Yau Sheng Tsai

Mitochondrial Fission Contributes to Mitochondrial Dysfunction and Insulin Resistance in Skeletal Muscle

ABSTRACT Mitochondrial dysfunction in skeletal muscle has been implicated in the development of insulin resistance and type 2 diabetes. Considering the importance of mitochondrial dynamics in mitochondrial and cellular functions, we hypothesized that obesity and excess energy intake shift the balance of mitochondrial dynamics, further contributing to mitochondrial dysfunction and metabolic deterioration in skeletal muscle. First, we revealed that excess palmitate (PA), but not hyperglycemia, hyperinsulinemia, or elevated tumor necrosis factor alpha, induced mitochondrial fragmentation and increased mitochondrion-associated Drp1 and Fis1 in differentiated C2C12 muscle cells. This fragmentation was associated with increased oxidative stress, mitochondrial depolarization, loss of ATP production, and reduced insulin-stimulated glucose uptake. Both genetic and pharmacological inhibition of Drp1 attenuated PA-induced mitochondrial fragmentation, mitochondrial depolarization, and insulin resistance in C2C12 cells. Furthermore, we found smaller and shorter mitochondria and increased mitochondrial fission machinery in the skeletal muscle of mice with genetic obesity and those with diet-induced obesity. Inhibition of mitochondrial fission improved the muscle insulin signaling and systemic insulin sensitivity of obese mice. Our findings indicated that aberrant mitochondrial fission is causally associated with mitochondrial dysfunction and insulin resistance in skeletal muscle. Thus, disruption of mitochondrial dynamics may underlie the pathogenesis of muscle insulin resistance in obesity and type 2 diabetes.

Hypertension and abnormal fat distribution but not insulin resistance in mice with P465L PPARγ

Peroxisome proliferator-activated receptor gamma (PPARgamma), the molecular target of a class of insulin sensitizers, regulates adipocyte differentiation and lipid metabolism. A dominant negative P467L mutation in the ligand-binding domain of PPARgamma in humans is associated with severe insulin resistance and hypertension. Homozygous mice with the equivalent P465L mutation die in utero. Heterozygous mice grow normally and have normal total adipose tissue weight. However, they have reduced interscapular brown adipose tissue and intra-abdominal fat mass, and increased extra-abdominal subcutaneous fat, compared with wild-type mice. They have normal plasma glucose levels and insulin sensitivity, and increased glucose tolerance. However, during high-fat feeding, their plasma insulin levels are mildly elevated in association with a significant increase in pancreatic islet mass. They are hypertensive, and expression of the angiotensinogen gene is increased in their subcutaneous adipose tissues. The effects of P465L on blood pressure, fat distribution, and insulin sensitivity are the same in both male and female mice regardless of diet and age. Thus the P465L mutation alone is sufficient to cause abnormal fat distribution and hypertension but not insulin resistance in mice. These results provide genetic evidence for a critical role for PPARgamma in blood pressure regulation that is not dependent on altered insulin sensitivity.

Interference With PPARγ Signaling Causes Cerebral Vascular Dysfunction, Hypertrophy, and Remodeling

The transcription factor PPARγ is expressed in endothelium and vascular muscle where it may exert antiinflammatory and antioxidant effects. We tested the hypothesis that PPARγ plays a protective role in the vasculature by examining vascular structure and function in heterozygous knockin mice expressing the P465L dominant negative mutation in PPARγ (L/+). In L/+ aorta, responses to the endothelium-dependent agonist acetylcholine (ACh) were not affected, but there was an increase in contraction to serotonin, PGF2α, and endothelin-1. In cerebral blood vessels both in vitro and in vivo, ACh produced dilation that was markedly impaired in L/+ mice. Superoxide levels were elevated in cerebral arterioles from L/+ mice and responses to ACh were restored to normal with a scavenger of superoxide. Diameter of maximally dilated cerebral arterioles was less, whereas wall thickness and cross-sectional area was greater in L/+ mice, indicating cerebral arterioles underwent hypertrophy and remodeling. Thus, interference with PPARγ signaling produces endothelial dysfunction via a mechanism involving oxidative stress and causes vascular hypertrophy and inward remodeling. These findings indicate that PPARγ has vascular effects which are particularly profound in the cerebral circulation and provide genetic evidence that PPARγ plays a critical role in protecting blood vessels.

Ligand-Activated Peroxisome Proliferator–Activated Receptor-γ Protects Against Ischemic Cerebral Infarction and Neuronal Apoptosis by 14-3-3ε Upregulation

Background— Thiazolidinediones have been reported to protect against ischemia-reperfusion injury. Their protective actions are considered to be peroxisome proliferator–activated receptor-γ (PPAR-γ)–dependent; however, it is unclear how PPAR-γ activation confers resistance to ischemia-reperfusion injury. Methods and Results— We evaluated the effects of rosiglitazone or PPAR-γ overexpression on cerebral infarction in a rat model and investigated the antiapoptotic actions in the N2-A neuroblastoma cell model. Rosiglitazone or PPAR-γ overexpression significantly reduced infarct volume. The protective effect was abrogated by PPAR-γ small interfering RNA. In mice with knock-in of a PPAR-γ dominant-negative mutant, infarct volume was enhanced. Proteomic analysis revealed that brain 14-3-3&egr; was highly upregulated in rats treated with rosiglitazone. Upregulation of 14-3-3&egr; was abrogated by PPAR-γ small interfering RNA or antagonist. Promoter analysis and chromatin immunoprecipitation revealed that rosiglitazone induced PPAR-γ binding to specific regulatory elements on the 14-3-3&egr; promoter and thereby increased 14-3-3&egr; transcription. 14-3-3&egr; Small interfering RNA abrogated the antiapoptotic actions of rosiglitazone or PPAR-γ overexpression, whereas 14-3-3&egr; recombinant proteins rescued brain tissues and N2-A cells from ischemia-induced damage and apoptosis. Elevated 14-3-3&egr; enhanced binding of phosphorylated Bad and protected mitochondrial membrane potential. Conclusions— Ligand-activated PPAR-γ confers resistance to neuronal apoptosis and cerebral infarction by driving 14-3-3&egr; transcription. 14-3-3&egr; Upregulation enhances sequestration of phosphorylated Bad and thereby suppresses apoptosis.

Modulation of energy deficiency in Huntington's disease via activation of the peroxisome proliferator-activated receptor gamma

Huntingtons disease (HD) is a neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. Here, we report that the transcript of the peroxisome proliferator-activated receptor-γ (PPARγ), a transcription factor that is critical for energy homeostasis, was markedly downregulated in multiple tissues of a mouse model (R6/2) of HD and in lymphocytes of HD patients. Therefore, downregulation of PPARγ seems to be a pathomechanism of HD. Chronic treatment of R6/2 mice with an agonist of PPARγ (thiazolidinedione, TZD) rescued progressive weight loss, motor deterioration, formation of mutant Htt aggregates, jeopardized global ubiquitination profiles, reduced expression of two neuroprotective proteins (brain-derived neurotrophic factor and Bcl-2) and shortened life span exhibited by these mice. By reducing HTT aggregates and, thus, ameliorating the recruitment of PPARγ into HTT aggregates, chronic TZD treatment also elevated the availability of the PPARγ protein and subsequently normalized the expression of two of its downstream genes (the glucose transporter type 4 and PPARγ coactivator-1 alpha genes). The protective effects described above appear to have been exerted, at least partially, via direct activation of PPARγ in the brain, as TZD was detected in the brains of mice treated with TZD and because a PPARγ agonist (rosiglitazone) protected striatal cells from mHTT-evoked energy deficiency and toxicity. We demonstrated that the systematic downregulation of PPARγ seems to play a critical role in the dysregulation of energy homeostasis observed in HD, and that PPARγ is a potential therapeutic target for this disease.

Toll-like receptor 2 deficiency improves insulin sensitivity and hepatic insulin signalling in the mouse

Aims/hypothesisSubstantial evidence suggests a link between elevated inflammation and development of insulin resistance. Toll-like receptor 2 (TLR2) recognises a large number of lipid-containing molecules and transduces inflammatory signalling in a variety of cell types, including insulin-responsive cells. Considering the contribution of the fatty acid composition in TLR2-depedent signalling, we hypothesised that the inflammatory signals transduced by TLR2 contribute to insulin resistance.MethodsMice deficient in TLR2 were used to investigate the in vivo roles of TLR2 in initiating and maintaining inflammation-associated insulin resistance and energy homeostasis.ResultsWe first recapitulated the observation with elevated expression of TLR2 and inflammatory cytokines in white adipose tissue and liver of ob/ob mice. Aged or high-fat-fed TLR2-deficient mice were protected from obesity and adipocyte hypertrophy compared with wild-type mice. Moreover, mice lacking TLR2 exhibited improved glucose tolerance and insulin sensitivity regardless of feeding them regular chow or a high-fat diet. This is accompanied by reductions in expression of inflammatory cytokines and activation of extracellular signal-regulated kinase (ERK) in a liver-specific manner. The attenuated hepatic inflammatory cytokine expression and related signalling are correlated with increased insulin action specifically in the liver in TLR2-deficient mice, reflected by increased insulin-stimulated protein kinase B (Akt) phosphorylation and IRS1 tyrosine phosphorylation and increased insulin-suppressed hepatocyte glucose production.Conclusions/interpretationThe absence of TLR2 attenuates local inflammatory cytokine expression and related signalling and increases insulin action specifically in the liver. Thus, our work has identified TLR2 as a key mediator of hepatic inflammation-related signalling and insulin resistance.

Dominant-Negative Loss of PPARγ Function Enhances Smooth Muscle Cell Proliferation, Migration, and Vascular Remodeling

Objective—The peroxisome proliferator activated receptor-gamma (PPAR&ggr;) protein is a nuclear transcriptional activator with importance in diabetes management as the molecular target for the thiazolidinedione (TZD) family of drugs. Substantial evidence indicates that the TZD family of PPAR&ggr; agonists may retard the development of atherosclerosis. However, recent clinical data have suggested that at least one TZD may increase the risk of myocardial infarction and death from cardiovascular disease. In this study, we used a genetic approach to disrupt PPAR&ggr; signaling to probe the proteins role in smooth muscle cell (SMC) responses that are important for atherosclerosis. Methods and Results—SMC isolated from transgenic mice harboring the dominate-negative P465L mutation in PPAR&ggr; (PPAR&ggr;L/+) exhibited greater proliferation and migration then did wild-type cells. Upregulation of ETS-1, but not ERK activation, correlated with enhanced proliferative and migratory responses PPAR&ggr;L/+ SMCs. After arterial injury, PPAR&ggr;L/+ mice had a ≈4.3-fold increase in the development of intimal hyperplasia. Conclusion—These findings are consistent with a normal role for PPAR&ggr; in inhibiting SMC migration and proliferation in the context of restenosis or atherosclerosis.

Overweight worsens apoptosis, neuroinflammation and blood-brain barrier damage after hypoxic ischemia in neonatal brain through JNK hyperactivation.

BackgroundApoptosis, neuroinflammation and blood-brain barrier (BBB) damage affect the susceptibility of the developing brain to hypoxic-ischemic (HI) insults. c-Jun N-terminal kinase (JNK) is an important mediator of insulin resistance in obesity. We hypothesized that neonatal overweight aggravates HI brain damage through JNK hyperactivation-mediated upregulation of neuronal apoptosis, neuroinflammation and BBB leakage in rat pups.MethodsOverweight (OF) pups were established by reducing the litter size to 6, and control (NF) pups by keeping the litter size at 12 from postnatal (P) day 1 before HI on P7. Immunohistochemistry and immunoblotting were used to determine the TUNEL-(+) cells and BBB damage, cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP), and phospho-JNK and phospho-BimEL levels. Immunofluorescence was performed to determine the cellular distribution of phospho-JNK.ResultsCompared with NF pups, OF pups had a significantly heavier body-weight and greater fat deposition on P7. Compared with the NF-HI group, the OF-HI group showed significant increases of TUNEL-(+) cells, cleaved levels of caspase-3 and PARP, and ED1-(+) activated microglia and BBB damage in the cortex 24 hours post-HI. Immunofluorescence of the OF-HI pups showed that activated-caspase 3 expression was found mainly in NeuN-(+) neurons and RECA1-(+) vascular endothelial cells 24 hours post-HI. The OF-HI group also had prolonged escape latency in the Morris water maze test and greater brain-volume loss compared with the NF-HI group when assessed at adulthood. Phospho-JNK and phospho-BimEL levels were higher in OF-HI pups than in NF-HI pups immediately post-HI. JNK activation in OF-HI pups was mainly expressed in neurons, microglia and vascular endothelial cells. Inhibiting JNK activity by AS601245 caused more attenuation of cleaved caspase-3 and PARP, a greater reduction of microglial activation and BBB damage post-HI, and significantly reduced brain damage in OF-HI than in NF-HI pups.ConclusionsNeonatal overweight increased HI-induced neuronal apoptosis, microglial activation and BBB damage, and aggravated HI brain damage in rat pups through JNK hyperactivation.

Increased Aortic Stiffness and Attenuated Lysyl Oxidase Activity in Obesity

Objective—One potential mechanism through which obesity exerts adverse effects on the vascular system is by increasing aortic stiffness, a change known to be predictive of increased cardiovascular mortality. The aim of this study was to investigate the pathophysiology that links obesity to aortic stiffening. Approach and Results—Obese (ob/ob) mice were used to examine physical, morphological, and molecular changes in the aorta in response to obesity. ob/ob mice had increased aortic pulse wave velocity and tissue rigidity. ob/ob aorta exhibited decreases of lysyl oxidase (LOX) activity and cross-linked elastin, and increases of elastin fragmentation and elastolytic activity. The aortas of ob/ob mice were surrounded by a significant amount of proinflammatory and pro-oxidative perivascular adipose tissue. In vitro studies revealed that the conditioned medium from differentiated adipocytes or the perivascular adipose tissue of ob/ob mice attenuated LOX activity. Furthermore, inhibition of LOX in wild-type lean mice caused elastin fragmentation and induced a significant increase in pulse wave velocity. Finally, we found that obese humans had stiffer arteries and lower serum LOX levels than do normal-weight humans. Conclusion—Our results demonstrated that obesity resulted in aortic stiffening in both humans and mice, and established a causal relationship between LOX downregulation and aortic stiffening in obesity.

Smoking, habitual tea drinking and metabolic syndrome in elderly men living in rural community: the Tianliao old people (TOP) study 02.

The literature shows an inconsistent relationship between lifestyle behaviors and metabolic syndrome (MetS), especially in the elderly. We designed this study to investigate the interrelationships among cigarette smoking, tea drinking and MetS, and to verify the factors associated with MetS in elderly males dwelling in rural community. In July 2010, with a whole community sampling method, 414 male subjects aged over 65 dwelling in Tianliao township were randomly sampled. The response rate was 60.8%. Each subject completed the structured questionnaires including sociodemographic characteristics, habitual behaviors (including cigarette smoking and tea drinking habits) and medical history. After an overnight fast, the laboratory and anthropometric data were obtained. MetS was confirmed according to the criteria defined by the modified NCEP ATP III for the male Chinese population. Subjects were split into either non-MetS or MetS groups for further analysis. Of the 361 subjects with complete data, 132 (36.6%) elderly men were classified as having MetS. Using binary logistic regression, body mass index, serum uric acid, high sensitivity C-reactive protein, HOMA index, current smokers (OR = 2.72, 95%CI: 1.03 ∼ 7.19), total smoking amount > = 30 (OR = 2.78, 95%CI: 1.31 ∼ 5.90) and more than 20 cigarettes daily (OR = 2.54, 95%CI: 1.24 ∼ 5.18) were positively associated with MetS. Current un- or partial fermented tea drinker (OR = 0.42, 95%CI: 0.22 ∼ 0.84), tea drinking habit for 1–9 years (OR = 0.36, 95%CI: 0.15 ∼ 0.90) and more than 240cc daily (OR = 0.35, 95%CI: 0.17 ∼ 0.72) were negatively associated with MetS. In conclusion, this study suggests that smoking habit was positively associated with MetS, but tea drinking habit was negatively associated with MetS in elderly men dwelling in rural community.