized on insulin granules. Hence, autophagy also plays a role in regulating insulin granules number and total insulin content.