Lawrence J. Mandarino

Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: Potential role of PGC1 and NRF1

Type 2 diabetes mellitus (DM) is characterized by insulin resistance and pancreatic β cell dysfunction. In high-risk subjects, the earliest detectable abnormality is insulin resistance in skeletal muscle. Impaired insulin-mediated signaling, gene expression, glycogen synthesis, and accumulation of intramyocellular triglycerides have all been linked with insulin resistance, but no specific defect responsible for insulin resistance and DM has been identified in humans. To identify genes potentially important in the pathogenesis of DM, we analyzed gene expression in skeletal muscle from healthy metabolically characterized nondiabetic (family history negative and positive for DM) and diabetic Mexican–American subjects. We demonstrate that insulin resistance and DM associate with reduced expression of multiple nuclear respiratory factor-1 (NRF-1)-dependent genes encoding key enzymes in oxidative metabolism and mitochondrial function. Although NRF-1 expression is decreased only in diabetic subjects, expression of both PPARγ coactivator 1-α and-β (PGC1-α/PPARGC1 and PGC1-β/PERC), coactivators of NRF-1 and PPARγ-dependent transcription, is decreased in both diabetic subjects and family history-positive nondiabetic subjects. Decreased PGC1 expression may be responsible for decreased expression of NRF-dependent genes, leading to the metabolic disturbances characteristic of insulin resistance and DM.

Insulin resistance differentially affects the PI 3-kinase– and MAP kinase–mediated signaling in human muscle

The broad nature of insulin resistant glucose metabolism in skeletal muscle of patients with type 2 diabetes suggests a defect in the proximal part of the insulin signaling network. We sought to identify the pathways compromised in insulin resistance and to test the effect of moderate exercise on whole-body and cellular insulin action. We conducted euglycemic clamps and muscle biopsies on type 2 diabetic patients, obese nondiabetics and lean controls, with and without a single bout of exercise. Insulin stimulation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway, as measured by phosphorylation of the insulin receptor and IRS-1 and by IRS protein association with p85 and with PI 3-kinase, was dramatically reduced in obese nondiabetics and virtually absent in type 2 diabetic patients. Insulin stimulation of the MAP kinase pathway was normal in obese and diabetic subjects. Insulin stimulation of glucose-disposal correlated with association of p85 with IRS-1. Exercise 24 hours before the euglycemic clamp increased phosphorylation of insulin receptor and IRS-1 in obese and diabetic subjects but did not increase glucose uptake or PI 3-kinase association with IRS-1 upon insulin stimulation. Thus, insulin resistance differentially affects the PI 3-kinase and MAP kinase signaling pathways, and insulin-stimulated IRS-1-association with PI 3-kinase defines a key step in insulin resistance.

Effect of Pioglitazone on Abdominal Fat Distribution and Insulin Sensitivity in Type 2 Diabetic Patients

We examined the effect of pioglitazone on abdominal fat distribution to elucidate the mechanisms via which pioglitazone improves insulin resistance in patients with type 2 diabetes mellitus. Thirteen type 2 diabetic patients (nine men and four women; age, 52 +/- 3 yr; body mass index, 29.0 +/- 1.1 kg/m(2)), who were being treated with a stable dose of sulfonylurea (n = 7) or with diet alone (n = 6), received pioglitazone (45 mg/d) for 16 wk. Before and after pioglitazone treatment, subjects underwent a 75-g oral glucose tolerance test (OGTT) and two-step euglycemic insulin clamp (insulin infusion rates, 40 and 160 mU/m(2).min) with [(3)H]glucose. Abdominal fat distribution was evaluated using magnetic resonance imaging at L4-5. After 16 wk of pioglitazone treatment, fasting plasma glucose (179 +/- 10 to 140 +/- 10 mg/dl; P < 0.01), mean plasma glucose during OGTT (295 +/- 13 to 233 +/- 14 mg/dl; P < 0.01), and hemoglobin A(1c) (8.6 +/- 0.4% to 7.2 +/- 0.5%; P < 0.01) decreased without a change in fasting or post-OGTT insulin levels. Fasting plasma FFA (674 +/- 38 to 569 +/- 31 microEq/liter; P < 0.05) and mean plasma FFA (539 +/- 20 to 396 +/- 29 microEq/liter; P < 0.01) during OGTT decreased after pioglitazone. In the postabsorptive state, hepatic insulin resistance [basal endogenous glucose production (EGP) x basal plasma insulin concentration] decreased from 41 +/- 7 to 25 +/- 3 mg/kg fat-free mass (FFM).min x microU/ml; P < 0.05) and suppression of EGP during the first insulin clamp step (1.1 +/- 0.1 to 0.6 +/- 0.2 mg/kg FFM.min; P < 0.05) improved after pioglitazone treatment. The total body glucose MCR during the first and second insulin clamp steps increased after pioglitazone treatment [first MCR, 3.5 +/- 0.5 to 4.4 +/- 0.4 ml/kg FFM.min (P < 0.05); second MCR, 8.7 +/- 1.0 to 11.3 +/- 1.1 ml/kg FFM(.)min (P < 0.01)]. The improvement in hepatic and peripheral tissue insulin sensitivity occurred despite increases in body weight (82 +/- 4 to 85 +/- 4 kg; P < 0.05) and fat mass (27 +/- 2 to 30 +/- 3 kg; P < 0.05). After pioglitazone treatment, sc fat area at L4-5 (301 +/- 44 to 342 +/- 44 cm(2); P < 0.01) increased, whereas visceral fat area at L4-5 (144 +/- 13 to 131 +/- 16 cm(2); P < 0.05) and the ratio of visceral to sc fat (0.59 +/- 0.08 to 0.44 +/- 0.06; P < 0.01) decreased. In the postabsorptive state hepatic insulin resistance (basal EGP x basal immunoreactive insulin) correlated positively with visceral fat area (r = 0.55; P < 0.01). The glucose MCRs during the first (r = -0.45; P < 0.05) and second (r = -0.44; P < 0.05) insulin clamp steps were negatively correlated with the visceral fat area. These results demonstrate that a shift of fat distribution from visceral to sc adipose depots after pioglitazone treatment is associated with improvements in hepatic and peripheral tissue sensitivity to insulin.

Interaction between glucose and free fatty acid metabolism in human skeletal muscle.

The mechanism by which FFA metabolism inhibits intracellular insulin-mediated muscle glucose metabolism in normal humans is unknown. We used the leg balance technique with muscle biopsies to determine how experimental maintenance of FFA during hyperinsulinemia alters muscle glucose uptake, oxidation, glycolysis, storage, pyruvate dehydrogenase (PDH), or glycogen synthase (GS). 10 healthy volunteers had two euglycemic insulin clamp experiments. On one occasion, FFA were maintained by lipid emulsion infusion; on the other, FFA were allowed to fall. Leg FFA uptake was monitored with [9,10-3H]-palmitate. Maintenance of FFA during hyperinsulinemia decreased muscle glucose uptake (1.57 +/- 0.31 vs 2.44 +/- 0.39 mumol/min per 100 ml tissue, P < 0.01), leg respiratory quotient (0.86 +/- 0.02 vs 0.93 +/- 0.02, P < 0.05), contribution of glucose to leg oxygen consumption (53 +/- 6 vs 76 +/- 8%, P < 0.05), and PDH activity (0.328 +/- 0.053 vs 0.662 +/- 0.176 nmol/min per mg, P < 0.05). Leg lactate balance was increased. The greatest effect of FFA replacement was reduced muscle glucose storage (0.36 +/- 0.20 vs 1.24 +/- 0.25 mumol/min per 100 ml, P < 0.01), accompanied by decreased GS fractional velocity (0.129 +/- 0.26 vs 0.169 +/- 0.033, P < 0.01). These results confirm in human skeletal muscle the existence of competition between glucose and FFA as oxidative fuels, mediated by suppression of PDH. Maintenance of FFA levels during hyperinsulinemia most strikingly inhibited leg muscle glucose storage, accompanied by decreased GS activity.

Adiponectin receptors gene expression and insulin sensitivity in non-diabetic Mexican Americans with or without a family history of Type 2 diabetes.

Aims/hypothesisThe recent discovery of two adiponectin receptors (AdipoR1 and AdipoR2) will improve our understanding of the molecular mechanisms underlying the insulin-sensitising effect of adiponectin. The aim of this study was to determine for the first time whether skeletal muscle AdipoR1 and/or AdipoR2 gene expression levels are associated with insulin resistance.MethodsUsing RT-PCR and northern analysis we measured AdipoR1 and AdipoR2 gene expression in skeletal muscle from healthy Mexican Americans with normal glucose tolerance who had (n=8) or did not have (n=10) a family history of Type 2 diabetes.ResultsGene expression profiling indicated that the AdipoR1 and AdipoR2 isoforms are highly expressed in human skeletal muscle, unlike in mice where AdipoR2 expression was highest in the liver, and AdipoR1 was highest in skeletal muscle. In the study subjects, the expression levels of AdipoR1 (p=0.004) and AdipoR2 (p=0.04), as well as plasma adiponectin concentration (p=0.03) were lower in people with a family history of Type 2 diabetes than in those with no family history of the disease. Importantly, the expression levels of both receptors correlated positively with insulin sensitivity (r=0.64, p=0.004 and r=0.47, p=0.048 respectively).Conclusions/interpretationCollectively, these data indicate that both isoforms of the adiponectin receptor play a role in the insulin-sensitising effect of adiponectin.

Lipid Infusion Decreases the Expression of Nuclear Encoded Mitochondrial Genes and Increases the Expression of Extracellular Matrix Genes in Human Skeletal Muscle

The association between elevated plasma free fatty acid (FFA) concentrations and insulin resistance is well known. Although the cause and effect relationship between FFAs and insulin resistance is complex, plasma FFA is negatively correlated with the expression of peroxisome proliferator activated receptor-γ cofactor-1 (PGC-1) and nuclear encoded mitochondrial genes. To test whether this association is causal, we infused a triglyceride emulsion (or saline as control) into healthy subjects to increase plasma FFA for 48 h followed by muscle biopsies, microarray analysis, quantitative real time PCR, and immunoblots. Lipid infusion increased plasma FFA concentration from 0.48 ± 0.02 to 1.73 ± 0.43 mm and decreased insulin-stimulated glucose disposal from 8.82 ± 0.69 to 6.67 ± 0.66 mg/kg·min, both with p < 0.05. PGC-1 mRNA, along with mRNAs for a number of nuclear encoded mitochondrial genes, were reduced by lipid infusion (p < 0.05). Microarray analysis also revealed that lipid infusion caused a significant overexpression of extracellular matrix genes and connective tissue growth factor. Quantitative reverse transcription PCR showed that the mRNA expression of collagens and multiple extracellular matrix genes was higher after the lipid infusion (p < 0.05). Immunoblot analysis revealed that lipid infusion also increased the expression of collagens and the connective tissue growth factor protein. These data suggest that an experimental increase in FFAs decreases the expression of PGC-1 and nuclear encoded mitochondrial genes and also increases the expression of extracellular matrix genes in a manner reminiscent of inflammation.

Tumor necrosis factor α and insulin resistance in obese type 2 diabetic patients

The relationship between basal serum tumor necrosis factor alpha (TNFα) levels and peripheral tissue (muscle) sensitivity to insulin was examined in 63 subjects with normal glucose tolerance (NGT), 18 subjects with impaired glucose tolerance (IGT), and 123 patients with type 2 diabetes mellitus (T2DM). The BMI was similar in NGT (28.8±0.7 kg/m2), IGT (31.1±1.0), and T2DM (30.0±0.4) groups. The fasting serum TNFα concentration in T2DM (4.4±0.2 pg/ml) was significantly higher than in NGT (3.1±0.2) and IGT (3.4±0.2; both P<0.05). In T2DM the fasting plasma glucose (FPG=183±5 mg/dl) and insulin (FPI=17±1 µU/ml) concentrations were significantly higher than in NGT (FPG=95±1; FPI=10±1) and IGT (FPG=100±2; FPI=13±1; all P<0.01). The rate of total body insulin-mediated glucose disposal (Rd; 40 mU/m2 min euglycemic insulin clamp in combination with 3H-glucose) was reduced in T2DM (102±3 mg/m2 min) compared with NGT (177±10) and IGT (151±14; both P<0.01). The serum TNFα concentration was inversely correlated with Rd (r=−0.47, P<0.0001) and positively correlated with both FPG (r=0.32, P=0.004) and FPI (r=0.32, P=0.004) in NGT plus IGT. No correlation was observed between serum TNFα and Rd (r=−0.02), FPG (r=0.15), or FPI (r=0.15) in T2DM. In stepwise multiple regression analysis using age, sex, BMI, FPG, FPI and serum TNFα concentration as independent variables, only BMI and serum TNFα concentration were significant and independent predictors of Rd (r2=0.29, P<0.0001) in the NGT plus IGT group, while FPG and FPI were significant and independent predictors of Rd (r2=0.13, P<0.0001) in T2DM. These results suggest that: (i) an increase in circulating TNFα concentration is associated with peripheral insulin resistance and increased plasma glucose and insulin levels prior to the onset of type 2 diabetes; and (ii) the further deterioration in peripheral insulin resistance in T2DM (compared with NGT and IGT) is unrelated to the increase in serum TNFα concentration.

The mitochondrial rhomboid protease PSARL is a new candidate gene for type 2 diabetes

Aims/hypothesisThis study aimed to identify genes that are expressed in skeletal muscle, encode proteins with functional significance in mitochondria, and are associated with type 2 diabetes.MethodsWe screened for differentially expressed genes in skeletal muscle of Psammomys obesus (Israeli sand rats), and prioritised these on the basis of genomic localisation and bioinformatics analysis for proteins with likely mitochondrial functions.ResultsWe identified a mitochondrial intramembrane protease, known as presenilins-associated rhomboid-like protein (PSARL) that is associated with insulin resistance and type 2 diabetes. Expression of PSARL was reduced in skeletal muscle of diabetic Psammomys obesus, and restored after exercise training to successfully treat the diabetes. PSARL gene expression in human skeletal muscle was correlated with insulin sensitivity as assessed by glucose disposal during a hyperinsulinaemic–euglycaemic clamp. In 1,031 human subjects, an amino acid substitution (Leu262Val) in PSARL was associated with increased plasma insulin concentration, a key risk factor for diabetes. Furthermore, this variant interacted strongly with age to affect insulin levels, accounting for 5% of the variation in plasma insulin in elderly subjects.Conclusions/interpretationVariation in PSARL sequence and/or expression may be an important new risk factor for type 2 diabetes and other components of the metabolic syndrome.

Regulation of hexokinase II activity and expression in human muscle by moderate exercise

A single bout of exercise increases the rate of insulin-stimulated glucose uptake and metabolism in skeletal muscle. Exercise also increases insulin-stimulated glucose 6-phosphate in skeletal muscle, suggesting that exercise increases hexokinase activity. Within 3 h, exercise increases hexokinase II (HK II) mRNA and activity in skeletal muscle from rats. It is not known, however, if a single bout of moderate-intensity exercise increases HK II expression in humans. The present study was undertaken to answer this question. Six subjects had percutaneous biopsies of the vastus lateralis muscle before and 3 h after a single 3-h session of moderate-intensity aerobic (60% of maximal oxygen consumption) exercise. Glycogen synthase, HK I, and HK II activities as well as HK I and HK II mRNA content were determined from the muscle biopsy specimens. The fractional velocity of glycogen synthase was increased by 446 +/- 84% after exercise (P < 0.005). Hexokinase II activity in the soluble fraction of the homogenates increased from 1.2 +/- 0.4 to 4.5 +/- 1.6 pmol.min-1.microgram-1 (P < 0.05) but was unchanged in the particulate fraction (4.3 +/- 1.3 vs. 5.3 +/- 1.5). HK I activity in neither the soluble nor particulate fraction changed after exercise. Relative to a 28S rRNA control signal, HK II mRNA increased from 0.091 +/- 0.02 to 0.195 +/- 0.037 (P < 0.05), whereas HK I mRNA was unchanged (0.414 +/- 0.061 vs. 0.498 +/- 0.134, P < 0.20). The increase in HK II activity after moderate exercise in healthy subjects could be one factor responsible for the enhanced rate of insulin-stimulated glucose uptake seen after exercise.

Seven Days of Euglycemic Hyperinsulinemia Induces Insulin Resistance for Glucose Metabolism but Not Hypertension, Elevated Catecholamine Levels, or Increased Sodium Retention in Conscious Normal Rats

Epidemiological studies have suggested an association among chronic hyperinsulinemia, insulin resistance, and hypertension. However, the causality of this relationship remains uncertain. In this study, chronically catheterized conscious rats were made hyperinsulinemic for 7 days (∼90 mU/1, i.e., threefold over basal), while strict euglycemia was maintained (∼130 mg/dl, coefficient of variation <10%) by using a modification of the insulin/glucose clamp technique. Control rats received vehicle infusion. Baseline mean arterial pressure and heart rate were 125 ± 5 mmHg and 427 ± 12 beats/min and remained unchanged during the 7-day infusion of insulin (127 ± 7 mmHg; 401 ± 12 beats/min) or vehicle (133 ± 4 mmHg; 411 ± 10 beats/min). Baseline plasma epinephrine (88 ± 15 pg/ml), norepinephrine (205 ± 31 pg/ml), and sodium balance (0.34 ± 0.09 mmol) remained constant during the 7-day insulin or vehicle infusion. After 7 days of insulin or vehicle infusion, in vivo insulin action was determined in all rats using a 2-h hyperinsulinemic (1 mU/min) euglycemic clamp with [3-3H]glucose infusion to quantitate whole-body glucose uptake, glycolysis, glucose storage (total glucose uptake minus glycolysis), and hepatic glucose production. Compared with vehicle-treated rats, 7 days of sustained hyperinsulinemia resulted in a reduction (P < 0.01) in insulin-mediated glucose uptake, glucose storage, and glycolysis by 39, 62, and 26%, respectively. Hepatic glucose production was normally suppressed after 7 days of hyperinsulinemia. Neither insulin-stimulated glucose uptake nor glucose storage correlated with blood pressure or heart rate. In conclusion, 7 days of euglycemic hyperinsulinemia induces severe insulin resistance with respect to whole-body glucose metabolism but does not increase blood pressure, catecholamine levels, or sodium retention. This indicates that hyperinsulinemia-induced insulin resistance is not associated with the development of hypertension in rats who do not have a genetic predisposition for hypertension. Because hyperinsulinemia was initiated in normal rats under euglycemic conditions, additional (inherited or acquired) factors may be necessary to observe an effect of hyperinsulinemia and/or insulin resistance to increase blood pressure.