SUMMARY Mitofusin 2 (Mfn2) plays critical roles in both mitochondrial fusion and the establishment of mitochondria-endoplasmic reticulum (ER) interactions. Hypothalamic ER stress has emerged as a causative factor for the development of leptin resistance, but the underlying mechanisms are largely unknown. Here we show that mitochondria-ER contacts in anorexigenic pro-opiomelanocortin (POMC) neurons in the hypothalamus are decreased in diet-induced obesity. POMC-specific ablation of Mfn2 resulted in loss of mitochondria-ER contacts, defective POMC processing, ER stress-induced leptin resistance, hyperphagia, reduced energy expenditure and obesity. Pharmacological relieve of hypothalamic ER stress reversed these metabolic alterations. Our data establishes Mfn2 in POMC neurons as an essential regulator of systemic energy balance by fine-tuning the mitochondrial-ER axis homeostasis and function. This previously unrecognized role for Mfn2 argues for a crucial involvement in mediating ER stress-induced leptin resistance.

Exercise results in rapid increases in expression of the transcription coactivator peroxisome proliferator-activated receptor ␥ coactivator-1␣ (PGC-1␣) and in mitochondrial biogenesis in skeletal muscle. PGC-1␣ regulates and coordinates mitochondrial biogenesis, and overexpression of PGC-1␣ in muscle cells results in increases in mitochondrial content. In this context, it has been proposed that the increase in PGC-1␣ protein expression mediates the exercise-induced increase in mitochondrial biogenesis. However, we found that mitochondrial proteins with a short half-life increase as rapidly as, or more rapidly than, PGC-1␣ protein. This finding led us to hypothesize that activation, rather than increased expression, of PGC-1␣ mediates the initial phase of the exercise-induced increase in mitochondria. In this study, we found that most of the PGC-1␣ in resting skeletal muscle is in the cytosol. Exercise resulted in activation of p38 MAPK and movement of PGC-1␣ into the nucleus. In support of our hypothesis, binding of the transcription factor nuclear respiratory factor 1 (NRF-1) to the cytochrome c promoter and NRF-2 to the cytochrome oxidase subunit 4 promoter increased in response to exercise prior to an increase in PGC-1␣ protein. Furthermore, exercise-induced increases in the mRNAs of cytochrome c, ␦-aminolevulinate synthase, and citrate synthase also occurred before an increase in PGC-1 protein. Thus, it appears that activation of PGC-1␣ may mediate the initial phase of the exercise-induced adaptive increase in muscle mitochondria, whereas the subsequent increase in PGC-1␣ protein sustains and enhances the increase in mitochondrial biogenesis.

Skeletal muscle contraction increases intracellular ATP turnover, calcium flux, and mechanical stress, initiating signal transduction pathways that modulate peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)-dependent transcriptional programmes. The purpose of this study was to determine if the intensity of exercise regulates PGC-1α expression in human skeletal muscle, coincident with activation of signalling cascades known to regulate PGC-1α transcription. Eight sedentary males expended 400 kcal (1674 kj) during a single bout of cycle ergometer exercise on two separate occasions at either 40% (LO) or 80% (HI) ofV O 2 peak . Skeletal muscle biopsies from the m. vastus lateralis were taken at rest and at +0, +3 and +19 h after exercise. Energy expenditure during exercise was similar between trials, but the high intensity bout was shorter in duration (LO, 69.9 ± 4.0 min; HI, 36.0 ± 2.2 min, P < 0.05) and had a higher rate of glycogen utilization (P < 0.05). PGC-1α mRNA abundance increased in an intensity-dependent manner +3 h after exercise (LO, 3.8-fold; HI, 10.2-fold, P < 0.05). AMP-activated protein kinase (AMPK) (2.8-fold, P < 0.05) and calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation (84%, P < 0.05) increased immediately after HI but not LO. p38 mitogen-activated protein kinase (MAPK) phosphorylation increased after both trials (∼2.0-fold, P < 0.05), but phosphorylation of the downstream transcription factor, activating transcription factor-2 (ATF-2), increased only after HI (2.4-fold, P < 0.05). Cyclic-AMP response element binding protein (CREB) phosphorylation was elevated at +3 h after both trials (∼80%, P < 0.05) and class IIa histone deacetylase (HDAC) phosphorylation increased only after HI (2.0-fold, P < 0.05). In conclusion, exercise intensity regulates PGC-1α mRNA abundance in human skeletal muscle in response to a single bout of exercise. This effect is mediated by differential activation of multiple signalling pathways, with ATF-2 and HDAC phosphorylation proposed as key intensity-dependent mediators.

A number of studies have reported that a high-fat diet induces increases in mitochondrial fatty acid oxidation enzymes in muscle. In contrast, in two recent studies raising plasma free fatty acids (FFA) resulted in a decrease in mitochondria. In this work, we reevaluated the effects of raising FFA on muscle mitochondrial biogenesis and capacity for fat oxidation. Rats were fed a high-fat diet and given daily injections of heparin to raise FFA. This treatment induced an increase in mitochondrial biogenesis in muscle, as evidenced by increases in mitochondrial enzymes of the fatty acid oxidation pathway, citrate cycle, and respiratory chain, with an increase in the capacity to oxidize fat, as well as an increase in mitochondrial DNA copy number. Raising FFA also resulted in an increase in binding of peroxisome proliferator-activated receptor (PPAR) ␦ to the PPAR response element on the carnitine palmitoyltransferase 1 promoter. We interpret our results as evidence that raising FFA induces an increase in mitochondrial biogenesis in muscle by activating PPAR␦.peroxisome proliferator-activated receptor (PPAR)␦ ͉ high-fat diet ͉ fatty acid oxidation I t has been reported that high-fat diets induce increased expression of mitochondrial fatty acid oxidation (FAO) enzymes in skeletal muscle, and it has been suggested that this adaptation results in an increase in the capacity to oxidize fatty acids (1-6). Similarly, exposure of cardiac myocytes to moderately high concentrations of fatty acids activates transcription of the muscle carnitine palmitoyltransferase (mCPT) gene (7). The peroxisome proliferator-activated receptors PPAR␣ and PPAR␤/␦ (PPAR␦) are ligand-activated nuclear receptors that activate transcription of genes encoding FAO enzymes as well as the uncoupling proteins (8-11). Both PPAR␣ and PPAR␦ are expressed in skeletal muscle, with PPAR␦ being the predominant isoform (12, 13). The PPARs are activated by long-chain fatty acids (14, 15). It, therefore, seemed likely that if raising plasma free fatty acids (FFA) does increase muscle FAO enzymes, this effect is mediated by PPAR␦ and ␣. However, in contrast to the earlier reports, two recent studies have reported that raising plasma FFA with a lipid emulsion (16) or a high-fat diet (17) results in decreases in muscle mitochondrial enzymes.In this context, the present study was undertaken to determine whether raising plasma FFA does induce an increase in expression of FAO enzymes in skeletal muscle and, if it does, to obtain evidence regarding whether the increase in FFA activates PPAR␦. A second aim, if raising FFA was found to increase FAO enzymes, was to determine whether an isolated increase in FAO enzymes results in an increase in the capacity to oxidize fatty acids or whether the citrate cycle and respiratory chain are rate-limiting. ResultsPlasma FFA Concentrations. Plasma FFA were markedly elevated in the rats fed the high-fat diet and given heparin (2.06 Ϯ 0.42 mM; range 1.22-3.84 mM) for six rats given heparin compared with 0.25 Ϯ 0.02 mM for six chow-f...