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Mitochondrial Conference

March 2014

Mitofusin-2 determines mitochondrial network architecture and mitochondrial metabolism. A novel regulatory mechanism altered in obesity.

In many cells and specially in muscle, mitochondria form elongated filaments or a branched reticulum. We show that Mfn2 (mitofusin 2), a mitochondrial membrane protein that participates in mitochondrial fusion in mammalian cells, is induced during myogenesis and contributes to the maintenance and operation of the mitochondrial network. Repression of Mfn2 caused morphological and functional fragmentation of the mitochondrial network into independent clusters. Concomitantly, repression of Mfn2 reduced glucose oxidation, mitochondrial membrane potential, cell respiration, and mitochondrial proton leak. We also show that the Mfn2-dependent mechanism of mitochondrial control is disturbed in obesity by reduced Mfn2 expression. In all, our data indicate that Mfn2 expression is crucial in mitochondrial metabolism through the maintenance of the mitochondrial network architecture, and reduced Mfn2 expression may explain some of the metabolic alterations associated with obesity.

J Biol Chem. 2003 May 9;278(19):17190-7

Expression of Mfn2, the Charcot-Marie-Tooth neuropathy type 2A gene, in human skeletal muscle: effects of type 2 diabetes, obesity, weight loss, and the regulatory role of tumor necrosis factor alpha and interleukin-6.

The primary gene mutated in Charcot-Marie-Tooth type 2A is mitofusin-2 (Mfn2). Mfn2 encodes a mitochondrial protein that participates in the maintenance of the mitochondrial network and that regulates mitochondrial metabolism and intracellular signaling. The potential for regulation of human Mfn2 gene expression in vivo is largely unknown. Based on the presence of mitochondrial dysfunction in insulin-resistant conditions, we have examined whether Mfn2 expression is dysregulated in skeletal muscle from obese or nonobese type 2 diabetic subjects, whether muscle Mfn2 expression is regulated by body weight loss, and the potential regulatory role of tumor necrosis factor (TNF)alpha or interleukin-6. We show that mRNA concentration of Mfn2 is decreased in skeletal muscle from both male and female obese subjects. Muscle Mfn2 expression was also reduced in lean or in obese type 2 diabetic patients. There was a strong negative correlation between the Mfn2 expression and the BMI in nondiabetic and type 2 diabetic subjects. A positive correlation between the Mfn2 expression and the insulin sensitivity was also detected in nondiabetic and type 2 diabetic subjects. To determine the effect of weight loss on Mfn2 mRNA expression, six morbidly obese subjects were subjected to weight loss by bilio-pancreatic diversion. Mean expression of muscle Mfn2 mRNA increased threefold after reduction in body weight, and a positive correlation between muscle Mfn2 expression and insulin sensitivity was again detected. In vitro experiments revealed an inhibitory effect of TNFalpha or interleukin-6 on Mfn2 expression in cultured cells. We conclude that body weight loss upregulates the expression of Mfn2 mRNA in skeletal muscle of obese humans, type 2 diabetes downregulates the expression of Mfn2 mRNA in skeletal muscle, Mfn2 expression in skeletal muscle is directly proportional to insulin sensitivity and is inversely proportional to the BMI, TNFalpha and interleukin-6 downregulate Mfn2 expression and may participate in the dysregulation of Mfn2 expression in obesity or type 2 diabetes, and the in vivo modulation of Mfn2 mRNA levels is an additional level of regulation for the control of muscle metabolism and could provide a molecular mechanism for alterations in mitochondrial function in obesity or type 2 diabetes.

Diabetes. 2005 Sep;54(9):2685-93

Mitofusin 2 (Mfn2) links mitochondrial and endoplasmic reticulum function with insulin signaling and is essential for normal glucose homeostasis.

Mitochondria are dynamic organelles that play a key role in energy conversion. Optimal mitochondrial function is ensured by a quality-control system tightly coupled to fusion and fission. In this connection, mitofusin 2 (Mfn2) participates in mitochondrial fusion and undergoes repression in muscle from obese or type 2 diabetic patients. Here, we provide in vivo evidence that Mfn2 plays an essential role in metabolic homeostasis. Liver-specific ablation of Mfn2 in mice led to numerous metabolic abnormalities, characterized by glucose intolerance and enhanced hepatic gluconeogenesis. Mfn2 deficiency impaired insulin signaling in liver and muscle. Furthermore, Mfn2 deficiency was associated with endoplasmic reticulum stress, enhanced hydrogen peroxide concentration, altered reactive oxygen species handling, and active JNK. Chemical chaperones or the antioxidant N-acetylcysteine ameliorated glucose tolerance and insulin signaling in liver-specific Mfn2 KO mice. This study provides an important description of a unique unexpected role of Mfn2 coordinating mitochondria and endoplasmic reticulum function, leading to modulation of insulin signaling and glucose homeostasis in vivo.

Proc Natl Acad Sci U S A. 2012 Apr 3;109(14):5523-8

Subjects with early-onset type 2 diabetes show defective activation of the skeletal muscle PGC-1{alpha}/Mitofusin-2 regulatory pathway in response to physical activity.

OBJECTIVE Type 2 diabetes is associated with insulin resistance and skeletal muscle mitochondrial dysfunction. We have found that subjects with early-onset type 2 diabetes show incapacity to increase Vo(2max) in response to chronic exercise. This suggests a defect in muscle mitochondrial response to exercise. Here, we have explored the nature of the mechanisms involved. RESEARCH DESIGN AND METHODS Muscle biopsies were collected from young type 2 diabetic subjects and obese control subjects before and after acute or chronic exercise protocols, and the expression of genes and/or proteins relevant to mitochondrial function was measured. In particular, the regulatory pathway peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha/mitofusin-2 (Mfn2) was analyzed. RESULTS At baseline, subjects with diabetes showed reduced expression (by 26%) of the mitochondrial fusion protein Mfn2 and a 39% reduction of the alpha-subunit of ATP synthase. Porin expression was unchanged, consistent with normal mitochondrial mass. Chronic exercise led to a 2.8-fold increase in Mfn2, as well as increases in porin, and the alpha-subunit of ATP synthase in muscle from control subjects. However, Mfn2 was unchanged after chronic exercise in individuals with diabetes, whereas porin and alpha-subunit of ATP synthase were increased. Acute exercise caused a fourfold increase in PGC-1alpha expression in muscle from control subjects but not in subjects with diabetes. CONCLUSIONS Our results demonstrate alterations in the regulatory pathway that controls PGC-1alpha expression and induction of Mfn2 in muscle from patients with early-onset type 2 diabetes. Patients with early-onset type 2 diabetes display abnormalities in the exercise-dependent pathway that regulates the expression of PGC-1alpha and Mfn2.

Diabetes Care. 2010 Mar;33(3):645-51

Dynamics of fat cell turnover in humans.

Obesity is increasing in an epidemic manner in most countries and constitutes a public health problem by enhancing the risk for cardiovascular disease and metabolic disorders such as type 2 diabetes. Owing to the increase in obesity, life expectancy may start to decrease in developed countries for the first time in recent history. The factors determining fat mass in adult humans are not fully understood, but increased lipid storage in already developed fat cells (adipocytes) is thought to be most important. Here we show that adipocyte number is a major determinant for the fat mass in adults. However, the number of fat cells stays constant in adulthood in lean and obese individuals, even after marked weight loss, indicating that the number of adipocytes is set during childhood and adolescence. To establish the dynamics within the stable population of adipocytes in adults, we have measured adipocyte turnover by analysing the integration of 14C derived from nuclear bomb tests in genomic DNA. Approximately 10% of fat cells are renewed annually at all adult ages and levels of body mass index. Neither adipocyte death nor generation rate is altered in early onset obesity, suggesting a tight regulation of fat cell number in this condition during adulthood. The high turnover of adipocytes establishes a new therapeutic target for pharmacological intervention in obesity.

Nature. 2008 Jun 5;453(7196):783-7

Mitophagy in neurodegeneration and aging.

Macroautophagy is a cellular catabolic process that involves the sequestration of cytoplasmic constituents into double-membrane vesicles known as autophagosomes, which subsequently fuse with lysosomes, where they deliver their cargo for degradation. The main physiological role of autophagy is to recycle intracellular components, under conditions of nutrient deprivation, so as to supply cells with vital materials and energy. Selective autophagy also takes place in nutrient-rich conditions to rid the cell of damaged organelles or protein aggregates that would otherwise compromise cell viability. Mitophagy is a selective type of autophagy, whereby damaged or superfluous mitochondria are eliminated to maintain proper mitochondrial numbers and quality control. While mitophagy shares key regulatory factors with the general macroautophagy pathway, it also involves distinct steps, specific for mitochondrial elimination. Recent findings indicate that parkin and the phosphatase and tensin homolog-induced putative kinase protein 1 (PINK1), which have been implicated in the pathogenesis of neurodegenerative diseases such as Parkinson’s disease, also regulate mitophagy and function to maintain mitochondrial homeostasis. Here, we survey the molecular mechanisms that govern the process of mitophagy and discuss its involvement in the onset and progression of neurodegenerative diseases during aging.

Front Genet. 2012 Dec 19;3:297

Autophagy and ageing: insights from invertebrate model organisms.

Ageing in diverse species ranging from yeast to humans is associated with the gradual, lifelong accumulation of molecular and cellular damage. Autophagy, a conserved lysosomal, self-destructive process involved in protein and organelle degradation, plays an essential role in both cellular and whole-animal homeostasis. Accumulating evidence now indicates that autophagic degradation declines with age and this gradual reduction of autophagy might have a causative role in the functional deterioration of biological systems during ageing. Indeed, loss of autophagy gene function significantly influences longevity. Moreover, genetic or pharmacological manipulations that extend lifespan in model organisms often activate autophagy. Interestingly, conserved signalling pathways and environmental factors that regulate ageing, such as the insulin/IGF-1 signalling pathway and oxidative stress response pathways converge on autophagy. In this article, we survey recent findings in invertebrates that contribute to advance our understanding of the molecular links between autophagy and the regulation of ageing. In addition, we consider related mechanisms in other organisms and discuss their similarities and idiosyncratic features in a comparative manner.

Ageing Res Rev. 2013 Jan;12(1):413-28

Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome.

Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy independent of SIRT1 in human and yeast cells as well as in nematodes. Although resveratrol and spermidine ignite autophagy through distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant modifications of the acetylproteome. Both agents favor convergent deacetylation and acetylation reactions in the cytosol and in the nucleus, respectively. Both resveratrol and spermidine were able to induce autophagy in cytoplasts (enucleated cells). Moreover, a cytoplasm-restricted mutant of SIRT1 could stimulate autophagy, suggesting that cytoplasmic deacetylation reactions dictate the autophagic cascade. At doses at which neither resveratrol nor spermidine stimulated autophagy alone, these agents synergistically induced autophagy. Altogether, these data underscore the importance of an autophagy regulatory network of antagonistic deacetylases and acetylases that can be pharmacologically manipulated.

J Cell Biol. 2011 Feb 21;192(4):615-29

Spermidine promotes stress resistance in Drosophila melanogaster through autophagy-dependent and -independent pathways.

The naturally occurring polyamine spermidine (Spd) has recently been shown to promote longevity across species in an autophagy-dependent manner. Here, we demonstrate that Spd improves both survival and locomotor activity of the fruit fly Drosophila melanogaster upon exposure to the superoxide generator and neurotoxic agent paraquat. Although survival to a high paraquat concentration (20 mM) was specifically increased in female flies only, locomotor activity and survival could be rescued in both male and female animals when exposed to lower paraquat levels (5 mM). These effects are dependent on the autophagic machinery, as Spd failed to confer resistance to paraquat-induced toxicity and locomotor impairment in flies deleted for the essential autophagic regulator ATG7 (autophagy-related gene 7). Spd treatment did also protect against mild doses of another oxidative stressor, hydrogen peroxide, but in this case in an autophagy-independent manner. Altogether, this study establishes that the protective effects of Spd can be exerted through different pathways that depending on the oxidative stress scenario do or do not involve autophagy.

Cell Death Dis. 2012 Oct 11;3:e401

Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage.

OBJECTIVE: The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function. METHODS AND RESULTS: Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide-induced damage. TERT increases overall respiratory chain activity, which is most pronounced at complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H(2)O(2)-induced apoptosis. Lung fibroblasts from 6-month-old TERT(-/-) mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT in vivo. CONCLUSIONS: Mitochondrial TERT exerts a novel protective function by binding to mitochondrial DNA, increasing respiratory chain activity and protecting against oxidative stress-induced damage.

Arterioscler Thromb Vasc Biol. 2009 Jun;29(6):929-35