Ketogenic essential amino acids replacement diet ameliorated hepatosteatosis with altering autophagy-associated molecules

Ketogenic amino acid (KAA) replacement diet has been shown to cure hepatic steatosis, a serious liver disease associated with diverse metabolic defects. In this study, we investigated the effects of KAA replacement diet on nutrition sensing signaling pathway and analyzed whether induction of hepatic autophagy was involved. Mice are fed with high fat diet (HFD) or KAA replacement in high-fat diet (30% fat in food; HFD)-fed (HFD^KAAR) and sacrificed at 8, 12, 16 weeks after initiation of experimental food. Hepatic autophagy was analyzed in protein expression of several autophagy-associated molecules and in light chain-3 green fluorescent protein (LC-3 GFP) transgenic mice. HFD^KAAR showed increased AMP-activated protein kinase (AMPK) phosphorylation and enhanced liver kinase B1 (LKB1) expression compared to control HFD-fed mice. The KAA-HFD-induced activation of AMPK was associated with an increased protein expression of sirtuin 1 (Sirt1), decreased forkhead box protein O3a (Foxo3a) level, and suppression of mammalian target of rapamycin (mTOR) phosphorylation compared with the HFD-fed mice. The intervention study revealed that a KAA-replacement diet also ameliorated all the established metabolic and autophagy defects in the HFD-fed mice, suggesting that a KAA-replacement diet can be used therapeutically in established diseases. These results indicate that KAA replacement in food could be a novel strategy to combat hepatic steatosis and metabolic abnormalities likely involvement of an induction of autophagy.     

Carbon monoxide improves cardiac function and mitochondrial population quality in a mouse model of metabolic syndrome

Aims

Metabolic syndrome induces cardiac dysfunction associated with mitochondria abnormalities. As low levels of carbon monoxide (CO) may improve myocardial and mitochondrial activities, we tested whether a CO-releasing molecule (CORM-3) reverses metabolic syndrome-induced cardiac alteration through changes in mitochondrial biogenesis, dynamics and autophagy.

Methods and Results

Mice were fed with normal diet (ND) or high-fat diet (HFD) for twelve weeks. Then, mice received two intraperitoneal injections of CORM-3 (10 mg.kg⁻¹), with the second one given 16 hours after the first. Contractile function in isolated hearts and mitochondrial parameters were evaluated 24 hours after the last injection. Mitochondrial population was explored by electron microscopy. Changes in mitochondrial dynamics, biogenesis and autophagy were assessed by western-blot and RT-qPCR. Left ventricular developed pressure was reduced in HFD hearts. Mitochondria from HFD hearts presented reduced membrane potential and diminished ADP-coupled respiration. CORM-3 restored both cardiac and mitochondrial functions. Size and number of mitochondria increased in the HFD hearts but not in the CORM-3–treated HFD group. CORM-3 modulated HFD-activated mitochondrial fusion and biogenesis signalling. While autophagy was not activated in the HFD group, CORM-3 increased the autophagy marker LC3-II. Finally, ex vivo experiments demonstrated that autophagy inhibition by 3-methyladenine abolished the cardioprotective effects of CORM-3.

Conclusion

CORM-3 may modulate pathways controlling mitochondrial quality, thus leading to improvements of mitochondrial efficiency and HFD-induced cardiac dysfunction.     

Garcinia cambogia Extract Ameliorates Visceral Adiposity in C57BL/6J Mice Fed on a High-Fat Diet

The aim of present study is to evaluate the effects of Garcinia cambogia on the mRNA levels of the various genes involved in adipogenesis, as well as on body weight gain, visceral fat accumulation, and other biochemical markers of obesity in obesity-prone C57BL/6J mice. Consumption of the Garcinia cambogia extract effectively lowered the body weight gain, visceral fat accumulation, blood and hepatic lipid concentrations, and plasma insulin and leptin levels in a high-fat diet (HFD)-induced obesity mouse model. The Garcinia cambogia extract reversed the HFD-induced changes in the expression pattern of such epididymal adipose tissue genes as adipocyte protein aP2 (aP2), sterol regulatory element-binding factor 1c (SREBP1c), peroxisome proliferator-activated receptor γ2 (PPARγ2), and CCAT/enhancer-binding protein α (C/EBPα). These findings suggest that the Garcinia cambogia extract ameliorated HFD-induced obesity, probably by modulating multiple genes associated with adipogenesis, such as aP2, SREBP1c, PPARγ2, and C/EBPα in the visceral fat tissue of mice.     

Hypoxia mimetic induces lipid accumulation through mitochondrial dysfunction and stimulates autophagy in murine preadipocyte cell line

BackgroundHypoxia occurs within adipose tissue of obese human and mice. However, its role in adipose tissue regulation is still controversial.MethodsWe used murine preadipocyte 3T3-L1 cells and hypoxia was induced by using hypoxia mimetic agents, as CoCl₂. To study adipocyte differentiation, we evaluated the adipocyte markers (PPARγ, C/EBPα and aP2), and a preadipocyte marker (pref-1) by qPCR, western blotting and immunofluorescence. Lipid accumulation was evaluated by Oil red-O assay and perilipin levels by western blotting and immunofluorescence. The effect of CoCl₂ in microRNA, miR-27a and miR-27b, levels was evaluated by qPCR. We also assessed the mitochondrial membrane potential and reactive oxygen species (ROS), superoxide and ATP production. The effect of hypoxia mimetic in autophagy was determined by LC3B and p62 level evaluation by western blotting.ResultsOur results show that the hypoxia mimetic cobalt chloride increases lipid accumulation with no expression of PPARγ2. Furthermore, using qPCR we observed that the hypoxia mimetic increases microRNAs miR-27a and miR-27b, which are known to block PPARγ2 expression. In contrast, cobalt chloride induces mitochondrial dysfunction, and increases ROS production and autophagy. Moreover, an antioxidant agent, glutathione, prevents lipid accumulation induced by hypoxia mimetic indicating that ROS are responsible for hypoxia-induced lipid accumulation.ConclusionsAll these results taken together suggest that hypoxia mimetic blocks differentiation and induces autophagy. Hypoxia mimetic also induces lipid accumulation through mitochondrial dysfunction and ROS accumulation.General significanceThis study highlights the importance of adipocyte response to hypoxia, which might impair adipocyte metabolism and compromise adipose tissue function.     

CD38 deficiency suppresses adipogenesis and lipogenesis in adipose tissues through activating Sirt1/PPARγ signaling pathway

It has been recently reported that CD38 was highly expressed in adipose tissues from obese people and CD38‐deficient mice were resistant to high‐fat diet (HFD)‐induced obesity. However, the role of CD38 in the regulation of adipogenesis and lipogenesis is unknown. In this study, to explore the roles of CD38 in adipogenesis and lipogenesis in vivo and in vitro, obesity models were generated with male CD38^?/? and WT mice fed with HFD. The adipocyte differentiations were induced with MEFs from WT and CD38^?/? mice, 3T3‐L1 and C3H10T1/2 cells in vitro. The lipid accumulations and the alternations of CD38 and the genes involved in adipogenesis and lipogenesis were determined with the adipose tissues from the HFD‐fed mice or the MEFs, 3T3‐L1 and C3H10T1/2 cells during induction of adipocyte differentiation. The results showed that CD38^?/? male mice were significantly resistant to HFD‐induced obesity. CD38 expressions in adipocytes were significantly increased in WT mice fed with HFD, and the similar results were obtained from WT MEFs, 3T3‐L1 and C3H10T1/2 during induction of adipocyte differentiation. The expressions of PPARγ, AP2 and C/EBPα were markedly attenuated in adipocytes from HFD‐fed CD38^?/? mice and CD38^?/? MEFs at late stage of adipocyte differentiation. Moreover, the expressions of SREBP1 and FASN were also significantly decreased in CD38^?/? MEFs. Finally, the CD38 deficiency‐mediated activations of Sirt1 signalling were up‐regulated or down‐regulated by resveratrol and nicotinamide, respectively. These results suggest that CD38 deficiency impairs adipogenesis and lipogenesis through activating Sirt1/PPARγ‐FASN signalling pathway during the development of obesity.     

Astaxanthin reduces hepatic lipid accumulations in high-fat-fed C57BL/6J mice via activation of peroxisome proliferator-activated receptor (PPAR) alpha and inhibition of PPAR gamma and Akt

We have previously reported that astaxanthin (AX), a dietary carotenoid, directly interacts with peroxisome proliferator-activated receptors PPARα and PPARγ, activating PPARα while inhibiting PPARγ, and thus reduces lipid accumulation in hepatocytes in vitro. To investigate the effects of AX in vivo, high-fat diet (HFD)-fed C57BL/6J mice were orally administered AX (6 or 30 mg/kg body weight) or vehicle for 8 weeks. AX significantly reduced the levels of triglyceride both in plasma and in liver compared with the control HFD mice. AX significantly improved liver histology and thus reduced both steatosis and inflammation scores of livers with hematoxylin and eosin staining. The number of inflammatory macrophages and Kupffer cells were reduced in livers by AX administration assessed with F4/80 staining. Hepatic PPARα-responsive genes involved in fatty acid uptake and β-oxidation were upregulated, whereas inflammatory genes were downregulated by AX administration. In vitro radiolabeled assays revealed that hepatic fatty acid oxidation was induced by AX administration, whereas fatty acid synthesis was not changed in hepatocytes. In mechanism studies, AX inhibited Akt activity and thus decreased SREBP1 phosphorylation and induced Insig-2a expression, both of which delayed nuclear translocation of SREBP1 and subsequent hepatic lipogenesis. Additionally, inhibition of the Akt-mTORC1 signaling axis by AX stimulated hepatic autophagy that could promote degradation of lipid droplets. These suggest that AX lowers hepatic lipid accumulation in HFD-fed mice via multiple mechanisms. In addition to the previously reported differential regulation of PPARα and PPARγ, inhibition of Akt activity and activation of hepatic autophagy reduced hepatic steatosis in mouse livers.     

Sulforaphane attenuates obesity by inhibiting adipogenesis and activating the AMPK pathway in obese mice

Obesity is associated with metabolic disorders. Sulforaphane, an isothiocyanate, inhibits adipogenesis and the occurrence of cardiovascular disease. In this study, we investigated whether sulforaphane could prevent high-fat diet (HFD)-induced obesity in C57BL/6N mice. Mice were fed a normal diet (ND), HFD or HFD plus 0.1% sulforaphane (SFN) for 6 weeks. Food efficiency ratios and body weight were lower in HFD-SFN-fed mice than in HFD-fed mice. SFN attenuated HFD-induced visceral adiposity, adipocyte hypertrophy and fat accumulation in the liver. Serum total cholesterol and leptin, and liver triglyceride levels were lower in HFD-SFN-fed mice than in HFD-fed mice. SFN decreased the expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and leptin in the adipose tissue of HFD-SFN mice and increased adiponectin expression. Phosphorylation of AMP-activated protein kinase α (AMPKα) and acetyl-CoA carboxylase in the adipose tissue of HFD-SFN-fed mice was elevated, and HMG-CoA reductase expression was decreased compared with HFD-fed mice. Thus, these results suggest that SFN may induce antiobesity activity by inhibiting adipogenesis through down-regulation of PPARγ and C/EBPα and by suppressing lipogenesis through activation of the AMPK pathway.     

Milk fat globule membrane and its component phosphatidylcholine induce adipose browning both in vivo and in vitro

The functional induction of brown-like adipocytes in white adipose tissue (WAT) provides a defense against obesity. The aim of this study was to analyze the effects of milk fat globule membrane (MFGM) and its component phosphatidylcholine (PC) on the brown remodeling of WAT. Male C57BL/6 J mice were fed a high-fat diet (HFD) for 8 weeks and then fed HFD for another 8 weeks with MFGM. In vitro studies were performed in C3H10T1/2 pluripotent stem cells, 3T3-L1 pre-adipocytes and differentiated inguinal WAT stromal vascular cells (SVCs) to determine the role of MFGM and PC on the formation of brown-like adipocytes. MFGM decreased fasting glucose and serum insulin levels in HFD-fed mice. MFGM improved glucose tolerance and insulin sensitivity, and induced browning of inguinal WAT. MFGM and its component PC stimulated transformation of brown-like adipocytes in C3H10T1/2 pluripotent stem cells, 3T3-L1 adipocytes and SVCs by increasing the protein expression of UCP1, PGC-1α, PRDM16 as well as the mRNA expression of other thermogenic genes and beige cell markers. MFGM and PC also increased mitochondrial DNA (mtDNA) copy number, mitochondrial density and oxygen consumption rate and up-regulated the mRNA expression of mitochondria-biogenesis-related genes in vitro. PPARα inhibitor GW6471 treatment or knockdown of PPARα using lentivirus-expressing shRNA inhibited the PC-induced increase in the protein expression of UCP1, PGC-1α and PRDM16 in C3H10T1/2 pluripotent stem cells and 3T3-L1 adipocytes, indicating the potential role of PPARα in PC-mediated brown-like adipocyte formation. In conclusion, MFGM and milk PC induced adipose browning, which has major protective effects against obesity and metabolic dysfunction.     

CCAAT/Enhancer-Binding Protein β is not Affected by Tetrachlorodibenzo-p-dioxin (TCDD) Inhibition of 3T3-L1 Preadipocyte Differentiation

The differentiation of 3T3-L1 preadipocytes is induced by the coordinate activation of trans-acting factors in response to inducers. Depending on the time of treatment, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was effective in inhibiting 3T3-L1 preadipocyte differentiation and the expression of differentiation-dependent trans-acting factors. Based on glycerol-3-phosphate dehydrogenase activity, the differentiation of 3T3-L1 cells was decreased by 70% in cells treated with TCDD before the induction of differentiation, 25% during induction, and not at all after induction. This time-dependent inhibition of cell differentiation by TCDD was correlated with the levels of aryl hydrocarbon receptor (AhR). TCDD treatment decreased the mRNA levels of C/EBPα and PPAR-γ2 but did not affect the mRNA levels of RXRα and RARα. Furthermore, TCDD did not change the mRNA or protein levels of C/EBPβ, which is thought to play a role in inducing C/EBPα and PPARγ2 expression. These results suggest that TCDD inhibited 3T3-L1 preadipocyte differentiation through the AhR pathway, and the change of C/EBPβ mRNA and protein was not involved in reducing mRNA expression of C/EBPα and PPARγ2.     

Chemerin‐induced mitochondrial dysfunction in skeletal muscle

Chemerin is a novel adipocyte‐derived factor that induces insulin resistance in skeletal muscle. However, the effect of chemerin on skeletal muscle mitochondrial function has received little attention. In the present study, we investigated whether mitochondrial dysfunction is involved in the pathogenesis of chemerin‐mediated insulin resistance. In this study, we used recombinant adenovirus to express murine chemerin in C57BL/6 mice. The mitochondrial function and structure were evaluated in isolated soleus muscles from mice. The oxidative mechanism of mitochondrial dysfunction in cultured C2C12 myotubes exposed to recombinant chemerin was analysed by western blotting, immunofluorescence and quantitative real‐time polymerase chain reaction. The overexpression of chemerin in mice reduced the muscle mitochondrial content and increased mitochondrial autophagy, as determined by the increased conversion of LC3‐I to LC3‐II and higher expression levels of Beclin1 and autophagy‐related protein‐5 and 7. The chemerin treatment of C2C12 myotubes increased the generation of mitochondrial reactive oxygen species, concomitant with a reduced mitochondrial membrane potential and increased the occurrence of mitochondrial protein carbonyls and mitochondrial DNA deletions. Knockdown of the expression of chemokine‐like receptor 1 or the use of mitochondria‐targeting antioxidant Mito‐TEMPO restored the mitochondrial dysfunction induced by chemerin. Furthermore, chemerin exposure in C2C12 myotubes not only reduced the insulin‐stimulated phosphorylation of protein kinase B (AKT) but also dephosphorylated forkhead box O3α (FoxO3α). Chemerin‐induced mitochondrial autophagy likely through an AKT‐FoxO3α‐dependent signalling pathway. These findings provide direct evidence that chemerin may play an important role in regulating mitochondrial remodelling and function in skeletal muscle.     

Trans‐repression of NFκB pathway mediated by PPARγ improves vascular endothelium insulin resistance

Previous study has shown that thiazolidinediones (TZDs) improved endothelium insulin resistance (IR) induced by high glucose concentration (HG)/hyperglycaemia through a PPARγ‐dependent‐NFκB trans‐repression mechanism. However, it is unclear, whether changes in PPARγ expression affect the endothelium IR and what the underlying mechanism is. In the present study, we aimed to address this issue. HG‐treated human umbilical vascular endothelial cells (HUVEC) were transfected by either PPARγ‐overexpressing (Ad‐PPARγ) or PPARγ‐shRNA‐containing (Ad‐PPARγ‐shRNA) adenoviral vectors. Likewise, the rats fed by high‐fat diet (HFD) were infected by intravenous administration of Ad‐PPARγ or Ad‐PPARγ‐shRNA. The levels of nitric oxide (NO), endothelin‐1 (ET‐1) and cytokines (TNFα, IL‐6, sICAM‐1 and sVCAM‐1) and the expression levels of PPARγ, eNOS, AKT, p‐AKT, IKKα/β and p‐IKKα/β and IκBα were examined; and the interaction between PPARγ and NFκB‐P65 as well as vascular function were evaluated. Our present results showed that overexpression of PPARγ notably increased the levels of NO, eNOS, p‐AKT and IκBα as well as the interaction of PPARγ and NFκB‐P65, and decreased the levels of ET‐1, p‐IKKα/β, TNFα, IL‐6, sICAM‐1 and sVCAM‐1. In contrast, down‐expression of PPARγ displayed the opposite effects. The results demonstrate that the overexpression of PPARγ improves while the down‐expression worsens the endothelium IR via a PPARγ‐mediated NFκB trans‐repression dependent manner. The findings suggest PPARγ is a potential therapeutic target for diabetic vascular complications.     

High‐fat diet affects skeletal muscle mitochondria comparable to pressure overload‐induced heart failure

In heart failure, high‐fat diet (HFD) may exert beneficial effects on cardiac mitochondria and contractility. Skeletal muscle mitochondrial dysfunction in heart failure is associated with myopathy. However, it is not clear if HFD affects skeletal muscle mitochondria in heart failure as well. To induce heart failure, we used pressure overload (PO) in rats fed normal chow or HFD. Interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) from gastrocnemius were isolated and functionally characterized. With PO heart failure, maximal respiratory capacity was impaired in IFM but increased in SSM of gastrocnemius. Unexpectedly, HFD affected mitochondria comparably to PO. In combination, PO and HFD showed additive effects on mitochondrial subpopulations which were reflected by isolated complex activities. While PO impaired diastolic as well as systolic cardiac function and increased glucose tolerance, HFD did not affect cardiac function but decreased glucose tolerance. We conclude that HFD and PO heart failure have comparable effects leading to more severe impairment of IFM. Glucose tolerance seems not causally related to skeletal muscle mitochondrial dysfunction. The additive effects of HFD and PO may suggest accelerated skeletal muscle mitochondrial dysfunction when heart failure is accompanied with a diet containing high fat.     

Exercise Protects against Diet-Induced Insulin Resistance through Downregulation of Protein Kinase Cβ in Mice

Physical exercise is an important and effective therapy for diabetes. However, its underlying mechanism is not fully understood. Protein kinase Cβ (PKCβ) has been suggested to be involved in the pathogenesis of obesity and insulin resistance, but the role of PKCβ in exercise-induced improvements in insulin resistance is completely unknown. In this study, we evaluated the involvement of PKCβ in exercise-attenuated insulin resistance in high-fat diet (HFD)-fed mice. PKCβ^-/- and wild-type mice were fed a HFD with or without exercise training. PKC protein expression, body and tissue weight change, glucose and insulin tolerance, metabolic rate, mitochondria size and number, adipose inflammation, and AKT activation were determined to evaluate insulin sensitivity and metabolic changes after intervention. PKCβ expression decreased in both skeletal muscle and liver tissue after exercise. Exercise and PKCβ deficiency can alleviate HFD-induced insulin resistance, as evidenced by improved insulin tolerance. In addition, fat accumulation and mitochondrial dysfunction induced by HFD were also ameliorated by both exercise and PKCβ deficiency. On the other hand, exercise had little effect on PKCβ^-/- mice. Further, our data indicated improved activation of AKT, the downstream signal molecule of insulin, in skeletal muscle and liver of exercised mice, whereas PKCβ deficiency blunted the difference between sedentary and exercised mice. These results suggest that downregulation of PKCβ contributes to exercise-induced improvement of insulin resistance in HFD-fed mice.     

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered ¹³C₃, ¹⁵N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.     

Differential structural features in soleus and gastrocnemius of carnitine-treated cancer cachectic rats

Muscle wasting is associated with chronic diseases and cancer. Elucidation of the biological mechanism involved in the process of muscle mass loss and cachexia may help identify therapeutic targets. We hypothesized that l -carnitine treatment may differentially revert muscle fiber atrophy and other structural alterations in slow- and fast-twitch limb muscles of rats bearing the Yoshida ascites hepatoma. In soleus and gastrocnemius of tumor-bearing rats (10⁸ AH-130 Yoshida ascites hepatoma cells inoculated intraperitoneally) with and without treatment with l -carnitine (1 g/kg body weight for 7 days, intragastric), food intake, body and muscle weights, fiber typing and morphometry, morphological features, redox balance, autophagy and proteolytic, and signaling markers were explored. Levels of carnitine palmitoyl transferase were also measured in all the study muscles. l - Carnitine treatment ameliorated the atrophy of both slow- and fast-twitch fibers (gastrocnemius particularly), muscle structural alterations (both muscles), and attenuated oxidative stress, proteolytic and signaling markers (gastrocnemius). Despite that carnitine palmitoyl transferase-1 levels increased in both muscle types in a similar fashion, l -carnitine ameliorated muscle atrophy and proteolysis in a muscle-specific manner in cancer-induced cachexia. These data reveal the need to study muscles of different fiber type composition and function to better understand whereby l -carnitine exerts its beneficial effects on the myofibers in muscle wasting processes. These findings also have potential clinical implications, since combinations of various exercise and muscle training modalities with l -carnitine should be specifically targeted for the muscle groups to be trained.     

Pharmacological restoration of autophagy reduces hypertension-related stroke occurrence

ABSTRACT

The identification of the mechanisms predisposing to stroke may improve its preventive and therapeutic strategies in patients with essential hypertension. The role of macroautophagy/autophagy in the development of hypertension-related stroke needs to be clarified. We hypothesized that a defective autophagy may favor hypertension-related spontaneous stroke by promoting mitochondrial dysfunction. We studied autophagy in the stroke-prone spontaneously hypertensive (SHRSP) rat, which represents a clinically relevant model of stroke associated with high blood pressure. We assessed autophagy, mitophagy and NAD⁺:NADH levels in brains of SHRSP and stroke-resistant SHR fed with high salt diet. Vascular smooth muscle cells silenced for the mitochondrial complex I subunit Ndufc2 gene (NADH:ubiquinone oxidoreductase subunit C2) and cerebral endothelial cells isolated from SHRSP were also used to assess autophagy/mitophagy and mitochondrial function in response to high salt levels. We found a reduction of autophagy in brains of high salt-fed SHRSP. Autophagy impairment was associated with NDUFC2 downregulation, mitochondrial dysfunction and NAD⁺ depletion. Restoration of NAD⁺ levels by nicotinamide administration reactivated autophagy and reduced stroke development in SHRSP. A selective reactivation of autophagy/mitophagy by Tat-Beclin 1 also reduced stroke occurrence, restored autophagy/mitophagy and improved mitochondrial function. Endothelial progenitor cells (EPCs) from subjects homozygous for the thymine allele variant at NDUFC2/rs11237379, which is associated with NDUFC2 deficiency and increased stroke risk, displayed an impairment of autophagy and increased senescence in response to high salt levels. EPC senescence was rescued by Tat-Beclin 1. Pharmacological activation of autophagy may represent a novel therapeutic strategy to reduce stroke occurrence in hypertension.     

Gender-dimorphic regulation of muscular proteins in response to high fat diet and sex steroid hormones

To investigate the roles of gender-dependent obesity, we investigated the effects of high fat diet (HFD) and sex steroid hormones on the fiber-type dependent expression of contractile and metabolic regulatory pathway proteins in gastrocnemius (G) and soleus (S) muscle tissue of male and female rats. The results revealed that estrogen (E2) negatively influences body weight gain, whereas testosterone (DHT) has positive effects. Additionally, E2 appeared to play an essential role in initiating muscle contraction and mediating glucose and lipid metabolism events via AMPK and AKT pathways. The elevated expression of ERα contributed to the expression of muscular proteins in a fiber-type and gender-dependent manner. E2 treatment increased the protein levels of AMPK, thereby activating downstream lipid metabolic proteins such as PPARγ, ACSL1, LPL, and A-FABP. Such cooperatively activating proteins enhanced fatty acid oxidation, attenuating TG accumulation. E2 stimulated AKT and AMPK activation suggests that these proteins enhanced GLUT4 expression. More importantly, the S muscle of HFD-fed control females showed higher expressions of MYH, TPM1α, and TnI, while only MYH and TnI were upregulated in males treated with E2, indicating that females may be more resistant to HFD-increased metabolic complications. Similarly, E2 treatment enhanced metabolic regulatory proteins in males, indicating that they are more susceptible to metabolic dysregulation than females. To the best our knowledge, this is the first report to distinguish the fatty acid uptake and oxidation between two types of muscle in both genders.