Mitochondrial Functional Capacity Research Articles

Mir-204-5p alleviates mitochondrial dysfunction by targeting IGFBP5 in diabetic cataract.

Cataract contributes to visual impairment worldwide, and diabetes mellitus accelerates the formation and progression of cataract. Here we found that the expression level of miR-204-5p was diminished in the lens epithelium with anterior lens capsule of cataract patients compared to normal donors, and decreased more obviously in those of diabetic cataract (DC) patients. However, the contribution and mechanism of miR-204-5p during DC development remain elusive. The mitochondrial membrane potential (MMP) was reduced in the lens epithelium with anterior lens capsule of DC patients and the H2O2-induced human lens epithelial cell (HLEC) cataract model, suggesting impaired mitochondrial functional capacity. Consistently, miR-204-5p knockdown by the specific inhibitor also attenuated the MMP in HLECs. Using bioinformatics and a luciferase assay, further by immunofluorescence staining and Western blot, we identified IGFBP5, an insulin-like growth factor binding protein, as a direct target of miR-204-5p in HLECs. IGFBP5 expression was upregulated in the lens epithelium with anterior lens capsule of DC patients and in the HLEC cataract model, and IGFBP5 knockdown could reverse the mitochondrial dysfunction in the HLEC cataract model. Our results demonstrate that miR-204-5p maintains mitochondrial functional integrity through repressing IGFBP5, and reveal IGFBP5 may be a new therapeutic target and prognostic factor for DC.

Deficits in mitochondrial function and glucose metabolism seen in sporadic and familial Alzheimer’s disease derived Astrocytes are ameliorated by increasing hexokinase 1

AbstractBackgroundAstrocytes have multiple roles including providing neurons with metabolic substrates and maintaining neurotransmitters at neuronal synapses. Astrocyte glucose metabolism plays a key role in learning and memory with astrocytic glycogen a key substrate supporting memory encoding. The homeostatic role the astrocyte provides for neurons leads to metabolic demands, meaning that abnormalities in the function of astrocyte mitochondria and glycolysis could affect this relationship. Changes to cellular metabolism are seen early in Alzheimer’s disease (AD). Understanding cellular metabolism changes in AD astrocytes could be exploited as a new biomarker or synergistic therapeutic agent when combined with anti‐amyloid treatments in AD.MethodsIn this project, we characterised mitochondrial and glycolytic dysfunction in astrocytes derived from patients with sporadic (n = 6) and familial (PSEN1, n = 3) AD, and associated controls (n = 9). Astrocytes were derived using direct reprogramming technology. Astrocyte metabolic outputs; ATP, and extracellular lactate levels were measured using luminescent and fluorescent protocols. Mitochondrial respiration and glycolytic function were measured using a Seahorse XF Analyzer. Hexokinase deficits identified where corrected by transfecting astrocytes with an adenovirus viral vector containing the hexokinase 1 gene.ResultsIn sAD astrocytes a 20% reduction (p = 0.05) and in fAD a 48% (p<0.01) reduction in total cellular ATP was seen. A 44% reduction (p<0.05), and 80% reduction in Mitochondrial spare capacity was seen in sAD and fAD respectively. Reactive oxygen species (ROS) where increased in both AD astrocyte types (p = 0.05). Mitochondrial complex I and II was significantly increased in sAD (p<0.05) but not in fAD. Astrocyte glycolytic reserve and extracellular lactate was significantly reduced when compared to controls in both sAD and fAD (p<0.05). We identified that astrocytes had a deficit in the glycolytic pathway enzyme hexokinase, and that correcting this deficit restored total cellular ATP levels, extracellular lactate and reduced ROS in sAD but not fAD astrocytes.ConclusionAD astrocytes have abnormalities in functional capacity of mitochondria and the process of glycolysis. These deficits are likely to impede metabolic support for neurons. These functional deficits can be improved by correcting hexokinase deficits. This suggests that hexokinase 1 deficiency could potentially be exploited as a new therapeutic target for AD.

Uncoupled respiration stability of isolated pancreatic acini as a novel functional test for cell vitality

Background. Assessment of cell viability is crucial in cell studies. Testing plasma membrane integrity is a traditional approach of evaluating cell viability. Mitochondrial functional capacity closely correlates with plasma membrane integrity and overall cell health. This study aimed to investigate whether any aspect of mitochondrial adaptive capacity in isolated pancreatic acini is associated with the quality of isolated pancreatic acini preparations, as determined by the dye exclusion method. Materials and Methods. Experiments were carried out on male Wistar rats weig­hing 250–300 g. A suspension of isolated pancreatic acini was obtained using collagenase. The rate of oxygen consumption of rat isolated pancreatic acini was measured with Clark oxygen electrode. Basal respiration of isolated pancreatic acini was recorded for approximately 2 min. Afterwards, the mitochondrial adaptive capacity was examined using FCCP in concentrations from 0.5 to 2 μM. Uncoupled respiratory stability was calculated as a ratio of respiration rate at high and low FCCP concentrations. Plasma membrane integrity was assessed with trypan blue staining. A total of 74 preparations of isolated pancreatic acini were used in this study. Results. In all experiments, 92–99 % of pancreatic acinar cells exhibited negative trypan blue staining, indicating intact plasma membranes. The basal and maximal uncoupled respiration rates were not affected by the fraction of trypan-negative cells. However, acini preparations with <less than 95 % plasma membrane integrity had significantly lower uncoupled respiration rates when exposed to a high concentration of FCCP (2 µM), indicating reduced stability of uncoupled respiration. Conclusions. Results of the study suggest that the stability of uncoupled respiration can serve as a novel metabolic functional test to complement the existing methods for assessing cell vitality.

Prospective association between maternal allostatic load during pregnancy and child mitochondrial content and bioenergetic capacity

BackgroundMitochondria are multifunctional energy-producing and signaling organelles that support life and contribute to stress adaptation. There is a growing understanding of the dynamic relationship between stress exposure and mitochondrial biology; however, the influence of stress on key domains of mitochondrial biology during early-life, particularly the earliest phases of intra-uterine/prenatal period remains largely unknown. Thus, the goal of this study was to examine the impact of fetal exposure to stress (modeled as the biological construct allostatic load) upon mitochondrial biology in early childhood. MethodsIn n = 30 children (range: 3.5–6 years, 53% male), we quantified mitochondrial content via citrate synthase (CS) activity and mtDNA copy number (mtDNAcn), and measured mitochondrial bioenergetic capacity via respiratory chain enzyme activities (complexes I (CI), II (CII), and IV (CIV)) in platelet-depleted peripheral blood mononuclear cells (PBMCs). In a cohort of healthy pregnant women, maternal allostatic load was operationalized as a latent variable (sum of z-scores) representing an aggregation of early-, mid- and late-gestation measures of neuroendocrine (cortisol), immune (interleukin-6, C-reactive protein), metabolic (homeostasis model assessment of insulin resistance, free fatty acids), and cardiovascular (aggregate systolic and diastolic blood pressure) systems, as well as an anthropometric indicator (pre-pregnancy body mass index [BMI]). ResultsAn interquartile increase in maternal allostatic load during pregnancy was associated with higher mitochondrial content (24% and 15% higher CS and mtDNAcn), and a higher mitochondrial bioenergetic capacity (16%, 23%, and 25% higher CI, CII and CIV enzymatic activities) in child leukocytes. The positive association between maternal allostatic load during pregnancy and child mitochondrial content and bioenergetic capacity remained significant after accounting for the effects of key pre- and post-natal maternal and child covariates (p’s < 0.05, except CI p = 0.073). ConclusionWe report evidence that prenatal biological stress exposure, modeled as allostatic load, was associated with elevated child mitochondrial content and bioenergetic capacity in early childhood. This higher mitochondrial content and bioenergetic capacity (per leukocyte) may reflect increased energetic demands at the immune or organism level, and thus contribute to wear-and-tear and pathophysiology, and/or programmed pro-inflammatory phenotypes. These findings provide potential mechanistic insight into the cellular processes underlying developmental programming, and support the potential role that changes in mitochondrial content and bioenergetic functional capacity may play in altering life-long susceptibility for health and disease.

Neuronal Dysfunction Associated with Colorectal Cancer Augments Sensitivity to Chemotherapy-induced Neuropathic Pain

Neuropathic pain is a common and debilitating side effect of many cancer treatments. The platinum-based chemotherapeutic agent oxaliplatin is widely used in treatment regimens for colorectal cancer, but its side effects (including numbness, tingling or sharp, stabbing, 'electrical' pain) are so severe as to become dose limiting in a subset of patients. We set out to determine if the systemic immune system alterations in colon cancer could influence subsequent sensitivity to oxaliplatin therapy. Using two orthotopic mouse models of colon cancer, we perform immunohistochemistry, behavioral analysis, functional calcium imaging, plasma cytokine/chemokine array analysis and mitochondrial functional capacity to determine the extent to which cancer-associated inflammation drives neuronal dysfunction in response to chemotherapy. We found that two distinct colorectal cancer cell lines in two immunocompetent mouse strains induced impaired mitochondrial function in dorsal root ganglion neurons in vitro. This was accompanied by a significant reduction in intra-epidermal nerve fiber density in the plantar hindpaw skin. Multi-electrode array and ratiometric calcium imaging show abnormalities in spontaneous firing and cytosolic calcium concentration in dorsal root ganglion neurons. Finally, we show that tumor-bearing mice show a greater degree of tactile and cold sensitivity in response to a sub-maximal dose of oxaliplatin, despite there being no overt symptoms of neuropathy in tumor-bearing mice that did not receive oxaliplatin. We conclude that colorectal cancer induces 'sub-clinical' neuronal injury that predisposes to neuropathic pain hypersensitivity upon subsequent treatment with oxaliplatin. Future experiments will focus on identifying the soluble factor(s) responsible, and the generalizability of this phenomenon. Grant support from Rita Allen Foundation.

Development of heart failure with preserved ejection fraction in type 2 diabetic mice is ameliorated by preserving vascular function

AimsHeart failure with preserved ejection fraction (HFpEF) is associated with endothelial dysfunction and is frequent in people with type 2 diabetes mellitus. In diabetic patients, increased levels of the eicosanoid 12-hydroxyeicosatetraenoic acid (12-HETE) are linked to vascular dysfunction. Here, we aimed to identify the importance of 12-HETE in type 2 diabetic patients exhibiting diastolic dysfunction, and mice exhibiting HFpEF and whether targeting 12-HETE is a means to ameliorate HFpEF progression by improving vascular function in diabetes. Material and methodsSubjects with diagnosed type 2 diabetes mellitus and reported diastolic dysfunction or healthy controls were recruited and 12(S)-HETE levels determined by ELISA. 12(S)-HETE levels were determined in type 2 diabetic, leptin receptor deficient mice (LepRdb/db) and HFpEF verified by echocardiography. Mitochondrial function, endothelial function and capillary density were assessed using Seahorse technique, pressure myography and immunohistochemistry in LepRdb/db or non-diabetic littermate controls. 12/15Lo generation was inhibited using ML351 and 12(S)-HETE action by using the V1-cal peptide. Key findingsEndothelium-dependent vasodilation and mitochondrial functional capacity both improved in response to either application of ML351 or the V1-cal peptide. Correlating to improved vascular function, mice treated with either pharmacological agent exhibited improved diastolic filling and left ventricular relaxation that correlated with increased myocardial capillary density. SignificanceOur results suggest that 12-HETE may serve as a biomarker indicating endothelial dysfunction and the resulting cardiovascular consequences such as HFpEF in type 2 diabetic patients. Antagonizing 12-HETE is a potent means to causally control HFpEF development and progression in type 2 diabetes by preserving vascular function.

1133 Chronic intrauterine hypoxia shifts the balance of mitochondrial dynamics in the fetal guinea pig forebrain

Mitochondrial ATP production facilitates proper fetal neurodevelopment and is dependent on mitochondrial dynamics consisting of fission and fusion. We hypothesize in the setting of chronic intrauterine hypoxia, which is associated poor neurodevelopmental outcomes, that mitochondrial dynamics are dysregulated in a manner dependent on fetal sex and the timing of hypoxia onset. Using a guinea pig model of chronic intrauterine hypoxia, we characterized the expression of mitochondrial fission and fusion proteins in fetal forebrains after exposure to early or late-onset hypoxia (EO-HPX and LO-HPX, respectively). Pregnant guinea pigs were exposed to either normoxia (NMX) or hypoxia (ambient O2 10.5%) starting at 28 d (EO-HPX) or 50 d (LO-HPX) gestation until term (65d). Male and female fetuses were extracted from anesthetized sows and forebrains were excised (n=6). Protein expression of mitochondrial fission (Drp-1) and fusion (Mfn-2) proteins were measured by Western Blot. Results of the hypoxic effects are means +/- SEM, with statistical comparison to NMX by one-tailed T-Test. EO-HPX significantly increased Drp-1 expression levels in male fetal forebrains compared to NMX. There was no significant effect of LO-HPX on Drp-1 in the male fetal brain (Fig 1A). Both EO-HPX and LO-HPX increased Drp-1 expression in female forebrains compared to NMX (Fig1B). Mfn-2 was significantly lower in male fetal forebrain tissues exposed to EO-HPX as compared to NMX (Fig 2A). In contrast, LO-HPX had no significant effect on Mfn-2 in male fetuses compared to NMX. Neither EO-HPX nor LO-HPX had any effect on Mfn-2 in female forebrains compared to NMX (Fig 2B). Hypoxia exposure at mid-gestation shifted the balance of mitochondrial dynamics in a sex-dependent manner toward fission. Hypoxia both decreased fusion and increased fission in the male forebrain, In contrast, hypoxia had no effect on fusion but increased fission within females. . Therefore, altered mitochondrial functional capacity can be an underlying mechanism contributing to altered fetal brain development.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Inhibiting miR-205 Alleviates Cardiac Ischemia/Reperfusion Injury by Regulating Oxidative Stress, Mitochondrial Function, and Apoptosis.

Background miR-205 is important for oxidative stress, mitochondrial dysfunction, and apoptosis. The roles of miR-205 in cardiac ischemia/reperfusion (I/R) injury remain unknown. The aim of this research is to reveal whether miR-205 could regulate cardiac I/R injury by focusing upon the oxidative stress, mitochondrial function, and apoptosis. Methods Levels of miR-205 and Rnd3 were examined in the hearts with I/R injury. Myocardial infarct size, cardiac function, oxidative stress, mitochondria function, and cardiomyocyte apoptosis were detected in mice with myocardial ischemia/reperfusion (MI/R) injury. The primary neonatal cardiomyocytes underwent hypoxia/reoxygenation (H/R) to simulate MI/R injury. Results miR-205 levels were significantly elevated in cardiac tissues from I/R in comparison with those from Sham. In comparison with controls, levels of Rnd3 were significantly decreased in the hearts from mice with MI/R injury. Furthermore, inhibiting miR-205 alleviated MI/R-induced apoptosis, reduced infarct size, prevented oxidative stress increase and mitochondrial fragmentation, and improved mitochondrial functional capacity and cardiac function. Consistently, overexpression of miR-205 increased infarct size and promoted apoptosis, oxidative stress, and mitochondrial dysfunction in mice with MI/R injury. In cultured mouse neonatal cardiomyocytes, downregulation of miR-205 reduced oxidative stress in H/R-treated cardiomyocytes. Finally, inhibiting Rnd3 ablated the cardioprotective effects of miR-205 inhibitor in MI/R injury. Conclusions We conclude that inhibiting miR-205 reduces infarct size, improves cardiac function, and suppresses oxidative stress, mitochondrial dysfunction, and apoptosis by promoting Rnd3 in MI/R injury. miR-205 inhibitor-induced Rnd3 activation is a valid target to treat MI/R injury.

Placental mitochondria central to gestational diabetes pathogenesis?

Placental mitochondria central to gestational diabetes pathogenesis?

The Functional Impact of Mitochondrial Structure Across Subcellular Scales.

Mitochondria are key determinants of cellular health. However, the functional role of mitochondria varies from cell to cell depending on the relative demands for energy distribution, metabolite biosynthesis, and/or signaling. In order to support the specific needs of different cell types, mitochondrial functional capacity can be optimized in part by modulating mitochondrial structure across several different spatial scales. Here we discuss the functional implications of altering mitochondrial structure with an emphasis on the physiological trade-offs associated with different mitochondrial configurations. Within a mitochondrion, increasing the amount of cristae in the inner membrane improves capacity for energy conversion and free radical-mediated signaling but may come at the expense of matrix space where enzymes critical for metabolite biosynthesis and signaling reside. Electrically isolating individual cristae could provide a protective mechanism to limit the spread of dysfunction within a mitochondrion but may also slow the response time to an increase in cellular energy demand. For individual mitochondria, those with relatively greater surface areas can facilitate interactions with the cytosol or other organelles but may be more costly to remove through mitophagy due to the need for larger phagophore membranes. At the network scale, a large, stable mitochondrial reticulum can provide a structural pathway for energy distribution and communication across long distances yet also enable rapid spreading of localized dysfunction. Highly dynamic mitochondrial networks allow for frequent content mixing and communication but require constant cellular remodeling to accommodate the movement of mitochondria. The formation of contact sites between mitochondria and several other organelles provides a mechanism for specialized communication and direct content transfer between organelles. However, increasing the number of contact sites between mitochondria and any given organelle reduces the mitochondrial surface area available for contact sites with other organelles as well as for metabolite exchange with cytosol. Though the precise mechanisms guiding the coordinated multi-scale mitochondrial configurations observed in different cell types have yet to be elucidated, it is clear that mitochondrial structure is tailored at every level to optimize mitochondrial function to meet specific cellular demands.

Abstract IA08: Elevated endogenous SDHA drives pathologic metabolism in highly metastatic uveal melanoma

Abstract Purpose: Metastatic uveal melanoma (UM) has a very poor prognosis and no effective therapy. Despite remarkable advances in treatment of cutaneous melanoma, UM remains recalcitrant to chemotherapy, small-molecule kinase inhibitors, and immune-based therapy. Experimental Design: We assessed two sets of oxidative phosphorylation (OxPhos) genes within 9,858 tumors across 31 cancer types. An OxPhos inhibitor was used to characterize differential metabolic programming of highly metastatic monosomy 3 (M3) UM. Seahorse analysis and global metabolomics profiling were done to identify metabolic vulnerabilities. Analyses of UM TCGA data set were performed to determine expressions of key OxPhos effectors in M3 and non M3 UM. We used targeted knockdown of succinate dehydrogenase A (SDHA) to determine the role of SDHA in M3 UM in conferring resistance to OxPhos inhibition. Results: We identified UM as having among the highest median OxPhos levels and showed that M3 UM exhibits a distinct metabolic profile. M3 UM shows markedly low succinate levels and has highly increased levels of SDHA, the enzyme that couples the TCA cycle with OxPhos by oxidizing (lowering) succinate. We show that SDHA-high M3 UM has elevated expression of key OxPhos molecules, exhibits abundant mitochondrial reserve respiratory capacity, and is resistant to OxPhos antagonism, which can be reversed by SDHA knockdown. Conclusions: Our study has identified a critical metabolic program within poor prognostic M3 UM. In addition to the heightened mitochondrial functional capacity due to elevated SDHA, M3 UM SDHA-high mediates resistance to therapy that is reversible with targeted treatment. Citation Format: Chandrani Chattopadhyay, Junna Oba, Jason Roszik, Scott E. Woodman, Elizabeth A. Grimm. Elevated endogenous SDHA drives pathologic metabolism in highly metastatic uveal melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA08.

Development and thyroid hormone dependence of skeletal muscle mitochondrial function towards birth.

Key points Skeletal muscle energy requirements increase at birth but little is known regarding the development of mitochondria that provide most of the cellular energy as ATP.Thyroid hormones are known regulators of adult metabolism and are important in driving several aspects of fetal development, including muscle fibre differentiation.Mitochondrial density and the abundance of mitochondrial membrane proteins in skeletal muscle increased during late gestation. However, mitochondrial functional capacity, measured as oxygen consumption rate, increased primarily after birth.Fetal hypothyroidism resulted in significant reductions in mitochondrial function and density in skeletal muscle before birth.Mitochondrial function matures towards birth and is dependent on the presence of thyroid hormones, with potential implications for the health of pre‐term and hypothyroid infants. Birth is a significant metabolic challenge with exposure to a pro‐oxidant environment and the increased energy demands for neonatal survival. This study investigated the development of mitochondrial density and activity in ovine biceps femoris skeletal muscle during the perinatal period and examined the role of thyroid hormones in these processes. Muscle capacity for oxidative phosphorylation increased primarily after birth but was accompanied by prepartum increases in mitochondrial density and the abundance of electron transfer system (ETS) complexes I–IV and ATP‐synthase as well as by neonatal upregulation of uncoupling proteins. This temporal disparity between prepartum maturation and neonatal upregulation of mitochondrial oxidative capacity may protect against oxidative stress associated with birth while ensuring energy availability to the neonate. Fetal thyroid hormone deficiency reduced oxidative phosphorylation and prevented the prepartum upregulation of mitochondrial density and ETS proteins in fetal skeletal muscle. Overall, the data show that mitochondrial function matures over the perinatal period and is dependent on thyroid hormones, with potential consequences for neonatal viability and adult metabolic health.

Elevated Endogenous SDHA Drives Pathological Metabolism in Highly Metastatic Uveal Melanoma.

PurposeMetastatic uveal melanoma (UM) has a very poor prognosis and no effective therapy. Despite remarkable advances in treatment of cutaneous melanoma, UM remains recalcitrant to chemotherapy, small-molecule kinase inhibitors, and immune-based therapy.MethodsWe assessed two sets of oxidative phosphorylation (OxPhos) genes within 9858 tumors across 31 cancer types. An OxPhos inhibitor was used to characterize differential metabolic programming of highly metastatic monosomy 3 (M3) UM. Seahorse analysis and global metabolomics profiling were done to identify metabolic vulnerabilities. Analyses of UM TCGA data set were performed to determine expressions of key OxPhos effectors in M3 and non-M3 UM. We used targeted knockdown of succinate dehydrogenase A (SDHA) to determine the role of SDHA in M3 UM in conferring resistance to OxPhos inhibition.ResultsWe identified UM to have among the highest median OxPhos levels and showed that M3 UM exhibits a distinct metabolic profile. M3 UM shows markedly low succinate levels and has highly increased levels of SDHA, the enzyme that couples the tricarboxylic acid cycle with OxPhos by oxidizing (lowering) succinate. We showed that SDHA-high M3 UM have elevated expression of key OxPhos molecules, exhibit abundant mitochondrial reserve respiratory capacity, and are resistant to OxPhos antagonism, which can be reversed by SDHA knockdown.ConclusionsOur study has identified a critical metabolic program within poor prognostic M3 UM. In addition to the heightened mitochondrial functional capacity due to elevated SDHA, M3 UM SDHA-high mediate resistance to therapy that is reversible with targeted treatment.

Hyperglycemia-Driven Inhibition of AMP-Activated Protein Kinase α2 Induces Diabetic Cardiomyopathy by Promoting Mitochondria-Associated Endoplasmic Reticulum Membranes In Vivo.

Fundc1 (FUN14 domain containing 1), an outer mitochondrial membrane protein, is important for mitophagy and mitochondria-associated endoplasmic reticulum membranes (MAMs). The roles of Fundc1 and MAMs in diabetic hearts remain unknown. The aims of this study, therefore, were to determine whether the diabetes mellitus-induced Fundc1 expression could increase MAM formation, and whether disruption of MAM formation improves diabetic cardiac function. Levels of FUNDC1 were examined in the hearts from diabetic patients and nondiabetic donors. Levels of Fundc1-induced MAMs and mitochondrial and heart function were examined in mouse neonatal cardiomyocytes exposed to high glucose (HG, 30 mmol/L d-glucose for 48 hours), and in streptozotocin-treated cardiac-specific Fundc1 knockout mice and cardiac-specific Fundc1 knockout diabetic Akita mice, as well. FUNDC1 levels were significantly elevated in cardiac tissues from diabetic patients in comparison with those from nondiabetic donors. In cultured mouse neonatal cardiomyocytes, HG conditions increased levels of Fundc1, the inositol 1,4,5-trisphosphate type 2 receptor (Ip3r2), and MAMs. Genetic downregulation of either Fundc1 or Ip3r2 inhibited MAM formation, reduced endoplasmic reticulum-mitochondrial Ca2+ flux, and improved mitochondrial function in HG-treated cardiomyocytes. Consistently, adenoviral overexpression of Fundc1 promoted MAM formation, mitochondrial Ca2+ increase, and mitochondrial dysfunction in cardiomyocytes exposed to normal glucose (5.5 mmol/L d-glucose). In comparison with nondiabetic controls, levels of Fundc1, Ip3r2, and MAMs were significantly increased in hearts from streptozotocin-treated mice and Akita mice. Furthermore, in comparison with control hearts, diabetes mellitus markedly increased coimmunoprecipitation of Fundc1 and Ip3r2. The binding of Fundc1 to Ip3r2 inhibits Ip3r2 ubiquitination and proteasome-mediated degradation. Cardiomyocyte-specific Fundc1 deletion ablated diabetes mellitus-induced MAM formation, prevented mitochondrial Ca2+ increase, mitochondrial fragmentation, and apoptosis with improved mitochondrial functional capacity and cardiac function. In mouse neonatal cardiomyocytes, HG suppressed AMP-activated protein kinase activity. Furthermore, in cardiomyocytes of Prkaa2 knockout mice, expression of Fundc1, MAM formation, and mitochondrial Ca2+ levels were significantly increased. Finally, adenoviral overexpression of a constitutively active mutant AMP-activated protein kinase ablated HG-induced MAM formation and mitochondrial dysfunction. We conclude that diabetes mellitus suppresses AMP-activated protein kinase, initiating Fundc1-mediated MAM formation, mitochondrial dysfunction, and cardiomyopathy, suggesting that AMP-activated protein kinase-induced Fundc1 suppression is a valid target to treat diabetic cardiomyopathy.

Hyperglycemia‐Driven Inhibition of AMP‐Activated Protein Kinase a2 Induces Diabetic Cardiomyopathy by Promoting Mitochondria‐Associated Endoplasmic Reticulum Membranes in Vivo

BackgroundFUN14 domain containing 1 (Fundc1), an outer mitochondrial membrane protein, is important for mitophagy and mitochondria‐associated endoplasmic reticulum (ER) membranes (MAMs). The roles of Fundc1 and MAMs in diabetic hearts remain unknown. The aims of this study therefore, were to determine if the diabetes‐induced Fundc1 expression could increase MAM formation, and whether disruption of MAM formation improves diabetic cardiac function.MethodsLevels of FUNDC1 were examined in the hearts from diabetic patients and non‐diabetic donors. Levels of Fundc1‐induced MAMs, and mitochondrial and heart function were examined in mouse neonatal cardiomyocytes exposed to high glucose (HG, 30 mmol/L D‐glucose for 48 h), as well as in streptozotocin (STZ)‐treated cardiac‐specific Fundc1 knockout (KO) mice and cardiac‐specific Fundc1 KO diabetic Akita mice.ResultsFUNDC1 levels were significantly elevated in cardiac tissues from diabetic patients compared to those in non‐diabetic donors. In cultured mouse neonatal cardiomyocytes, HG conditions increased levels of Fundc1, the inositol 1,4,5‐trisphosphate type 2 receptor (Ip3r2), and MAMs. Genetic downregulation of either Fundc1 or Ip3r2 inhibited MAM formation, reduced ER‐mitochondrial Ca2+ flux, and improved mitochondrial function in HG‐treated cardiomyocytes. Consistently, adenoviral overexpression of Fundc1 promoted MAM formation, mitochondrial Ca2+ overload, and mitochondrial dysfunction in cardiomyocytes exposed to normal glucose (5.5 mmol/L D‐glucose). Compared with non‐diabetic controls, levels of Fundc1, Ip3r2, and MAMs were significantly increased in hearts from STZ‐treated mice and Akita mice. Further, compared with control hearts, diabetes markedly increased co‐immunoprecipitation of Fundc1 and Ip3r2. The binding of Fundc1 to Ip3r2 inhibits Ip3r2 ubiquitination and proteasome‐mediated degradation. Cardiomyocyte‐specific Fundc1 deletion ablated diabetes‐induced MAM formation, prevented mitochondrial Ca2+ overload, mitochondrial fragmentation, and apoptosis with improved mitochondrial functional capacity and cardiac function. In mouse neonatal cardiomyocytes, HG suppressed AMP‐activated protein kinase (Ampk) activity. Furthermore, in cardiomyocytes of Prkaa2 KO mice, expression of Fundc1, MAM formation, and mitochondrial Ca2+ levels were significantly increased. Finally, adenoviral overexpression of a constitutively active mutant Ampk ablated HG‐induced MAM formation, mitochondrial Ca2+ overload, and mitochondrial dysfunction.ConclusionsWe conclude that HG conditions in diabetes suppress Ampk, initiating Fundc1‐mediated MAM formation, mitochondrial dysfunction, and cardiomyopathy, suggesting that Ampk‐induced Fundc1 suppression is a valid target to treat diabetic cardiomyopathy.Support or Funding InformationThis study was supported by funding from the following agencies: NHLBI (HL079584, HL080499, HL089920, HL110488, HL128014, HL132500, HL137371, HL142287, and HL140954), NCI (CA213022), NIA (AG047776), and AHA (16GRANT29590003). Dr. Zou is the Eminent Scholar in Molecular and Translational Medicine of Georgia Research Alliance.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Body weight influences genes related to energy metabolism in human skeletal muscle

Over one third of adults in the United States are obese. Skeletal muscle is a major site of metabolic activity and the most abundant tissue in the human body. For skeletal muscle to function at optimal levels, the efficient activation of processes that regulate muscle development, growth, regeneration and metabolism is required. Mitochondria play a key role in ensuring adequate levels of ATP needed for skeletal muscle contraction. In addition, the capacity of skeletal muscle to contribute to whole‐body energy expenditure is related to the fact that muscle makes up ~40% of total body mass and accounts for 20–30% of total resting oxygen uptake. This indicates that mitochondrial functional capacity is likely to directly affect muscle metabolic function and have a significant impact on whole‐body metabolism. Mitochondria are also important for ensuring muscle is able to modulate substrate oxidation (e.g. lipid and carbohydrate oxidation) following a meal. The objective of this study was to determine if there was an effect of body weight on genes related to mitochondrial function in skeletal muscle from normal weight (NW) and obese/overweight (OW) participants. Muscle biopsies were taken from OW (n=31, ages 48±19) and NW (n=12, ages 43±18) adults. Gene expression was measured using real‐time PCR. There was an increase (p&lt;0.05) in the expression of both PGC1α and PPARγ, which are regulators of mitochondrial biogenesis, in OW vs NW participants. Expression of UCP2, a key regulator involved in ATP synthesis, was higher (p&lt;0.05) in OW participants. In addition, genes related to mitochondrial respiration, mitochondrial DNA replication and transcription, NRF1 and Tfam, had higher (p&lt;0.05) expression in OW. Cpt1a, a gene encoding carnitine palmitoyltransferase 1A, which is necessary for fatty acid oxidation, had lower (p&lt;0.05) expression in OW compared with NW. Conclusively, these data provide a preliminary view of differences in energy metabolism gene expression in obese/overweight versus normal weight individuals.Support or Funding InformationArkansas Biosciences InstituteThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A natural product composition targeting oxidative stress induced mitochondrial dysfunction in Alzheimer's Disease

Mitochondria regulates cellular metabolism and apoptosis. Oxidative stress induced mitochondrial dysfunction, appears to play a role in the pathogenesis of Alzheimer's disease. Natural antioxidants have been known to play an important role in alleviating the deleterious effects induced by free radicals by blocking the initiation and propagation of oxidizing chain reactions. Hence, the study hypothesizes the effect of natural product composition (combinatorial extract of Eclipta alba, Morinda pubescens and Withania somnifera) on restoring the mitochondrial functional capacity and mitochondrial enzyme levels. The individual and combinatorial methanolic extracts of dried whole plants of Eclipta alba (MEEA), dried fruits of Morinda pubescens (MEMP) and dried roots of Withania somnifera (MEWS) and MECE were prepared. The extracts were screened for in vitro antioxidant activity by DPPH assay. A rodent behavioural model to assess the effect of the extracts on short term memory was performed subsequent to scopolamine hydrobromide induced amnesia and oxidative stress. The brain homogenates from these rodents were subjected to screening in vivo antioxidaint activity using a battery of assays. The effect of extracts on restoring mitochondrial functional capacity (using MTT assay) and the levels of key mitochondrial enzymes succinate dehydrogenase and isocitrate dehydrogenase; was performed using synaptosomal fractions prepared from rodent brain samples.MEEA, MEMP, MEWS and MECE compared with ascorbic acid exhibited 50% scavenging effect at 69.34 μg/ml, 72.19 μg/ml, 65.84 μg/ml, 45.09μg/ml and 57.64 μg/ml, respectively by DPPH assay. Pretreatment with MEEA, MEMP and MEWS at the doses 50,100 and 200 mg/kg and MECE at 50 and 100mg/kg for 8 days followed by scopolamine hydrobromide treatment significantly (p&lt;0.001) improved the antioxidant defense system, the mitochondrial functional capacity and mitochondrial enzyme levels.The natural product composition strengthened the endogenous antioxidant defense system, restored the mitochondrial functional capacity and enzyme levels. Hence, it has a potential neuroprotective role in Alzheimer's disease which needs to be investigated further in a clinical setting.Support or Funding InformationNMIMS UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolism, Inflammation, and Tumor Progression

Altered tumor cell metabolism is now firmly established as a hallmark of human cancer. Downstream of oncogenic events, metabolism is re‐wired to support cellular energetics and supply the building blocks for biomass. Rapid, uncontrolled proliferation results in tumor growth beyond the reach of existing vasculature and triggers cellular adaptations to overcome limiting nutrient and oxygen delivery. However, oncogenic activation and metabolic re‐programming also elicit cell intrinsic stresses, independent of the tumor microenvironment. To ensure metabolic robustness and stress resistance, pro‐growth signals downstream of oncogene activation or tumor suppressor loss simultaneously activate homeostatic processes. Here, I will summarize adaptive mechanisms co‐opted by common oncogenes, including mTOR, MYC, and RAS. Recurrent themes include: (1) coordination of oncogene‐induced changes in protein and lipid metabolism to sustain endoplasmic reticulum homeostasis, (2) maintenance of mitochondrial functional capacity to support anabolic metabolism, (3) adaptations to sustain intracellular metabolite concentrations required for growth, and (4) prevention of oxidative stress. Ultimately, an understanding of the adaptations required by both tumor cells and recruited inflammatory cells could reveal targetable metabolic vulnerabilities.Support or Funding InformationR35 CA197602 (Outstanding Investigator Award)Title: “Metabolic Tumor Suppressors in Renal Cancer: Unprecedented Roles in Disease Progression”3 P01 CA 104838Title: “Cancer Cell Adaptation to Metabolic Stress”This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Mitochondrial Health Index Sensitive to Mood and Caregiving Stress

BackgroundChronic life stress, such as the stress of caregiving, can promote pathophysiology, but the underlying cellular mechanisms are not well understood. Chronic stress may induce recalibrations in mitochondria leading to changes either in mitochondrial content per cell, or in mitochondrial functional capacity (i.e., quality). MethodsHere we present a functional index of mitochondrial health (MHI) for human leukocytes that can distinguish between these two possibilities. The MHI integrates nuclear and mitochondrial DNA–encoded respiratory chain enzymatic activities and mitochondrial DNA copy number. We then use the MHI to test the hypothesis that daily emotional states and caregiving stress influence mitochondrial function by comparing healthy mothers of a child with an autism spectrum disorder (high-stress caregivers, n = 46) with mothers of a neurotypical child (control group, n = 45). ResultsThe MHI outperformed individual mitochondrial function measures. Elevated positive mood at night was associated with higher MHI, and nightly positive mood was also a mediator of the association between caregiving and MHI. Moreover, MHI was correlated to positive mood on the days preceding, but not following the blood draw, suggesting for the first time in humans that mitochondria may respond to proximate emotional states within days. Correspondingly, the caregiver group, which had higher perceived stress and lower positive and greater negative daily affect, exhibited lower MHI. This effect was not explained by a mismatch between nuclear and mitochondrial genomes. ConclusionsDaily mood and chronic caregiving stress are associated with mitochondrial functional capacity. Mitochondrial health may represent a nexus between psychological stress and health.

UCP3 Ablation Exacerbates High-Salt Induced Cardiac Hypertrophy and Cardiac Dysfunction

Background/Aims: Excessive salt intake and left ventricular hypertrophy (LVH) are both critical for the development of hypertension and heart failure. The uncoupling protein 3 (UCP3) plays a cardio-protective role in early heart failure development. However, the potential role for UCP3 in salt intake and LVH is unclear. Methods: UCP3^-/- and C57BL/6 mice were placed on either a normal-salt (NS, 0.5%) or a high-salt (HS, 8%) diet for 24 weeks. The cardiac function, endurance capacity, energy expenditure, and mitochondrial functional capacity were measured in each group. Results: Elevated blood pressure was only observed in HS-fed UCP3^-/- mice. High salt induced cardiac hypertrophy and dysfunction were observed in both C57BL/6 and UCP3^-/- mice. However, the cardiac lesions were more profound in HS-fed UCP3^-/- mice. Furthermore, HS-fed UCP3^-/-mice experienced more severe mitochondrial respiratory dysfunction compared with HS-fed C57BL/6 mice, represented by the decreased volume of oxygen consumption and heat production at the whole-body level. Conclusion: UCP3 protein was involved in the incidence of high-salt induced hypertension and the progression of cardiac dysfunction in the early stages of heart failure. UCP3 ablation exacerbated high-salt-induced cardiac hypertrophy and cardiac dysfunction.