Mitochondrial Oxidative Capacity Research Articles

Adipose tissue macrophage populations and inflammation are associated with systemic inflammation and insulin resistance in obesity.

Obesity is accompanied by numerous systemic and tissue-specific derangements, including systemic inflammation, insulin resistance, and mitochondrial abnormalities in skeletal muscle. Despite growing recognition that adipose tissue dysfunction plays a role in obesity-related disorders, the relationship between adipose tissue inflammation and other pathological features of obesity is not well-understood. We assessed macrophage populations and measured the expression of inflammatory cytokines in abdominal adipose tissue biopsies in 39 nondiabetic adults across a range of body mass indexes (BMI 20.5-45.8 kg/m2). Skeletal muscle biopsies were used to evaluate mitochondrial respiratory capacity, ATP production capacity, coupling, and reactive oxygen species production. Insulin sensitivity (SI) and β cell responsivity were determined from test meal postprandial glucose, insulin, c-peptide, and triglyceride kinetics. We examined the relationships between adipose tissue inflammatory markers, systemic inflammatory markers, SI, and skeletal muscle mitochondrial physiology. BMI was associated with increased adipose tissue and systemic inflammation, reduced SI, and reduced skeletal muscle mitochondrial oxidative capacity. Adipose-resident macrophage numbers were positively associated with circulating inflammatory markers, including tumor necrosis factor-α (TNFα) and C-reactive protein (CRP). Local adipose tissue inflammation and circulating concentrations of TNFα and CRP were negatively associated with SI, and circulating concentrations of TNFα and CRP were also negatively associated with skeletal muscle oxidative capacity. These results demonstrate that obese humans exhibit increased adipose tissue inflammation concurrently with increased systemic inflammation, reduced insulin sensitivity, and reduced muscle oxidative capacity and suggest that adipose tissue and systemic inflammation may drive obesity-associated metabolic derangements.NEW AND NOTEWORTHY Adipose inflammation is proposed to be at the nexus of the systemic inflammation and metabolic derangements associated with obesity. The present study provides evidence to support adipose inflammation as a central feature of the pathophysiology of obesity. Adipose inflammation is associated with systemic and peripheral metabolic derangements, including increased systemic inflammation, reduced insulin sensitivity, and reduced skeletal muscle mitochondrial respiration.

Cardiac Energy Metabolism in Heart Failure.

Alterations in cardiac energy metabolism contribute to the severity of heart failure. However, the energy metabolic changes that occur in heart failure are complex and are dependent not only on the severity and type of heart failure present but also on the co-existence of common comorbidities such as obesity and type 2 diabetes. The failing heart faces an energy deficit, primarily because of a decrease in mitochondrial oxidative capacity. This is partly compensated for by an increase in ATP production from glycolysis. The relative contribution of the different fuels for mitochondrial ATP production also changes, including a decrease in glucose and amino acid oxidation, and an increase in ketone oxidation. The oxidation of fatty acids by the heart increases or decreases, depending on the type of heart failure. For instance, in heart failure associated with diabetes and obesity, myocardial fatty acid oxidation increases, while in heart failure associated with hypertension or ischemia, myocardial fatty acid oxidation decreases. Combined, these energy metabolic changes result in the failing heart becoming less efficient (ie, a decrease in cardiac work/O2 consumed). The alterations in both glycolysis and mitochondrial oxidative metabolism in the failing heart are due to both transcriptional changes in key enzymes involved in these metabolic pathways, as well as alterations in NAD redox state (NAD+ and nicotinamide adenine dinucleotide levels) and metabolite signaling that contribute to posttranslational epigenetic changes in the control of expression of genes encoding energy metabolic enzymes. Alterations in the fate of glucose, beyond flux through glycolysis or glucose oxidation, also contribute to the pathology of heart failure. Of importance, pharmacological targeting of the energy metabolic pathways has emerged as a novel therapeutic approach to improving cardiac efficiency, decreasing the energy deficit and improving cardiac function in the failing heart.

Increasing fatty acid oxidation elicits a sex-dependent response in failing mouse hearts

BackgroundReduced fatty acid oxidation (FAO) is a hallmark of metabolic remodeling in heart failure. Enhancing mitochondrial long-chain fatty acid uptake by Acetyl-CoA carboxylase 2 (ACC2) deletion increases FAO and prevents cardiac dysfunction during chronic stresses, but therapeutic efficacy of this approach has not been determined. MethodsMale and female ACC2 f/f-MCM (ACC2KO) and their respective littermate controls were subjected to chronic pressure overload by TAC surgery. Tamoxifen injection 3 weeks after TAC induced ACC2 deletion and increased FAO in ACC2KO mice with pathological hypertrophy. ResultsACC2 deletion in mice with pre-existing cardiac pathology promoted FAO in female and male hearts, but improved cardiac function only in female mice. In males, pressure overload caused a downregulation in the mitochondrial oxidative function. Stimulating FAO by ACC2 deletion caused unproductive acyl-carnitine accumulation, which failed to improve cardiac energetics. In contrast, mitochondrial oxidative capacity was sustained in female pressure overloaded hearts and ACC2 deletion improved myocardial energetics. Mechanistically, we revealed a sex-dependent regulation of PPARα signaling pathway in heart failure, which accounted for the differential response to ACC2 deletion. ConclusionMetabolic remodeling in the failing heart is sex-dependent which could determine the response to metabolic intervention. The findings suggest that both mitochondrial oxidative capacity and substrate preference should be considered for metabolic therapy of heart failure.

Insulin-like growth factor-1 short-period therapy stimulates bone marrow cells in obese swiss mice.

Bone marrow cells (BMCs) from obese Swiss mice fed with Western diet show mitochondrial dysfunction. Obesity interferes with BMCs disrupting energetic metabolism, stimulating apoptosis, and reducing cell proliferation since adipose tissue releases inflammatory adipokines into the medullar microenvironment. These changes lead to reduction of BMC differentiation capacity and hematopoiesis impairment, a process responsible for blood cell continuous production through hematopoietic stem cells (HSCs). This work aimed to analyze the effects of IGF-1 therapy on BMC viability in Western diet-induced obesity, in vivo. We observed that after only 1week of treatment, obese Swiss mice presented reduced body weight and visceral fat and increased mitochondrial oxidative capacity and coupling, indicating mitochondrial function improvement. In addition, IGF-1 was able to reduce apoptosis of total BMCs, stem cell subpopulations (hematopoietic and mesenchymal), and leukocytes, restoring all progenitor hematopoietic lineages. The treatment also contributed to increase proliferative capacity of hematopoietic stem cells and leukocytes, keeping the hematopoietic and immune systems balanced. Therefore, we conclude that IGF-1 short period therapy improved BMC survival, proliferation, and differentiation capacity in obese Swiss mice.

Impaired Skeletal Muscle Mitochondrial Efficiency in Smokers

Introduction Cigarette smoke can directly contribute to the development of skeletal muscle dysfunction. Mitochondrial oxidative capacity, a product of energy production efficiency and oxygen consumption, is important for skeletal muscle health. Whether mitochondrial energy production efficiency and muscle oxygen consumption play a role into skeletal muscle dysfunction in smokers has yet to be investigated. Thus, this study sought to test the hypothesis that mitochondrial oxidative capacity and mitochondrial efficiency are reduced in smokers compared to non-smokers. Methods 30 active smokers (Age: 33 ± 4 yrs, BMI: 30.8 ± 11.7 kg/m2) and 30 demographically matched healthy controls (Age: 33 ± 3 yrs, BMI: 32.5 ± 9.1 kg/m2) participated in this study. Participants self-reported the number of cigarettes smoked per day in the past 30 days. Near infrared spectroscopy (NIRS) was used to assess changes in oxygenated (O2Hb) and deoxygenated (HHb) hemoglobin/myoglobin of the gastrocnemius following 15s of electrical stimulation and multiple rapid arterial occlusions. The phosphocreatine (PCr) recovery rate constant (kPCr), an index of mitochondrial oxidative capacity, was calculated by fitting post-stimulation measurements to a monoexponential curve. Muscle oxygen consumption (mVO2 = abs[([ΔO2Hb – Δ HHb]/2] × 60)/(10 × 1.04) x4] × 22.4/1000) was calculated for each occlusion and the slopes of the total hemoglobin (tHb = O2Hb+HHb) signal following each occlusion were converted to muscle blood flow [mBF = 1/hemoglobin (mmol/L) × ΔtHb/Δtime]. Mitochondrial efficiency (MEFF) was calculated by the dividing the estimated mVO2 needed to achieve kPcr at 100% (mVO2estimated) efficiency by the observed mVO2 [Meff = (mVO2estimated/mVO2observed) × 100]. Results The average cigarettes smoked was 5 ± 3 cigarettes per day. kPcr was significantly (p = 0.022) reduced in smokers (1.99 ± 0.19 min-1) compared to non-smokers (2.54 ± 0.19 min-1). Compared to non-smokers (157.2 ± 45.5%), MEFF was significantly (p = 0.025) attenuated in smokers (46.5 ± 10.1%) and significantly associated with mVO2 (r = -0.409, p = 0.009) and kPcr (r = 0.461, p =0.003). Average mVO2 (p = 0.231) and mBF (p = 0.993)were not significantly different in smokers compared to non-smokers. Conclusion Muscle mitochondrial oxidative capacity and mitochondrial efficiency are reduced in smokers compared to non-smokers. However, skeletal muscle blood flow and oxygen consumption were not different between groups. Taken together, mitochondrial oxidative capacity may be reduced prior to alterations in blood flow and oxygen consumption or delivery in young smokers.

The Influence of High‐Fat Feeding and Exercise Training on Substrate‐Specific Mitochondrial Respiration in Skeletal Muscle of Female Mice

Objective High-fat diet (HFD) induced obesity is associated with excessive lipid supply which drives remodeling of skeletal muscle mitochondria. Exercise stimulates mitochondrial respiration but it is not clear if such adaptations are specific to lipids. Our objective was to determine if HFD-induced obesity would change the sensitivity and capacity for lipid oxidation, and if exercise modifies mitochondrial-specific respiration during HFD. Hypothesis We hypothesized that HFD-induced obesity would increase the sensitivity (intrinsic function) and capacity for lipid respiration while exercise would further augment overall capacity for skeletal muscle mitochondrial substrate oxidation in female mice. Methods We used a 2x2 designof diet and exercise in 10-week old female C57BL/6J mice (n=13-15 per group). Mice consumed diet (either 10% fat or 60% HFD) for 4 weeks then performed 8 weeks of treadmill training for 1 hour per day, 5 days per week or remained sedentary while continuing diet. HFD increased body-weight (P<0.01) by 4 weeks and fat mass measured by dual-energy x-ray absorptiometry at week 12 (P<0.01). We confirmed glucose and insulin intolerance at week 4 of HFD with no change after 8 weeks of treadmill training (P=0.435). Mice rested 24 hours prior to tissue collection. We isolated mitochondria from fresh quadriceps then performed high-resolution respirometry. We measured lipid sensitivity (Km) and capacity (Vmax) using titrations of octanoyl-carnitine (F-linked). Glutamate-succinate were used to determine non-lipid substrate oxidative phosphorylation capacity (N- and S-linked). Primary outcomes were analyzed using 2-way ANOVA (diet x exercise) with Tukey's multiple comparisons (α=0.05). Results The Km for lipid substrates was not different between diet (-7.7%, P=0.66) or exercise (+20.3%, P=0.26) groups. Absolute Vmax was 38% higher in HFD (P=0.018 versus low fat). There was a tendency for 27% higher Vmax after exercise (P=0.074 versus sedentary). For non-lipid substrates, HFD had 23% greater absolute N-linked respiration (P=0.04) and 19% greater S-linked respiration (P=0.02); however, no effects of exercise were detected. Normalizing respiration to mitochondrial protein content eliminated the HFD effects in all substrates. Conclusion We provide evidence that mitochondrial adaptations to HFD, in female mice, are driven primarily by increases in protein abundance rather than intrinsic factors as the normalization of respiration to mitochondrial protein content eliminated HFD effects for increased substrate respiration and there were no differences to lipid sensitivity between diet or exercise. Exercise did not appear to alter the mitochondrial respiratory response to HFD.

Vitamin D Promotes Skeletal Muscle Regeneration and Mitochondrial Health

Vitamin D is an essential nutrient for the maintenance of skeletal muscle and bone health. The vitamin D receptor (VDR) is present in muscle, as is CYP27B1, the enzyme that hydroxylates 25(OH)D to its active form, 1,25(OH)D. Furthermore, mounting evidence suggests that vitamin D may play an important role during muscle damage and regeneration. Muscle damage is characterized by compromised muscle fiber architecture, disruption of contractile protein integrity, and mitochondrial dysfunction. Muscle regeneration is a complex process that involves restoration of mitochondrial function and activation of satellite cells (SC), the resident skeletal muscle stem cells. VDR expression is strongly upregulated following injury, particularly in central nuclei and SCs in animal models of muscle injury. Mechanistic studies provide some insight into the possible role of vitamin D activity in injured muscle. In vitro and in vivo rodent studies show that vitamin D mitigates reactive oxygen species (ROS) production, augments antioxidant capacity, and prevents oxidative stress, a common antagonist in muscle damage. Additionally, VDR knockdown results in decreased mitochondrial oxidative capacity and ATP production, suggesting that vitamin D is crucial for mitochondrial oxidative phosphorylation capacity; an important driver of muscle regeneration. Vitamin D regulation of mitochondrial health may also have implications for SC activity and self-renewal capacity, which could further affect muscle regeneration. However, the optimal timing, form and dose of vitamin D, as well as the mechanism by which vitamin D contributes to maintenance and restoration of muscle strength following injury, have not been determined. More research is needed to determine mechanistic action of 1,25(OH)D on mitochondria and SCs, as well as how this action manifests following muscle injury in vivo. Moreover, standardization in vitamin D sufficiency cut-points, time-course study of the efficacy of vitamin D administration, and comparison of multiple analogs of vitamin D are necessary to elucidate the potential of vitamin D as a significant contributor to muscle regeneration following injury. Here we will review the contribution of vitamin D to skeletal muscle regeneration following injury.

Combined effects of a ketogenic diet and exercise training alter mitochondrial and peroxisomal substrate oxidative capacity in skeletal muscle.

Ketogenic diets (KDs) are reported to improve body weight, fat mass, and exercise performance in humans. Unfortunately, most rodent studies have used a low-protein KD, which does not recapitulate diets used by humans. Since skeletal muscle plays a critical role in responding to macronutrient perturbations induced by diet and exercise, the purpose of this study was to test if a normal-protein KD (NPKD) impacts shifts in skeletal muscle substrate oxidative capacity in response to exercise training (ExTr). A high fat, carbohydrate-deficient NPKD (16.1% protein, 83.9% fat, 0% carbohydrate) was given to C57BL/6J male mice for 6 wk, whereas controls (Con) received a low-fat diet with similar protein (15.9% protein, 11.9% fat, 72.2% carbohydrate). After 3 wk on the diet, mice began treadmill training 5 days/wk, 60 min/day for 3 wks. The NPKD increased body weight and fat mass, whereas ExTr negated a continued rise in adiposity. ExTr increased intramuscular glycogen, whereas the NPKD increased intramuscular triglycerides. Neither the NPKD nor ExTr alone altered mitochondrial content; however, in combination, the NPKD-ExTr group showed increases in PGC-1α and markers of mitochondrial fission/fusion. Pyruvate oxidative capacity was unchanged by either intervention, whereas ExTr increased leucine oxidation in NPKD-fed mice. Lipid metabolism pathways had the most notable changes as the NPKD and ExTr interventions both enhanced mitochondrial and peroxisomal lipid oxidation and many adaptations were additive or synergistic. Overall, these results suggest that a combination of a NPKD and ExTr induces additive and/or synergistic adaptations in skeletal muscle oxidative capacity.NEW & NOTEWORTHY A ketogenic diet with normal protein content (NPKD) increases body weight and fat mass, increases intramuscular triglyceride storage, and upregulates pathways related to protein metabolism. In combination with exercise training, a NPKD induces additive and/or synergistic activation of AMPK, PGC-1α, mitochondrial fission/fusion genes, mitochondrial fatty acid oxidation, and peroxisomal adaptations in skeletal muscle. Collectively, results from this study provide mechanistic insight into adaptations in skeletal muscle relevant to keto-adaptation.

POS-075 Decreased IFT88 causes cilia shortening and mitochondrial dysfunction in cisplatin-induced tubular injury

Renal primary cilia are associated with the pathogenesis of various diseases, including acute kidney injury (AKI). The length of the primary cilia dynamically change during the progression of diseases: tubular primary cilia shortened in cisplatin-induced AKI mouse model. However, its relevance in kidney disease and the underlying mechanism are largely unknown. Tubular damage in AKI closely links to mitochondrial dysfunction. Thus, we investigated the interaction between primary cilia and mitochondria in cisplatin-induced tubular injury. C57BL6 mice with cisplatin-induced AKI were euthanized at 72 h after disease induction to collect blood and kidney samples. In in vitro experiments, we used RPTEC/TERT1 cells, which are human proximal tubular epithelial cells that maintain the cilia length at high cell densities, and knocked down IFT88 (IFT88-KD), a cilia maintenance protein, by siRNA. Cisplatin-treated or IFT88-KD cells were assessed for the cellular phenotypic changes or mitochondrial metabolic function using the Flux Analyzer. We found that the expression of protein IFT88 was decreased in damaged tubules of cisplatin-induced AKI mice. These data were consistent with that IFT88 expression decreased in the cilia of cisplatin-treated RPTEC/TERT1 at mRNA and protein levels in association with shortening of the primary cilia, suggesting the pathogenic link between tubular damage and IFT88-mediated cilia alteration. Interestingly, IFT88-KD cells significantly exhibited shorter cilia as compared to control siRNA-transfected cells and showed downregulation of mitochondrial oxidative phosphorylation capacity and decreased ATP production, indicating the contribution of IFT88 to mitochondrial homeostasis. In addition, the knockdown of IFT88 decreases fatty acid oxidation (FAO) in the same direction as cisplatin treatment. Our findings suggest that tubular mitochondrial dysfunction in cisplatin-induced AKI is mediated by a decreased IFT88 with shortening cilia, at least in part. Tubular mitochondrial damage followed by tubular injury in AKI may occur by the alteration of IFT88 expression, and subsequent cilia shortening in the tubular epithelial cells.

Short-Term High-Fat Feeding Does Not Alter Mitochondrial Lipid Respiratory Capacity but Triggers Mitophagy Response in Skeletal Muscle of Mice.

Lipid overload of the mitochondria is linked to the development of insulin resistance in skeletal muscle which may be a contributing factor to the progression of type 2 diabetes during obesity. The targeted degradation of mitochondria through autophagy, termed mitophagy, contributes to the mitochondrial adaptive response to changes in dietary fat. Our previous work demonstrates long-term (2-4 months) consumption of a high-fat diet increases mitochondrial lipid oxidation capacity but does not alter markers of mitophagy in mice. The purpose of this study was to investigate initial stages of mitochondrial respiratory adaptations to high-fat diet and the activation of mitophagy. C57BL/6J mice consumed either a low-fat diet (LFD, 10% fat) or high-fat diet (HFD, 60% fat) for 3 or 7 days. We measured skeletal muscle mitochondrial respiration and protein markers of mitophagy in a mitochondrial-enriched fraction of skeletal muscle. After 3 days of HFD, mice had lower lipid-supported oxidative phosphorylation alongside greater electron leak compared with the LFD group. After 7 days, there were no differences in mitochondrial respiration between diet groups. HFD mice had greater autophagosome formation potential (Beclin-1) and greater activation of mitochondrial autophagy receptors (Bnip3, p62) in isolated mitochondria, but no difference in downstream autophagosome (LC3II) or lysosome (Lamp1) abundance after both 3 and 7 days compared with the LFD groups. In cultured myotubes, palmitate treatment decreased mitochondrial membrane potential and hydrogen peroxide treatment increased accumulation of upstream mitophagy markers. We conclude that several days of high-fat feeding stimulated upstream activation of skeletal muscle mitophagy, potentially through lipid-induced oxidative stress, without downstream changes in respiration.

Excessive exercise training causes mitochondrial functional impairment and decreases glucose tolerance in healthy volunteers

Exercise training positively affects metabolic health through increased mitochondrial oxidative capacity and improved glucose regulation and is the first line of treatment in several metabolic diseases. However, the upper limit of the amount of exercise associated with beneficial therapeutic effects has not been clearly identified. Here, we used a training model with a progressively increasing exercise load during an intervention over 4weeks. We closely followed changes in glucose tolerance, mitochondrial function and dynamics, physical exercise capacity, and whole-body metabolism. Following the week with the highest exercise load, we found a striking reduction in intrinsic mitochondrial function that coincided with a disturbance in glucose tolerance and insulin secretion. We also assessed continuous blood glucose profiles in world-class endurance athletes and found that they had impaired glucose control compared with a matched control group.

The administration of an extract from Berberis microphylla stimulates energy expenditure, thermogenesis and mitochondrial dynamics in mice brown adipose tissue

Obesity is defined as excess fat accumulation in white adipose tissue. In opposition to this storage function, brown adipose tissue (BAT) counters obesity, by consuming fat through thermogenesis. Obese individuals display lower BAT mitochondrial oxidative capacity and altered mitochondrial morphology. A promising strategy to fight obesity is dietary polyphenols, which increase BAT mass and function, stimulating energy expenditure (EE). Calafate, a polyphenol-rich Chilean native fruit, has anti-inflammatory and antioxidant characteristics. The effect of a Calafate extract (50 mg [total polyphenols]/kg body weight/day) on EE and mitochondrial function and morphology in BAT from obese mice was assessed. Adult male C57BL/6J mice were subdivided into four treatments for 18 weeks: control diet (C), control diet + Calafate (CC), high-fat diet (HF), high-fat diet + Calafate (HFC). Calafate extract decreased high-fat diet-induced body weight gain from week 6 of treatment (p<0,05) and increased EE at rest (p = 0.03). In BAT, Calafate extract reversed the decrease in UCP-1 protein levels generated by the high-fat diet (p = 0.004). Also, Calafate extract improved mitochondrial transmembrane potential (p = 0.04). The extract did not substantially modify mitochondrial morphology, although it increased the expression of optic atrophy protein 1 (p = 0.01), a mitochondrial fusion-related protein. In sum, consumption of a polyphenol-rich Calafate extract prevents high-fat diet-induced obesity, concomitant with higher energy expenditure, and improved BAT mitochondrial function in obese mice.

New insights into muscle function in chronic kidney disease and metabolic acidosis.

: Sarcopenia, defined as decreased muscle mass or function, is prevalent in chronic kidney disease (CKD) increasing the risk of mobility impairment and frailty. CKD leads to metabolic acidosis (MA) and retention of uremic toxins contributing to insulin resistance and impaired muscle mitochondrial energetics. Here we focus on the central role of muscle mitochondrial metabolism in muscle function. : Mitochondrial dysfunction underlies muscle wasting and poor physical endurance in CKD. Uremic toxins accumulate in muscle disrupting mitochondrial respiration and enzymes. Changes in mitochondrial quantity, quality, and oxidative capacity contribute to mobility impairment in CKD. Major determinants of muscle mitochondrial function are kidney function, inflammation, and oxidative stress. In CKD, MA is the major determinant of muscle mitochondrial function. Metabolomics reveals defects in pathways linked to mitochondrial energy metabolism and acid-base homeostasis underlying insulin resistance in CKD. : Decreased mitochondrial capacity and quality control can impair muscle function contributing to decreased physical endurance. MA augments insulin resistance perpetuating the catabolic state underlying muscle wasting in CKD. Further studies are needed to investigate if targeting of MA improves muscle mitochondrial function and insulin resistance translating into meaningful improvements in physical endurance.

Exercise, CaMKII, and type 2 diabetes.

Individuals who exercise regularly are protected from type 2 diabetes and other metabolic syndromes, in part by enhanced gene transcription and induction of many signaling pathways crucial in correcting impaired metabolic pathways associated with a sedentary lifestyle. Exercise activates Calmodulin-dependent protein kinase (CaMK)II, resulting in increased mitochondrial oxidative capacity and glucose transport. CaMKII regulates many health beneficial cellular functions in individuals who exercise compared with those who do not exercise. The role of exercise in the regulation of carbohydrate, lipid metabolism, and insulin signaling pathways are explained at the onset. Followed by the role of exercise in the regulation of glucose transporter (GLUT)4 expression and mitochondrial biogenesis are explained. Next, the main functions of Calmodulin-dependent protein kinase and the mechanism to activate it are illustrated, finally, an overview of the role of CaMKII in regulating GLUT4 expression, mitochondrial biogenesis, and histone modification are discussed.

Deletion of Von Willebrand A Domain Containing Protein (VWA8) raises activity of mitochondrial electron transport chain complexes in hepatocytes

VWA8 (Von Willebrand A Domain Containing Protein 8) is a AAA+ ATPase that is localized to the mitochondrial matrix and is widely expressed in highly energetic tissues. Originally found to be higher in abundance in livers of mice fed a high fat diet, deletion of the VWA8 gene in differentiated mouse AML12 hepatocytes unexpectedly produced a phenotype of higher mitochondrial and nonmitochondrial oxidative metabolism, higher ROS (reactive oxygen species) production mainly from NADPH oxidases, and increased HNF4a expression. The purposes of this study were first, to determine whether higher mitochondrial oxidative capacity in VWA8 null hepatocytes is the product of higher capacity in all aspects of the electron transport chain and oxidative phosphorylation, and second, the density of cristae in mitochondria and mitochondrial content was measured to determine if higher mitochondrial oxidative capacity is accompanied by greater cristae area and mitochondrial abundance. Electron transport chain complexes I, II, III, and IV activities all were higher in hepatocytes in which the VWA8 gene had been deleted using CRISPR/Cas9. A comparison of abundance of proteins in electron transport chain complexes I, III and ATP synthase previously determined using an unbiased proteomics approach in hepatocytes in which VWA8 had been deleted showed agreement with the activity assays. Mitochondrial cristae, the site where electron transport chain complexes are located, were quantified using electron microscopy and stereology. Cristae density, per mitochondrial area, was almost two-fold higher in the VWA8 null cells (P < 0.01), and mitochondrial area was two-fold higher in the VWA8 null cells (P < 0.05). The results of this study allow us to conclude that despite sustained, higher ROS production in VWA8 null cells, a global mitochondrial compensatory response was maintained, resulting in overall higher mitochondrial oxidative capacity.

Post-exercise intramuscular O2 supply is tightly coupled with a higher proximal-to-distal ATP synthesis rate in human tibialis anterior.

Key points The post‐exercise recovery of phosphocreatine, a measure of the oxidative capacity of muscles, as assessed by 31P MR spectroscopy, shows a striking increase from distal to proximal along the human tibialis anterior muscle.To investigate why this muscle exhibits a greater oxidative capacity proximally, we tested whether the spatial variation in phosphocreatine recovery rate is related to oxygen supply, muscle fibre type or type of exercise.We revealed that oxygen supply also increases from distal to proximal along the tibialis anterior, and that it strongly correlated with phosphocreatine recovery. Carnosine level, a surrogate measure for muscle fibre type was not different between proximal and distal, and type of exercise did not affect the gradient in phosphocreatine recovery rate.Taken together, the findings of this study suggest that the post‐exercise spatial gradients in oxygen supply and phosphocreatine recovery are driven by a higher intrinsic mitochondrial oxidative capacity proximally. Phosphorus magnetic resonance spectroscopy (31P MRS) of human tibialis anterior (TA) revealed a strong proximo‐distal gradient in the post‐exercise phosphocreatine (PCr) recovery rate constant (k PCr), a measure of muscle oxidative capacity. The aim of this study was to investigate whether this k PCr gradient is related to O2 supply, resting phosphorylation potential, muscle fibre type, or type of exercise. Fifteen male volunteers performed continuous isometric ankle dorsiflexion at 30% maximum force until exhaustion. At multiple locations along the TA, we measured the oxidative PCr resynthesis rate (V PCr = k PCr × PCr depletion) by 31P MRS, the oxyhaemoglobin recovery rate constant (k O2Hb) by near infrared spectroscopy, and muscle perfusion with MR intravoxel incoherent motion imaging. The k O2Hb, k PCr, V PCr and muscle perfusion depended on measurement location (P < 0.001, P < 0.001, P = 0.032 and P = 0.003, respectively), all being greater proximally. The k O2Hb and muscle perfusion correlated with k PCr (r = 0.956 and r = 0.852, respectively) and V PCr (r = 0.932 and r = 0.985, respectively), the latter reflecting metabolic O2 consumption. Resting phosphorylation potential (PCr/inorganic phosphate) was also higher proximally (P < 0.001). The surrogate for fibre type, carnosine content measured by 1H MRS, did not differ between distal and proximal TA (P = 0.884). Performing intermittent exercise to avoid exercise ischaemia, still led to larger k PCr proximally than distally (P = 0.013). In conclusion, the spatial k PCr gradient is strongly associated with the spatial variation in O2 supply. It cannot be explained by exercise‐induced ischaemia nor by fibre type. Our findings suggest it is driven by a higher proximal intrinsic mitochondrial oxidative capacity, apparently to support contractile performance of the TA.

Skeletal muscle maximal mitochondrial activity in ambulatory children with cerebral palsy.

To compare skeletal muscle mitochondrial enzyme activity and mitochondrial content between independently ambulatory children with cerebral palsy (CP) and typically developing children. Gracilis biopsies were obtained from 12 children during surgery (n=6/group, children with CP: one female, five males, mean age 13y 4mo, SD 5y 1mo, 4y 1mo-17y 10mo; typically developing children: three females, three males, mean age 16y 5mo, SD 1y 4mo, 14y 6mo-18y 2mo). Spectrophotometric enzymatic assays were used to evaluate the activity of mitochondrial electron transport chain complexes. Mitochondrial content was evaluated using citrate synthase assay, mitochondrial DNA copy number, and immunoblots for specific respiratory chain proteins. Maximal enzyme activity was significantly (50-80%) lower in children with CP versus typically developing children, for complex I (11nmol/min/mg protein, standard error of the mean [SEM] 1.7 vs 20.7nmol/min/mg protein, SEM 4), complex II (6.9nmol/min/mg protein, SEM 1.2 vs 21nmol/min/mg protein, SEM 2.7), complex III (31.9nmol/min/mg protein, SEM 7.4 vs 72.7nmol/min/mg protein, SEM 7.2), and complex I+III (7.4nmol/min/mg protein, SEM 2.5 vs 31.8nmol/min/mg protein, SEM 9.3). Decreased electron transport chain activity was not the result of lower mitochondrial content. Skeletal muscle mitochondrial electron transport chain enzymatic activity but not mitochondrial content is reduced in independently ambulatory children with CP. Decreased mitochondrial oxidative capacity might explain reported increased energetics of movement and fatigue in ambulatory children with CP. What this paper adds Skeletal muscle mitochondrial electron transport chain enzymatic activity is reduced in independently ambulatory children with cerebral palsy (CP). Mitochondrial content appears to be similar between children with CP and typically developing children.

BN-PAGE-Based Approach to Study Thyroid Hormones and Mitochondrial Function.

In recent years, a number of advancements have been made in the study of entire mitochondrial proteomes in both physiological and pathological conditions. Naturally occurring iodothyronines (i.e., T3 and T2) greatly influence mitochondrial oxidative capacity, directly or indirectly affecting the structure and function of the respiratory chain components. Blue native PAGE (BN-PAGE) can be used to isolate enzymatically active oxidative phosphorylation (OXPHOS) complexes in one step, allowing the clinical diagnosis of mitochondrial metabolism by monitoring OXPHOS catalytic and/or structural features. Protocols for isolating mammalian liver mitochondria and subsequent one-dimensional (1D) BN-PAGE will be described in relation to the impact of thyroid hormones on mitochondrial bioenergetics.

Energetski metabolizam u pankreasu tekunica (Spermophilus citellus) tokom produženog izlaganja hladnoći i u hibernaciji

Mammalian hibernators undergo a host of biochemical adaptations that allow them to survive the harsh cold environment and food restriction. Since the energy metabolism of the pancreas during hibernation remains unknown, we investigated the molecular basis of mitochondrial energy-producing pathways in line with their regulating mechanisms, as well as the (re)organization of antioxidative defence in the pancreas during the prehibernation period and in the hibernating state. To this end, male ground squirrels (Spermophilus citellus) were divided into two groups, the control group kept at room temperature (22±1 °C) and the group exposed to low temperature (4±1 °C). Active animals from the cold exposed group were sacrificed after 1, 3, 7, 12, and 21 days; animals that entered hibernation were sacrificed after 2-5 days of torpor. Our results showed that the protein levels of respiratory complexes I, II, IV and cytochrome c were increased in response to prolonged cold exposure (from day 12) and that such expression profiles were maintained during hibernation. In parallel, AMP-activated protein kinase a (AMPKa) and nuclear respiratory factor 1 (NRF-1) were shown to be upregulated. Moreover, prolonged cold exposure and hibernation induced an increase in the protein expression of antioxidative defence enzymes copper-zinc superoxide dismutase (CuZnSOD) and glutathione peroxidase (GSH-Px). In conclusion, these results point to a controlled metabolic remodeling in the pancreas of ground squirrels during prolonged cold exposure and in hibernation, which includes an improvement of mitochondrial oxidative capacity along with a proportional upregulation of antioxidative defence.

Therapeutic Efficiency of Lowering Branched-Chain Amino Acid Levels in Patients with Type 2 Diabetes Using Sodium-Phenylbutyrate: &lt;i&gt;A Randomized Placebo Controlled Clinical Intervention Study&lt;/i&gt;

Background: Elevations in plasma branched-chain amino acid (BCAA) levels associate with insulin resistance and type 2 diabetes (T2D). Pre-clinical models have suggested that lowering BCAA levels improve glucose tolerance, but data in humans are lacking. In the present study, we used sodium phenylbutyrate (NaPB), an accelerator of BCAA catabolism, as a tool to lower plasma BCAA levels in patients with T2D, and to evaluate its effect on patients’ metabolic health. Methods: This trial had a randomized, placebo-controlled, double-blind cross-over design and was performed in the Netherlands. Patients were eligible for the trial if they were 40-75years, BMI of 25-38 kg/m², relatively well-controlled T2D (HbA1C<8.5%) and treated with oral glucose-lowering medication. Participants were randomly assigned to receive either the NaPB 4.8g/m²/day or placebo three times a day for two weeks, separated by a washout period of six to eight weeks. The primary outcome was peripheral insulin sensitivity. Secondary outcomes were ex vivo muscle mitochondrial oxidative capacity, whole-body substrate oxidation, ectopic fat accumulation. Fasting blood samples were collected to determine levels of BCAA and their intermediates. The trial is registered with Netherlands Trial Register, NTR 7426. Findings: Between February 2019 and February 2020, sixteen patients were included. NaPB effectively led to a robust improvement in peripheral insulin sensitivity (ΔRd:13.2±1.8 vs. 9.6±1.8 µmol/kg/min, p=0.0155), carbohydrate-driven muscle mitochondrial oxidative capacity (O2-flux:74.0±4.1 vs. 67.1±4.3 pmol/(s*mg), p=0.0417), whole-body insulin-stimulated carbohydrate oxidation (10.9±0.6 vs. 9.6±0.7 µmol/kg/min, p=0.0246), and reduced plasma BCAA (549.2±4.5 vs. 598.4±5.0 µmol/L, p=0.0125) and oxidation-derived metabolites (KIV:13.4±0.1 vs. 15.2±0.1 µmol/L, p=0.0154; 3-HIB:36.94±0.7 vs. 43.5±0.6 µmol/L, p=0.0194:). No adverse events were reported. Interpretation: In patients with T2D, NaPB treatment significantly improved peripheral insulin sensitivity and glucose oxidation. These data establish the proof-of-concept in humans that targeting the BCAA pathway may represent a potential new treatment strategy for patients with T2D. Trial Registration: The trial has been registered with number NTR 7426, direct link to the trial: https://www.trialregister.nl/trial/7227. Funding: Dutch Diabetes Foundation Declaration of Interest: The authors declare no conflicts of interest. Ethical Approval: The study was conducted at the Maastricht University Medical Center (MUMC+), the Netherlands. The protocol was reviewed and approved by the Medical Review Ethics Committee of Maastricht University and Medical Centre.