Dapagliflozin, a Sodium-Glucose Co-Transporter 2 Inhibitor, Acutely Reduces Energy Expenditure in BAT via Neural Signals in Mice.

Selective sodium glucose cotransporter-2 inhibitor (SGLT2i) treatment promotes urinary glucose excretion, thereby reducing blood glucose as well as body weight. However, only limited body weight reductions are achieved with SGLT2i treatment. Hyperphagia is reportedly one of the causes of this limited weight loss. However, the effects of SGLT2i treatment on systemic energy expenditure have not been fully elucidated. Herein, we investigated the acute effects of dapagliflozin, a SGLT2i, on systemic energy expenditure in mice. Eighteen hours after dapagliflozin treatment oxygen consumption and brown adipose tissue (BAT) expression of ucp1, a thermogenesis-related gene, were significantly decreased as compared to those after vehicle treatment. In addition, dapagliflozin significantly suppressed norepinephrine (NE) turnover in BAT and c-fos expression in the rostral raphe pallidus nucleus (rRPa) which contains the sympathetic premotor neurons responsible for thermogenesis. These findings indicate that the dapagliflozin-mediated acute decrease in energy expenditure involves a reduction in BAT thermogenesis via decreased sympathetic nerve activity from the rRPa. Furthermore, common hepatic branch vagotomy abolished the reductions in ucp1 expression and NE contents in BAT and c-fos expression in the rRPa. In addition, alterations in hepatic carbohydrate metabolism, such as decreases in glycogen contents and upregulation of phosphoenolpyruvate carboxykinase, manifested prior to the suppression of BAT thermogenesis, e.g. 6 hours after dapagliflozin treatment. Collectively, these results suggest that SGLT2i treatment acutely suppresses energy expenditure in BAT via regulation of an inter-organ neural network consisting of the common hepatic vagal branch and sympathetic nerves.

Increased Energy Expenditure, Ucp1 Expression, and Resistance to Diet-induced Obesity in Mice Lacking Nuclear Factor-Erythroid-2-related Transcription Factor-2 (Nrf2).

The NRF2 (also known as NFE2L2) transcription factor is a critical regulator of genes involved in defense against oxidative stress. Previous studies suggest thatNrf2plays a role in adipogenesisin vitro, and deletion of theNrf2gene protects against diet-induced obesity in mice. Here, we demonstrate that resistance to diet-induced obesity inNrf2(-/-)mice is associated with a 20-30% increase in energy expenditure. Analysis of bioenergetics revealed thatNrf2(-/-)white adipose tissues exhibit greater oxygen consumption. White adipose tissue showed a >2-fold increase inUcp1gene expression. Oxygen consumption is also increased nearly 2.5-fold inNrf2-deficient fibroblasts. Oxidative stress induced by glucose oxidase resulted in increasedUcp1expression. Conversely, antioxidant chemicals (such asN-acetylcysteine and Mn(III)tetrakis(4-benzoic acid)porphyrin chloride) and SB203580 (a known suppressor ofUcp1expression) decreasedUcp1and oxygen consumption inNrf2-deficient fibroblasts. These findings suggest that increasing oxidative stress by limitingNrf2function in white adipocytes may be a novel means to modulate energy balance as a treatment of obesity and related clinical disorders.

Assessment of protein synthesis in highly aerobic canine species at the onset and during exercise training.

Canis lupus familiaris, the domesticated dog, is capable of extreme endurance performance. The ability to perform sustained aerobic exercise is dependent on a well-developed mitochondrial reticulum. In this study we examined the cumulative muscle protein and DNA synthesis in groups of athletic dogs at the onset of an exercise training program and following a strenuous exercise training program. We hypothesized that both at the onset and during an exercise training program there would be greater mitochondrial protein synthesis rates compared with sedentary control with no difference in mixed or cytoplasmic protein synthesis rates. Protein synthetic rates of three protein fractions and DNA synthesis were determined over 1 wk using (2)H2O in competitive Alaskan Huskies and Labrador Retrievers trained for explosive device detection. Both groups of dogs had very high rates of skeletal muscle protein synthesis in the sedentary state [Alaskan Huskies: Mixed = 2.28 ± 0.12, cytoplasmic (Cyto) = 2.91 ± 0.10, and mitochondrial (Mito) = 2.62 ± 0.07; Labrador Retrievers: Mixed = 3.88 ± 0.37, Cyto = 3.85 ± 0.06, and Mito = 2.92 ± 0.20%/day]. Mitochondrial (Mito) protein synthesis rates did not increase at the onset of an exercise training program. Exercise-trained dogs maintained Mito protein synthesis during exercise training when mixed (Mixed) and cytosolic (Cyto) fractions decreased, and this coincided with a decrease in p-RpS6 but also a decrease in p-ACC signaling. Contrary to our hypothesis, canines did not have large increases in mitochondrial protein synthesis at the onset or during an exercise training program. However, dogs have a high rate of protein synthesis compared with humans that perhaps does not necessitate an extra increase in protein synthesis at the onset of aerobic exercise training.

Nonclinical and pharmacokinetic assessments to evaluate the potential of tedizolid and linezolid to affect mitochondrial function.

Prolonged treatment with the oxazolidinone linezolid is associated with myelosuppression, lactic acidosis, and neuropathies, toxicities likely caused by impairment of mitochondrial protein synthesis (MPS). To evaluate the potential of the novel oxazolidinone tedizolid to cause similar side effects, nonclinical and pharmacokinetic assessments were conducted. In isolated rat heart mitochondria, tedizolid inhibited MPS more potently than did linezolid (average [± standard error of the mean] 50% inhibitory concentration [IC50] for MPS of 0.31 ± 0.02 µM versus 6.4 ± 1.2 µM). However, a rigorous 9-month rat study comparing placebo and high-dose tedizolid (resulting in steady-state area under the plasma concentration-time curve values about 8-fold greater than those with the standard therapeutic dose in humans) showed no evidence of neuropathy. Additional studies explored why prolonged, high-dose tedizolid did not cause these mitochondriopathic side effects despite potent MPS inhibition by tedizolid. Murine macrophage (J774) cell fractionation studies found no evidence of a stable association of tedizolid with eukaryotic mitochondria. Monte Carlo simulations based on population pharmacokinetic models showed that over the course of a dosing interval using standard therapeutic doses, free plasma concentrations fell below the respective MPS IC50 in 84% of tedizolid-treated patients (for a median duration of 7.94 h) and 38% of linezolid-treated patients (for a median duration of 0 h). Therapeutic doses of tedizolid, but not linezolid, may therefore allow for mitochondrial recovery during antibacterial therapy. The overall results suggest that tedizolid has less potential to cause myelosuppression and neuropathy than that of linezolid during prolonged treatment courses. This, however, remains a hypothesis that must be confirmed in clinical studies.

Mex3c mutation reduces adiposity and increases energy expenditure.

The function of MEX3C, the mammalian homolog of Caenorhabditis elegans RNA-binding protein muscle excess 3 (MEX-3), was unknown until our recent report that MEX3C is necessary for normal postnatal growth and enhances the expression of local bone Igf1 expression. Here we report the pivotal role of Mex3c in energy balance regulation. Mex3c mutation caused leanness in both heterozygous and homozygous transgenic mice, as well as a more beneficial blood glucose and lipid profile in homozygous transgenic mice, in both sexes. Although transgenic mice showed normal food intake and fecal lipid excretion, they had increased energy expenditure independent of physical activity. Mutant mice had normal body temperature, Ucp1 expression in brown adipose tissue, and muscle and liver fatty acid oxidation. Mex3c is expressed in neurons and is detectable in the arcuate nucleus, the ventromedial nucleus, and the dorsomedial nucleus of the hypothalamus. Mex3c was not detected in NPY or POMC neurons but was detected in leptin-responsive neurons in the ventromedial nucleus. Mex3c and Leptin double mutant mice were growth retarded and obese and had blood profiles similar to those of ob/ob mice but showed none of the steatosis observed in ob/ob mice. Our data show that Mex3c is involved in energy balance regulation.

Yeast mitochondrial leucyl-tRNA synthetase CP1 domain has functionally diverged to accommodate RNA splicing at expense of hydrolytic editing.

The yeast mitochondrial leucyl-tRNA synthetase (ymLeuRS) performs dual essential roles in group I intron splicing and protein synthesis. A specific LeuRS domain called CP1 is responsible for clearing noncognate amino acids that are misactivated during aminoacylation. The ymLeuRS CP1 domain also plays a critical role in splicing. Herein, the ymLeuRS CP1 domain was isolated from the full-length enzyme and was active in RNA splicing in vitro. Unlike its Escherichia coli LeuRS CP1 domain counterpart, it failed to significantly hydrolyze misaminoacylated tRNA(Leu). In addition and in stark contrast to the yeast domain, the editing-active E. coli LeuRS CP1 domain failed to recapitulate the splicing activity of the full-length E. coli enzyme. Although LeuRS-dependent splicing activity is rooted in an ancient adaptation for its aminoacylation activity, these results suggest that the ymLeuRS has functionally diverged to confer a robust splicing activity. This adaptation could have come at some expense to the protein's housekeeping role in aminoacylation and editing.

A comprehensive assessment of mitochondrial protein synthesis and cellular proliferation with age and caloric restriction.

It is proposed that caloric restriction (CR) increases mitochondrial biogenesis. However, it is not clear why CR increases an energetically costly biosynthetic process. We hypothesized that 40% CR would decrease mitochondrial protein synthesis and would be regulated by translational rather than transcriptional mechanisms. We assessed cumulative mitochondrial protein synthesis over 6 weeks and its transcriptional and translational regulation in the liver, heart, and skeletal muscle of young (6 month), middle (12 month), and old (24 month) male B6D2F1 mice that were lifelong CR or ad lib (AL) controls. Mitochondrial protein synthesis was not different between AL and CR (fractional synthesis over 6 weeks (range): liver, 91-100%; heart, 74-85%; skeletal muscle, 53-72%) despite a decreased cellular proliferation in liver and heart with CR. With CR, there was an increase in AMP-activated protein kinase phosphorylation/total (P:T) in heart and liver, and an increase in peroxisome proliferator-activated receptor gamma coactivator 1-α mRNA in all tissues, but not protein. Ribosomal protein S6 was decreased with CR. In conclusion, CR maintained mitochondrial protein synthesis while decreasing cellular proliferation during a time of energetic stress, which is consistent with the concept that CR increases somatic maintenance. Alternative mechanisms to global translation initiation may be responsible for selective translation of mitochondrial proteins.

Brain-derived neurotrophic factor (BDNF) in the hypothalamic ventromedial nucleus increases energy expenditure.

Brain-derived neurotrophic factor (BDNF) decreases food intake and body weight, but few central sites of action have been identified for its effect on energy expenditure. The hypothalamic ventromedial nucleus (VMH) is important in regulating energy metabolism. Our previous work indicated that BDNF in the VMH reduced food intake. The purposes of the study were to determine: 1) if BDNF in the VMH increases energy expenditure (EE); 2) if BDNF-enhanced thermogenesis results from increased spontaneous physical activity (SPA) and resting metabolic rate (RMR); and 3) if VMH BDNF thermogenic effects are mediated by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT). BDNF (0.5 microg) was injected into the VMH of male Sprague-Dawley rats and oxygen consumption, carbon dioxide production, food intake and SPA were measured for 24h in an indirect calorimeter. Animals were sacrificed 4h after BDNF injection, and BAT UCP1 gene expression was measured with quantitative real-time polymerase chain reaction. BDNF significantly decreased food and water intake, and body weight gain. Heat production and RMR were significantly elevated for 9h immediately after BDNF injection. BDNF increased SPA and EE during SPA (aEE) within 9h after injection although BDNF had no effect on 0-24h SPA and aEE. BDNF did not induce a significant increase in BAT UCP1 expression. In conclusion, VMH BDNF reduces body weight by decreasing food intake and increasing EE consequent to increased SPA and RMR, suggesting that the VMH is an important site of BDNF action to influence energy balance.

Genetic and functional mitochondrial assessment of HIV-infected patients developing HAART-related hyperlactatemia.

BACKGROUND: Mitochondrial damage of HIV and antiretrovirals, especially nucleoside-analogue interference on mitochondrial DNA (mtDNA) replication, is reported to underlay highly active antiretroviral therapy (HAART)-related hyperlactatemia, but scarce approaches have been performed to correlate clinical manifestations and mitochondrial abnormalities. METHODS: We obtained lymphocytes and monocytes of 26 HIV-infected and treated patients who developed hyperlactatemia and after recovery, 28 nonhyperlactatemic HIV subjects on HAART, 31 naive individuals, and 20 uninfected controls. Mitochondrial replication and transcription analysis were performed by quantitative real-time PCR, mitochondrial translation quantification by western blot and mitochondrial enzymatic activities by spectrophotometry. RESULTS: Mitochondrial parameters decreased during hyperlactatemia and improved at recovery. Mitochondrial replication and transcription species were reduced (P = 0.16 and P = 0.71), but the most significant decay was observed on mitochondrial protein content (P < 0.05) and mitochondrial complexes III and IV activities (P < 0.01 and P < 0.001). During hyperlactatemia lactate level correlated complexes III and IV function (P < 0.05). After recovery mitochondrial parameters achieved values of nonhyperlactatemic HIV individuals, which were lower than ranges of naive subjects and uninfected controls. CONCLUSIONS: HIV and HAART-related hyperlactatemia is associated with a general mitochondrial impairment which reverts after recovery. Mitochondrial biochemistry show a better correlation with lactate levels than mitochondrial genetics suggesting that mitochondrial function could be a better marker of hyperlactatemia development than mtDNA content.