Blood based bioenergetic profiling strategies are emerging as potential reporters of systemic mitochondrial function; however, the extent to which these measures reflect the bioenergetic capacity of other tissues is not known. The premise of this work is that highly metabolically active tissues, such as skeletal and cardiac muscle, are susceptible to differences in systemic bioenergetic capacity. Therefore, we tested whether the respiratory capacity of blood cells, monocytes and platelets, are related to contemporaneous respirometric assessments of skeletal and cardiac muscle mitochondria. 18 female vervet/African green monkeys (Chlorocebus aethiops sabaeus) of varying age and metabolic status were examined for this study. Monocyte and platelet maximal capacity correlated with maximal oxidative phosphorylation capacity of permeabilized skeletal muscle (R=0.75, 95% confidence interval [CI]: 0.38-0.97; R=0.51, 95%CI: 0.05-0.81; respectively), isolated skeletal muscle mitochondrial respiratory control ratio (RCR; R=0.70, 95%CI: 0.35-0.89; R=0.64, 95%CI: 0.23-0.98; respectively), and isolated cardiac muscle mitochondrial RCR (R=0.55, 95%CI: 0.22-0.86; R=0.58, 95%CI: 0.22-0.85; respectively). These results suggest that blood based bioenergetic profiling may be used to report on the bioenergetic capacity of muscle tissues. Blood cell respirometry represents an attractive alternative to tissue based assessments of mitochondrial function in human studies based on ease of access and the minimal participant burden required by these measures.

Inner mitochondrial membrane peptidase 2-like (IMMP2L) protein is a mitochondrial inner membrane peptidase that cleaves the signal peptide sequences of cytochrome c1 (CYC1) and mitochondrial glycerol phosphate dehydrogenase (GPD2). Immp2l mutant mice show infertility and early signs of aging. It is unclear whether mitochondrial respiratory deficiency underlies this phenotype. Here we show that the intermediate forms of GPD2 and CYC1 have normal expression levels and enzymatic function in Immp2l mutants. Mitochondrial respiration is not diminished in isolated mitochondria and cells from mutant mice. Our data suggest that respiratory deficiency is not the cause of the observed Immp2l mutant phenotypes.

Objective

Obesity exists with and without accompanying cardiometabolic disease, termed metabolically unhealthy obesity (MUO) and healthy obesity (MHO), respectively. Underlying differences in the ability of subcutaneous (SQ) fat to respond to nutrient excess are emerging as a key pathway. This study aimed to document the first spontaneous animal model of MHO and MUO and differences in SQ adipose tissue.

Methods

Vervet monkeys (Chlorocebus aethiops; N = 171) were screened for metabolic syndrome. A subset of MHO and MUO monkeys (n = 6/group) had SQ fat biopsies collected for histological evaluations and examination of key mitochondrial proteins.

Results

Obesity was seen in 20% of monkeys, and within this population, 31% were healthy, which mirrors human prevalence estimates. MUO monkeys had more than 60% lower adiponectin concentrations despite similar fat cell size, uncoupling protein 3, and activated macrophage abundance. However, alternatively activated/anti-inflammatory macrophages were 70% lower. Deficiencies of 50% or more in mitochondrial quality control regulators and selected mitochondrial fission and fusion markers were observed in the SQ fat of MUO monkeys despite comparable mitochondrial content.

Conclusions

A novel and translatable spontaneously obese animal model of MHO and MUO, occurring independently of dietary factors, was characterized. Differences in mitochondrial quality and inflammatory cell populations of subcutaneous fat may underpin divergent metabolic health.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 μg of mitochondria indicate that 1.0 μg is sufficient to measure respiration and that 5.0 μg provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Background

Mitochondrial function declines with age; however, the relationship between adiposity and mitochondrial function among older adults is unclear. This study examined relationships between skeletal muscle mitochondrial content and electron transport chain complex 2 driven respiration with whole body and thigh composition, body fat distribution, and insulin sensitivity in older adults.

Methods

25 healthy, sedentary, weight-stable men (N = 13) and women (N = 12) >65 years of age, with a BMI range of 18-35 kg/m(2), participated in this study. Vastus lateralis biopsies were analyzed for citrate synthase (CS) activity and succinate mediated respiration of isolated mitochondria. Whole body and thigh composition were measured by DXA and CT. HOMA-IR was calculated using fasting glucose and insulin as an estimate of insulin sensitivity.

Results

Similar to reports in middle-aged adults, skeletal muscle CS activity was negatively correlated with BMI (R = -0.43) in our cohort of older adults. Higher total and thigh adiposity were correlated with lower CS activity independent of BMI (R = -0.50 and -0.71 respectively). Maximal complex 2 driven mitochondrial respiration was negatively correlated with lower body adiposity in males (R = -0.66). In this cohort of non-diabetic older adults, both HOMA-IR and insulin were positively correlated with CS activity when controlling for BMI (R = 0.57 and 0.66 respectively).

Conclusions

Adiposity and body composition are correlated with skeletal muscle mitochondrial content and electron transport chain function in healthy, sedentary, community dwelling, older adults. Specific relationships of mitochondrial bioenergetics with gender and insulin sensitivity are also apparent.

Trial registration

ClinicalTrials.gov identifier NCT01049698.

Bariatric surgeries including vertical sleeve gastrectomy (VSG) ameliorate obesity and diabetes. Weight loss and accompanying increases to insulin sensitivity contribute to improved glycemia after surgery; however, studies in humans also suggest weight-independent actions of bariatric procedures to lower blood glucose, possibly by improving insulin secretion. To evaluate this hypothesis, we compared VSG-operated mice with pair-fed, sham-surgical controls (PF-Sham) 2 weeks after surgery. This paradigm yielded similar postoperative body weight and insulin sensitivity between VSG and calorically restricted PF-Sham animals. However, VSG improved glucose tolerance and markedly enhanced insulin secretion during oral nutrient and i.p. glucose challenges compared with controls. Islets from VSG mice displayed a unique transcriptional signature enriched for genes involved in Ca2+ signaling and insulin secretion pathways. This finding suggests that bariatric surgery leads to intrinsic changes within the islet that alter function. Indeed, islets isolated from VSG mice had increased glucose-stimulated insulin secretion and a left-shifted glucose sensitivity curve compared with islets from PF-Sham mice. Isolated islets from VSG animals showed corresponding increases in the pulse duration of glucose-stimulated Ca2+ oscillations. Together, these findings demonstrate a weight-independent improvement in glycemic control following VSG, which is, in part, driven by improved insulin secretion and associated with substantial changes in islet gene expression. These results support a model in which β cells play a key role in the adaptation to bariatric surgery and the improved glucose tolerance that is typical of these procedures.

Blood-based bioenergetic profiling provides a minimally invasive assessment of mitochondrial health shown to be related to key features of aging. Previous studies show that blood cells recapitulate mitochondrial alterations in the central nervous system under pathological conditions, including the development of Alzheimer's disease. In this study of nonhuman primates, we focus on mitochondrial function and bioenergetic capacity assessed by the respirometric profiling of monocytes, platelets, and frontal cortex mitochondria. Our data indicate that differences in the maximal respiratory capacity of brain mitochondria are reflected by CD14+ monocyte maximal respiratory capacity and platelet and monocyte bioenergetic health index. A subset of nonhuman primates also underwent [¹⁸F] fluorodeoxyglucose positron emission tomography (FDG-PET) imaging to assess brain glucose metabolism. Our results indicate that platelet respiratory capacity positively correlates to measures of glucose metabolism in multiple brain regions. Altogether, the results of this study provide early evidence that blood-based bioenergetic profiling is related to brain mitochondrial metabolism. While these measures cannot substitute for direct measures of brain metabolism, provided by measures such as FDG-PET, they may have utility as a metabolic biomarker and screening tool to identify individuals exhibiting systemic bioenergetic decline who may therefore be at risk for the development of neurodegenerative diseases.

Mitochondrial reactive oxygen species (ROS) are essential regulators of cellular signaling, metabolism and epigenetics underlying the pathophysiology of numerous diseases. Despite the critical function of redox regulation in mitochondria, currently there are limited methods available to monitor protein oxidation in this key subcellular organelle. Here, we describe compounds for imaging sulfenylated proteins in mitochondria: DCP-NEt2-Coumarin (DCP-NEt2C) and rhodamine-based DCP-Rho1. Side-by-side comparison studies are presented on the reactivity of DCP-NEt2C and DCP-Rho1 with a model protein sulfenic acid (AhpC-SOH) and mitochondrial localization to identify optimized experimental conditions for labeling and visualization of protein sulfenylation that would be independent of mitochondria membrane potential and would not impact mitochondrial function. These probes are applied to image mitochondrial protein sulfenylation under conditions of serum starvation and in a cell culture model of lung cancer exposed to ionizing radiation and silver nanoparticles, agents serving dual functions as environmental stressors and cancer therapeutics.

Introduction

APOL1 G1 and G2 nephropathy-risk variants cause mitochondrial dysfunction and contribute to kidney disease. Analyses were performed to determine the genetic regulation of APOL1 and elucidate potential mechanisms in APOL1-nephropathy.

Methods

A global gene expression analysis was performed in human primary renal tubule cell lines derived from 50 African American individuals. Follow-up gene knock out, cell-based rescue, and microscopy experiments were performed.

Results

APOL1 genotypes did not alter APOL1 expression levels in the global gene expression analysis. Expression quantitative trait locus (eQTL) analysis in polyinosinic-polycytidylic acid (poly IC)-stimulated renal tubule cells revealed that single nucleotide polymorphism (SNP) rs513349 adjacent to BAK1 was a trans eQTL for APOL1 and a cis eQTL for BAK1; APOL1 and BAK1 were co-expressed in cells. BAK1 knockout in a human podocyte cell line resulted in diminished APOL1 protein, supporting a pivotal effect for BAK1 on APOL1 expression. Because BAK1 is involved in mitochondrial dynamics, mitochondrial morphology was examined in primary renal tubule cells and HEK293 Tet-on cells of various APOL1 genotypes. Mitochondria in APOL1 wild-type (G0G0) tubule cells maintained elongated morphology when stimulated by low-dose poly IC, whereas those with G1G1, G2G2, and G1G2 genotypes appeared to fragment. HEK293 Tet-on cells overexpressing APOL1 G0, G1, and G2 were created; G0 cells appeared to promote mitochondrial fusion, whereas G1 and G2 induced mitochondrial fission. The mitochondrial dynamic regulator Mdivi-1 significantly preserved cell viability and mitochondrial cristae structure and reversed mitochondrial fission induced by overexpression of G1 and G2.

Conclusion

Results suggest the mitochondrial fusion/fission pathway may be a therapeutic target in APOL1-nephropathy.