Mouse Mitochondria Research Articles

High-throughput identification of calcium-regulated proteins across diverse proteomes

Calcium ions play important roles in nearly every biological process, yet whole-proteome analysis of calcium effectors has been hindered by a lack of high-throughput, unbiased, and quantitative methods to identify protein-calcium engagement. To address this, we adapted protein thermostability assays in budding yeast, human cells, and mouse mitochondria. Based on calcium-dependent thermostability, we identified 2,884 putative calcium-regulated proteins across human, mouse, and yeast proteomes. These data revealed calcium engagement of signaling hubs and cellular processes, including metabolic enzymes and the spliceosome. Cross-species comparison of calcium-protein engagement and mutagenesis experiments identified residue-specific cation engagement, even within well-known EF-hand domains. Additionally, we found that the dienoyl-coenzyme A (CoA) reductase DECR1 binds calcium at physiologically relevant concentrations with substrate-specific affinity, suggesting direct calcium regulation of mitochondrial fatty acid oxidation. These discovery-based proteomic analyses of calcium effectors establish a key resource to dissect cation engagement and its mechanistic effects across multiple species and diverse biological processes.

Mitochondrial OPA1 Deficiency Is Associated to Reversible Defects in Spatial Memory Related to Adult Neurogenesis in Mice.

Mitochondria are integrative hubs central to cellular adaptive pathways. Such pathways are critical in highly differentiated postmitotic neurons, the plasticity of which sustains brain function. Consequently, defects in mitochondria and in their dynamics appear instrumental in neurodegenerative diseases and may also participate in cognitive impairments. To directly test this hypothesis, we analyzed cognitive performances in a mouse mitochondria-based disease model, because of haploinsufficiency in the mitochondrial optic atrophy type 1 (OPA1) protein involved in mitochondrial dynamics. In males, we evaluated adult hippocampal neurogenesis parameters using immunohistochemistry. We performed a battery of tests to assess basal behavioral characteristics and cognitive performances, and tested putative treatments. While in dominant optic atrophy (DOA) mouse models, the known main symptoms are late onset visual deficits, we discovered early impairments in hippocampus-dependent spatial memory attributable to defects in adult neurogenesis. Moreover, less connected adult-born hippocampal neurons showed a decrease in mitochondrial content. Remarkably, voluntary exercise or pharmacological treatment targeting mitochondrial dynamics restored spatial memory in DOA mice. Altogether, our study identifies a crucial role for OPA1-dependent mitochondrial functions in adult neurogenesis, and thus in hippocampal-dependent cognitive functions. More generally, our findings show that adult neurogenesis is highly sensitive to mild mitochondrial defects, generating impairments in spatial memory that can be detected at an early stage and counterbalanced by physical exercise and pharmacological targeting of mitochondrial dynamics. Thus, amplification of mitochondrial function at an early stage appears beneficial for late-onset neurodegenerative diseases.

Low-dose atorvastatin protects skeletal muscle mitochondria in high-fat diet-fed mice with mitochondrial autophagy inhibition and fusion enhancement

Despite the great clinical benefits of statins in cardiovascular diseases, their widespread use may lead to adverse muscle reactions associated with mitochondrial dysfunction. Some studies have demonstrated that statins provide substantial improvement to skeletal muscle health in mice. Our previous study found that oral treatment with atorvastatin (Ator, 3 mg/kg) protected myocardial mitochondria in high-fat diet (HFD)-fed mice. Therefore, this study aimed to explore the influence of low-dose Ator (3 mg/kg) on mitochondria in skeletal muscle under cholesterol overload. Male C57BL/6J mice were fed a HFD for 18 weeks and orally administered Ator (3 mg/kg) during the last 12 weeks. Ator treatment had no effects on elevated serum cholesterol and glucose levels in HFD-fed mice. Serum creatine kinase levels and the cross-sectional area of muscle cells were not affected by HFD feeding or Ator treatment. Increased expression of PINK1-LC3 II (activated mitophagy), MFN2 (fusion), and PGC-1α (biogenesis) proteins was induced in the skeletal muscles of HFD-fed mice. Treatment with Ator inhibited PINK1 and LC3 II protein expression, but further promoted MFN1, MFN2, and OPA1 expression. The impairments in mitochondrial quality and morphology in HFD-fed mice were attenuated by treatment with Ator. Furthermore, Ator treatment enhanced glucose oxidation capacity and restored ATP production in the skeletal muscles of HFD-fed mice. The study reveals that low-dose Ator has a protective effect on muscle mitochondria in mice, likely through inhibiting mitophagy and enhancing mitochondrial fusion. This suggests that skeletal muscle mitochondria may be one of low-dose Ator-mediated protective targets.

Loss and recovery of myocardial mitochondria in mice under different tail suspension time: Apoptosis and mitochondrial fission, fusion and autophagy.

Long-term weightlessness in animals can cause changes in myocardial structure and function, in which mitochondria play an important role. Here, a tail suspension (TS) Kunming mouse (Mus musculus) model was used to simulate the effects of weightlessness on the heart. We investigated the effects of 2 and 4weeks of TS (TS2 and TS4) on myocardial mitochondrial ultrastructure and oxidative respiratory function and on the molecular mechanisms of apoptosis and mitochondrial fission, autophagy and fusion-related signalling. Our study revealed significant changes in the ultrastructural features of cardiomyocytes in response to TS. The results showed: (1) mitochondrial swelling and disruption of cristae in TS2, but mitochondrial recovery and denser cristae in TS4; (2) an increase in the total number of mitochondria and number of sub-mitochondria in TS4; (3) no significant changes in the nuclear ultrastructure or DNA fragmentation among the two TS groups and the control group; (4) an increase in the bax/bcl-2 protein levels in the two TS groups, indicating increased activation of the bax-mediated apoptosis pathway; (5) no change in the phosphorylation ratio of dynamin-related protein 1 in the two TS groups; (6) an increase in the protein levels of optic atrophy 1 and mitofusin 2 in the two TS groups; and (7) in comparison to the TS2 group, an increase in the phosphorylation ratio of parkin and the ratio of LC3II to LC3I in TS4, suggesting an increase in autophagy. Taken together, these findings suggest that mitochondrial autophagy and fusion levels increased after 4weeks of TS, leading to a restoration of the bax-mediated myocardial apoptosis pathway observed after 2weeks of TS. NEW FINDINGS: What is the central question of this study? What are the effects of 2 and 4weeks of tail suspension on myocardial mitochondrial ultrastructure and oxidative respiratory function and on the molecular mechanisms of apoptosis and mitochondrial fission, autophagy and fusion-related signalling? What is the main finding and its importance? Increased mitochondrial autophagy and fusion levels after 4weeks of tail suspension help to reshape the morphology and increase the number of myocardial mitochondria.

Efficacy and Safety of 5-Aminolevulinic Acid Combined with Iron on Skeletal Muscle Mass Index and Physical Performance of Patients with Sarcopenia: A Multicenter, Double-Blinded, Randomized-Controlled Trial (ALADDIN Study).

Sarcopenia is a geriatric syndrome characterized by decreased physical performance, muscle mass, and strength. Since the intake of 5-aminolevulinic acid (ALA) with iron can increase muscle mass and mitochondria in mice and elevate physical exercise performance in humans, the beneficial effects of ALA in patients with sarcopenia are expected, but this remains unexplored in the literature. This study aimed to assess the efficacy and dose dependency of ALA combined with iron in sarcopenia by measuring skeletal muscle mass index (SMI). Subjects with sarcopenia were enrolled and randomized into the ALA and sodium ferrous citrate (SFC) intake groups (ALA50/SFC29, ALA100/SFC29, ALA150/SFC29, ALA 100/SFC57, and ALA0/SFC29 placebo) and ingested the assigned study food for 12 weeks. The primary endpoint, the change in SMI from baseline to week 12, did not differ significantly between the groups. Hand grip significantly increased or tended to increase from baseline after 12 weeks with all doses of ALA or SFC compared with the placebo group. No consistent changes were observed in the other endpoints, including calf circumference, physical function, or quality of life (QOL). Although this study suggests safe administration and the possibility of ALA improving hand grip strength in patients with sarcopenia, further investigation is required.

193-OR: Muscle α-Parvin Deletion Results in Insulin Resistance by Impairing GLUT4 Translocation and Hexokinase II Association with Mitochondria in Mice

Genetic deletions of specific integrin receptor a-subunits and the integrin-like kinase (ILK) have no effect on insulin action in lean C57Bl/6J mice but are required for insulin resistance in diet-induced obese (DIO) mice. ILK binds to a-parvin through which it controls the rho kinase pathway and cytoskeleton thereby altering cellular compartmentation and dynamics. The hypothesis tested was that integrin signaling is required for insulin resistance in DIO mice due to disruption of the critical interaction between ILK and a-parvin. We predicted that preventing this interaction in lean mice would recapitulate the insulin resistance observed in DIO mice. Lean and DIO mice with muscle-specific a-parvin (mPar) knockout (KO) and wild type littermates (WT) were studied. Insulin clamps were performed in mPar KO and WT mice. A muscle-specific index of glucose uptake (Rg) was measured isotopically during insulin clamps. Lean mPar KO had a 50% reduction in muscle Rg that was associated with a similar deficit in GLUT4 translocation to the cell membrane and a decrease in hexokinase II-associated with mitochondria. Mitochondria oxidative capacity was reduced by 50% in lean mPar KO mice compared to lean WT mice. The insulin resistance in DIO WT mice was no worse in DIO mPar KO mice. Insulin signaling was not impaired by mPar KO in lean mice. However, lean mPar KO had impaired a-actinin, Rac1, p-cofilin/cofilin, and p-MLC/MLC compared to lean WT mice during insulin stimulation. These changes affect cytoskeletal dynamics and provide a basis for the reduction in GLUT4 translocation, hexokinase II localization to mitochondria, and impaired mitochondria oxidation. In conclusion, a-parvin is a key node in coupling integrin signaling as it causes muscle insulin resistance in otherwise healthy lean mice. Deletion of muscle a-parvin does not affect insulin signaling but rather attenuates insulin-stimulated glucose uptake by impairing cytoskeletal dynamics. Disclosure D.A.Cappel: None. D.H.Wasserman: None. J.W.Deaver: None. L.Cozzani: None. D.Bracy: None. L.Lantier: None. X.Dong: None. R.Printz: None. R.Zent: None. A.Pozzi: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK050277, R01DK054902, R24DK059637)

Dietary Folic Acid Supplementation Inhibits HighFat DietInduced Body Weight Gain through Gut Microbiota-Associated Branched-Chain Amino Acids and Mitochondria in Mice.

The effects of folic acid on body weight gain in obesity and gut microbiota-associated branched-chain amino acids (BCAAs) and mitochondrial function were investigated. Three- to four-wk-old male C57BL/6J conventional (CV) and germ-free (GF) mice were fed a high-fat diet (HD), folic acid-supplemented HD (FSHD) and a normal-fat diet (ND) for 25 wk. In CV mice, the HD-induced increases in body weight and plasma BCAA concentrations, downregulated expression of genes related to BCAA catabolism (Bcat2, Bckdha, or Ppm1k), mitochondrial biogenesis (Pgc-1α, Cox1, Nd1 or Nd6), fusion (Mfn1, Mfn2 or Opa1) and mitophagy (Pink1 or Park2), and upregulated expression of the fission-associated gene Drp1 in epididymal fat were reversely corrected with FSHD feeding. In contrast, the expression of these genes in the liver was the opposite under HD feeding or folic acid supplementation. In GF mice, plasma BCAA concentrations were much less affected by HD feeding and were reduced by FSHD feeding, with almost no alterations in the expression of genes associated with BCAA catabolism and mitochondrial function. Further analysis indicated a correlation between adipose and hepatic Mt C/N and plasma BCAA concentrations, and the latter had a close association with specific gut bacteria. Therefore, dietary folic acid supplementation differentially affected body weight gain, BCAA catabolism, and mitochondrial dynamics and metabolism under HD feeding between CV and GF mice, suggesting that gut bacteria-altered BCAAs and mitochondria might partially share the responsibility for the beneficial effects of dietary folic acid on obesity.

Transgenic type2 diabetes mouse models for in vivo redox measurement of hepatic mitochondrial oxidative stress

BackgroundOxidative stress is involved in the progression of diabetes and its associated complications. However, it is unclear whether increased oxidative stress plays a primary role in the onset of diabetes or is a secondary indicator caused by tissue damage. Previous methods of analyzing oxidative stress have involved measuring the changes in oxidative stress biomarkers. Our aim is to identify a novel approach to clarify whether oxidative stress plays a primary role in the onset of diabetes. MethodsWe constructed transgenic type 2 diabetes mouse models expressing redox-sensitive green fluorescent proteins (roGFPs) that distinguished between mitochondria and whole cells. Pancreas, liver, skeletal muscle, and kidney redox states were measured in vivo. ResultsHepatic mitochondrial oxidation increased when the mice were 4 weeks old and continued to increase in an age-dependent manner. The increase in hepatic mitochondrial oxidation occurred simultaneously with weight gain and increased blood insulin levels before the blood glucose levels increased. Administering the oxidative stress inducer acetaminophen increased the vulnerability of the liver mitochondria to oxidative stress. ConclusionsThis study demonstrates that oxidative stress in liver mitochondria in mice begins at the onset of diabetes rather than after the disease has progressed. General significanceRoGFP-expressing transgenic type 2 diabetes mouse models are effective and convenient tools for measuring hepatic mitochondrial redox statuses in vivo. These models may be used to assess mitochondria-targeting antioxidants and establish the role of oxidative stress in type 2 diabetes.

Mitochondrial Triglyceride Dysregulation in Optic Nerves Following Indirect Traumatic Optic Neuropathy.

The purpose of this work is to identify mitochondrial optic nerve (ON) lipid alterations associated with sonication-induced traumatic optic neuropathy (TON). Briefly, a mouse model of indirect TON was generated using sound energy concentrated focally at the entrance of the optic canal using a laboratory sonifier (Branson Digital Sonifier 450, Danbury, CT, USA) with a microtip probe. We performed an analysis of a previously generated dataset from high-performance liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS). We analyzed lipids from isolated mitochondria from the ON at 1 day, 7 days, and 14 days post-sonication compared to non-sonicated controls. Lipid abundance alterations in post-sonicated ON mitochondria were evaluated with 1-way ANOVA (FDR-adjusted significant p-value < 0.01), debiased sparse partial correlation (DSPC) network modeling, and partial least squares-discriminant analysis (PLS-DA). We find temporal alterations in triglyceride metabolism are observed in ON mitochondria of mice following sonication-induced optic neuropathy with notable depletions of TG(18:1/18:2/18:2), TG(18:1/18:1/18:1), and TG(16:0/16:0/18:1). Depletion of mitochondrial triglycerides may mediate ON damage in indirect traumatic optic neuropathy through loss energy substrates for neuronal metabolism.

The Effect of Aerobic Exercise on the Oxidative Capacity of Skeletal Muscle Mitochondria in Mice with Impaired Glucose Tolerance.

Methods Male C57BL/6J mice were randomly divided into six different experimental groups (8 animals/group): (1) normal group (NOR), (2) normal control group (NC), (3) normal + exercise group (NE), (4) IGT group (IGT), (5) IGT control group (IC), and (6) IGT+ exercise group (IE).The exercise group received aerobic exercise for 8 weeks. After the intervention, a blood glucose meter was used to detect the level of glucose tolerance in the mouse's abdominal cavity; a biochemical kit was used to detect serum lipid metabolism indicators, malondialdehyde, and superoxide dismutase levels; the ELISA method was used to detect serum insulin and mouse gastrocnemius homogenate LDH, PDH, SDH, and CCO levels. Western blot method was used to detect the protein expression levels of NOX4, PGC-1α, and Mfn2 in the gastrocnemius muscle of mice. Results (1) Mice with high-fat diet for 30 weeks showed impaired glucose tolerance, insulin resistance, and lipid metabolism disorders. The level of LDH, PDH, SDH, and CCO in the gastrocnemius homogenate of mice was reduced. The expressions of NOX4 protein were significantly upregulated, while the expressions of PGC-1α and Mfn2 proteins were significantly downregulated. (2) 8-week aerobic exercise improved the disorders of glucose and lipid metabolism in IGT mice and increased homogenized LDH, PDH, SDH, and CCO levels, and the expressions of NOX4, PGC-1α, and Mfn2 proteins in the gastrocnemius muscle of mice were reversed. It is speculated that aerobic exercise can accelerate energy metabolism. Conclusion (1) C57BL/6 mice were fed high fat for 30 weeks and successfully constructed a mouse model of reduced diabetes; the mice with reduced diabetes have impaired glucose tolerance, insulin resistance, and lipid metabolism disorders; (2) 8 weeks of aerobic exercise improve glucose tolerance, reduce glucose tolerance in mice, reduce insulin resistance, improve lipid metabolism disorders, and reduce oxidative stress; (3) 8-week aerobic exercise reduces skeletal muscle NOX4 expression and increases glucose tolerance; reduces the expression of LDH, PDH, SDH, and CCO in mouse skeletal muscle; increases the expression level of mitochondrial fusion protein 2 and PGC-1α; improves glucose tolerance; reduces energy metabolism of mouse skeletal muscle; reduces oxidative stress; and reduces insulin resistance. It is speculated that aerobic exercise can accelerate energy metabolism. This process may involve two aspects: firstly, increase the expression level of oxidative metabolism enzymes and promote the tricarboxylic acid cycle; secondly, increase the expression of Mfn2 and accelerate mitochondria fission or fusion to regulate energy metabolism, thereby reducing oxidative stress and insulin resistance.

TGFβ Induces Changes in Mitochondrial Homeostasis in Skeletal Muscle of Mice with Bone Metastases

Cancer cachexia is a common neoplastic syndrome of advanced cancers characterized by a loss of skeletal muscle mass and decreased function. Patients with cancer cachexia have higher chances of morbidity and mortality. We have shown that mice inoculated with human MDA‐MB‐231 breast cancer cells, which metastasize to bone, have skeletal muscle weakness, cachexia, and altered skeletal muscle metabolism. Mice with bone metastases have increased serum levels of transforming growth factor beta (TGFβ). Serum TGFβ decreased after treatment with the bone anti‐resorptive agent, zoledronic acid (ZA), suggesting TGFβ is, in part, bone‐derived. Additionally, mice with bone metastases treated with ZA had increased skeletal muscle function. TGFβ has also been shown to decrease the master regulator of mitochondrial biogenesis, peroxisome‐activated receptor γ coactivator 1α (PGC1α), in C2C12 myotubes. Mitochondria provide muscle with energy (ATP) via oxidative phosphorylation, so we measured oxygen consumption and energy production using indirect calorimetry in mice with bone metastases. Oxygen consumption and energy production were both decreased in these mice suggesting a decrease in mitochondrial function. We then examined skeletal muscle mitochondria using transmission electron microscopy and western blotting. We found highly dysmorphic mitochondria and altered protein markers of mitochondrial biogenesis and dynamics. Additionally, LC3B protein was increased in skeletal muscle mitochondria demonstrating increased mitophagy in skeletal muscle of mice with bone metastases. From our previous knowledge and data of TGFβ and its role in mice with bone metastases, we hypothesize that TGFβ causes mitochondrial dysfunction in skeletal muscle of mice with bone metastases. We have shown that a 48‐hour treatment with TGFβ decreases protein expression in markers of mitochondrial biogenesis and dynamics in C2C12 myotubes. This data suggests that TGFβ could be causing the mitochondrial dysfunction and dysmorphia we see in our mouse model, but further experiments are needed to prove this hypothesis. Our data demonstrates the importance of mitochondrial function in skeletal muscle cells and skeletal muscle of mice with bone metastases. Understanding the role TGFβ plays in changes of skeletal muscle mitochondria in mice with bone metastases will help contribute to defining treatments for patients with cancer cachexia.

Targeting Diabetic Cardiomyopathy: LncRNA Kcnq1ot1 Rescues Mitochondrial ATP Synthase via Sponging of MiR‐378a‐5p

Diabetes mellitus has been linked to an increase in mitochondrial microRNA‐378a (miR‐378a) content. Twenty‐five‐week‐old miR‐378a knockout Db/Db mice display preserved mt‐ATP6 and ATP synthase protein content, ATP synthase activity, and preserved cardiac function, supporting the hypothesis that miR‐378a inhibition may be a therapeutic option for maintaining ATP synthase functionality during diabetes mellitus. Evidence also suggests that long non‐coding RNAs (lncRNAs), including lncRNA potassium voltage‐gated channel subfamily Q member 1 overlapping transcript 1 (Kcnq1ot1), participate in regulatory axes with microRNAs (miRs). Prediction analyses indicate that Kcnq1ot1 has the potential to bind miR‐378a. This study aimed to ascertain whether Kcnq1ot1 interacts with miR‐378a to impact ATP synthase functionality by preserving mt‐ATP6 levels. While miR‐378a was confirmed to be significantly higher in type 2 diabetes mellitus (T2DM) patient and twenty‐five‐week‐old Db/Db mouse mitochondria (P < 0.05), mitochondrial mt‐ATP6 and Kcnq1ot1 levels were significantly reduced when compared to controls (P < 0.05). Assessments following Kcnq1ot1 overexpression in HL‐1 cardiomyocytes overexpressing miR‐378a revealed Kcnq1ot1 binding significantly reduced miR‐378a levels, as indicated by reduced luciferase activity following transfection (P < 0.05). Further, Kcnq1ot1 overexpression reduced miR‐378a levels (P < 0.05), and rescued mt‐ATP6 and ATP synthase protein content (P < 0.05). Together, these data suggest that Kcnq1ot1 and miR‐378a may act as constituents in an axis that regulates mt‐ATP6 content, and that manipulation of this axis may provide benefit to ATP synthase functionality in the heart during type 2 diabetic insult.

Manipulation of the miR-378a/mt-ATP6 regulatory axis rescues ATP synthase in the diabetic heart and offers a novel role for lncRNA Kcnq1ot1.

Diabetes mellitus has been linked to an increase in mitochondrial microRNA-378a (miR-378a) content. Enhanced miR-378a content has been associated with a reduction in mitochondrial genome-encoded mt-ATP6 abundance, supporting the hypothesis that miR-378a inhibition may be a therapeutic option for maintaining ATP synthase functionality during diabetes mellitus. Evidence also suggests that long noncoding RNAs (lncRNAs), including lncRNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (Kcnq1ot1), participate in regulatory axes with microRNAs (miRs). Prediction analyses indicate that Kcnq1ot1 has the potential to bind miR-378a. This study aimed to determine if loss of miR-378a in a genetic mouse model could ameliorate cardiac dysfunction in type 2 diabetes mellitus (T2DM) and to ascertain whether Kcnq1ot1 interacts with miR-378a to impact ATP synthase functionality by preserving mt-ATP6 levels. MiR-378a was significantly higher in patients with T2DM and 25-wk-old Db/Db mouse mitochondria, whereas mt-ATP6 and Kcnq1ot1 levels were significantly reduced when compared with controls. Twenty-five-week-old miR-378a knockout Db/Db mice displayed preserved mt-ATP6 and ATP synthase protein content, ATP synthase activity, and preserved cardiac function, implicating miR-378a as a potential therapeutic target in T2DM. Assessments following overexpression of the 500-bp Kcnq1ot1 fragment in established mouse cardiomyocyte cell line (HL-1) cardiomyocytes overexpressing miR-378a revealed that Kcnq1ot1 may bind and significantly reduce miR-378a levels, and rescue mt-ATP6 and ATP synthase protein content. Together, these data suggest that Kcnq1ot1 and miR-378a may act as constituents in an axis that regulates mt-ATP6 content, and that manipulation of this axis may provide benefit to ATP synthase functionality in type 2 diabetic heart.

Astaxanthin Protects Against Heat-induced Mitochondrial Alterations in Mouse Hypothalamus

The hypothalamus plays an essential role in regulating whole-body energy and temperature homeostasis when adapting to environmental changes. We previously reported that heat exposure causes mitochondrial dysfunction and apoptosis in mouse skeletal muscle, and pretreatment with astaxanthin (AST), an antioxidant, prevents this effect. How the hypothalamus responds to heat stress remains largely unexplored. In this study, we investigated the effects of heat exposure on hypothalamic mitochondria in mice with and without AST pretreatment. During heat exposure, both vehicle and AST-treated mice had a hyperthermic response though no significant differences in peak core body temperature were noted between the two groups. Heat exposure induced mitochondrial fission in the hypothalamus, as manifested by increased mitochondrial fragmentation and expression of both total and phosphorylated dynamin-related protein 1. In addition, transmission electron microscopy revealed damaged and degraded mitochondria in the hypothalamus of heat-exposed mice. Heat induced apoptosis and mitophagy were further confirmed by increased formation of reactive oxygen species, activation of caspase 3/7 and expression of LC3 proteins. Moreover, heat exposure increased the expression of PINK1 and Parkin in mouse hypothalamus. In contrast, pretreatment with AST reduced these effects. These results demonstrate that heat stress-induced hypothalamic apoptosis is associated with altered mitochondrial dynamics favoring fission and mitophagy. AST protects the hypothalamus against heat-induced injury by preserving redox homeostasis and mitochondrial integrity.

768-P: MSDC-0602 Is a Direct Mitochondrial Pyruvate Carrier Inhibitor That Modulates Central Carbon Metabolism in Mice and Humans

Mitochondrial pyruvate carrier (MPC) disruption attenuates type 2 diabetes NAFLD in mice. MSDC-0602, a PPARγ-sparing, TZD-like molecule that inhibits the MPC, was recently tested in a phase 2B trial for treating NASH, which reported beneficial effects on glucose and insulin homeostasis. Yet, the broad, in vivo metabolomic effects of MSDC-0602 treatment have remained undefined. Here, we tested MSDC-062 metabolic mechanisms of action in vitro and in vivo, spanning from mitochondrial pyruvate uptake assays in isolated mouse mitochondria, through cultured mouse liver cells, in vivo in mice, and in humans participating in the EMMINENCE trial. First, we examined MSDC-0602’s effect in isolated mouse liver mitochondria: Pyruvate-oxidation was decreased by MSDC-0602, and 2.5 µM MSDC-0602 was sufficient to decrease mitochondrial pyruvate uptake by >50%. We extended these findings to AML12 mouse liver cells and mouse liver in vivo. In AML12 cells, MSDC-0602 treatment increased pyruvate and lactate and decreased alanine. These results were comparable but not identical to results from parallel testing of the specific MPC inhibitor UK5099. 14-day treatment of NCD and HFD fed mice with MSDC-0602 decreased liver alanine and branched chain amino acids. As we previously observed with MPC liver-specific knockout mice, the HFD-induced liver TCA intermediate pool size expansion was mitigated by MSDC-0602. Accordingly, MSDC-0602-dependent metabolomic changes were more pronounced in HFD- versus NCD-fed mice. Human EMMINENCE trial participants receiving MSDC-0602 showed decreased circulating alanine, ketone bodies, and glucose. Similarly, circulating and liver glucose were decreased in HFD mice treated with MSDC-0602. Together, these data are consistent with MSDC-0602 modulating in vivo metabolism in a partially MPC-dependent manner and by additional mechanisms that remain to be understood. Disclosure A. J. Rauckhorst: None. D. J. Pape: None. J. R. Colca: Board Member; Self; Metabolic Solutions Development Company, LLC, Employee; Self; Cirius Therapeutics. E. B. Taylor: None. Funding American Diabetes Association (1-18-PDF-060 to A.J.R.); National Institute of Diabetes and Digestive and Kidney Diseases (DK104998); Cirius Therapeutics

Quantitative Analyses of Foot Processes, Mitochondria, and Basement Membranes by Structured Illumination

IntroductionFoot process effacement and mitochondrial fission associate with kidney disease pathogenesis. Electron microscopy is the gold-standard method for their visualization, but the observable area of electron microscopy is smaller than light microscopy. It is important to develop alternative ways to quantitatively evaluate these microstructural changes because the lesion site of renal diseases can be focal.MethodsWe analyzed elastica-Masson trichrome (EMT) and periodic acid-Schiff (PAS) stained kidney sections using structured illumination microscopy (SIM).ResultsEMT staining revealed three-dimensional (3D) structures of foot process, whereas ponceau xylidine acid fuchsin azophloxine solution induced fluorescence. Conversion of foot process images into their constituent frequencies by Fourier transform showed that the concentric square of (1/4)2-(1/16)2 in the power spectra (PS) included information for normal periodic structures of foot processes. Foot process integrity, assessed by PS, negatively correlated with proteinuria. EMT-stained sections revealed fragmented mitochondria in mice with mitochondrial injuries and patients with tubulointerstitial nephritis; Fourier transform quantified associated mitochondrial injury. Quantified mitochondrial damage in patients with immunoglobulin A (IgA) nephropathy predicted a decline in estimated glomerular filtration rate (eGFR) after kidney biopsy but did not correlate with eGFR at biopsy. PAS-stained sections, excited by a 640 nm laser, combined with the coefficient of variation values, quantified subtle changes in the basement membranes of patients with membranous nephropathy stage I.ConclusionsKidney microstructures are quantified from sections prepared in clinical practice using SIM.

Triggering mitochondrial plasticity in the liver of rodents is key for successful acclimatization to hypoxia

Liver is the most metabolically active organ in mice and rats, accounting for up to 50% of the body's energy expenditure. As such, this organ is highly sensitive to the reduction in O2 availability occurring in hypoxic conditions such as high-altitude. Keeping in mind that mice, but not rats, successfully colonized habitats over 2,500 masl, we showed previously that mice only increase their metabolic rates under chronic hypoxia. Accordingly, we hypothesized here that mice and rats have divergent mitochondrial and molecular features in the liver that determine an adequate energy balance in mice (not rats) during the process of acclimatization to hypoxia. To test this hypothesis, we used saponin-permeabilized samples of liver from male adult FVB mice and SD rats after exposure to hypoxia (12% O2, for 0, 1, 7 or 21 days) to measure the mitochondrial O2 consumption rate (OCR) and the activation of the N (complex I-linked), S (complex II-linked), and NS (complex I&II-linked) electron-transfer pathways in the high-resolution respirometer O2K (OROBOROS instruments). Additional experiments were performed to evaluate the activity of representative enzymes from the glycolytic (hexokinase [HK]), aerobic (pyruvate dehydrogenase [PDH]), and anaerobic (lactate dehydrogenase [LDH]) metabolic pathways by spectrometric methods. Our results show that liver mitochondria in mice increase the OCR and improve the activation of the electron-transfer pathways S (complex II-linked) and NS (complexes I&II-linked). in line, the glycolytic and aerobic metabolic enzymatic activities (HK and PDH) increased, while the anaerobic metabolism (LDH) reduced after seven-days hypoxia. On the other hand, rats were shown to be unable of altering liver mitochondrial respiration but augmented glycolytic and anaerobic metabolic activities (HK and LDH) and concomitantly reduced the aerobic metabolism (PDH). These results show that liver mitochondria of mice display plastic responses in the utilization of oxygen during the process of acclimatization to hypoxia and that these changes are supported by adjustments in the glycolytic, aerobic, and anaerobic metabolic pathways. Contrastingly, together with the inability to adjust their mitochondrial activity, rats depend heavily on anaerobic pathways (known to be less effective) to produce energy. Our results contribute to explain why mice, but not rats, are present in high-altitude environments.

Flufenoxuron suppresses the proliferation of testicular cells by targeting mitochondria in mice

Flufenoxuron is a benzoylurea pesticide that is used to eradicate insects and acarids in the farmland. Even though it specifically works on target animals, the possibilities of its bioaccumulation and harmful effects on non-target animals cannot be denied. As the usage and application of pesticides increases, exposure to them also increases through ingestion of food residues, inhalation, or dermal contact. Pesticides could also be considered as endocrine disruptor chemicals; however, the reproductive toxicity and cellular mechanisms of flufenoxuron have not been identified. Our results indicate that flufenoxuron inhibits cellular proliferation and hampers calcium homeostasis, especially by targeting mitochondria. We also confirmed the induction of endoplasmic reticulum (ER) stress and ER-mitochondrial contact signaling. Using pharmacological inhibitors, we also observed that the mitogen-activated protein kinase and Akt signaling pathways were upregulated by flufenoxuron. Further, by oral administration of flufenoxuron (100 mg/kg/bw) to C57BL/6 male mice, we observed transcriptional changes in the testis-related genes. Collectively, we demonstrated that flufenoxuron inhibits cell proliferation and alters gene expression in mouse testis cells and induces testicular dysfunction in mice. These results indicate that flufenoxuron may be harmful to male reproduction and fertility in the early stages of pregnancy.

Bone metastases induce metabolic changes and mitophagy in mice.

What is the central question of this study? Cachexia causes severe changes in skeletal muscle metabolism and function and is a key predictor of negative outcomes in cancer patients: what are the changes in whole animal energy metabolism and mitochondria in skeletal muscle? What is the main finding and its importance? There is decreased whole animal energy expenditure in mice with cachexia. They displayed highly dysmorphic mitochondria and mitophagy in skeletal muscle. Cachexia causes changes in skeletal muscle metabolism. Mice with MDA-MB-231 breast cancer bone metastases and cachexia have decreased whole animal energy metabolism and increased skeletal muscle mitophagy. We examined whole animal energy metabolism by indirect calorimetry in mice with MDA-MB-231 breast cancer bone metastases, and showed decreased energy expenditure. We also examined skeletal muscle mitochondria and found that mitochondria in mice with MDA-MB-231 bone metastases are highly dysmorphic and have altered protein markers of mitochondrial biogenesis and dynamics. In addition, LC3B protein was increased in mitochondria of skeletal muscle from cachectic mice, and colocalized with the mitochondrial protein Tom20. Our data demonstrate the importance of mitophagy in cachexia. Understanding these changes will help contribute to defining treatments for cancer cachexia.

Drp1/Mff signaling pathway is involved in fluoride-induced abnormal fission of hepatocyte mitochondria in mice

Fluoride, a toxic substance, is widely distributed in the environment and causes serious damage to the body. This study was performed to investigate the effects of fluoride on mitochondrial fission in mouse hepatocytes. A total of 48 mice were equally divided into four groups and admisnistered with NaF in drinking water at fluorine ion concentrations of 0, 25, 50 and 100 mg/L for 70 days. The pathomorphology and ultrastructurre of hepatocytes were then observed. The mitochondrial lesion parameters (number, length, width and vacuolization area) are evaluated. The expression of Drp1, Mff, Fis1, MiD49, MiD51 and Dyn2, which are associated with mitochondrial fission, was determined by quantitative real-time PCR and Western blot analysis. Apoptosis was detected by using TUNEL assay. Results showed that fluoride causes notable changes in the pathological morphology of liver tissues and severely damages the ultrastructure of hepatocytes. Damage manifested as nuclear condensation, nuclear membrane breakdown, mitochondrial vacuolation, increased fragmentation, and mitochondrial fission. Moreover, mRNA and protein expression levels were significantly upregulated in the Drp1/Mff signaling pathway. The mRNA expression levels of Cyt c, caspase 9 and 3 markedly increased in the fluoride treated groups in a dose-dependent manner. The percentage of TUNEL-positive nuclei in the liver remarkably increased after fluoride treatment. Overall, the results indicate that excessive fluoride exposure can increase mitochondrial fission via the Drp1/Mff signaling pathway, severely damage the mitochondrial structure, and lead to apoptosis of hepatocytes.