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1.  Elevated Muscle TLR4 Expression and Metabolic Endotoxemia in Human Aging 
Aging is associated with alterations in glucose metabolism and sarcopenia that jointly contribute to a higher risk of developing type 2 diabetes. Because aging is considered as a state of low-grade inflammation, in this study we examined whether older, healthy (lean, community-dwelling) participants have altered signaling flux through toll-like receptor 4 (TLR4), a key mediator of innate and adaptive immune responses. We also examined whether a 4-month aerobic exercise program would have an anti-inflammatory effect by reducing TLR4 expression and signaling. At baseline, muscle TLR4, nuclear factor κB p50 and nuclear factor κB p65 protein content, and c-Jun N-terminal kinase phosphorylation were significantly elevated in older versus young participants. The plasma concentration of the TLR4 agonist lipopolysaccharide and its binding protein also were significantly elevated in older participants, indicative of metabolic endotoxemia, which is a recently described phenomenon of increased plasma endotoxin level in metabolic disease. These alterations in older participants were accompanied by decreased insulin sensitivity, quadriceps muscle volume, and muscle strength. The exercise training program increased insulin sensitivity, without affecting quadriceps muscle volume or strength. Muscle TLR4, nuclear factor κB, and c-Jun N-terminal kinase, and plasma lipopolysaccharide and lipopolysaccharide binding protein were not changed by exercise. In conclusion, insulin resistance and sarcopenia of aging are associated with increased TLR4 expression/signaling, which may be secondary to metabolic endotoxemia.
PMCID: PMC4311182  PMID: 24846769
Aging; LPS; TLR4; NFκB; JNK; Sarcopenia
2.  Diabetes and Cardiovascular Disease in Older Adults: Current Status and Future Directions 
Diabetes  2014;63(8):2578-2589.
The prevalence of diabetes increases with age, driven in part by an absolute increase in incidence among adults aged 65 years and older. Individuals with diabetes are at higher risk for cardiovascular disease, and age strongly predicts cardiovascular complications. Inflammation and oxidative stress appear to play some role in the mechanisms underlying aging, diabetes, cardiovascular disease, and other complications of diabetes. However, the mechanisms underlying the age-associated increase in risk for diabetes and diabetes-related cardiovascular disease remain poorly understood. Moreover, because of the heterogeneity of the older population, a lack of understanding of the biology of aging, and inadequate study of the effects of treatments on traditional complications and geriatric conditions associated with diabetes, no consensus exists on the optimal interventions for older diabetic adults. The Association of Specialty Professors, along with the National Institute on Aging, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Heart, Lung, and Blood Institute, and the American Diabetes Association, held a workshop, summarized in this Perspective, to discuss current knowledge regarding diabetes and cardiovascular disease in older adults, identify gaps, and propose questions to guide future research.
PMCID: PMC4113072  PMID: 25060886
3.  Pharmacological TLR4 Inhibition Protects against Acute and Chronic Fat-Induced Insulin Resistance in Rats 
PLoS ONE  2015;10(7):e0132575.
To evaluate whether pharmacological TLR4 inhibition protects against acute and chronic fat-induced insulin resistance in rats.
Materials and Methods
For the acute experiment, rats received a TLR4 inhibitor [TAK-242 or E5564 (2x5 mg/kg i.v. bolus)] or vehicle, and an 8-h Intralipid (20%, 8.5 mg/kg/min) or saline infusion, followed by a two-step hyperinsulinemic-euglycemic clamp. For the chronic experiment, rats were subcutaneously implanted with a slow-release pellet of TAK-242 (1.5 mg/d) or placebo. Rats then received a high fat diet (HFD) or a low fat control diet (LFD) for 10 weeks, followed by a two-step insulin clamp.
Acute experiment; the lipid-induced reduction (18%) in insulin-stimulated glucose disposal (Rd) was attenuated by TAK-242 and E5564 (the effect of E5564 was more robust), suggesting improved peripheral insulin action. Insulin was able to suppress hepatic glucose production (HGP) in saline- but not lipid-treated rats. TAK-242, but not E5564, partially restored this effect, suggesting improved HGP. Chronic experiment; insulin-stimulated Rd was reduced ~30% by the HFD, but completely restored by TAK-242. Insulin could not suppress HGP in rats fed a HFD and TAK-242 had no effect on HGP.
Pharmacological TLR4 inhibition provides partial protection against acute and chronic fat-induced insulin resistance in vivo.
PMCID: PMC4510579  PMID: 26196892
4.  Baseline Adiponectin Levels Do Not Influence the Response to Pioglitazone in ACT NOW 
Diabetes Care  2014;37(6):1706-1711.
Plasma adiponectin levels are reduced in type 2 diabetes mellitus (T2DM) and other insulin-resistant states. We examined whether plasma adiponectin levels at baseline and after pioglitazone treatment in impaired glucose tolerance (IGT) subjects were associated with improved insulin sensitivity (SI) and glucose tolerance status.
A total of 602 high-risk IGT subjects in ACT NOW were randomized to receive pioglitazone or placebo with a median follow-up of 2.4 years.
Pioglitazone reduced IGT conversion to diabetes by 72% in association with improved β-cell function by 64% (insulin secretion/insulin resistance index) and increased tissue sensitivity by 88% (Matsuda index). In pioglitazone-treated subjects, plasma adiponectin concentration increased threefold from 13 ± 0.5 to 38 ± 2.5 μg/mL (P < 0.001) and was strongly correlated with the improvement in SI (r = 0.436, P < 0.001) and modestly correlated with glucose area under the curve during oral glucose tolerance test (r = 0.238, P < 0.005) and insulin secretion/insulin resistance index (r = 0.306, P < 0.005). The increase in adiponectin was a strong predictor of reversion to normal glucose tolerance and prevention of T2DM. In the placebo group, plasma adiponectin did not change and was not correlated with changes in glucose levels. There was an inverse association between baseline plasma adiponectin concentration and progression to diabetes in the placebo group but not in the pioglitazone group.
Baseline adiponectin does not predict the response to pioglitazone. The increase in plasma adiponectin concentration after pioglitazone therapy in IGT subjects is strongly related to improved glucose tolerance status and enhanced tissue sensitivity to insulin.
PMCID: PMC4179517  PMID: 24705615
5.  APPL1 Potentiates Insulin Sensitivity by Facilitating the Binding of IRS1/2 to the Insulin Receptor 
Cell reports  2014;7(4):1227-1238.
Binding of insulin receptor substrate proteins 1 and 2 (IRS1/2) to the insulin receptor (IR) is essential for the regulation of insulin sensitivity and energy homeostasis. However, the mechanism of IRS1/2 recruitment to the IR remains elusive. Here, we identify adaptor protein APPL1 as a critical molecule that promotes IRS1/2-IR interaction. APPL1 forms a complex with IRS1/2 under basal conditions, and this complex is then recruited to the IR in response to insulin or adiponectin stimulation. The interaction between APPL1 and IR depends on insulin- or adiponectin-stimulated APPL1 phosphorylation, which is greatly reduced in insulin target tissues in obese mice. appl1 deletion in mice consistently leads to systemic insulin resistance and a significant reduction in insulin-stimulated IRS1/2, but not IR, tyrosine phosphorylation, indicating that APPL1 sensitizes insulin signaling by acting at a site downstream of the IR. Our study uncovers a mechanism regulating insulin signaling and crosstalk between the insulin and adiponectin pathways.
PMCID: PMC4380268  PMID: 24813896
6.  Antenatal Corticosteroids Alter Insulin Signaling Pathways in Fetal Baboon Skeletal Muscle 
The Journal of endocrinology  2014;221(2):253-260.
We hypothesize that prenatal exposure to glucocorticoids (GCs) will negatively alter the insulin signal transduction pathway and has differing effects on the fetus according to gestational age at exposure.
Twenty-three fetal baboons were delivered from twenty-three healthy, non-diabetic mothers. Twelve preterm (0.67 gestational age) and eleven near term (0.95 gestational age) baboons were euthanized immediately after delivery. Half of the pregnant baboons at each gestation received two doses of intramuscular betamethasone 24-hours apart (170 μ−1) before delivery, while the other half received no intervention. Vastus lateralis muscle was obtained from postnatal animals to measure protein content and gene expression of insulin receptor (IR)-β, IR-β Tyr 1361 phosphorylation (pIR-β), IR substate-1 (IRS-1), IRS-1 tyrosine phosphorylation (pIRS-1), p85 subunit of PI3-kinase (p85), Akt (Protein Kinase B), phospho-Akt Ser473 (pAkt), Akt-1, Akt-2, and glucose transporters (GLUT1 and GLUT4).
Skeletal muscle from preterm baboons exposed to glucocorticoids had markedly reduced protein content of Akt and Akt-1 (respectively, 73% and 72% from 0.67 gestational age Control, P<0.001); IR-β and pIR-β were decreased (respectively, 94% and 85%, P<0.01) in the muscle of premature GC exposed fetuses, but not in term fetuses. GLUT1 and GLUT4 tended to increase with GC exposure in preterm animals (P=0.09), while GLUT4 increased 6.0 fold in term animals after GC exposure (P<0.05).
Exposure to a single course of antenatal GCs during fetal life alters the insulin-signaling pathway in fetal muscle in a manner dependent on the stage of gestation.
PMCID: PMC4347920  PMID: 24756099
insulin; fetus; primate; muscle; glucocorticoid; corticosteroid; preterm
7.  A sustained increase in plasma NEFA upregulates the Toll-like receptor network in human muscle 
Diabetologia  2013;57(3):582-591.
Insulin-sensitive tissues (muscle, liver) of individuals with obesity and type 2 diabetes mellitus are in a state of low-grade inflammation, characterised by increased Toll-like receptor (TLR) expression and TLR-driven signalling. However, the cause of this mild inflammatory state is unclear. We tested the hypothesis that a prolonged mild increase in plasma NEFA will increase TLR expression and TLR-driven signalling (nuclear factor κB [NFκB] and mitogen-activated kinase [MAPK]) and impair insulin action in muscle of lean healthy individuals.
Twelve lean, normal-glucose-tolerant participants were randomised to receive a 48 h infusion (30 ml/h) of saline or Intralipid followed by a euglycaemic–hyperinsulinaemic clamp. Vastus lateralis muscle biopsies were performed before and during the clamp.
Lipid infusion impaired insulin-stimulated IRS-1 tyrosine phosphorylation and reduced peripheral insulin sensitivity (p < 0.01). The elevation in circulating NEFA increased expression of TLR3, TLR4 and TLR5, and several MAPK (MAPK8, MAP4K4, MAP2K3) and inhibitor of κB kinase-NFκB (CHUK [IKKA], c-REL [REL] and p65 [RELA, NFKB3,p65]) signalling genes (p < 0.05). The lipid infusion also increased extracellular signal-regulated kinase (ERK) phosphorylation (p < 0.05) and tended to reduce the content of nuclear factor of light polypeptide gene enhancer in B cells inhibitor α (p = 0.09). The muscle content of most diacyglycerol, ceramide and acylcarnitine species was unaffected. In summary, insulin resistance induced by prolonged low-dose lipid infusion occurs together with increased TLR-driven inflammatory signalling and impaired insulin-stimulated IRS-1 tyrosine phosphorylation.
A sustained, mild elevation in plasma NEFA is sufficient to increase TLR expression and TLR-driven signalling (NFκB and MAPK) in lean individuals. The activation of this pathway by NEFA may be involved in the pathogenesis of insulin resistance in humans.
PMCID: PMC3945433  PMID: 24337154
Acylcarnitine; Ceramide; Diacylglycerol; Inflammation; Insulin resistance
8.  Genetic Disruption of SOD1 Gene Causes Glucose Intolerance and Impairs β-Cell Function 
Diabetes  2013;62(12):4201-4207.
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.
PMCID: PMC3837066  PMID: 24009256
9.  Prediction of Diabetes Based on Baseline Metabolic Characteristics in Individuals at High Risk 
Diabetes Care  2013;36(11):3607-3612.
Individuals with impaired glucose tolerance (IGT) are at high risk for developing type 2 diabetes mellitus (T2DM). We examined which characteristics at baseline predicted the development of T2DM versus maintenance of IGT or conversion to normal glucose tolerance.
We studied 228 subjects at high risk with IGT who received treatment with placebo in ACT NOW and who underwent baseline anthropometric measures and oral glucose tolerance test (OGTT) at baseline and after a mean follow-up of 2.4 years.
In a univariate analysis, 45 of 228 (19.7%) IGT individuals developed diabetes. After adjusting for age, sex, and center, increased fasting plasma glucose, 2-h plasma glucose, ∆G0–120 during OGTT, HbA1c, adipocyte insulin resistance index, ln fasting plasma insulin, and ln ∆I0–120, as well as family history of diabetes and presence of metabolic syndrome, were associated with increased risk of diabetes. At baseline, higher insulin secretion (ln [∆I0–120/∆G0–120]) during the OGTT was associated with decreased risk of diabetes. Higher β-cell function (insulin secretion/insulin resistance or disposition index; ln [∆I0–120/∆G0–120 × Matsuda index of insulin sensitivity]; odds ratio 0.11; P < 0.0001) was the variable most closely associated with reduced risk of diabetes.
In a stepwise multiple-variable analysis, only HbA1c and β-cell function (ln insulin secretion/insulin resistance index) predicted the development of diabetes (r = 0.49; P < 0.0001).
PMCID: PMC3816921  PMID: 24062330
10.  Prevention of Diabetes With Pioglitazone in ACT NOW 
Diabetes  2013;62(11):3920-3926.
We examined the metabolic characteristics that attend the development of type 2 diabetes (T2DM) in 441 impaired glucose tolerance (IGT) subjects who participated in the ACT NOW Study and had complete end-of-study metabolic measurements. Subjects were randomized to receive pioglitazone (PGZ; 45 mg/day) or placebo and were observed for a median of 2.4 years. Indices of insulin sensitivity (Matsuda index [MI]), insulin secretion (IS)/insulin resistance (IR; ΔI0–120/ΔG0–120, ΔIS rate [ISR]0–120/ΔG0–120), and β-cell function (ΔI/ΔG × MI and ΔISR/ΔG × MI) were calculated from plasma glucose, insulin, and C-peptide concentrations during oral glucose tolerance tests at baseline and study end. Diabetes developed in 45 placebo-treated vs. 15 PGZ-treated subjects (odds ratio [OR] 0.28 [95% CI 0.15–0.49]; P < 0.0001); 48% of PGZ-treated subjects reverted to normal glucose tolerance (NGT) versus 28% of placebo-treated subjects (P < 0.005). Higher final glucose tolerance status (NGT > IGT > T2DM) was associated with improvements in insulin sensitivity (OR 0.61 [95% CI 0.54–0.80]), IS (OR 0.61 [95% CI 0.50–0.75]), and β-cell function (ln IS/IR index and ln ISR/IR index) (OR 0.26 [95% CI 0.19–0.37]; all P < 0.0001). Of the factors measured, improved β-cell function was most closely associated with final glucose tolerance status.
PMCID: PMC3806596  PMID: 23863810
11.  Acute insulin resistance stimulates and insulin sensitization attenuates vascular smooth muscle cell migration and proliferation 
Physiological Reports  2014;2(8):e12123.
Differential activation/deactivation of insulin signaling, PI‐3K and MAP‐K pathways by high glucose and palmitate, with/out the insulin sensitizer pioglitazone (PIO), have been previously shown in vascular smooth muscle cells (VSMCs). To determine the biological impact of these molecular changes, we examined VSMC migration and proliferation (“M”&”P”) patterns in similar conditions. VSMCs from healthy human coronary arteries were incubated in growth medium and “M”&”P” were analyzed after exposure to high glucose (25 mmol/L) ± palmitate (200 μmol/L) and ± PIO (8 μmol/L) for 5 h. “M”&”P” were assessed by: (1) polycarbonate membrane barrier with chemo‐attractants and extended cell protrusions quantified by optical density (OD595 nm); (2) % change in radius area (2D Assay) using inverted microscopy images; and (3) cell viability assay expressed as cell absorbance (ABS) in media. “M” in 25 mmol/L glucose media increased by ~25% from baseline and % change in radius area rose from ~20% to ~30%. The addition of PIO was accompanied by a significant decrease in “M” from 0.25 ± 0.02 to 0.19 ± 0.02; a comparable decline from 0.25 ± 0.02 to 0.18 ± 0.02 was also seen with 25 mmol/L of glucose +200 μmol/L of palmitate. When PIO was coincubated with high glucose plus palmitate there was a 50% reduction in % change in radius. A ~10% increase in ABS, reflecting augmented “P” in media with 25 mmol/L glucose versus control was documented. The addition of PIO reduced ABS from 0.208 ± 0.03 to 0.183 ± 0.06. Both high glucose and palmitate showed ABS of ~0.140 ± 0.02, which decreased with PIO to ~0.120 ± 0.02, indicating “P” was reduced. Conclusion: These results confirm that high glucose and palmitate stimulate VSMCs migration and proliferation in vitro, which is attenuated by coincubation with the insulin sensitizer PIO. Although, we cannot ascertain whether these functional changes are coincident with the activation/deactivation of signal molecules, our findings are consistent with the theory that differential regulation of insulin signaling pathways in VSMCs in insulin‐resistant states plays an important role in inflammation, arterial wall thickening, and plaque formation during development of atherosclerosis.
This article describes findings on the degree of stimulation of migration and proliferation rates of VSMC derived from human coronary arteries in vitro. Exposure to high glucose and palmitate increases, whereas pioglitazone, an insulin sensitizing agent reduces migration and proliferation rates. Combined with previous demonstration that insulin signaling pathways are differentially regulated, these results suggest that insulin may be involved in vascular plaque formation in insulin‐resistant states, including type 2 diabetes
PMCID: PMC4246575  PMID: 25138792
Cell proliferation; hyperinsulinemia; inflammation; insulin signaling; migration; vascular dysfunction; vascular smooth muscle cells
12.  Methionine sulfoxide reductase A affects insulin resistance by protecting insulin receptor function 
Oxidative stress plays a significant role in the development of insulin resistance; however, the cellular targets of oxidation that cause insulin resistance have yet to be fully elucidated. Methionine sulfoxide reductases (Msr) reduce oxidized methionine residues, thereby repairing and protecting proteins from oxidation. Recently, several genome-wide analyses have found human obesity to be strongly correlated with polymorphisms near the methionine sulfoxide reductase A (MsrA) locus. In this study, we tested whether modulation of MsrA expression significantly alters the development of obesity and/or insulin resistance in mice. We show that mice lacking MsrA (MsrA−/−) are prone to the development of high fat diet-induced insulin resistance and a reduced physiological insulin response compared to high fat-fed wild type mice. We also show that oxidative stress in C2C12 cell cultures reduces both insulin-stimulated phosphorylation and autophosphorylation of the insulin receptor. Tissues from high fat-fed mice show similar reduction in insulin receptor function and the lack of MsrA further diminishes these functions. Together, these data demonstrate for the first time that MsrA plays a role in the regulation of glucose homeostasis. In addition, these data support a novel hypothesis that obesity-induced insulin resistance is caused in part by reduced function of insulin signaling proteins arising from protein oxidation.
PMCID: PMC3578155  PMID: 23089224
oxidative stress; methionine sulfoxide; diabetes; obesity; glucose homeostasis
13.  Pioglitazone slows progression of atherosclerosis in prediabetes independent of changes in cardiovascular risk factors 
To determine whether changes in standard and novel risk factors during the ACT NOW trial explained the slower rate of CIMT progression with pioglitazone treatment in persons with prediabetes.
Methods and Results
CIMT was measured in 382 participants at the beginning and up to three additional times during follow-up of the ACT NOW trial. During an average follow-up of 2.3 years, the mean unadjusted annual rate of CIMT progression was significantly (P=0.01) lower with pioglitazone treatment (4.76 × 10−3 mm/year, 95% CI, 2.39 × 10−3 – 7.14 × 10−3 mm/year) compared with placebo (9.69 × 10−3 mm/year, 95% CI, 7.24 × 10−3 – 12.15 × 10−3 mm/year). High-density lipoprotein cholesterol, fasting and 2-hour glucose, HbA1c, fasting insulin, Matsuda insulin sensitivity index, adiponectin and plasminogen activator inhibitor-1 levels improved significantly with pioglitazone treatment compared with placebo (P < 0.001). However, the effect of pioglitazone on CIMT progression was not attenuated by multiple methods of adjustment for traditional, metabolic and inflammatory risk factors and concomitant medications, and was independent of changes in risk factors during pioglitazone treatment.
Pioglitazone slowed progression of CIMT, independent of improvement in hyperglycemia, insulin resistance, dyslipidemia and systemic inflammation in prediabetes. These results suggest a possible direct vascular benefit of pioglitazone.
PMCID: PMC3908828  PMID: 23175674
Carotid atherosclerosis progression; Impaired glucose tolerance; Insulin resistance; Inflammation; Pioglitazone
14.  Disruption of Growth Factor Receptor–Binding Protein 10 in the Pancreas Enhances β-Cell Proliferation and Protects Mice From Streptozotocin-Induced β-Cell Apoptosis 
Diabetes  2012;61(12):3189-3198.
Defects in insulin secretion and reduction in β-cell mass are associated with type 2 diabetes in humans, and understanding the basis for these dysfunctions may reveal strategies for diabetes therapy. In this study, we show that pancreas-specific knockout of growth factor receptor–binding protein 10 (Grb10), which is highly expressed in pancreas and islets, leads to elevated insulin/IGF-1 signaling in islets, enhanced β-cell mass and insulin content, and increased insulin secretion in mice. Pancreas-specific disruption of Grb10 expression also improved glucose tolerance in mice fed with a high-fat diet and protected mice from streptozotocin-induced β-cell apoptosis and body weight loss. Our study has identified Grb10 as an important regulator of β-cell proliferation and demonstrated that reducing the expression level of Grb10 could provide a novel means to increase β-cell mass and reduce β-cell apoptosis. This is critical for effective therapeutic treatment of both type 1 and 2 diabetes.
PMCID: PMC3501856  PMID: 22923474
15.  Fat-Specific DsbA-L Overexpression Promotes Adiponectin Multimerization and Protects Mice From Diet-Induced Obesity and Insulin Resistance 
Diabetes  2012;61(11):2776-2786.
The antidiabetic and antiatherosclerotic effects of adiponectin make it a desirable drug target for the treatment of metabolic and cardiovascular diseases. However, the adiponectin-based drug development approach turns out to be difficult due to extremely high serum levels of this adipokine. On the other hand, a significant correlation between adiponectin multimerization and its insulin-sensitizing effects has been demonstrated, suggesting a promising alternative therapeutic strategy. Here we show that transgenic mice overexpressing disulfide bond A oxidoreductase-like protein in fat (fDsbA-L) exhibited increased levels of total and the high-molecular-weight form of adiponectin compared with wild-type (WT) littermates. The fDsbA-L mice also displayed resistance to diet-induced obesity, insulin resistance, and hepatic steatosis compared with WT control mice. The protective effects of DsbA-L overexpression on diet-induced insulin resistance, but not increased body weight and fat cell size, were significantly decreased in adiponectin-deficient fDsbA-L mice (fDsbA-L/Ad−/−). In addition, the fDsbA-L/Ad−/− mice displayed greater activity and energy expenditure compared with adiponectin knockout mice under a high-fat diet. Taken together, our results demonstrate that DsbA-L protects mice from diet-induced obesity and insulin resistance through adiponectin-dependent and independent mechanisms. In addition, upregulation of DsbA-L could be an effective therapeutic approach for the treatment of obesity and its associated metabolic disorders.
PMCID: PMC3478538  PMID: 22807031
16.  Reduced AMPK-ACC and mTOR signaling in muscle from older men, and effect of resistance exercise 
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.
PMCID: PMC3631591  PMID: 23000302
Aging; Skeletal muscle; AMPK; mTOR; Resistance exercise
17.  Energy Expenditure Evaluation in Humans and Non-Human Primates by SenseWear Armband. Validation of Energy Expenditure Evaluation by SenseWear Armband by Direct Comparison with Indirect Calorimetry 
PLoS ONE  2013;8(9):e73651.
The purpose of this study was to compare and validate the use of SenseWear Armband (SWA) placed on the arm (SWA ARM) and on the back (SWA BACK) in healthy humans during resting and a cycle-ergometer exercise and to evaluate the SWA to estimate Resting Energy Expenditure (REE) and Total Energy Expenditure (TEE) in healthy baboons.
We studied 26 (15F/11M) human subjects wearing SWA in two different anatomical sites (arm and back) during resting and a cycle-ergometer test and directly compared these results with indirect calorimetry evaluation (IC), performed at the same time. We then inserted the SWA in a metabolic jacket for baboons and evaluated the TEE and REE in free living condition for 6 days in 21 (8F/13M) non-human primates.
In humans we found a good correlation between SWA place on the ARM and on the BACK with IC during the resting experiment (1.1±0.3 SWAs, 1±0.2 IC kcal/min) and a slight underestimation in the SWAs data compared with IC during the cycle-ergometer exercise (5±1.9 SWA ARM, 4.5±1.5 SWA BACK and 5.4±2.1 IC kcal/min). In the non-human primate (baboons) experiment SWA estimated a TEE of 0.54±0.009 kcal/min during free living and a REE of 0.82±0.06 kcal/min.
SWA, an extremely simple and inexpensive apparatus, provides quite accurate measurements of energy expenditure in humans and in baboons. Energy expenditure data obtained with SWA are highly correlated with the data obtained with “gold standard”, IC, in humans.
PMCID: PMC3777938  PMID: 24069218
18.  Tribbles 3 Mediates Endoplasmic Reticulum Stress-Induced Insulin Resistance in Skeletal Muscle 
Nature communications  2013;4:1871.
Endoplasmic Reticulum (ER) stress has been linked to insulin resistance in multiple tissues but the role of ER stress in skeletal muscle has not been explored. ER stress has also been reported to increase tribbles 3 (TRB3) expression in multiple cell lines. Here, we report that high fat feeding in mice, and obesity and type 2 diabetes in humans significantly increases TRB3 and ER stress markers in skeletal muscle. Overexpression of TRB3 in C2C12 myotubes and mouse tibialis anterior muscles significantly impairs insulin signaling. Incubation of C2C12 cells and mouse skeletal muscle with ER stressors thapsigargin and tunicamycin increases TRB3 and impairs insulin signaling and glucose uptake, effects reversed in cells overexpressing RNAi for TRB3 and in muscles from TRB3 knockout mice. Furthermore, TRB3 knockout mice are protected from high fat diet-induced insulin resistance in skeletal muscle. These data demonstrate that TRB3 mediates ER stress-induced insulin resistance in skeletal muscle.
PMCID: PMC3707125  PMID: 23695665
19.  Effect of Lipopolysaccharide on Inflammation and Insulin Action in Human Muscle 
PLoS ONE  2013;8(5):e63983.
Accumulating evidence from animal studies suggest that chronic elevation of circulating intestinal-generated lipopolysaccharide (LPS) (i.e., metabolic endotoxemia) could play a role in the pathogenesis of insulin resistance. However, the effect of LPS in human muscle is unclear. Moreover, it is unknown whether blockade/down regulation of toll-like receptor (TLR)4 can prevent the effect of LPS on insulin action and glucose metabolism in human muscle cells. In the present study we compared plasma LPS concentration in insulin resistant [obese non-diabetic and obese type 2 diabetic (T2DM)] subjects versus lean individuals. In addition, we employed a primary human skeletal muscle cell culture system to investigate the effect of LPS on glucose metabolism and whether these effects are mediated via TLR4. Obese non-diabetic and T2DM subjects had significantly elevated plasma LPS and LPS binding protein (LBP) concentrations. Plasma LPS (r = −0.46, P = 0.005) and LBP (r = −0.49, P = 0.005) concentrations negatively correlated with muscle insulin sensitivity (M). In human myotubes, LPS increased JNK phosphorylation and MCP-1 and IL-6 gene expression. This inflammatory response led to reduced insulin-stimulated IRS-1, Akt and AS160 phosphorylation and impaired glucose transport. Both pharmacologic blockade of TLR4 with TAK-242, and TLR4 gene silencing, suppressed the inflammatory response and insulin resistance caused by LPS in human muscle cells. Taken together, these findings suggest that elevations in plasma LPS concentration found in obese and T2DM subjects could play a role in the pathogenesis of insulin resistance and that antagonists of TLR4 may improve insulin action in these individuals.
PMCID: PMC3660322  PMID: 23704966
20.  Short-Term Exercise Training Improves Insulin Sensitivity but Does Not Inhibit Inflammatory Pathways in Immune Cells from Insulin-Resistant Subjects 
Journal of Diabetes Research  2013;2013:107805.
Background. Exercise has an anti-inflammatory effect against, and immune cells play critical roles in the development, of insulin resistance and atherosclerotic vascular disease (AVD). Thus, the goal of this study was to determine whether exercise improves insulin sensitivity in insulin-resistant subjects by downregulating proinflammatory signaling in immune cells. Methods. Seventeen lean, 8 obese nondiabetic, and 11 obese type 2 diabetic individuals underwent an aerobic exercise program for 15 days and an insulin clamp before and after exercise. Peripheral mononuclear cells (PMNC) were obtained for determination of Toll-like receptor (TLR) 2 and 4 protein content and mitogen-activated protein kinase phosphorylation. Results. Compared with that in lean individuals, TLR4 protein content was increased by 4.2-fold in diabetic subjects. This increase in TLR4 content was accompanied by a 3.0-fold increase in extracellular signal-regulated kinase (ERK) phosphorylation. Exercise improved insulin sensitivity in the lean, obese, and type 2 diabetes groups. However, exercise did not affect TLR content or ERK phosphorylation. Conclusions. TLR4 content and ERK phosphorylation are increased in PMNC of type 2 diabetic individuals. While exercise improves insulin sensitivity, this effect is not related to changes in TLR2/TLR4 content or ERK phosphorylation in PMNC of type 2 diabetic individuals.
PMCID: PMC3647562  PMID: 23671849
21.  TAK-242, a small-molecule inhibitor of Toll-like receptor 4 signalling, unveils similarities and differences in lipopolysaccharide- and lipidinduced inflammation and insulin resistance in muscle cells 
Bioscience Reports  2012;33(1):e00004.
Emerging evidence suggests that TLR (Toll-like receptor) 4 and downstream pathways [MAPKs (mitogen-activated protein kinases) and NF-κB (nuclear factor κB)] play an important role in the pathogenesis of insulin resistance. LPS (lipopolysaccharide) and saturated NEFA (non-esterified fatty acids) activate TLR4, and plasma concentrations of these TLR4 ligands are elevated in obesity and Type 2 diabetes. Our goals were to define the role of TLR4 on the insulin resistance caused by LPS and saturated NEFA, and to dissect the independent contribution of LPS and NEFA to the activation of TLR4-driven pathways by employing TAK-242, a specific inhibitor of TLR4. LPS caused robust activation of the MAPK and NF-κB pathways in L6 myotubes, along with impaired insulin signalling and glucose transport. TAK-242 completely prevented the inflammatory response (MAPK and NF-κB activation) caused by LPS, and, in turn, improved LPS-induced insulin resistance. Similar to LPS, stearate strongly activated MAPKs, although stimulation of the NF-κB axis was modest. As seen with LPS, the inflammatory response caused by stearate was accompanied by impaired insulin action. TAK-242 also blunted stearate-induced inflammation; yet, the protective effect conferred by TAK-242 was partial and observed only on MAPKs. Consequently, the insulin resistance caused by stearate was only partially improved by TAK-242. In summary, TAK-242 provides complete and partial protection against LPS- and NEFA-induced inflammation and insulin resistance, respectively. Thus, LPS-induced insulin resistance depends entirely on TLR4, whereas NEFA works through TLR4-dependent and -independent mechanisms to impair insulin action.
PMCID: PMC3522475  PMID: 23050932
endotoxin; mitogen-activated protein kinase (MAPK); nuclear factor κB (NF-κB); saturated non-esterified fatty acid (NEFA); Toll-like receptor 4 (TLR4).; AP-1, activator protein-1; 2-DG, 2-deoxy-D[1,2-3H]glucose; DAG, diacylglycerol; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK, glycogen synthase kinase; IκB, inhibitory κB; IKK, IκB kinase; IL, interleukin; iNOS, inducible nitric oxide synthase; IRAK, IL-1-receptor-associated kinase; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MCP1, monocyte chemoattractant protein-1; MEMα, minimum essential medium α; MyD88, myeloid differentiation factor 88; NEFA, non-esterified fatty acid(s); NF-κB, nuclear factor κB; NOD2, nucleotide-binding oligomerization domain-2; PKC, protein kinase C; RT–PCR, reverse transcription–PCR; TIR, Toll/IL-1 receptor; TIRAP, TIR domain-containing adaptor protein; TLR, Toll-like receptor; TNF, tumour necrosis factor; TRAF-6, TNF-receptor-associated factor-6
22.  Reduction in Reactive Oxygen Species Production by Mitochondria From Elderly Subjects With Normal and Impaired Glucose Tolerance 
Diabetes  2011;60(8):2051-2060.
Aging increases the risk of developing impaired glucose tolerance (IGT) and type 2 diabetes. It has been proposed that increased reactive oxygen species (ROS) generation by dysfunctional mitochondria could play a role in the pathogenesis of these metabolic abnormalities. We examined whether aging per se (in subjects with normal glucose tolerance [NGT]) impairs mitochondrial function and how this relates to ROS generation, whether older subjects with IGT have a further worsening of mitochondrial function (lower ATP production and elevated ROS generation), and whether exercise reverses age-related changes in mitochondrial function.
Mitochondrial ATP and ROS production were measured in muscle from younger individuals with NGT, older individuals with NGT, and older individuals with IGT. Measurements were performed before and after 16 weeks of aerobic exercise.
ATP synthesis was lower in older subjects with NGT and older subjects with IGT versus younger subjects. Notably, mitochondria from older subjects (with NGT and IGT) displayed reduced ROS production versus the younger group. ATP and ROS production were similar between older groups. Exercise increased ATP synthesis in the three groups. Mitochondrial ROS production also increased after training. Proteomic analysis revealed downregulation of several electron transport chain proteins with aging, and this was reversed by exercise.
Old mitochondria from subjects with NGT and IGT display mitochondrial dysfunction as manifested by reduced ATP production but not with respect to increased ROS production. When adjusted to age, the development of IGT in elderly individuals does not involve changes in mitochondrial ATP and ROS production. Lastly, exercise reverses the mitochondrial phenotype (proteome and function) of old mitochondria.
PMCID: PMC3142073  PMID: 21677280
23.  Does Reduced IGF-1R Signaling in Igf1r+/− Mice Alter Aging? 
PLoS ONE  2011;6(11):e26891.
Mutations in insulin/IGF-1 signaling pathway have been shown to lead to increased longevity in various invertebrate models. Therefore, the effect of the haplo- insufficiency of the IGF-1 receptor (Igf1r+/−) on longevity/aging was evaluated in C57Bl/6 mice using rigorous criteria where lifespan and end-of-life pathology were measured under optimal husbandry conditions using large sample sizes. Igf1r+/− mice exhibited reductions in IGF-1 receptor levels and the activation of Akt by IGF-1, with no compensatory increases in serum IGF-1 or tissue IGF-1 mRNA levels, indicating that the Igf1r+/− mice show reduced IGF-1 signaling. Aged male, but not female Igf1r+/− mice were glucose intolerant, and both genders developed insulin resistance as they aged. Female, but not male Igf1r+/− mice survived longer than wild type mice after lethal paraquat and diquat exposure, and female Igf1r+/− mice also exhibited less diquat-induced liver damage. However, no significant difference between the lifespans of the male Igf1r+/− and wild type mice was observed; and the mean lifespan of the Igf1r+/− females was increased only slightly (less than 5%) compared to wild type mice. A comprehensive pathological analysis showed no significant difference in end-of-life pathological lesions between the Igf1r+/− and wild type mice. These data show that the Igf1r+/− mouse is not a model of increased longevity and delayed aging as predicted by invertebrate models with mutations in the insulin/IGF-1 signaling pathway.
PMCID: PMC3223158  PMID: 22132081
24.  Beyond AICA Riboside: In Search of New Specific AMP-activated Protein Kinase Activators 
IUBMB life  2009;61(1):18-26.
5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICA riboside) has been extensively used in vitro and in vivo to activate the AMP-activated protein kinase (AMPK), a metabolic sensor involved in both cellular and whole body energy homeostasis. However, it has been recently highlighted that AICA riboside also exerts AMPK-independent effects, mainly on AMP-regulated enzymes and mitochondrial oxidative phosphorylation (OXPHOS), leading to the conclusion that new compounds with reduced off target effects are needed to specifically activate AMPK. Here, we review recent findings on newly discovered AMPK activators, notably on A-769662, a nonnucleoside compound from the thienopyridone family. We also report that A-769662 is able to activate AMPK and stimulate glucose uptake in both L6 cells and primary myotubes derived from human satellite cells. In addition, A-769662 increases AMPK activity and phosphorylation of its main downstream targets in primary cultured rat hepatocytes but, by contrast with AICA riboside, does neither affect mitochondrial OXPHOS nor change cellular AMP:ATP ratio. We conclude that A-769662 could be one of the new promising chemical agents to activate AMPK with limited AMPK-independent side effects.
PMCID: PMC2845387  PMID: 18798311
AMPK; AICA riboside; 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; ZMP; A-769662; glucose uptake; hepatocytes; mitochondria
25.  Effect of Acute Exercise on AMPK Signaling in Skeletal Muscle of Subjects With Type 2 Diabetes 
Diabetes  2007;56(3):836-848.
Activation of AMP-activated protein kinase (AMPK) by exercise induces several cellular processes in muscle. Exercise activation of AMPK is unaffected in lean (BMI ~25 kg/m2) subjects with type 2 diabetes. However, most type 2 diabetic subjects are obese (BMI >30 kg/m2), and exercise stimulation of AMPK is blunted in obese rodents. We examined whether obese type 2 diabetic subjects have impaired exercise stimulation of AMPK, at different signaling levels, spanning from the upstream kinase, LKB1, to the putative AMPK targets, AS160 and peroxisome proliferator–activated receptor coactivator (PGC)-1α, involved in glucose transport regulation and mitochondrial biogenesis, respectively. Twelve type 2 diabetic, eight obese, and eight lean subjects exercised on a cycle ergometer for 40 min. Muscle biopsies were done before, during, and after exercise. Subjects underwent this protocol on two occasions, at low (50% VO2max) and moderate (70% VO2max) intensities, with a 4–6 week interval. Exercise had no effect on LKB1 activity. Exercise had a time- and intensity-dependent effect to increase AMPK activity and AS160 phosphorylation. Obese and type 2 diabetic subjects had attenuated exercise-stimulated AMPK activity and AS160 phosphorylation. Type 2 diabetic subjects had reduced basal PGC-1 gene expression but normal exercise-induced increases in PGC-1 expression. Our findings suggest that obese type 2 diabetic subjects may need to exercise at higher intensity to stimulate the AMPK-AS160 axis to the same level as lean subjects.
PMCID: PMC2844111  PMID: 17327455

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