Jakob D. Wikstrom

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.

The Histone Deacetylase Sirt6 Regulates Glucose Homeostasis via Hif1α

SIRT6 is a member of a highly conserved family of NAD(+)-dependent deacetylases with various roles in metabolism, stress resistance, and life span. SIRT6-deficient mice develop normally but succumb to a lethal hypoglycemia early in life; however, the mechanism underlying this hypoglycemia remained unclear. Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a corepressor of the transcription factor Hif1alpha, a critical regulator of nutrient stress responses. Consistent with this notion, SIRT6-deficient cells exhibit increased Hif1alpha activity and show increased glucose uptake with upregulation of glycolysis and diminished mitochondrial respiration. Our studies uncover a role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis and may provide the basis for novel therapeutic approaches against metabolic diseases, such as diabetes and obesity.

Dual role of proapoptotic BAD in insulin secretion and beta cell survival

The proapoptotic BCL-2 family member BAD resides in a glucokinase-containing complex that regulates glucose-driven mitochondrial respiration. Here, we present genetic evidence of a physiologic role for BAD in glucose-stimulated insulin secretion by beta cells. This novel function of BAD is specifically dependent upon the phosphorylation of its BH3 sequence, previously defined as an essential death domain. We highlight the pharmacologic relevance of phosphorylated BAD BH3 by using cell-permeable, hydrocarbon-stapled BAD BH3 helices that target glucokinase, restore glucose-driven mitochondrial respiration and correct the insulin secretory response in Bad-deficient islets. Our studies uncover an alternative target and function for the BAD BH3 domain and emphasize the therapeutic potential of phosphorylated BAD BH3 mimetics in selectively restoring beta cell function. Furthermore, we show that BAD regulates the physiologic adaptation of beta cell mass during high-fat feeding. Our findings provide genetic proof of the bifunctional activities of BAD in both beta cell survival and insulin secretion.

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.

Mitochondrial autophagy in cells with mtDNA mutations results from synergistic loss of transmembrane potential and mTORC1 inhibition

Autophagy has emerged as a key cellular process for organellar quality control, yet this pathway apparently fails to eliminate mitochondria containing pathogenic mutations in mitochondrial DNA (mtDNA) in patients with a variety of human diseases. In order to explore how mtDNA-mediated mitochondrial dysfunction interacts with endogenous autophagic pathways, we examined autophagic status in a panel of human cytoplasmic hybrid (cybrid) cell lines carrying a variety of pathogenic mtDNA mutations. We found that both genetic- and chemically induced loss of mitochondrial transmembrane potential (Δψ(m)) caused recruitment of the pro-mitophagic factor Parkin to mitochondria. Strikingly, however, the loss of Δψ(m) alone was insufficient to prompt delivery of mitochondria to the autophagosome (mitophagy). We found that mitophagy could be induced following treatment with the mTORC1 inhibitor rapamycin in cybrids carrying either large-scale partial deletions of mtDNA or complete depletion of mtDNA. Further, we found that the level of endogenous Parkin is a crucial determinant of mitophagy. These results suggest a two-hit model, in which the synergistic induction of both (i) mitochondrial recruitment of Parkin following the loss of Δψ(m) and (ii) mTORC1-controlled general macroautophagy is required for mitophagy. It appears that mitophagy can be accomplished by the endogenous autophagic machinery, but requires the full engagement of both of these pathways.

What can mitochondrial heterogeneity tell us about mitochondrial dynamics and autophagy

A growing body of evidence shows that mitochondria are heterogeneous in terms of structure and function. Increased heterogeneity has been demonstrated in a number of disease models including ischemia-reperfusion and nutrient-induced beta cell dysfunction and diabetes. Subcellular location and proximity to other organelles, as well as uneven distribution of respiratory components have been considered as the main contributors to the basal level of heterogeneity. Recent studies point to mitochondrial dynamics and autophagy as major regulators of mitochondrial heterogeneity. While mitochondrial fusion mixes the content of the mitochondrial network, fission dissects the mitochondrial network and generates depolarized segments. These depolarized mitochondria are segregated from the networking population, forming a pre-autophagic pool contributing to heterogeneity. The capacity of a network to yield a depolarized daughter mitochondrion by a fission event is fundamental to the generation of heterogeneity. Several studies and data presented here provide a potential explanation, suggesting that protein and membranous structures are unevenly distributed within the individual mitochondrion and that inner membrane components do not mix during a fusion event to the same extent as the matrix components do. In conclusion, mitochondrial subcellular heterogeneity is a reflection of the mitochondrial lifecycle that involves frequent fusion events in which components may be unevenly mixed and followed by fission events generating disparate daughter mitochondria, some of which may fuse again, others will remain solitary and join a pre-autophagic pool.

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.

β-Cell Mitochondria Exhibit Membrane Potential Heterogeneity That Can Be Altered by Stimulatory or Toxic Fuel Levels

OBJECTIVE—β-Cell response to glucose is characterized by mitochondrial membrane potential (ΔΨ) hyperpolarization and the production of metabolites that serve as insulin secretory signals. We have previously shown that glucose-induced mitochondrial hyperpolarization accompanies the concentration-dependent increase in insulin secretion within a wide range of glucose concentrations. This observation represents the integrated response of a large number of mitochondria within each individual cell. However, it is currently unclear whether all mitochondria within a single β-cell represent a metabolically homogenous population and whether fuel or other stimuli can recruit or silence sizable subpopulations of mitochondria. This study offers insight into the different metabolic states of β-cell mitochondria. RESULTS—We show that mitochondria display a wide heterogeneity in ΔΨ and a millivolt range that is considerably larger than the change in millivolts induced by fuel challenge. Increasing glucose concentration recruits mitochondria into higher levels of homogeneity, while an in vitro diabetes model results in increased ΔΨ heterogeneity. Exploration of the mechanism behind heterogeneity revealed that temporary changes in ΔΨ of individual mitochondria, ATP-hydrolyzing mitochondria, and uncoupling protein 2 are not significant contributors to ΔΨ heterogeneity. We identified BAD, a proapoptotic BCL-2 family member previously implicated in mitochondrial recruitment of glucokinase, as a significant factor influencing the level of heterogeneity. CONCLUSIONS—We suggest that mitochondrial ΔΨ heterogeneity in β-cells reflects a metabolic reservoir recruited by an increased level of fuels and therefore may serve as a therapeutic target.

A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets.

Background The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets. Methodology/Principal Findings The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets. Conclusions/Significance The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.