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Pioglitazone Induces Mitochondrial Biogenesis in Human Subcutaneous Adipose Tissue In Vivo Iwona Bogacka , Hui Xie , George A. Bray and Steven R. Smith From the Molecular Endocrinology Laboratory, Pennington Biomedical Research Center, Baton Rouge, Louisiana Address correspondence and reprint requests to Iwona Bogacka, Pennington Biomedical Research Center, 6400 Perkins Rd., Baton Rouge, LA. E-mail: bogackiu{at}pbrc.edu Abstract Thiazolidenediones such as pioglitazone improve insulin sensitivity in diabetic patients by several mechanisms, including increased uptake and metabolism of free fatty acids in adipose tissue. The purpose of the present study was to determine the effect of pioglitazone on mitochondrial biogenesis and expression of genes involved in fatty acid oxidation in subcutaneous fat. Patients with type 2 diabetes were randomly divided into two groups and treated with placebo or pioglitazone (45 mg/day) for 12 weeks. Mitochodrial DNA copy number and expression of genes involved in mitochondrial biogenesis were quantified by real-time PCR. Pioglitazone treatment significantly increased mitochondrial copy number and expression of factors involved in mitochondrial biogenesis, including peroxisome proliferator–activated receptor (PPAR)-γ coactivator-1α and mitochondrial transcription factor A. Treatment with pioglitazone stimulated the expression of genes in the fatty acid oxidation pathway, including carnitine palmitoyltransferase-1, malonyl-CoA decarboxylase, and medium-chain acyl-CoA dehydrogenase. The expression of PPAR-α, a transcriptional regulator of genes encoding mitochondrial enzymes involved in fatty acid oxidation, was higher after pioglitazone treatment. Finally, the increased mitochondrial copy number and the higher expression of genes involved in fatty acid oxidation in human adipocytes may contribute to the hypolipidemic effects of pioglitazone. ADRB3, adrenergic β-3 receptor CPT-1, carnitine palmitoylotransferase I FPG, fasting plasma glucose MCAD, medium-chain acyl-CoA dehydrogenase MLYCD, malonyl-CoA decarboxylase mtDNA, mitochondrial DNA mtTFA, mitochondrial transcription factor A NRF, nuclear respiratory factor PGC-1α, peroxisome proliferator–activated receptor coactivator-1α PPAR, peroxisome proliferator–activated receptor TZD, thiazolidinedione UCP, uncoupling protein Footnotes I.B. has received grant/research support from Takeda Pharmaceuticals. H.X. has received grant/research support from Takeda Pharmaceuticals. G.A.B. has served on an advisory board of, has received honoraria from, and has received grant/research support from Takeda Pharmaceuticals. S.R.S. has received honoraria/consulting fees and research support from Takeda Pharmaceuticals. Accepted February 17, 2005. Received June 23, 2004. DIABETES

Background: Associations between prenatal household air pollution (HAP) exposure or cookstove intervention to reduce HAP and cord blood mononuclear cell (CBMC) mitochondrial deoxyribonucleic acid copy number (mtDNAcn), an oxidative stress biomarker, are unknown. Materials and Methods: Pregnant women were recruited and randomized to one of two cookstove interventions, including a clean-burning liquefied petroleum gas (LPG) stove, or control. Prenatal HAP exposure was determined by serial, personal carbon monoxide (CO) measurements. CBMC mtDNAcn was measured by quantitative polymerase chain reaction. Multivariable linear regression determined associations between prenatal CO and cookstove arm on mtDNAcn. Associations between mtDNAcn and birth outcomes and effect modification by infant sex were explored. Results: LPG users had the lowest CO exposures (p = 0.02 by ANOVA). In boys only, average prenatal CO was inversely associated with mtDNAcn (β = -14.84, SE = 6.41, p = 0.03, per 1ppm increase in CO). When examined by study arm, LPG cookstove had the opposite effect in all children (LPG β = 19.34, SE = 9.72, p = 0.049), but especially boys (β = 30.65, SE = 14.46, p = 0.04), as compared to Control. Increased mtDNAcn was associated with improved birth outcomes. Conclusions: Increased prenatal HAP exposure reduces CBMC mtDNAcn, suggesting cumulative prenatal oxidative stress injury. An LPG stove intervention may reverse this effect. Boys appear most susceptible.

Background. The effect of nonthymidine nucleoside reverse-transcriptase inhibitors (NRTIs) on fat mitochondrial DNA (mtDNA) content and function is unclear. Methods. A5202 randomized antiretroviral therapy—naive human immunodeficiency virus—infected subjects to abacavir-lamivudine (ABC/3TC) versus tenofovir DF—emtricitabine (TDF/FTC) with efavirenz (EFV) or atazanavir-ritonavir (ATV/r). A5224s, substudy of A5202, enrolled 269 subjects with fat measurements by dual-energy x-ray absorptiometry and computed tomography. A subset of subjects underwent fat biopsies at baseline and week 96 for mtDNA content (real-time polymerase chain reaction) and oxidative phosphorylation nicotinamide adenine dinucleotide (reduced) dehydrogenase (complex I) and cytochrome c oxidase (complex IV) activity levels (immunoassays). Intent-to-treat analyses were performed using analysis of variance and paired t tests. Results. Fifty-six subjects (87% male; median age, 39 years) were included; their median body mass index, CD4 cell count, and fat mtDNA level were 26 kg/m 2 , 227 cells/μL, and 1197 copies/cell, respectively. Fat mtDNA content decreased within the ABC/3TC and TDF/FTC groups (combining EFV and ATV/r arms; median change, -341 [interquartile range, -848 to 190; P = .03] and -400 [-661 to -221; P < .001] copies/cell, respectively), but these changes did not differ significantly between the 2 groups (P = .57). Complex I and IV activity decreased significantly in the TDF/FTC group (median change, -12.45 [interquartile range, -24.70 to 2.90; P = .003] and -8.25 [-13.90 to -1.30; P < .001], optical density × 10 3 /μg, respectively) but not the ABC/3TC group. Differences between the ABC/3TC and TDF/FTC groups were significant for complex I (P = .03). Conclusions. ABC/3TC and TDF/FTC significantly and similarly decreased fat mtDNA content, but only TDF/FTC decreased complex I and complex IV activity levels. Clinical Trials Registration. NCT00118898.

•We examine the effect of red ginseng on mitochondrial function, hormones and metabolic syndrome in men with metabolic syndrome.•We show that red ginseng increase growth hormone and mitochondria DNA copy number after 4 weeks treatment.•We did not show an improvement of metabolic syndrome component except diastolic BP, after 4 weeks treatment. It has been observed that mitochondrial dysfunction is associated with an increased risk of metabolic syndrome. There is growing evidence that hyperactivity of the hypothalamus-pituitary-adrenal (HPA) axis and hormone (testosterone and growth hormone) deficiency may lead to metabolic syndrome. Recent studies have reported that ginseng treatment improves mitochondrial and HPA-axis function and increases anabolic hormone secretion. The objective of this study was to investigate the effect of red ginseng (RG) on metabolic syndrome, hormones, and mitochondrial function using leukocyte mitochondrial DNA copy number in men with metabolic syndrome. We performed a randomized, double-blind, placebo-controlled study in 62 subjects who were not taking drugs that could affect their metabolic function. A total of 62 men with metabolic syndrome were randomly assigned to either an RG group (3.0g/day) or a placebo group for 4 weeks. We analyzed changes in metabolic syndrome components, leukocyte mitochondrial DNA copy number, hormones (total testosterone, IGF-1, cortisol, and DHEAS) and inflammatory markers (C-reactive protein, ferritin) from baseline to 4 weeks. Significant improvement in mitochondrial function (95% CI −44.9 to −1.3) and an increase in total testosterone (95% CI −70.1 to −1.0) and IGF-1(P=0.01) levels were observed in the RG group when compared with the placebo group. Diastolic blood pressure (95% CI 2.0–9.4) and serum cortisol (95% CI 1.1–5.5) significantly decreased in the RG group. We found evidence that RG had a favorable effect on mitochondrial function and hormones in men with metabolic syndrome.

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system 1 – 6 . The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS 7 – 17 , and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease. Inhibitors of mitochondrial transcription that target human mitochondrial RNA polymerase provide a chemical biology tool for studying the role of mitochondrial DNA expression in a wide range of pathologies.

Background. Stavudine is widely used in developing countries. Lipoatrophy and mitochondrial toxicity have been linked to stavudine use, but it is unclear whether switching to a lower dose can reduce these toxicities while maintaining human immunodeficiency virus (HIV) suppression. Methods. HIV-infected subjects receiving standard-dose stavudine with undetectable HIV type 1 RNA for ⩾6 months were randomized (ratio, 3:2) to receive one-half of the stavudine dose (switch arm) or to maintain the dose (continuation arm) while continuing to receive all other prescribed antiretrovirals. The following measurements were obtained at baseline and week 48: fasting lactate, pyruvate, and lipid levels; results of whole-body dual-energy x-ray absorptiometry; and mitochondrial DNA (mtDNA) measurements in fat and peripheral blood mononuclear cells. Change from baseline to week 48 was compared within and between groups. Results. Twenty-four patients (79% of whom were men and 79% of whom were African American; median age, 45 years) were enrolled in the study, 15 were enrolled in the switch arm, and 9 were enrolled in the continuation arm. The median duration of stavudine treatment was 55 months (range, 21–126 months). The median CD4 cell count was 558 cells/mm3 (range, 207–1698 cells/mm3). At baseline, the study arms had similar demographic characteristics and laboratory indices, except for body mass index, total lean body mass, and triglyceride levels (all of which were higher in the switch arm). Three patients (2 in the switch arm) discontinued the study because of study-unrelated reasons. CD4 cell counts remained unchanged. At 48 weeks, 6 patients (4 [27%] in the switch arm and 2 [22%] in the continuation arm) had detectable HIV RNA levels (median, 972 copies/mL; range, 60–49,400 copies/mL). All patients with detectable HIV RNA levels reported significant lapses in treatment adherence; none exhibited mutations in HIV genotype. After the treatment switch, significant changes from study entry to week 48 were noted only for lactate level (median change, −0.27 mmol/L; range, −1.2 to 0.25 mmol/L; P=.02) and fat mtDNA (median change, 40 copies/cell; range, −49 to 261 copies/cell; P=.02). In the continuation arm, a significant loss of bone mineral density was seen at week 48 (median change, −1.7%; range, −6.3% to 0.8%; P=.02). The only significant between-group difference was the change in bone mineral density from baseline (P=.003). Conclusions. Reducing stavudine dose by one-half increased fat mtDNA and decreased lactate levels, suggesting improvement in mitochondrial indices while preserving HIV suppression in subjects who maintained adherence. A significant loss of bone mineral density was seen in patients receiving standard-dose stavudine but not in those receiving low-dose stavudine. These results suggest that switching to low-dose stavudine may improve mitochondrial indices while maintaining virological suppression.

The naturally occurring compound urolithin A has been found to promote mitophagy, thereby increasing lifespan in worms and improving skeletal muscle activity in rodents. The biological effects of urolithins remain poorly characterized, despite wide-spread human exposure via the dietary consumption of their metabolic precursors, the ellagitannins, which are found in the pomegranate fruit, as well as in nuts and berries. We identified urolithin A (UA) as a first-in-class natural compound that induces mitophagy both in vitro and in vivo following oral consumption. In C. elegans , UA prevented the accumulation of dysfunctional mitochondria with age and extended lifespan. Likewise, UA prolonged normal activity during aging in C. elegans , including mobility and pharyngeal pumping, while maintaining mitochondrial respiratory capacity. These effects translated to rodents, where UA improved exercise capacity in two different mouse models of age-related decline of muscle function, as well as in young rats. Our findings highlight the health benefits of urolithin A and its potential application in strategies to improve mitochondrial and muscle function.

Although the development of mitochondrial therapies has largely focused on diseases caused by mutations in mitochondrial DNA or in nuclear genes encoding mitochondrial proteins, it has been found that mitochondrial dysfunction also contributes to the pathology of many common disorders, including neurodegeneration, metabolic disease, heart failure, ischaemia-reperfusion injury and protozoal infections. Mitochondria therefore represent an important drug target for these highly prevalent diseases. Several strategies aimed at therapeutically restoring mitochondrial function are emerging, and a small number of agents have entered clinical trials. This Review discusses the opportunities and challenges faced for the further development of mitochondrial pharmacology for common pathologies.

Cells respond to environmental stressors through several key pathways, including response to reactive oxygen species (ROS), nutrient and ATP sensing, DNA damage response (DDR), and epigenetic alterations. Mitochondria play a central role in these pathways not only through energetics and ATP production but also through metabolites generated in the tricarboxylic acid cycle, as well as mitochondria-nuclear signaling related to mitochondria morphology, biogenesis, fission/fusion, mitophagy, apoptosis, and epigenetic regulation. We investigated the concept of bidirectional interactions between mitochondria and cellular pathways in response to environmental stress with a focus on epigenetic regulation, and we examined DNA repair and DDR pathways as examples of biological processes that respond to exogenous insults through changes in homeostasis and altered mitochondrial function. The National Institute of Environmental Health Sciences sponsored the Workshop on Mitochondria, Energetics, Epigenetics, Environment, and DNA Damage Response on 25-26 March 2013. Here, we summarize key points and ideas emerging from this meeting. A more comprehensive understanding of signaling mechanisms (cross-talk) between the mitochondria and nucleus is central to elucidating the integration of mitochondrial functions with other cellular response pathways in modulating the effects of environmental agents. Recent studies have highlighted the importance of mitochondrial functions in epigenetic regulation and DDR with environmental stress. Development and application of novel technologies, enhanced experimental models, and a systems-type research approach will help to discern how environmentally induced mitochondrial dysfunction affects key mechanistic pathways. Understanding mitochondria-cell signaling will provide insight into individual responses to environmental hazards, improving prediction of hazard and susceptibility to environmental stressors.

Major depressive disorder (MDD) has been linked to mitochondrial defects, which could manifest in mitochondrial DNA (mtDNA) polymorphisms or mutations. Additionally, copy number of mtDNA (mtDNA-cn) can be quantified in peripheral blood mononuclear cells (PBMC)s, indirectly reflecting cellular energetics, or in the circulating cell-free mtDNA (ccf-mtDNA) levels, which may reflect a fraction of the mitochondrial genome released during cellular stress. Few studies have examined ccf-mtDNA in MDD, and no studies have tested its relationship with intracellular mtDNA-cn or with antidepressant treatment response. Here, mtDNA levels were quantified in parallel from: (i) PBMCs and (ii) cell-free plasma of 50 unmedicated MDD subjects and 55 controls, in parallel with PBMC telomere length (TL) and antioxidant enzyme glutathione peroxidase (GpX) activity. MtDNA measures were repeated in 19 MDD subjects after 8 weeks of open-label SSRI treatment. In analyses adjusted for age, sex, BMI, and smoking, MDD subjects had significantly elevated levels of ccf-mtDNA (F = 20.6, p = 0.00002). PBMC mtDNA-cn did not differ between groups (p > 0.4). In preliminary analyses, we found that changes in ccf-mtDNA with SSRI treatment differed between SSRI responders and non-responders (F = 6.47, p = 0.02), with the non-responders showing an increase in ccf-mtDNA and responders not changing. Baseline ccf-mtDNA was positively correlated with GpX (r = 0.32, p = 0.001), and PBMC mtDNA correlated positively with PBMC TL (r = 0.38, p = 0.0001). These data suggest that plasma ccf-mtDNA and PBMC mtDNA-cn reflect different cellular processes and that the former may be more reflective of certain aspects of MDD pathophysiology and of the response to SSRI antidepressants.