No abstract

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1β (PGC-1β) has been implicated in important metabolic processes. A mouse lacking PGC-1β (PGC1βKO) was generated and phenotyped using physiological, molecular, and bioinformatic approaches. PGC1βKO mice are generally viable and metabolically healthy. Using systems biology, we identified a general defect in the expression of genes involved in mitochondrial function and, specifically, the electron transport chain. This defect correlated with reduced mitochondrial volume fraction in soleus muscle and heart, but not brown adipose tissue (BAT). Under ambient temperature conditions, PGC-1β ablation was partially compensated by up-regulation of PGC-1α in BAT and white adipose tissue (WAT) that lead to increased thermogenesis, reduced body weight, and reduced fat mass. Despite their decreased fat mass, PGC1βKO mice had hypertrophic adipocytes in WAT. The thermogenic role of PGC-1β was identified in thermoneutral and cold-adapted conditions by inadequate responses to norepinephrine injection. Furthermore, PGC1βKO hearts showed a blunted chronotropic response to dobutamine stimulation, and isolated soleus muscle fibres from PGC1βKO mice have impaired mitochondrial function. Lack of PGC-1β also impaired hepatic lipid metabolism in response to acute high fat dietary loads, resulting in hepatic steatosis and reduced lipoprotein-associated triglyceride and cholesterol content. Altogether, our data suggest that PGC-1β plays a general role in controlling basal mitochondrial function and also participates in tissue-specific adaptive responses during metabolic stress.

Mitochondrial dysfunction may play a role in the pathogenesis of several renal diseases. Although functional roles and metabolic demands differ among tubule segments, relatively little is known about the properties of mitochondria in different parts of the nephron. Clinically, the proximal tubule seems particularly vulnerable to mitochondrial toxicity. In this study, we used multiphoton imaging of live rat kidney slices to investigate differences in mitochondrial function along the nephron. The mitochondrial membrane potential was markedly higher in distal than proximal tubules. Inhibition of respiration rapidly collapsed the membrane potential in proximal tubules, but potential was better maintained in distal tubules. Inhibition of the F 1 F o -ATPase abolished this difference, suggesting that maintenance of potential via ATPase activity is more effective in distal than proximal tubules. Immunostaining revealed that the ratio of the expression of ATPase to IF1, an endogenous inhibitor of the mitochondrial ATPase, was lower in proximal tubules than in distal tubules. Production of reactive oxygen species was higher in proximal than distal cells, but inhibition of NADPH oxidase eliminated this difference. Glutathione levels were higher in proximal tubules. Overall, mitochondria in the proximal tubules were in a more oxidized state than those in the distal tubules. In summary, there are axial differences in mitochondrial function along the nephron, which may contribute to the pattern and pathophysiology of some forms of renal injury.

No abstract

Leak of protons into the mitochondrial matrix during substrate oxidation partially uncouples electron transport from phosphorylation of ADP, but the functions and source of basal and inducible proton leak in vivo remain controversial. In the present study we describe an endogenous activation of proton conductance in mitochondria isolated from rat and mouse skeletal muscle following addition of respiratory substrate. This endogenous activation increased with time, required a high membrane potential and was diminished by high concentrations of serum albumin. Inhibition of this endogenous activation by GDP [classically considered specific for UCPs (uncoupling proteins)], carboxyatractylate and bongkrekate (considered specific for the adenine nucleotide translocase) was examined in skeletal muscle mitochondria from wild-type and Ucp3-knockout mice. Proton conductance through endogenously activated UCP3 was calculated as the difference in leak between mitochondria from wild-type and Ucp3-knockout mice, and was found to be inhibited by carboxyatractylate and bongkrekate, but not GDP. Proton conductance in mitochondria from Ucp3-knockout mice was strongly inhibited by carboxyatractylate, bongkrekate and partially by GDP. We conclude the following: (i) at high protonmotive force, an endogenously generated activator stimulates proton conductance catalysed partly by UCP3 and partly by the adenine nucleotide translocase; (ii) GDP is not a specific inhibitor of UCP3, but also inhibits proton translocation by the adenine nucleotide translocase; and (iii) the inhibition of UCP3 by carboxyatractylate and bongkrekate is likely to be indirect, acting through the adenine nucleotide translocase.

Nonalcoholic steatohepatitis (NASH) is a common feature of the metabolic syndrome and toxic reactions to pharmacological drugs. Tamoxifen, (TMX) a widely used anti-breast cancer drug, can induce NASH and changes in plasma cholesterol levels through mechanisms that are unclear. We studied primary actions of TMX using a short-term treatment (5 days) that induces microvesicular hepatic steatosis and marked hypercholesterolemia in male rats. Using a combined approach of gene expression profiling and NMR-based metabolite analysis, we found that TMX-treated livers have increased saturated fatty acid content despite changes in gene expression, indicating decreased de novo lipogenesis and increased fatty acid oxidation. Our results show that TMX predominantly down-regulates FAS expression and activity as indicated by the accumulation of malonyl-CoA, a known inhibitor of mitochondrial beta-oxidation. In the face of a continued supply of exogenous free fatty acids, the blockade of fatty acid oxidation produced by elevated malonyl-CoA is likely to be the major factor leading to steatosis. Use of a combination of metabolomic and transcriptomic analysis has allowed us to identify mechanisms underlying important metabolic side effects of a widely prescribed drug. Given the broader importance of hepatic steatosis, the novel molecular mechanism revealed in this study should be examined in other forms of steatosis and nonalcoholic steatohepatitis.

The mitochondrial Ca 2+ uniporter (MCU) has been characterized for several decades [1], but the molecule responsible for this transport activity is currently unknown. Therefore, a recent Nature Cell Biology paper by Trenker et al. [2] reporting a role for uncoupling proteins (UCP2/3) in mitochondrial Ca 2+ uniport generated much excitement in the field. Subsequently, the authors contend that Ca 2+ transport accounts for most physiologic effects assigned to UCPs [3].The defining characteristic of mitochondrial Ca 2+ uptake is that mitochondrial matrix Ca 2+ ([Ca 2+ ] m ) responds to changes in cytosolic Ca 2+ at a rate dependent on both the driving force (the electrochemical gradient comprising membrane potential (Δψ m ) plus Ca 2+ concentration gradient), and the transporter activity [4]. The steady-state free [Ca 2+ ] m depends on the balance between Ca 2+ uptake and efflux [4]. Trenker et al. report that UCP overexpression increased the free [Ca 2+ ] m attained upon agonist stimulation (Fig. 1c in [2]), apparently without effect on the rate of Ca 2+ uptake. When mitochondrial Ca 2+ efflux via the Na + /Ca 2+ exchanger was blocked using CGP37157, and the driving force for Ca 2+ entry (Δψ m ) was collapsed by oligomycin plus FCCP, agonist-stimulated free [Ca 2+ ] m was again higher in UCP overexpressing cells, with no discernible effect on Ca 2+ uptake rate (Fig. 1f in [2]). In such a system, where both Ca 2+ uptake and efflux are inactive, an effect of UCP expression on steady-state free [Ca 2+ ] m levels cannot be due to changes in Ca 2+ transport. Since overexpression of UCPs can sometimes result in a non-functional uncoupled phenotype thought to be due to protein misfolding [5], and the apparent lack of effect of UCP overexpression on Δψ m (supplement Fig. 1d ]), the significance of these data is also difficult to assess.Alternative to the MCU, the Na + /Ca 2+ or H + /Ca 2+ exchangers could serve a mitochondrial Ca 2+ uptake function, in which UCPs may play a role. However, the V max of these systems is 2 or 18 nmol Ca 2+ /min/mg protein for liver or heart mitochondria respectively, i.e. 2-3 orders of magnitude lower than the V max of the MCU (1700 nmol Ca 2+ /min/mg protein) [6,7]. Thus UCPs cannot significantly affect mitochondrial Ca 2+ uptake through such mechanisms.In Fig. 3 of [2], Ca 2+ uptake by isolated murine liver mitochondria was only partially inhibited by 100 nM ruthenium red (RR), although it is not clear whether crude RR was used (IC 50 ~1 µM for MCU activity) or its purified derivative Ru360 (IC 50 5 nM) [4,8]. Using this experimental system, a decrease in Ca 2+ uptake by liver mitochondria of Ucp2 −/− mice was observed relative to wild-type controls. However, liver mitochondria contain exceedingly small amounts of UCP2 (<1 ng UCP2/mg protein, vs. 25 or 75 ng UCP2/mg protein in mouse kidney or spleen mitochondria respectively; V. Azzu and M.D. Brand unpublished observations). Furthermore, the mitochondrial inner membrane surface area is 5 × 10 10 µm 2 / mg protein [9] and each µm 2 cont...

Background: The human 6-16 and ISG12 genes are transcriptionally upregulated in a variety of cell types in response to type I interferon (IFN). The predicted products of these genes are small (12.9 and 11.5 kDa respectively), hydrophobic proteins that share 36% overall amino acid identity. Gene disruption and over-expression studies have so far failed to reveal any biochemical or cellular roles for these proteins.