🌟 Ella Li, Ph.D. 👑

Skeletal muscle insulin resistance is a prominent early feature in the pathogenesis of type 2 diabetes (T2D). In attempt to overcome this defect, we generated mice overexpressing insulin receptors (IR) specifically in skeletal muscle (IRMOE). On normal chow, IRMOE mice have similar body weight as controls, but an increase in lean mass and glycolytic muscle fibers and reduced fat mass. IRMOE mice also show higher basal phosphorylation of IR, IRS-1 and Akt in muscle and improved glucose tolerance… Show more

Skeletal muscle insulin resistance is a prominent early feature in the pathogenesis of type 2 diabetes (T2D). In attempt to overcome this defect, we generated mice overexpressing insulin receptors (IR) specifically in skeletal muscle (IRMOE). On normal chow, IRMOE mice have similar body weight as controls, but an increase in lean mass and glycolytic muscle fibers and reduced fat mass. IRMOE mice also show higher basal phosphorylation of IR, IRS-1 and Akt in muscle and improved glucose tolerance compared to controls. When challenged with high fat diet (HFD), IRMOE mice are protected from diet-induced obesity. This is associated with reduced inflammation in fat and liver, improved glucose tolerance and improved systemic insulin sensitivity. Surprisingly, however, in both chow and HFD-fed mice, insulin stimulated Akt phosphorylation is significantly reduced in muscle of IRMOE mice, indicating post-receptor insulin resistance. RNA sequencing reveals downregulation of several post-receptor signaling proteins that contribute to this resistance. Thus, enhancing early insulin signaling in muscle by overexpression of the insulin receptor protects mice from diet-induced obesity and its effects on glucose metabolism. However, chronic overstimulation of this pathway leads to post-receptor desensitization, indicating the critical balance between normal signaling and hyperstimulation of the insulin signaling pathway. Show less

Nuclear factor (NF)κB is a transcription factor that controls immune and inflammatory signaling pathways. In skeletal muscle, NFκB has been implicated in the regulation of metabolic processes and tissue mass, yet its affects on mitochondrial function in this tissue are unclear. To investigate the role of NFκB on mitochondrial function and its relationship with muscle mass across the life span, we study a mouse model with muscle-specific NFκB suppression (muscle-specific IκBα super-repressor… Show more

Nuclear factor (NF)κB is a transcription factor that controls immune and inflammatory signaling pathways. In skeletal muscle, NFκB has been implicated in the regulation of metabolic processes and tissue mass, yet its affects on mitochondrial function in this tissue are unclear. To investigate the role of NFκB on mitochondrial function and its relationship with muscle mass across the life span, we study a mouse model with muscle-specific NFκB suppression (muscle-specific IκBα super-repressor [MISR] mice). In wild-type mice, there was a natural decline in muscle mass with aging that was accompanied by decreased mitochondrial function and mRNA expression of electron transport chain subunits. NFκB inactivation downregulated expression of PPARGC1A, and upregulated TFEB and PPARGC1B. NFκB inactivation also decreased gastrocnemius (but not soleus) muscle mass in early life (1–6 months old). Lower oxygen consumption rates occurred in gastrocnemius and soleus muscles from young MISR mice, whereas soleus (but not gastrocnemius) muscles from old MISR mice displayed increased oxygen consumption compared to age-matched controls. We conclude that the NFκB pathway plays an important role in muscle development and growth. The extent to which NFκB suppression alters mitochondrial function is age dependent and muscle specific. Finally, mitochondrial function and muscle mass are tightly associated in both genotypes and across the life span. Show less

Skeletal muscle insulin resistance, decreased phosphatidylinositol 3-kinase (PI3K) activation and altered mitochondrial function are hallmarks of type 2 diabetes. To determine the relationship between these abnormalities, we created mice with muscle-specific knockout of the p110α or p110β catalytic subunits of PI3K. We find that mice with muscle-specific knockout of p110α, but not p110β, display impaired insulin signaling and reduced muscle size due to enhanced proteasomal and autophagic… Show more

Skeletal muscle insulin resistance, decreased phosphatidylinositol 3-kinase (PI3K) activation and altered mitochondrial function are hallmarks of type 2 diabetes. To determine the relationship between these abnormalities, we created mice with muscle-specific knockout of the p110α or p110β catalytic subunits of PI3K. We find that mice with muscle-specific knockout of p110α, but not p110β, display impaired insulin signaling and reduced muscle size due to enhanced proteasomal and autophagic activity. Despite insulin resistance and muscle atrophy, M-p110αKO mice show decreased serum myostatin, increased mitochondrial mass, increased mitochondrial fusion, and increased PGC1α expression, especially PCG1α2 and PCG1α3. This leads to enhanced mitochondrial oxidative capacity, increased muscle NADH content, and higher muscle free radical release measured in vivo using pMitoTimer reporter. Thus, p110α is the dominant catalytic isoform of PI3K in muscle in control of insulin sensitivity and muscle mass, and has a unique role in mitochondrial homeostasis in skeletal muscle. Show less

Insulin deficiency and uncontrolled diabetes lead to a catabolic state with decreased muscle strength, contributing to disease-related morbidity. FoxO transcription factors are suppressed by insulin and thus are key mediators of insulin action. To study their role in diabetic muscle wasting, we created mice with muscle-specific triple knockout of FoxO1/3/4 and induced diabetes in these M-FoxO-TKO mice with streptozotocin (STZ). Muscle mass and myofiber area were decreased 20–30% in STZ-Diabetes… Show more

Insulin deficiency and uncontrolled diabetes lead to a catabolic state with decreased muscle strength, contributing to disease-related morbidity. FoxO transcription factors are suppressed by insulin and thus are key mediators of insulin action. To study their role in diabetic muscle wasting, we created mice with muscle-specific triple knockout of FoxO1/3/4 and induced diabetes in these M-FoxO-TKO mice with streptozotocin (STZ). Muscle mass and myofiber area were decreased 20–30% in STZ-Diabetes mice due to increased ubiquitin-proteasome degradation and autophagy alterations, characterized by increased LC3-containing vesicles, and elevated levels of phosphorylated ULK1 and LC3-II. Both the muscle loss and markers of increased degradation/autophagy were completely prevented in STZ FoxO-TKO mice. Transcriptomic analyses revealed FoxO-dependent increases in ubiquitin-mediated proteolysis pathways in STZ-Diabetes, including regulation of Fbxo32 (Atrogin1), Trim63 (MuRF1), Bnip3L, and Gabarapl. These same genes were increased 1.4- to 3.3-fold in muscle from humans with type 1 diabetes after short-term insulin deprivation. Thus, FoxO-regulated genes play a rate-limiting role in increased protein degradation and muscle atrophy in insulin-deficient diabetes. Show less

Brown adipose tissue (BAT) is rich in mitochondria and plays important roles in energy expenditure, thermogenesis, and glucose homeostasis. We find that levels of mitochondrial protein succinylation and malonylation are high in BAT and subject to physiological and genetic regulation. BAT-specific deletion of Sirt5, a mitochondrial desuccinylase and demalonylase, results in dramatic increases in global protein succinylation and malonylation. Mass spectrometry-based quantification of… Show more

Brown adipose tissue (BAT) is rich in mitochondria and plays important roles in energy expenditure, thermogenesis, and glucose homeostasis. We find that levels of mitochondrial protein succinylation and malonylation are high in BAT and subject to physiological and genetic regulation. BAT-specific deletion of Sirt5, a mitochondrial desuccinylase and demalonylase, results in dramatic increases in global protein succinylation and malonylation. Mass spectrometry-based quantification of succinylation reveals that Sirt5 regulates the key thermogenic protein in BAT, UCP1. Mutation of the two succinylated lysines in UCP1 to acyl-mimetic glutamine and glutamic acid significantly decreases its stability and activity. The reduced function of UCP1 and other proteins in Sirt5KO BAT results in impaired mitochondria respiration, defective mitophagy, and metabolic inflexibility. Thus, succinylation of UCP1 and other mitochondrial proteins plays an important role in BAT and in regulation of energy homeostasis.
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While the brain is now recognized as an insulin sensitive tissue, and impaired insulin signaling in brain can lead to many metabolic and behavioral abnormalities, effects of insulin on gene expression in the brain remain largely unknown. To understand now this aspect of insulin action may impact physiology in vivo, we performed hyperinsulinemic-euglycemic clamps at low (4 mU/kg/min) and high (12 mU/kg/min) physiological doses of insulin in C57BL/6 mice, and after 3 hours collected different… Show more

While the brain is now recognized as an insulin sensitive tissue, and impaired insulin signaling in brain can lead to many metabolic and behavioral abnormalities, effects of insulin on gene expression in the brain remain largely unknown. To understand now this aspect of insulin action may impact physiology in vivo, we performed hyperinsulinemic-euglycemic clamps at low (4 mU/kg/min) and high (12 mU/kg/min) physiological doses of insulin in C57BL/6 mice, and after 3 hours collected different brain regions and analyzed their transcriptomes using RNA Seq. In response to low dose insulin infusion, 1851 genes were up- or down-regulated in the hypothalamus by at least 50% (all P < 0.01). This was more than 4-fold as the number of genes regulated in liver or muscle under the same conditions. Other regions showed different responses with 2significantly regulated by at least 50% in hippocampus and 131 regulated in nucleus accumbens. The most upregulated pathway in all three regions was that for very long chain fatty acyl-CoA synthesis, indicating an important role of insulin action on brain fatty acid metabolism. Most other insulin responsive genes in these three brain regions were distinct. In the hypothalamus, insulin played a key role in modulating genes involved in neurotransmission in the hypothalamus, including enhancing the GABA-A receptor signaling pathway and suppressing neuropeptide signaling pathways. Insulin also modulated metabolism in the hypothalamus by suppressing the glycolysis and pentose phosphate pathways, while increasing the pyruvate dehydrogenase complex and cholesterol biosynthesis. Thus, insulin action in the brain acutely and potently regulates expression of genes involved in brain metabolism, neurotransmission and neuromodulation. In this way, insulin re-routes the carbon source to the biogenesis of plasma membrane for neuronal and glial function and synaptic remodeling. Show less

Complications of diabetes affect tissues throughout the body, including the central nervous system. Epidemiological studies show that diabetic patients have an increased risk of depression, anxiety, age-related cognitive decline, and Alzheimer’s disease. Mice lacking insulin receptor (IR) in the brain or on hypothalamic neurons display an array of metabolic abnormalities; however, the role of insulin action on astrocytes and neurobehaviors remains less well studied. Here, we demonstrate that… Show more

Complications of diabetes affect tissues throughout the body, including the central nervous system. Epidemiological studies show that diabetic patients have an increased risk of depression, anxiety, age-related cognitive decline, and Alzheimer’s disease. Mice lacking insulin receptor (IR) in the brain or on hypothalamic neurons display an array of metabolic abnormalities; however, the role of insulin action on astrocytes and neurobehaviors remains less well studied. Here, we demonstrate that astrocytes are a direct insulin target in the brain and that knockout of IR on astrocytes causes increased anxiety- and depressive-like behaviors in mice. This can be reproduced in part by deletion of IR on astrocytes in the nucleus accumbens. At a molecular level, loss of insulin signaling in astrocytes impaired tyrosine phosphorylation of Munc18c. This led to decreased exocytosis of ATP from astrocytes, resulting in decreased purinergic signaling on dopaminergic neurons. These reductions contributed to decreased dopamine release from brain slices. Central administration of ATP analogs could reverse depressive-like behaviors in mice with astrocyte IR knockout. Thus, astrocytic insulin signaling plays an important role in dopaminergic signaling, providing a potential mechanism by which astrocytic insulin action may contribute to increased rates of depression in people with diabetes, obesity, and other insulin-resistant states. Show less

NFκB is a transcription factor that controls immune and inflammatory processes. In muscle, NFκB has been implicated in regulation of muscle mass/function, metabolism, and mitochondrial function. Yet, NFκB’s role in aging-related changes to skeletal muscle and heart remains less defined. To investigate the role of NFκB in aging skeletal muscle and heart, we study a mouse model (MISR mice) with suppressed NFκB signaling in these tissues. Recently we found that these mice have exacerbated loss of… Show more

NFκB is a transcription factor that controls immune and inflammatory processes. In muscle, NFκB has been implicated in regulation of muscle mass/function, metabolism, and mitochondrial function. Yet, NFκB’s role in aging-related changes to skeletal muscle and heart remains less defined. To investigate the role of NFκB in aging skeletal muscle and heart, we study a mouse model (MISR mice) with suppressed NFκB signaling in these tissues. Recently we found that these mice have exacerbated loss of skeletal muscle mass during aging. Here, we report that NFκB suppression caused reductions in skeletal muscle mass as early as 1 month of age. Aging per se (in WT mice) lead to a global downregulation of mitochondrial function (reduced respiration and enhanced reactive oxygen species production) and expression of mitochondrial biogenesis genes in skeletal muscle. Furthermore, suppression of NFκB signaling in skeletal muscle resulted in mitochondrial function defects in early life (4 months old), whereas in aged mice (30-33 months old) it had the opposite effect. Contrary to findings in skeletal muscle, in the heart aging increased mitochondrial respiration and reactive oxygen species production (in WT mice) and NFκB suppression protected against the aging related changes to reactive oxygen species production. We conclude that suppression of the canonical NFκB pathway has antagonistic pleiotropic effects (detrimental in early life and advantageous in aging animals) on mitochondrial function in skeletal muscle, but is beneficial throughout the lifespan in the heart. Furthermore, canonical NFκB signaling regulates muscle development and cellular metabolism at the transcriptional and functional levels. Show less

Insulin and IGF1 signaling are important for adipose tissue development and function; however, their role in mature adipocytes is unclear. Mice with a tamoxifen-inducible knockout of insulin and/or IGF1 receptors (IR/IGF1R) demonstrate a rapid loss of white and brown fat due to increased lipolysis and adipocyte apoptosis. This results in insulin resistance, glucose intolerance, hepatosteatosis, islet hyperplasia with hyperinsulinemia, and cold intolerance. This phenotype, however, resolves over… Show more

Insulin and IGF1 signaling are important for adipose tissue development and function; however, their role in mature adipocytes is unclear. Mice with a tamoxifen-inducible knockout of insulin and/or IGF1 receptors (IR/IGF1R) demonstrate a rapid loss of white and brown fat due to increased lipolysis and adipocyte apoptosis. This results in insulin resistance, glucose intolerance, hepatosteatosis, islet hyperplasia with hyperinsulinemia, and cold intolerance. This phenotype, however, resolves over 10–30 days due to a proliferation of preadipocytes and rapid regeneration of both brown and white adipocytes as identified by mTmG lineage tracing. This cycle can be repeated with a second round of receptor inactivation. Leptin administration prior to tamoxifen treatment blocks development of the metabolic syndrome without affecting adipocyte loss or regeneration. Thus, IR is critical in adipocyte maintenance, and this loss of adipose tissue stimulates regeneration of brown/white fat and reversal of metabolic syndrome associated with fat loss. Show less

Circulating hormones must cross the vascular endothelium to elicit their actions in target tissues via either transcytosis or paracellular diffusion. Insulin receptors on endothelial cells are believed to mediate transcytosis of circulating insulin, but how this affects insulin action in vivo is unknown. Here, we demonstrate that knockout of insulin receptors on endothelial cells delays the kinetics of activation of insulin signaling in skeletal muscle, fat, and several regions of the brain but… Show more

Circulating hormones must cross the vascular endothelium to elicit their actions in target tissues via either transcytosis or paracellular diffusion. Insulin receptors on endothelial cells are believed to mediate transcytosis of circulating insulin, but how this affects insulin action in vivo is unknown. Here, we demonstrate that knockout of insulin receptors on endothelial cells delays the kinetics of activation of insulin signaling in skeletal muscle, fat, and several regions of the brain but not in liver or olfactory bulb. This alters the kinetics of insulin action in vivo and induces tissue-specific insulin resistance leading to dysregulated glucose and body weight homeostasis. Show less

Older adults universally suffer from sarcopenia and approximately 60–70% are diabetic or prediabetic. Nonetheless, the mechanisms underlying these aging‐related metabolic disorders are unknown. NFκB has been implicated in the pathogenesis of several aging‐related pathologies including sarcopenia and type 2 diabetes and has been proposed as a target against them. NFκB also is thought to mediate muscle wasting seen with disuse, denervation, and some systemic diseases (e.g., cancer, sepsis). We… Show more

Older adults universally suffer from sarcopenia and approximately 60–70% are diabetic or prediabetic. Nonetheless, the mechanisms underlying these aging‐related metabolic disorders are unknown. NFκB has been implicated in the pathogenesis of several aging‐related pathologies including sarcopenia and type 2 diabetes and has been proposed as a target against them. NFκB also is thought to mediate muscle wasting seen with disuse, denervation, and some systemic diseases (e.g., cancer, sepsis). We tested the hypothesis that lifelong inhibition of the classical NFκB pathway would protect against aging‐related sarcopenia and insulin resistance. Aged mice with muscle‐specific overexpression of a super‐repressor IκBα mutant (MISR) were protected from insulin resistance. However, MISR mice were not protected from sarcopenia; to the contrary, these mice had decreases in muscle mass and strength compared to wild‐type mice. In MISR mice, NFκB suppression also led to an increase in proteasome activity and alterations in several genes and pathways involved in muscle growth and atrophy (e.g., myostatin). We conclude that the mechanism behind aging‐induced sarcopenia is NFκB independent and differs from muscle wasting due to pathologic conditions. Our findings also indicate that, while suppressing NFκB improves insulin sensitivity in aged mice, this transcription factor is important for normal muscle mass maintenance and its sustained inhibition is detrimental to muscle function. Show less

Protein kinase C (PKC)δ has been shown to be increased in liver in obesity and plays an important role in the development of hepatic insulin resistance in both mice and humans. In the current study, we explored the role of PKCδ in skeletal muscle in the control of insulin sensitivity and glucose metabolism by generating mice in which PKCδ was deleted specifically in muscle using Cre-lox recombination. Deletion of PKCδ in muscle improved insulin signaling in young mice, especially at low insulin… Show more

Protein kinase C (PKC)δ has been shown to be increased in liver in obesity and plays an important role in the development of hepatic insulin resistance in both mice and humans. In the current study, we explored the role of PKCδ in skeletal muscle in the control of insulin sensitivity and glucose metabolism by generating mice in which PKCδ was deleted specifically in muscle using Cre-lox recombination. Deletion of PKCδ in muscle improved insulin signaling in young mice, especially at low insulin doses; however, this did not change glucose tolerance or insulin tolerance tests done with pharmacological levels of insulin. Likewise, in young mice, muscle-specific deletion of PKCδ did not rescue high-fat diet–induced insulin resistance or glucose intolerance. However, with an increase in age, PKCδ levels in muscle increased, and by 6 to 7 months of age, muscle-specific deletion of PKCδ improved whole-body insulin sensitivity and muscle insulin resistance and by 15 months of age improved the age-related decline in whole-body glucose tolerance. At 15 months of age, M-PKCδKO mice also exhibited decreased metabolic rate and lower levels of some proteins of the OXPHOS complex suggesting a role for PKCδ in the regulation of mitochondrial mass at older age. These data indicate an important role of PKCδ in the regulation of insulin sensitivity and mitochondrial homeostasis in skeletal muscle with aging. Show less

Oxidative stress has been associated with insulin resistance and type 2 diabetes. However, it is not clear whether oxidative damage is a cause or a consequence of the metabolic abnormalities present in diabetic subjects. The goal of this study was to determine whether inducing oxidative damage through genetic ablation of superoxide dismutase 1 (SOD1) leads to abnormalities in glucose homeostasis. We studied SOD1-null mice and wild-type (WT) littermates. Glucose tolerance was evaluated with… Show more

Oxidative stress has been associated with insulin resistance and type 2 diabetes. However, it is not clear whether oxidative damage is a cause or a consequence of the metabolic abnormalities present in diabetic subjects. The goal of this study was to determine whether inducing oxidative damage through genetic ablation of superoxide dismutase 1 (SOD1) leads to abnormalities in glucose homeostasis. We studied SOD1-null mice and wild-type (WT) littermates. Glucose tolerance was evaluated with intraperitoneal glucose tolerance tests. Peripheral and hepatic insulin sensitivity was quantitated with the euglycemic-hyperinsulinemic clamp. β-Cell function was determined with the hyperglycemic clamp and morphometric analysis of pancreatic islets. Genetic ablation of SOD1 caused glucose intolerance, which was associated with reduced in vivo β-cell insulin secretion and decreased β-cell volume. Peripheral and hepatic insulin sensitivity were not significantly altered in SOD1-null mice. High-fat diet caused glucose intolerance in WT mice but did not further worsen the glucose intolerance observed in standard chow–fed SOD1-null mice. Our findings suggest that oxidative stress per se does not play a major role in the pathogenesis of insulin resistance and demonstrate that oxidative stress caused by SOD1 ablation leads to glucose intolerance secondary to β-cell dysfunction. Show less

AMP-activated protein kinase (AMPK) is a key energy-sensitive enzyme that controls numerous metabolic and cellular processes. Mammalian target of rapamycin (mTOR) is another energy/nutrient-sensitive kinase that controls protein synthesis and cell growth. In this study we determined whether older versus younger men have alterations in the AMPK and mTOR pathways in skeletal muscle, and examined the effect of a long term resistance type exercise training program on these signaling intermediaries.… Show more

AMP-activated protein kinase (AMPK) is a key energy-sensitive enzyme that controls numerous metabolic and cellular processes. Mammalian target of rapamycin (mTOR) is another energy/nutrient-sensitive kinase that controls protein synthesis and cell growth. In this study we determined whether older versus younger men have alterations in the AMPK and mTOR pathways in skeletal muscle, and examined the effect of a long term resistance type exercise training program on these signaling intermediaries. Older men had decreased AMPKα2 activity and lower phosphorylation of AMPK and its downstream signaling substrate acetyl-CoA carboxylase (ACC). mTOR phosphylation also was reduced in muscle from older men. Exercise training increased AMPKα1 activity in older men, however, AMPKα2 activity, and the phosphorylation of AMPK, ACC and mTOR, were not affected. In conclusion, older men have alterations in the AMPK-ACC and mTOR pathways in muscle. In addition, prolonged resistance type exercise training induces an isoform-selective up regulation of AMPK activity. Show less

Effect of Aging and Resistance Exercise on AMPK Activity and Signaling in Human Muscle Aging is associated with an in Aging is associated with an increased incidence of type 2 diabetes (T2D). Alterations in glucose, lipid, and protein metabolism are thought to be responsible for the higher risk of T2D in the elderly. AMP-activated protein kinase (AMPK) is an energy/nutrient-sensitive Ser/Thr kinase whose activation enhances lipid oxidation in muscle by phosphorylating acetyl-CoA carboxylase… Show more

Effect of Aging and Resistance Exercise on AMPK Activity and Signaling in Human Muscle Aging is associated with an in Aging is associated with an increased incidence of type 2 diabetes (T2D). Alterations in glucose, lipid, and protein metabolism are thought to be responsible for the higher risk of T2D in the elderly. AMP-activated protein kinase (AMPK) is an energy/nutrient-sensitive Ser/Thr kinase whose activation enhances lipid oxidation in muscle by phosphorylating acetyl-CoA carboxylase (ACC) and promotes glucose uptake by increasing GLUT4 translocation through phosphorylation of TBC1D1/4. mTOR is another energy/nutrient-sensitive Ser/Thr kinase that enhances protein synthesis. Animal studies suggest aging leads to altered signaling through AMPK-ACC and mTOR in muscle, which have been implicated in the metabolic abnormalities with aging. Our goals were to determine whether: (1) aging leads to a downregulation of the AMPK-ACC and mTOR pathways in human muscle; and (2) resistance exercise can restore aging-related alterations in AMPK-ACC and mTOR signaling. Measurements were performed in vastus lateralis muscle from 32 healthy, elderly, nondiabetic, community-dwelling subjects and 32 healthy, younger, nondiabetic subjects . In older subjects, the muscle samples were obtained before and after 3 months of resistance training. RESULTS: Aging caused significant decreases in AMPK[alpha]2 activity, and in the phosphorylation of AMPK-mTOR Axis. AMPK[alpha]1 activity and the protein content of LKB1, an AMPK upstream kinase, were similar between groups. Resistance exercise caused an increase in AMPK[alpha]1 activity in older subjects by 1.4-fold (P[lt]0.05). However, AMPK[alpha]2 activity, LKB1 protein content, and the phosphorylation of AMPK, ACC, and mTOR were not affected by resistance training. SUMMARY: (i) alterations in the AMPK-ACC and mTOR pathways could explain some changes in glucose, lipid, and protein metabolism that occur with aging; and (ii) resistance... Show less

NF-κB is a transcription factor that controls the gene expression of several proinflammatory proteins. Cell culture and animal studies have implicated increased NF-κB activity in the pathogenesis of insulin resistance and muscle atrophy. However, it is unclear whether insulin-resistant human subjects have abnormal NF-κB activity in muscle. The effect that exercise has on NF-κB activity/signaling also is not clear. We measured NF-κB DNA-binding activity and the mRNA level of putative… Show more

NF-κB is a transcription factor that controls the gene expression of several proinflammatory proteins. Cell culture and animal studies have implicated increased NF-κB activity in the pathogenesis of insulin resistance and muscle atrophy. However, it is unclear whether insulin-resistant human subjects have abnormal NF-κB activity in muscle. The effect that exercise has on NF-κB activity/signaling also is not clear. We measured NF-κB DNA-binding activity and the mRNA level of putative NF-κB-regulated myokines interleukin (IL)-6 and monocyte chemotactic protein-1 (MCP-1) in muscle samples from T2DM, obese, and lean subjects immediately before, during (40 min), and after (210 min) a bout of moderate-intensity cycle exercise. At baseline, NF-κB activity was elevated 2.1- and 2.7-fold in obese nondiabetic and T2DM subjects, respectively. NF-κB activity was increased significantly at 210 min following exercise in lean (1.9-fold) and obese (2.6-fold) subjects, but NF-κB activity did not change in T2DM. Exercise increased MCP-1 mRNA levels significantly in the three groups, whereas IL-6 gene expression increased significantly only in lean and obese subjects. MCP-1 and IL-6 gene expression peaked at the 40-min exercise time point. We conclude that insulin-resistant subjects have increased basal NF-κB activity in muscle. Acute exercise stimulates NF-κB in muscle from nondiabetic subjects. In T2DM subjects, exercise had no effect on NF-κB activity, which could be explained by the already elevated NF-κB activity at baseline. Exercise-induced MCP-1 and IL-6 gene expression precedes increases in NF-κB activity, suggesting that other factors promote gene expression of these cytokines during exercise. Show less

Effect of Diabetes and Exercise on Gene Expression of Ubiquitin Ligases and Proteasomal Subunits in Human Muscle Diabetes has been associated Diabetes has been associated with muscle wasting. Atrogin and MuRF1 are ubiquitin ligases which promote breakdown of proteins by the proteasome system. The expression and function of atrogin and MuRF1 is upregulated in several animal models of muscle wasting, including diabetes, cancer, sepsis and disuse, suggesting a role for these ligases in muscle… Show more

Effect of Diabetes and Exercise on Gene Expression of Ubiquitin Ligases and Proteasomal Subunits in Human Muscle Diabetes has been associated Diabetes has been associated with muscle wasting. Atrogin and MuRF1 are ubiquitin ligases which promote breakdown of proteins by the proteasome system. The expression and function of atrogin and MuRF1 is upregulated in several animal models of muscle wasting, including diabetes, cancer, sepsis and disuse, suggesting a role for these ligases in muscle loss. Our aims were to examine: (1) whether the gene expression of atrogin and MuRF1 is increased in muscle from insulin resistant [obese and type 2 diabetic (T2D)] subjects; and (2) whether exercise downregulates atrogin and MuRF1 gene expression. A vastus lateralis muscle biopsy was performed in 11 lean, and 9 obese T2D (BMI=34.1±0.6; age=51±4, FPG=133±14, VO2max=15±1) before and after a short-term training program. The training program consisted of 14 consecutive days of cycle exercise for 40 min/day at 70% to 90% VO2max. Training increased VO2max by 19%, 17%, and 14% in lean, obese, and T2D subjects, respectively. In T2D subjects, atrogin and MuRF1 mRNA expression tended to be elevated by 22% and 31% (P=0.2), respectively. Training did not affect atrogin expression in lean and obese subjects; training decreased atrogin mRNA levels in the T2D group by 31% (P<0.05). Furthermore, exercise training decreased MuRF1 mRNA expression by 25% and 54% in muscle from lean and T2D subjects, respectively (P<0.05 in both groups). In summary: (1) atrogin and MuRF1 mRNA levels were not significantly elevated in muscle from obese and T2D subjects; and (2) short term exercise training decreases atrogin (T2D) and MuRF1 (lean and T2D) gene expression. The ability of training to decrease atrogin and MuRF1 expression may be a mechanism by which physical activity helps to prevent protein breakdown in conditions of muscle wasting. Show less

The molecular mechanisms by which thiazolidinediones improve insulin sensitivity in type 2 diabetes are not fully understood. We hypothesised that pioglitazone would activate the adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway and increase the expression of genes involved in adiponectin signalling, NEFA oxidation and mitochondrial function in human skeletal muscle.

The molecular mechanisms by which thiazolidinediones improve insulin sensitivity in type 2 diabetes are not fully understood. We hypothesised that pioglitazone would activate the adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway and increase the expression of genes involved in adiponectin signalling, NEFA oxidation and mitochondrial function in human skeletal muscle.
Methods: a randomised, double-blind, parallel study was performed in 26 drug-naive type 2 diabetes patients… Show more

The molecular mechanisms by which thiazolidinediones improve insulin sensitivity in type 2 diabetes are not fully understood. We hypothesised that pioglitazone would activate the adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway and increase the expression of genes involved in adiponectin signalling, NEFA oxidation and mitochondrial function in human skeletal muscle.
Methods: a randomised, double-blind, parallel study was performed in 26 drug-naive type 2 diabetes patients treated with: (1) pioglitazone (n=14) or (2) aggressive nutritional therapy (n=12) to reduce HbA1c to levels observed in the pioglitazone-treated group. Participants were assigned randomly to treatment using a table of random numbers. Before and after 6 months, patients reported to the Clinical Research Center of the Texas Diabetes Institute for a vastus lateralis muscle biopsy followed by a 180 min euglycaemic–hyperinsulinaemic (80 mU m−2 min−1) clamp. Conclusion: Pioglitazone increases plasma adiponectin levels, stimulates muscle AMPK signalling and increases the expression of genes involved in adiponectin signalling, mitochondrial function and fat oxidation. These changes may represent an important cellular mechanism by which thiazolidinediones improve skeletal muscle insulin sensitivity. Show less

S-Adenosylhomocysteine hydrolase (SAHH) regulates biomethylation and homocysteine metabolism and thus is an attractive target in drug design studies. SAHH has been shown to be a copper binding protein in vivo; however, the structure and catalytic mechanism of SAHH exclude a role for Cu2+. In the present work, we studied the mechanism of inhibition of SAHH activity by Cu2+. The experimental results showed that Cu2+ inhibited SAHH activity in a noncompetitive manner. Binding of Cu2+ to SAHH… Show more

S-Adenosylhomocysteine hydrolase (SAHH) regulates biomethylation and homocysteine metabolism and thus is an attractive target in drug design studies. SAHH has been shown to be a copper binding protein in vivo; however, the structure and catalytic mechanism of SAHH exclude a role for Cu2+. In the present work, we studied the mechanism of inhibition of SAHH activity by Cu2+. The experimental results showed that Cu2+ inhibited SAHH activity in a noncompetitive manner. Binding of Cu2+ to SAHH resulted in the release of NAD+ cofactors, explaining the loss of the enzymatic activity of SAHH. Further investigation by an ESR probe and computational simulation suggested that Cu2+ could bind at the central channel and interrupt the subunit interactions of SAHH, resulting in a large decrease in affinity to the NAD+ cofactor. This effect of Cu2+ resembled that of enzyme mutations at the C-terminal domain or Asp244 [Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) Effects of site-directed mutagenesis on structure and function of recombinant rat liver S-adenosylhomocysteine hydrolase. Crystal structure of D244E mutant enzyme, J. Biol. Chem. 275, 32147−32156]. The mechanism of action of Cu2+ on SAHH suggested a possible regulative role for Cu2+ on the intracellular activity of SAHH. This could be helpful in understanding the biological effects of copper compounds and suggest a potential coupling mechanism between biomethylation and the redox states of cells. Show less