Loss Of Mitochondrial Content Research Articles

Fibroblast Growth Factor 21 Signaling Activation by Selected Bioactive Compounds from Cocoa Shell Modulated Metabolism and Mitochondrial Function in Hepatocytes

Abstract Objectives To evaluate the effect of selected bioactive compounds from cocoa shell and its aqueous extract on the activation of fibroblast growth factor 21 (FGF21) signaling in hepatocytes and the resulting modulation of lipid and glucose metabolism, oxidative stress, and mitochondrial function. Methods The bioactive compounds from cocoa shells were extracted using water and characterized by UPLC-MS/MS. An in vitro model of non-alcoholic fatty liver disease was used: HepG2 cells were co-treated with either pure theobromine (TH), protocatechuic acid (PCA), procyanidin B2 (PB2), epicatechin (EPI), catechin (CAT) (10–50 µmol L−1), or the aqueous extract from cocoa shells (CAE, 20–100 µg mL−1) in the presence or absence of palmitic acid (PA, 500 µmol L−1). FGF21 receptor interaction with cocoa shell phytochemical was evaluated in silico. Biomarkers of cell metabolism were assessed 24 h after the co-treatment in cell supernatants and lysates using chemical, biochemical, and immunochemical techniques. Results Phytochemicals from cocoa shell were able to interact in silico with FGF21 receptor (biding energies from −6.0 to −11.0 kcal mol−1) denoting a potential interaction causing FGF21 receptor response. FGF21 hepatic signaling was activated (1.1 to 2.3-fold enhance in ERK1/2 phosphorylation). CAE, TH, PCA, PB2, EPI, and CAT diminished PA-elicited lipid accumulation (5–86%, P < 0.05) by reducing fatty acid synthase expression, and stimulated lipolysis (1–87%, P < 0.05) through the up-regulation of lipoprotein lipase expression. CAE and therein-bioactive compounds, mainly TH and PCA, promoted glucose uptake (4–35%, P < 0.05) via the increase in the phosphorylation of the insulin receptor (2.2-fold, P < 0.05), protein kinase B (2.8-fold, P < 0.05), AMPKα (2.0-fold, P < 0.05), and ERK1/2 (2.8-fold, P < 0.05) phosphorylation. Oxidative stress was prevented (50–100%, P < 0.05), and the loss of mitochondrial content (15–100%, P < 0.05) and function, measured as ATP production, alleviated (28–100%, P < 0.05). Conclusions Results demonstrated that the major bioactive compounds, primarily TH and PCA, found in cocoa shell could activate FGF21 signaling and regulated hepatic lipid and glucose metabolism, preventing insulin resistance, oxidative stress, and mitochondrial dysfunction. Funding Sources USDA-NIFA-HATCH, and the Spanish Ministry of Science and Innovation.

Molecular signalling towards mitochondrial breakdown is enhanced in skeletal muscle of patients with chronic obstructive pulmonary disease (COPD)

Loss of skeletal muscle mitochondrial oxidative capacity is well-established in patients with COPD, but the role of mitochondrial breakdown herein is largely unexplored. Currently, we studied if mitochondrial breakdown signalling is increased in skeletal muscle of COPD patients and associates with the loss of mitochondrial content, and whether it is affected in patients with iron deficiency (ID) or systemic inflammation. Therefore, mitophagy, autophagy, mitochondrial dynamics and content markers were analysed in vastus lateralis biopsies of COPD patients (N = 95, FEV1% predicted: 39.0 [31.0–53.6]) and healthy controls (N = 15, FEV1% predicted: 112.8 [107.5–125.5]). Sub-analyses were performed on patients stratified by ID or C-reactive protein (CRP). Compared with controls, COPD patients had lower muscle mitochondrial content, higher BNIP3L and lower FUNDC1 protein, and higher Parkin protein and gene-expression. BNIP3L and Parkin protein levels inversely correlated with mtDNA/gDNA ratio and FEV1% predicted. ID-COPD patients had lower BNIP3L protein and higher BNIP3 gene-expression, while high CRP patients had higher BNIP3 and autophagy-related protein levels. In conclusion, our data indicates that mitochondrial breakdown signalling is increased in skeletal muscle of COPD patients, and is related to disease severity and loss of mitochondrial content. Moreover, systemic inflammation is associated with higher BNIP3 and autophagy-related protein levels.

PPARα-independent effects of nitrate supplementation on skeletal muscle metabolism in hypoxia

Hypoxia is a feature of many disease states where convective oxygen delivery is impaired, and is known to suppress oxidative metabolism. Acclimation to hypoxia thus requires metabolic remodelling, however hypoxia tolerance may be aided by dietary nitrate supplementation. Nitrate improves tissue oxygenation and has been shown to modulate skeletal muscle tissue metabolism via transcriptional changes, including through the activation of peroxisome proliferator-activated receptor alpha (PPARα), a master regulator of fat metabolism. Here we investigated whether nitrate supplementation protects skeletal muscle mitochondrial function in hypoxia and whether PPARα is required for this effect. Wild-type and PPARα knockout (PPARα−/−) mice were supplemented with sodium nitrate via the drinking water or sodium chloride as control, and exposed to environmental hypoxia (10% O2) or normoxia for 4 weeks. Hypoxia suppressed mitochondrial respiratory function in mouse soleus, an effect partially alleviated through nitrate supplementation, but occurring independently of PPARα. Specifically, hypoxia resulted in 26% lower mass specific fatty acid-supported LEAK respiration and 23% lower pyruvate-supported oxidative phosphorylation capacity. Hypoxia also resulted in 24% lower citrate synthase activity in mouse soleus, possibly indicating a loss of mitochondrial content. These changes were not seen, however, in hypoxic mice when supplemented with dietary nitrate, indicating a nitrate dependent preservation of mitochondrial function. Moreover, this was observed in both wild-type and PPARα−/− mice. Our results support the notion that nitrate supplementation can aid hypoxia tolerance and indicate that nitrate can exert effects independently of PPARα.

Mitochondrial Adaptation in Insulin Resistant Human Skeletal Muscle

Introduction: The integrity, morphology, structure and functionality of skeletal muscle mitochondria in patients with type 2 diabetes (T2D) is heavily disputed. Thus, the purpose of this investigation was to characterize mitochondrial phenotypes across the insulin resistance spectrum. Methods: 30 sedentary adults aged 18-55 were grouped as lean (BMI 21±0.6, N=13), obese (BMI 32±0.9, N=12), or T2D (BMI 33±2.5, N=5) based on oral glucose tolerance tests and body mass index (BMI). All subjects completed a 3-day, inpatient stay consisting of cardio-metabolic testing, body composition analysis and a hyperinsulinemic-euglycemic clamp study. On the day of the clamp, muscle specimens were obtained basally and under insulin-stimulated conditions. Mitochondrial membrane potential (δψm) was measured by confocal microscopy using the cationic fluorophore TMRM. Oxidative phosphorylation (OXPHOS) was assessed in permeabilized fibers via high resolution respirometry (Oroboros Oxygraph-2k). Muscle mitochondrial ultrastructure and content were determined by transmission electron microscopy (TEM) of fixed muscle sections. Results: There was a significant, step-wise decrease in insulin-stimulated glucose uptake across the metabolic spectrum (P<0.001). No differences in resting δψm were observed, however, insulin stimulation depolarized δψm in all groups (P<0.05). Those with T2D exhibited increased basal complex III (P<0.05) and IV (P<0.05) activity vs. lean and obese controls. Furthermore, obese controls and T2D displayed augmented complex IV responsiveness to insulin stimulation (P<0.01). Threshold analysis of muscle sections revealed a progressive loss of mitochondrial content, as well as accumulation of lipid droplets across the metabolic spectrum (P<0.05). Conclusion: Collectively, these findings provide direct evidence of mitochondrial adaptation in insulin resistant and diabetic skeletal muscle. Disclosure C.L. Axelrod: None. C.E. Fealy: None. A. Mulya: None. E. Huang: None. H. Fujioka: None. B. Burguera: None. C.L. Hoppel: None. J.P. Kirwan: None.

Differential Effects of Superoxide Dismutase Mimetics after Mechanical Overload of Articular Cartilage.

Post-traumatic osteoarthritis can develop as a result of the initial mechanical impact causing the injury and also as a result of chronic changes in mechanical loading of the joint. Aberrant mechanical loading initiates excessive production of reactive oxygen species, oxidative damage, and stress that appears to damage mitochondria in the surviving chondrocytes. To probe the benefits of increasing superoxide removal with small molecular weight superoxide dismutase mimetics under severe loads, we applied both impact and overload injury scenarios to bovine osteochondral explants using characterized mechanical platforms with and without GC4403, MnTE-2-PyP, and MnTnBuOE-2-PyP. In impact scenarios, each of these mimetics provides some dose-dependent protection from cell death and loss of mitochondrial content while in repeated overloading scenarios only MnTnBuOE-2-PyP provided a clear benefit to chondrocytes. These results support the hypothesis that superoxide is generated in excess after impact injuries and suggest that superoxide production within the lipid compartment may be a critical mediator of responses to chronic overload. This is an important nuance distinguishing roles of superoxide, and thus superoxide dismutases, in mediating damage to cellular machinery in hyper-acute impact scenarios compared to chronic scenarios.

Exercise training normalizes mitochondrial respiratory capacity within the striatum of the R6/1 model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive cell loss in the striatum and cerebral cortex, leading to a decline in motor control and eventually death. The mechanisms promoting motor dysfunction are not known, however loss of mitochondrial function and content has been observed, suggesting that mitochondrial dysfunction may contribute to HD phenotype. Recent work has demonstrated that voluntary wheel running reduces hindlimb clasping in the R6/1 mouse model of HD, which we hypothesized may be due to preservation of mitochondrial content with exercise. Therefore, we investigated the role of chronic exercise training on preventing symptom progression and the loss of mitochondrial content in HD. Exercising R6/1 mice began training at 7 wks of age and continued for 10 or 20 wks. At 17 wks of age, R6/1 mice displayed a clasping phenotype without showing changes in mitochondrial respiration or protein content in either the cortex or striatum, suggesting mitochondrial dysfunction is not necessary for the progression of symptoms. At 27 wks of age, R6/1 mice demonstrated no additional changes in mitochondrial content or respiration within the cortex, but displayed loss of protein in complexes I and III of the striatum, which was not present in exercise-trained R6/1 mice. Mitochondrial respiration was also elevated in the striatum of R6/1 mice at 27 wks, which was prevented with exercise training. Together, the present study provides evidence that mitochondrial dysfunction is not necessary for the progression of hindlimb clasping in R6/1 mice, and that exercise partially prevents changes in mitochondrial content and function that occur late in HD.

Daily heat stress treatment rescues denervation-activated mitochondrial clearance and atrophy in skeletal muscle.

Traumatic nerve injury or nerve disease leads to denervation and severe muscle atrophy. Recent evidence shows that mitochondrial loss could be a key mediator of skeletal muscle atrophy. Here, we show that daily heat stress treatment rescues denervation-induced loss of mitochondria and concomitant muscle atrophy. We also found that denervation-activated autophagy-dependent mitochondrial clearance (mitophagy) was suppressed by daily heat stress treatment. The molecular basis of this observation is explained by our results showing that heat stress treatment attenuates the increase of key proteins that regulate the tagging step for mitochondrial clearance and the intermediate step of autophagosome formation in denervated muscle. These findings contribute to the better understanding of mitochondrial quality control in denervated muscle from a translational perspective and provide a mechanism behind the attenuation of muscle wasting by heat stress. Traumatic nerve injury or motor neuron disease leads to denervation and severe muscle atrophy. Recent evidence indicates that loss of mitochondria and the related reduction in oxidative capacity could be key mediators of skeletal muscle atrophy. As our previous study showed that heat stress increased the numbers of mitochondria in skeletal muscle, we evaluated whether heat stress treatment could have a beneficial impact on denervation-induced loss of mitochondria and subsequent muscle atrophy. Here, we report that daily heat stress treatment (mice placed in a chamber with a hot environment; 40°C, 30minday(-1) , for 7days) rescues the following parameters: (i)muscle atrophy (decreased gastrocnemius muscle mass); (ii)loss of mitochondrial content (decreased levels of ubiquinol-cytochrome c reductase core proteinII, cytochrome c oxidase subunitsI and IV and voltage-dependent anion channel protein); and (iii)reduction in oxidative capacity (reduced maximal activities of citrate synthase and 3-hydroxyacyl-CoA dehydrogenase) in denervated muscle (produced by unilateral sciatic nerve transection). In order to gain a better understanding of the above mitochondrial adaptations, we also examined the effects of heat stress on autophagy-dependent mitochondrial clearance (mitophagy). Daily heat stress normalized denervation-activated induction of mitophagy (increased mitochondrial microtubule-associated protein 1A/1B-light chain3-II (LC3-II) with and without blocker of autophagosome clearance). The molecular basis of this observation was explained by the results that heat stress attenuated the denervation-induced increase in key proteins that regulate the following steps: (i)the tagging step of mitochondrial clearance (increased mitochondrial Parkin, ubiquitin-conjugated, P62/sequestosome 1 (P62/SQSTM1)); and (ii)the elongation step of autophagosome formation (increased Atg5-Atg12 conjugate and Atg16L). Overall, our results contribute to the better understanding of mitochondrial quality control and the mechanisms behind the attenuation of muscle wasting by heat stress in denervated skeletal muscle.

T1620 Loss of Mitochondrial Content and Function Contribute to the Natural History of Nonalcoholic Fatty Liver Disease in a Hyperphagic Obese Rodent Model

T1620 Loss of Mitochondrial Content and Function Contribute to the Natural History of Nonalcoholic Fatty Liver Disease in a Hyperphagic Obese Rodent Model