Publications by authors named "Anthony J Molina"

34 Publications

Mitochondria-dependent synthetic small-molecule vaccine adjuvants for influenza virus infection.

Proc Natl Acad Sci U S A 2021 Jun;118(23)

Moores Cancer Center, University of California San Diego, La Jolla, CA 92093-0809;

Vaccine adjuvants enhance and prolong pathogen-specific protective immune responses. Recent reports indicate that host factors-such as aging, pregnancy, and genetic polymorphisms-influence efficacies of vaccines adjuvanted with Toll-like receptor (TLR) or known pattern-recognition receptor (PRR) agonists. Although PRR independent adjuvants (e.g., oil-in-water emulsion and saponin) are emerging, these adjuvants induce some local and systemic reactogenicity. Hence, new TLR and PRR-independent adjuvants that provide greater potency alone or in combination without compromising safety are highly desired. Previous cell-based high-throughput screenings yielded a small molecule 81 [-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide], which enhanced lipopolysaccharide-induced NF-κB and type I interferon signaling in reporter assays. Here compound 81 activated innate immunity in primary human peripheral blood mononuclear cells and murine bone marrow-derived dendritic cells (BMDCs). The innate immune activation by 81 was independent of TLRs and other PRRs and was significantly reduced in mitochondrial antiviral-signaling protein (MAVS)-deficient BMDCs. Compound 81 activities were mediated by mitochondrial dysfunction as mitophagy inducers and a mitochondria specific antioxidant significantly inhibited cytokine induction by 81. Both compound 81 and a derivative obtained via structure-activity relationship studies, 2F52 [-benzyl--(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] modestly increased mitochondrial reactive oxygen species and induced the aggregation of MAVS. Neither 81 nor 2F52 injected as adjuvants caused local or systemic toxicity in mice at effective concentrations for vaccination. Furthermore, vaccination with inactivated influenza virus adjuvanted with 2F52 demonstrated protective effects in a murine lethal virus challenge study. As an unconventional and safe adjuvant that does not require known PRRs, compound 2F52 could be a useful addition to vaccines.
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http://dx.doi.org/10.1073/pnas.2025718118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201894PMC
June 2021

Heterochronic Parabiosis: Old Blood Induces Changes in Mitochondrial Structure and Function of Young Mice.

J Gerontol A Biol Sci Med Sci 2021 02;76(3):434-439

Division of Geriatrics and Gerontology, Department of Medicine, University of California San Diego School of Medicine, La Jolla.

Heterochronic parabiosis models have been utilized to demonstrate the role of blood-borne circulating factors in systemic effects of aging. In previous studies, heterochronic parabiosis has shown positive effects across multiple tissues in old mice. More recently, a study demonstrated old blood had a more profound negative effect on muscle performance and neurogenesis of young mice. In this study, we used heterochronic parabiosis to test the hypothesis that circulating factors mediate mitochondrial bioenergetic decline, a well-established biological hallmark of aging. We examined mitochondrial morphology, expression of mitochondrial complexes, and mitochondrial respiration from skeletal muscle of mice connected as heterochronic pairs, as well as young and old isochronic controls. Our results indicate that young heterochronic mice had significantly lower total mitochondrial content and on average had significantly smaller mitochondria compared to young isochronic controls. Expression of complex IV followed a similar pattern: young heterochronic mice had a trend for lower expression compared to young isochronic controls. Additionally, respirometric analyses indicate that young heterochronic mice had significantly lower complex I, complex I + II, and maximal mitochondrial respiration and a trend for lower complex II-driven respiration compared to young isochronic controls. Interestingly, we did not observe significant improvements in old heterochronic mice compared to old isochronic controls, demonstrating the profound deleterious effects of circulating factors from old mice on mitochondrial structure and function. We also found no significant differences between the young and old heterochronic mice, demonstrating that circulating factors can be a driver of age-related differences in mitochondrial structure and function.
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http://dx.doi.org/10.1093/gerona/glaa299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8177798PMC
February 2021

An Acidic Environment Induces APOL1-Associated Mitochondrial Fragmentation.

Am J Nephrol 2020 31;51(9):695-704. Epub 2020 Aug 31.

Department of Internal Medicine, Section on Nephrology, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.

Background: Apolipoprotein L1 gene (APOL1) G1 and G2 kidney-risk variants (KRVs) cause CKD in African Americans, inducing mitochondrial dysfunction. Modifying factors are required, because a minority of individuals with APOL1 high-risk genotypes develop nephropathy. Given that APOL1 function is pH-sensitive and the pH of the kidney interstitium is <7, we hypothesized the acidic kidney interstitium may facilitate APOL1 KRV-induced mitochondrial dysfunction.

Methods: Human embryonic kidney (HEK293) cells conditionally expressing empty vector (EV), APOL1-reference G0, and G1 or G2 KRVs were incubated in media pH 6.8 or 7.4 for 4, 6, or 8 h. Genotype-specific pH effects on mitochondrial length (µm) were assessed using confocal microscopy in live cells and Fiji derivative of ImageJ software with MiNA plug-in. Lower mitochondrial length indicated fragmentation and early dysfunction.

Results: After 6 h doxycycline (Dox) induction in pH 6.8 media, G2-expressing cells had shorter mitochondria (6.54 ± 0.40) than cells expressing EV (7.65 ± 0.72, p = 0.02) or G0 (7.46 ± 0.31, p = 0.003). After 8 h Dox induction in pH 6.8 media, both G1- (6.21 ± 0.26) and G2-expressing cells had shorter mitochondria (6.46 ± 0.34) than cells expressing EV (7.13 ± 0.32, p = 0.002 and p = 0.008, respectively) or G0 (7.22 ± 0.45, p = 0.003 and p = 0.01, respectively). Mitochondrial length in cells incubated in pH 7.4 media were comparable after 8 h Dox induction regardless of genotype. APOL1 mRNA expression and cell viability were comparable regardless of pH or genotype after 8 h Dox induction.

Conclusion: Acidic pH facilitates early mitochondrial dysfunction induced by APOL1 G1 and G2 KRVs in HEK293 cells. We propose that the acidic kidney interstitium may play a role in APOL1-mediated mitochondrial pathophysiology and nephropathy.
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http://dx.doi.org/10.1159/000509989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511450PMC
July 2021

Kidney-Risk Variants Induce Mitochondrial Fission.

Kidney Int Rep 2020 Jun 30;5(6):891-904. Epub 2020 Mar 30.

Department of Internal Medicine, Section on Nephrology, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.

Introduction: G1 and G2 nephropathy-risk variants cause mitochondrial dysfunction and contribute to kidney disease. Analyses were performed to determine the genetic regulation of and elucidate potential mechanisms in -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: genotypes did not alter 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 was a eQTL for and a eQTL for ; and were co-expressed in cells. knockout in a human podocyte cell line resulted in diminished APOL1 protein, supporting a pivotal effect for BAK1 on expression. Because BAK1 is involved in mitochondrial dynamics, mitochondrial morphology was examined in primary renal tubule cells and HEK293 Tet-on cells of various genotypes. Mitochondria in 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 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 -nephropathy.
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http://dx.doi.org/10.1016/j.ekir.2020.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271005PMC
June 2020

Solute Carrier Family 37 Member 2 (SLC37A2) Negatively Regulates Murine Macrophage Inflammation by Controlling Glycolysis.

iScience 2020 May 4;23(5):101125. Epub 2020 May 4.

Department of Internal Medicine, Section of Molecular Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA; Department of Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA. Electronic address:

Increased flux of glucose through glycolysis is a hallmark of inflammatory macrophages and is essential for optimal effector functions. Solute carrier (SLC) 37A2 is an endoplasmic reticulum-anchored phosphate-linked glucose-6-phosphate transporter that is highly expressed in macrophages and neutrophils. We demonstrate that SLC37A2 plays a pivotal role in murine macrophage inflammatory activation and cellular metabolic rewiring. Toll-like receptor (TLR) 4 stimulation by lipopolysaccharide (LPS) rapidly increases macrophage SLC37A2 protein expression. SLC37A2 deletion reprograms macrophages to a hyper-glycolytic process and accelerates LPS-induced inflammatory cytokine production, which partially depends on nicotinamide adenine dinucleotide (NAD) biosynthesis. Blockade of glycolysis normalizes the differential expression of pro-inflammatory cytokines between control and SLC37A2 deficient macrophages. Conversely, overexpression of SLC37A2 lowers macrophage glycolysis and significantly reduces LPS-induced pro-inflammatory cytokine expression. In conclusion, our study suggests that SLC37A2 dampens murine macrophage inflammation by down-regulating glycolytic reprogramming as a part of macrophage negative feedback system to curtail acute innate activation.
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http://dx.doi.org/10.1016/j.isci.2020.101125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232099PMC
May 2020

Mitochondrial GTP Links Nutrient Sensing to β Cell Health, Mitochondrial Morphology, and Insulin Secretion Independent of OxPhos.

Cell Rep 2019 07;28(3):759-772.e10

Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06519, USA; Departments of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06519, USA. Electronic address:

Mechanisms coordinating pancreatic β cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of β cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phosphoenolpyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and β cell health.
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http://dx.doi.org/10.1016/j.celrep.2019.06.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713209PMC
July 2019

Skeletal Muscle Mitochondrial Respiration Is Elevated in Female Cynomolgus Macaques Fed a Western Compared with a Mediterranean Diet.

J Nutr 2019 09;149(9):1493-1502

Section on Gerontology and Geriatrics, Department of Internal Medicine, Winston-Salem, NC.

Background: Western diets are associated with increased incidences of obesity, hypertension, diabetes, and hypercholesterolemia, whereas Mediterranean diets, richer in polyphenols, monounsaturated fats, fruits, vegetables, poultry, and fish, appear to have cardiometabolic health benefits. Previous work has included population-based studies with limited evidence for causation or animal studies focused on single macro- or micronutrients; therefore, primate animal models provide an opportunity to determine potential mechanisms underlying the effects of dietary patterns on health and disease.

Objective: The aim of this study was to determine the effects of whole dietary patterns, either a Western or Mediterranean diet, on skeletal muscle mitochondrial bioenergetics in cynomolgus macaques.

Methods: In this study, 22 adult female cynomolgus macaques (∼11-14 y by dentition) were fed either a Western or Mediterranean diet for 30 mo. The Western diet was designed to mimic the diet of a middle-aged American woman and the Mediterranean diet included key aspects of Mediterranean diets studied in humans, such as plant-based proteins and fat, complex carbohydrates, and fiber. Diets were matched on macronutrient composition (16% protein, 54% carbohydrate, and 31% fat) and cholesterol content. Skeletal muscle was collected for high-resolution respirometry, citrate synthase activity, and western blot measurements. Pearson correlation analysis between respirometry measures and measures of carbohydrate metabolism was also performed.

Results: We found that consumption of a Western diet resulted in significantly higher mitochondrial respiration with fatty acid oxidation (FAO) (53%), FAO + complex I (52%), complex I + II (31%), max electron transport system (ETS) (31%), and ETS rotenone sensitive (31%) than did consumption of a Mediterranean diet. In addition, measures of respiration in response to fatty acids were significantly and positively correlated with both insulin resistance and plasma insulin concentrations.

Conclusions: This study highlights the importance of dietary composition in mitochondrial bioenergetics and that diet can influence skeletal muscle mitochondrial respiration independently of other factors such as macronutrient composition.
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http://dx.doi.org/10.1093/jn/nxz092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736071PMC
September 2019

Blood-based bioenergetic profiling is related to differences in brain morphology in African Americans with Type 2 diabetes.

Clin Sci (Lond) 2018 12 5;132(23):2509-2518. Epub 2018 Dec 5.

Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest University Health Sciences, Winston-Salem, NC 27157, U.S.A.

Blood-based bioenergetic profiling has promising applications as a minimally invasive biomarker of systemic bioenergetic capacity. In the present study, we examined peripheral blood mononuclear cell (PBMC) mitochondrial function and brain morphology in a cohort of African Americans with long-standing Type 2 diabetes. Key parameters of PBMC respiration were correlated with white matter, gray matter, and total intracranial volumes. Our analyses indicate that these relationships are primarily driven by the relationship of systemic bioenergetic capacity with total intracranial volume, suggesting that systemic differences in mitochondrial function may play a role in overall brain morphology.
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http://dx.doi.org/10.1042/CS20180690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6512318PMC
December 2018

Mitochondria-targeted Probes for Imaging Protein Sulfenylation.

Sci Rep 2018 04 27;8(1):6635. Epub 2018 Apr 27.

Department of Internal Medicine, Section on Molecular Medicine, Wake Forest University Health Sciences, Winston-Salem, NC, 27157, USA.

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-NEt-Coumarin (DCP-NEtC) and rhodamine-based DCP-Rho1. Side-by-side comparison studies are presented on the reactivity of DCP-NEtC 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.
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http://dx.doi.org/10.1038/s41598-018-24493-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923234PMC
April 2018

Blood-Based Bioenergetic Profiling Reflects Differences in Brain Bioenergetics and Metabolism.

Oxid Med Cell Longev 2017 2;2017:7317251. Epub 2017 Oct 2.

Section on Gerontology and Geriatrics, Sticht Center for Healthy Aging and Alzheimer's Prevention & Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA.

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 [F] 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.
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http://dx.doi.org/10.1155/2017/7317251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643153PMC
July 2018

Blood-based bioenergetic profiling: A readout of systemic bioenergetic capacity that is related to differences in body composition.

Redox Biol 2017 10 30;13:418-420. Epub 2017 Jun 30.

J. Paul Sticht Center on Healthy Aging and Alzheimer's Prevention, Department of Internal Medicine Section on Gerontology and Geriatric Medicine, Wake Forest School of Medicine, United States. Electronic address:

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http://dx.doi.org/10.1016/j.redox.2017.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499103PMC
October 2017

Regulators of mitochondrial quality control differ in subcutaneous fat of metabolically healthy and unhealthy obese monkeys.

Obesity (Silver Spring) 2017 04 25;25(4):689-696. Epub 2017 Feb 25.

Sticht Center on Aging and Department of Internal Medicine, Section on Gerontology and Geriatrics, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.

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.
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http://dx.doi.org/10.1002/oby.21762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373959PMC
April 2017

Renal-Risk Variants Induce Mitochondrial Dysfunction.

J Am Soc Nephrol 2017 Apr 7;28(4):1093-1105. Epub 2016 Nov 7.

Department of Internal Medicine, Section on Nephrology,

G1 and G2 variants facilitate kidney disease in blacks. To elucidate the pathways whereby these variants contribute to disease pathogenesis, we established HEK293 cell lines stably expressing doxycycline-inducible (Tet-on) reference G0 or the G1 and G2 renal-risk variants, and used Illumina human HT-12 v4 arrays and Affymetrix HTA 2.0 arrays to generate global gene expression data with doxycycline induction. Significantly altered pathways identified through bioinformatics analyses involved mitochondrial function; results from immunoblotting, immunofluorescence, and functional assays validated these findings. Overexpression of by doxycycline induction in HEK293 Tet-on G1 and G2 cells led to impaired mitochondrial function, with markedly reduced maximum respiration rate, reserve respiration capacity, and mitochondrial membrane potential. Impaired mitochondrial function occurred before intracellular potassium depletion or reduced cell viability occurred. Analysis of global gene expression profiles in nondiseased primary proximal tubule cells from black patients revealed that the nicotinate phosphoribosyltransferase gene, responsible for NAD biosynthesis, was among the top downregulated transcripts in cells with two renal-risk variants compared with those without renal-risk variants; nicotinate phosphoribosyltransferase also displayed gene expression patterns linked to mitochondrial dysfunction in HEK293 Tet-on cell pathway analyses. These results suggest a pivotal role for mitochondrial dysfunction in -associated kidney disease.
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http://dx.doi.org/10.1681/ASN.2016050567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373457PMC
April 2017

Blood cell respirometry is associated with skeletal and cardiac muscle bioenergetics: Implications for a minimally invasive biomarker of mitochondrial health.

Redox Biol 2016 12 21;10:65-77. Epub 2016 Sep 21.

Sticht Center on Aging & Department of Internal Medicine, Section on Gerontology and Geriatrics, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA. Electronic address:

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045569PMC
http://dx.doi.org/10.1016/j.redox.2016.09.009DOI Listing
December 2016

Skeletal Muscle Mitochondrial Content, Oxidative Capacity, and Mfn2 Expression Are Reduced in Older Patients With Heart Failure and Preserved Ejection Fraction and Are Related to Exercise Intolerance.

JACC Heart Fail 2016 08 11;4(8):636-45. Epub 2016 May 11.

Cardiology Section, Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina. Electronic address:

Objectives: The aim of this study was to examine skeletal muscle mitochondria content, oxidative capacity, and the expression of key mitochondrial dynamics proteins in patients with heart failure with preserved ejection fraction (HFpEF), as well as to determine potential relationships with measures of exercise performance.

Background: Multiple lines of evidence indicate that severely reduced peak exercise oxygen uptake (peak VO2) in older patients with HFpEF is related to abnormal skeletal muscle oxygen utilization. Mitochondria are key regulators of skeletal muscle metabolism; however, little is known about how these organelles are affected in HFpEF.

Methods: Both vastus lateralis skeletal muscle citrate synthase activity and the expression of porin and regulators of mitochondrial fusion were examined in older patients with HFpEF (n = 20) and healthy, age-matched control subjects (n = 17).

Results: Compared with age-matched healthy control subjects, mitochondrial content assessed by porin expression was 46% lower (p = 0.01), citrate synthase activity was 29% lower (p = 0.01), and Mfn2 (mitofusin 2) expression was 54% lower (p <0.001) in patients with HFpEF. Expression of porin was significantly positively correlated with both peak VO2 and 6-min walk distance (r = 0.48, p = 0.003 and r = 0.33, p = 0.05, respectively). Expression of Mfn2 was also significantly positively correlated with both peak VO2 and 6-min walk distance (r = 0.40, p = 0.02 and r = 0.37, p = 0.03 respectively).

Conclusions: These findings suggest that skeletal muscle oxidative capacity, mitochondrial content, and mitochondrial fusion are abnormal in older patients with HFpEF and might contribute to their severe exercise intolerance.
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http://dx.doi.org/10.1016/j.jchf.2016.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967040PMC
August 2016

Relationships between mitochondrial content and bioenergetics with obesity, body composition and fat distribution in healthy older adults.

BMC Obes 2015 6;2:40. Epub 2015 Oct 6.

Sticht Center on Aging & Department of Internal Medicine, Section on Gerontology and Geriatrics, Wake Forest School of Medicine, Winston-Salem, NC 27157 USA.

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.
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http://dx.doi.org/10.1186/s40608-015-0070-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594906PMC
October 2015

Blood-cell bioenergetics are associated with physical function and inflammation in overweight/obese older adults.

Exp Gerontol 2015 Oct 29;70:84-91. Epub 2015 Jul 29.

Sticht Center on Aging and Department of Internal Medicine, Section on Gerontology and Geriatrics, Wake Forest School of Medicine, Winston-Salem, NC 27157, United States. Electronic address:

Background: Physical function and strength decline with age and lead to limited mobility and independence in older adults. Alterations in mitochondrial function are thought to underlie numerous age-related changes, including declining physical ability. Recent studies suggest that systemic changes in bioenergetic capacity may be reported by analyzing mitochondrial function in circulating cells. The objective of this study was to determine whether the bioenergetic capacity of peripheral blood mononuclear cells (PBMCs) is related to differences in physical function among older, overweight/obese, adults. To address this, we tested the hypothesis that greater PBMC respirometric capacity would be associated with better physical function, muscular strength, leg lean mass, and muscle quality. Furthermore, we tested whether the respirometric capacity of PBMCs is related to cellular composition and inflammatory status reported by interleukin-6 (IL-6).

Methods: Fasted PBMC respiration (pmol/min/500,000 cells), expanded short physical performance battery (Ex-SPPB), peak knee extensor (KE) strength (Nm), grip strength (kg), leg lean mass (kg, via dual energy X-ray absorptiometry [DXA]), muscle quality (Nm/kg), and plasma IL-6 (pg/mL) were analyzed in 15 well-functioning, community-dwelling, sedentary overweight/obese older men (n=9) and women (n=6) aged 65 to 78 (mean 68.3 ± 3.5 years). Pearson and partial correlations were calculated to determine associations between PBMC respiration and these variables.

Results: Higher maximal respiration of PBMCs was associated with better Ex-SPPB (r=0.58, p=0.02), greater KE strength (r=0.60, p=0.02), greater grip strength (r=0.52, p=0.05) and lower IL-6 (r=-0.58, p=0.04). Higher spare respiratory capacity was associated with better Ex-SPPB (r=0.59, p=0.02), greater KE strength (r=0.60, p=0.02), greater grip strength (r=0.54, p=0.04), greater leg muscle quality (r=0.56, p=0.04), and lower IL-6 (r=-0.55, p=0.05). Monocyte and lymphocyte counts were not related to PBMC respiratory capacity.

Conclusions: Our results indicate that respirometric profiles of readily obtainable blood cells are associated with physical function and strength. Future studies should be undertaken in order to determine whether blood-based bioenergetic profiling can provide an objective index of systemic mitochondrial health.
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http://dx.doi.org/10.1016/j.exger.2015.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471587PMC
October 2015

Preparation and respirometric assessment of mitochondria isolated from skeletal muscle tissue obtained by percutaneous needle biopsy.

J Vis Exp 2015 Feb 7(96). Epub 2015 Feb 7.

Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest School of Medicine;

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.
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http://dx.doi.org/10.3791/52350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354623PMC
February 2015

Energy metabolism in a matched model of radiation resistance for head and neck squamous cell cancer.

Radiat Res 2015 Mar 4;183(3):291-304. Epub 2015 Mar 4.

Sections on a Molecular Medicine, Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina 27157.

While radiation therapy is commonly used for treating cancer, radiation resistance can limit long-term control of the disease. In this study, we investigated the reprogramming of the energy metabolism in radiosensitive and radioresistant head and neck squamous cell carcinomas (HNSCC) using a preclinical matched model of radiation resistance. Our investigation found that radioresistant rSCC-61 cells: 1. They display increased glucose uptake and decreased fatty acid uptake; 2. They deviate from the classical Warburg effect by diverting the glycolytic flux into the pentose phosphate pathway; 3. They are more dependent on glucose than glutamine metabolism to support growth; 4. They have decreased mitochondrial oxidative phosphorylation; 5. They have enhanced fatty acid biosynthesis by increasing the expression of fatty acid synthase; and 6. They utilize endogenous fatty acids to meet the energy demands for proliferation. Inhibition of fatty acid synthase with orlistat or FASN siRNA resulted in increased cytotoxicity and sensitivity to radiation in rSCC-61 cells. These results demonstrate the potential of combination therapy using radiation and orlistat or other inhibitors of lipid and energy metabolism for treating radiation resistance in HNSCC.
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http://dx.doi.org/10.1667/RR13828.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4465128PMC
March 2015

Examination of bioenergetic function in the inner mitochondrial membrane peptidase 2-like (Immp2l) mutant mice.

Redox Biol 2014 28;2:1008-15. Epub 2014 Aug 28.

Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Medical Center Boulevard, Winston-Salem, NC 27157, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.redox.2014.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215389PMC
January 2018

Sequential actions of SIRT1-RELB-SIRT3 coordinate nuclear-mitochondrial communication during immunometabolic adaptation to acute inflammation and sepsis.

J Biol Chem 2015 Jan 17;290(1):396-408. Epub 2014 Nov 17.

From the Section of Molecular Medicine,

We reported that NAD(+)-dependent SIRT1, RELB, and SIRT6 nuclear proteins in monocytes regulate a switch from the glycolysis-dependent acute inflammatory response to fatty acid oxidation-dependent sepsis adaptation. We also found that disrupting SIRT1 activity during adaptation restores immunometabolic homeostasis and rescues septic mice from death. Here, we show that nuclear SIRT1 guides RELB to differentially induce SIRT3 expression and also increases mitochondrial biogenesis, which alters bioenergetics during sepsis adaptation. We constructed this concept using TLR4-stimulated THP1 human promonocytes, a model that mimics the initiation and adaptation stages of sepsis. Following increased expression, mitochondrial SIRT3 deacetylase activates the rate-limiting tricarboxylic acid cycle (TCA) isocitrate dehydrogenase 2 and superoxide dismutase 2, concomitant with increases in citrate synthase activity. Mitochondrial oxygen consumption rate increases early and decreases during adaptation, parallel with modifications to membrane depolarization, ATP generation, and production of mitochondrial superoxide and whole cell hydrogen peroxide. Evidence of SIRT1-RELB induction of mitochondrial biogenesis included increases in mitochondrial mass, mitochondrial-to-nuclear DNA ratios, and both nuclear and mitochondrial encoded proteins. We confirmed the SIRT-RELB-SIRT3 adaptation link to mitochondrial bioenergetics in both TLR4-stimulated normal and sepsis-adapted human blood monocytes and mouse splenocytes. We also found that SIRT1 inhibition ex vivo reversed the sepsis-induced changes in bioenergetics.
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http://dx.doi.org/10.1074/jbc.M114.566349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4281742PMC
January 2015

Respirometric Profiling of Muscle Mitochondria and Blood Cells Are Associated With Differences in Gait Speed Among Community-Dwelling Older Adults.

J Gerontol A Biol Sci Med Sci 2015 Nov 16;70(11):1394-9. Epub 2014 Jul 16.

Sticht Center on Aging and Department of Internal Medicine, Section on Gerontology and Geriatrics, Wake Forest School of Medicine, Winston-Salem, North Carolina.

Background: Gait speed provides an integrated measure of physical ability that is predictive of morbidity, disability, and mortality in older adults. Energy demands associated with walking suggest that mitochondrial bioenergetics may play a role in gait speed. Here, we examined the relationship between gait speed and skeletal muscle mitochondrial bioenergetics, and further evaluated whether blood-based bioenergetic profiling might have similar associations with gait speed.

Methods: Participants in this study were comprised of two subsets (n = 17 per subset) and were overweight/obese (body mass index, 30.9 ± 2.37), well-functioning, community-dwelling older adults (69.1 ± 3.69 years) without major comorbidity. Gait speeds were calculated from a fast-paced 400 m walk test. Respiratory control ratios were measured from mitochondria isolated from leg skeletal muscle biopsies from one subset. Maximal respiration and spare respiratory capacity were measured from peripheral blood mononuclear cells from the other subset.

Results: Individual differences in gait speed correlated directly with respiratory control ratio of mitochondria isolated from skeletal muscle (r = .536, p = .027) and with both maximal respiration and spare respiratory capacity of peripheral blood mononuclear cells (r = .585 and p = .014; r = .609 and p = .009, respectively).

Conclusions: The bioenergetic profile of mitochondria isolated from skeletal muscle is associated with gait speed in older adults. Blood-based bioenergetic profiling is also associated with gait speed and may provide an alternative measure of mitochondrial function.
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http://dx.doi.org/10.1093/gerona/glu096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731403PMC
November 2015

A faster, high resolution, mtPA-GFP-based mitochondrial fusion assay acquiring kinetic data of multiple cells in parallel using confocal microscopy.

J Vis Exp 2012 Jul 20(65):e3991. Epub 2012 Jul 20.

Department of Neuroscience, Center for Neuroscience Research, Tufts School of Medicine.

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment. Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary. Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment. A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP). Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE. The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity. In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15 nM TMRE in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.
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http://dx.doi.org/10.3791/3991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3476419PMC
July 2012

High glucose induces mitochondrial morphology and metabolic changes in retinal pericytes.

Invest Ophthalmol Vis Sci 2011 Nov 7;52(12):8657-64. Epub 2011 Nov 7.

Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

Purpose: Mitochondrial dysfunction is known to play a role in retinal vascular cell loss, a prominent lesion of diabetic retinopathy. High glucose (HG) has been reported to induce mitochondrial fragmentation and dysfunction in retinal endothelial cells, contributing to apoptosis. In this study, the effects of HG on mitochondrial morphology, membrane potential, and metabolic changes and whether they could contribute to HG-induced apoptosis in retinal pericytes were investigated.

Methods: Bovine retinal pericytes (BRPs) were grown in normal or HG medium for 7 days. Both sets of cells were double stained with mitochondrial membrane potential-independent dye and tetramethylrhodamine-ethyl-ester-perchlorate (TMRE) and imaged by confocal microscopy. The images were analyzed for average mitochondria shape, by using form factor and aspect ratio values, and membrane potential changes, by using the ratio between the red and green dye. BRPs grown in normal or HG medium were analyzed for transient changes in oxygen consumption and extracellular acidification with a flux analyzer and apoptosis by TUNEL assay.

Results: BRPs grown in HG media exhibited significant fragmentation of mitochondria and increased membrane potential heterogeneity compared with the BRPs grown in normal medium. Concomitantly, BRPs grown in HG showed reduced steady state and maximum oxygen consumption and reduced extracellular acidification. Number of TUNEL-positive pericytes was increased in HG condition as well.

Conclusions: In HG condition, mitochondria of retinal pericytes display significant fragmentation, metabolic dysfunction, and reduced extracellular acidification. The detrimental effects of HG on mitochondrial function and cellular metabolism could play a role in the accelerated apoptosis associated with the retinal pericytes in diabetic retinopathy.
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http://dx.doi.org/10.1167/iovs.11-7934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230288PMC
November 2011

High glucose disrupts mitochondrial morphology in retinal endothelial cells: implications for diabetic retinopathy.

Am J Pathol 2010 Jul 3;177(1):447-55. Epub 2010 Jun 3.

Department of Medicine, Boston University School of Medicine, 650 Albany Street, Boston 02118, USA.

Mitochondrial dysfunction has been implicated in diabetic complications; however, it is unknown whether hyperglycemia affects mitochondrial morphology and metabolic capacity during development of diabetic retinopathy. We investigated high glucose (HG) effects on mitochondrial morphology, membrane potential heterogeneity, cellular oxygen consumption, extracellular acidification, cytochrome c release, and apoptosis in retinal endothelial cells. Rat retinal endothelial cells grown in normal (5 mmol/L) or HG (30 mmol/L) medium and double-stained with MitoTracker Green and tetramethylrhodamine-ethyl-ester-perchlorate were examined live with confocal microscopy. Images were analyzed for mitochondrial shape change using Form Factor and Aspect Ratio values, and membrane potential heterogeneity, using deviation of fluorescence intensity values. Rat retinal endothelial cells grown in normal or HG medium were analyzed for transient changes in oxygen consumption and extracellular acidification using an XF-24 flux analyzer, cytochrome c release by Western blot, and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Rat retinal endothelial cells grown in HG medium exhibited increased mitochondrial fragmentation concurrent with membrane potential heterogeneity. Metabolic analysis showed increased extracellular acidification in HG with reduced steady state/maximal oxygen consumption. Cytochrome c and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were also increased in HG. Thus, HG-induced mitochondrial fragmentation with concomitant increase in membrane potential heterogeneity, reduced oxygen consumption, and cytochrome c release may underlie apoptosis of retinal endothelial cells as seen in diabetic retinopathy.
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http://dx.doi.org/10.2353/ajpath.2010.091029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2893686PMC
July 2010

Biophysical properties of mitochondrial fusion events in pancreatic beta-cells and cardiac cells unravel potential control mechanisms of its selectivity.

Am J Physiol Cell Physiol 2010 Aug 5;299(2):C477-87. Epub 2010 May 5.

Evans Biomedical Research Center, Boston University, Boston, Massachusetts 02118, USA.

Studies in various types of cells find that, on average, each mitochondrion becomes involved in a fusion event every 15 min, depending on the cell type. As most contact events do not result in mitochondrial fusion, it is expected that properties of the individual mitochondrion determine the likelihood of a fusion event. However, apart from membrane potential, the properties that influence the likelihood of entering a fusion event are not known. Here, we tag and track individual mitochondria in H9c2, INS1, and primary beta-cells and determine the biophysical properties that increase the likelihood of a fusion event. We found that the probability for fusion is independent of contact duration and organelle dimensions, but it is influenced by organelle motility. Furthermore, the history of a previous fusion event of the individual mitochondrion influenced both the likelihood for a subsequent fusion event, as well as the site on the mitochondrion at which the fusion occurred. These observations unravel the specific properties that distinguish mitochondria that will enter fusion events from the ones that will not. Altogether, these properties may help to elucidate the molecular mechanisms that regulate fusion at the level of the single mitochondrion.
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http://dx.doi.org/10.1152/ajpcell.00427.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2928626PMC
August 2010

Mitochondrial networking protects beta-cells from nutrient-induced apoptosis.

Diabetes 2009 Oct 6;58(10):2303-15. Epub 2009 Jul 6.

Department of Molecular Medicine, Obesity Research Center, Boston University School of Medicine, Boston, Massachusetts, USA.

Objective: Previous studies have reported that beta-cell mitochondria exist as discrete organelles that exhibit heterogeneous bioenergetic capacity. To date, networking activity, and its role in mediating beta-cell mitochondrial morphology and function, remains unclear. In this article, we investigate beta-cell mitochondrial fusion and fission in detail and report alterations in response to various combinations of nutrients.

Research Design And Methods: Using matrix-targeted photoactivatable green fluorescent protein, mitochondria were tagged and tracked in beta-cells within intact islets, as isolated cells and as cell lines, revealing frequent fusion and fission events. Manipulations of key mitochondrial dynamics proteins OPA1, DRP1, and Fis1 were tested for their role in beta-cell mitochondrial morphology. The combined effects of free fatty acid and glucose on beta-cell survival, function, and mitochondrial morphology were explored with relation to alterations in fusion and fission capacity.

Results: beta-Cell mitochondria are constantly involved in fusion and fission activity that underlies the overall morphology of the organelle. We find that networking activity among mitochondria is capable of distributing a localized green fluorescent protein signal throughout an isolated beta-cell, a beta-cell within an islet, and an INS1 cell. Under noxious conditions, we find that beta-cell mitochondria become fragmented and lose their ability to undergo fusion. Interestingly, manipulations that shift the dynamic balance to favor fusion are able to prevent mitochondrial fragmentation, maintain mitochondrial dynamics, and prevent apoptosis.

Conclusions: These data suggest that alterations in mitochondrial fusion and fission play a critical role in nutrient-induced beta-cell apoptosis and may be involved in the pathophysiology of type 2 diabetes.
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http://dx.doi.org/10.2337/db07-1781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2750232PMC
October 2009

Monitoring mitochondrial dynamics with photoactivatable [corrected] green fluorescent protein.

Methods Enzymol 2009 ;457:289-304

Department of Medicine, Boston University, Massachusetts, USA.

Mitochondria are dynamic organelles that undergo continuous cycles of fusion and fission. Monitoring and quantification of mitochondrial dynamics has proved to be challenging because these processes are distinctly different from movement and apposition. While the majority of contact events do not lead to fusion, fission can occur without translocation, leaving the two mitochondria juxtaposed. The advent of photoactivatable fluorescent proteins has enabled researchers to distinguish mitochondrial fusion and fission. These genetically encoded fluorophores can be targeted to the mitochondrial compartments of interest to visualize how these intermix and segregate between dynamic mitochondria over time. The PAGFPmt-based mitochondrial dynamics assay has proved to be a powerful technique for revealing the treatments and cellular processes that affect fusion and fission. By using this technique in combination with other parameters, such as measurements of mitochondrial membrane potential, we have begun to understand the processes that control fusion and fission as well as the significance of mitochondrial dynamics.
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http://dx.doi.org/10.1016/S0076-6879(09)05016-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967387PMC
July 2009

Dual role of proapoptotic BAD in insulin secretion and beta cell survival.

Nat Med 2008 Feb 27;14(2):144-53. Epub 2008 Jan 27.

Department of Pathology, Harvard Medical School, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA.

The proapoptotic BCL-2 family member BAD resides in a glucokinase-containing complex that regulates glucose-driven mitochondrial respiration. Here, we present genetic evidence of a physiologic role for BAD in glucose-stimulated insulin secretion by beta cells. This novel function of BAD is specifically dependent upon the phosphorylation of its BH3 sequence, previously defined as an essential death domain. We highlight the pharmacologic relevance of phosphorylated BAD BH3 by using cell-permeable, hydrocarbon-stapled BAD BH3 helices that target glucokinase, restore glucose-driven mitochondrial respiration and correct the insulin secretory response in Bad-deficient islets. Our studies uncover an alternative target and function for the BAD BH3 domain and emphasize the therapeutic potential of phosphorylated BAD BH3 mimetics in selectively restoring beta cell function. Furthermore, we show that BAD regulates the physiologic adaptation of beta cell mass during high-fat feeding. Our findings provide genetic proof of the bifunctional activities of BAD in both beta cell survival and insulin secretion.
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http://dx.doi.org/10.1038/nm1717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918232PMC
February 2008

Fission and selective fusion govern mitochondrial segregation and elimination by autophagy.

EMBO J 2008 Jan 17;27(2):433-46. Epub 2008 Jan 17.

Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA 02111, USA.

Accumulation of depolarized mitochondria within beta-cells has been associated with oxidative damage and development of diabetes. To determine the source and fate of depolarized mitochondria, individual mitochondria were photolabeled and tracked through fusion and fission. Mitochondria were found to go through frequent cycles of fusion and fission in a 'kiss and run' pattern. Fission events often generated uneven daughter units: one daughter exhibited increased membrane potential (delta psi(m)) and a high probability of subsequent fusion, while the other had decreased membrane potential and a reduced probability for a fusion event. Together, this pattern generated a subpopulation of non-fusing mitochondria that were found to have reduced delta psi(m) and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1(K38A) or FIS1 RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion. Pulse chase and arrest of autophagy at the pre-proteolysis stage reveal that before autophagy mitochondria lose delta psi(m) and OPA1, and that overexpression of OPA1 decreases mitochondrial autophagy. Together, these findings suggest that fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy.
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http://dx.doi.org/10.1038/sj.emboj.7601963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2234339PMC
January 2008
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