Rates Of Insulin-stimulated Glucose Uptake Research Articles

Increased plasma fatty acid clearance, not fatty acid concentration, is associated with muscle insulin resistance in people with obesity

BackgroundAlthough it is well-accepted that increased plasma free fatty acid (FFA) concentration causes lipid overload and muscle insulin resistance in people with obesity, plasma FFA concentration poorly predicts insulin-resistant glucose metabolism. It has been proposed that hyperinsulinemia in people with obesity sufficiently inhibits adipose tissue triglyceride lipolysis to prevent FFA-induced insulin resistance. However, we hypothesized enhanced FFA clearance in people with obesity, compared with lean people, prevents a marked increase in plasma FFA even when FFA appearance is high. MethodsWe assessed FFA kinetics during basal conditions and during a hyperinsulinemic-euglycemic clamp procedure in 14 lean people and 46 people with obesity by using [13C]palmitate tracer infusion. Insulin-stimulated muscle glucose uptake rate was evaluated by dynamic PET-imaging of skeletal muscles after [18F]fluorodeoxyglucose injection. ResultsPlasma FFA clearance was accelerated in participants with obesity and correlated negatively with muscle insulin sensitivity without a difference between lean and obese participants. Furthermore, insulin infusion increased FFA clearance and the increase was greater in obese than lean participants. ConclusionsOur findings suggest plasma FFA extraction efficiency, not just plasma FFA concentration, is an important determinant of the cellular fatty acid load and the stimulatory effect of insulin on FFA clearance counteracts some of its antilipolytic effect.

Heterogeneity in insulin-stimulated glucose uptake among different muscle groups in healthy lean people and people with obesity

It has been proposed that muscle fibre type composition and perfusion are key determinants of insulin-stimulated muscle glucose uptake, and alterations in muscle fibre type composition and perfusion contribute to muscle, and consequently whole-body, insulin resistance in people with obesity. The goal of the study was to evaluate the relationships among muscle fibre type composition, perfusion and insulin-stimulated glucose uptake rates in healthy, lean people and people with obesity. We measured insulin-stimulated whole-body glucose disposal and glucose uptake and perfusion rates in five major muscle groups (erector spinae, obliques, rectus abdominis, hamstrings, quadriceps) in 15 healthy lean people and 37 people with obesity by using the hyperinsulinaemic-euglycaemic clamp procedure in conjunction with [2H]glucose tracer infusion (to assess whole-body glucose disposal) and positron emission tomography after injections of [15O]H2O (to assess muscle perfusion) and [18F]fluorodeoxyglucose (to assess muscle glucose uptake). A biopsy from the vastus lateralis was obtained to assess fibre type composition. We found: (1) a twofold difference in glucose uptake rates among muscles in both the lean and obese groups (rectus abdominis: 67 [51, 78] and 32 [21, 55] μmol kg-1min-1 in the lean and obese groups, respectively; erector spinae: 134 [103, 160] and 66 [24, 129] μmol kg-1min-1, respectively; median [IQR]) that was unrelated to perfusion or fibre type composition (assessed in the vastus only); (2) the impairment in insulin action in the obese compared with the lean group was not different among muscle groups; and (3) insulin-stimulated whole-body glucose disposal expressed per kg fat-free mass was linearly related with muscle glucose uptake rate (r2 = 0.65, p < 0.05). Obesity-associated insulin resistance is generalised across all major muscles, and is not caused by alterations in muscle fibre type composition or perfusion. In addition, insulin-stimulated whole-body glucose disposal relative to fat-free mass provides a reliable index of muscle glucose uptake rate.

Docosahexaenoic and eicosapentaenoic fatty acids differentially regulate glucose and fatty acid metabolism in L6 rat skeletal muscle cells.

The objective of this study was to investigate whether the n-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) can directly regulate glucose and fat metabolism in skeletal muscle besides exerting anti-inflammatory effects. To accomplish this, L6 skeletal muscle cells were treated with 50 µM of either DHA or EPA for 1, 3, and 5 days. Here, we report that basal and insulin-stimulated rates of glucose uptake, glycogen synthesis, protein kinase B (AKT), and glycogen synthase kinase 3 (GSK3) phosphorylation were not affected by DHA or EPA. However, glucose and palmitate oxidation were consistently elevated by DHA treatment, whereas EPA only increased this variable transiently. Similarly, only DHA caused significant and sustained increases in AMP-activated protein kinase (AMPK) phosphorylation and protein levels of carnitine-palmitoyl transferase-1b (CPT1b) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in skeletal muscle cells. DHA also caused a larger anti-inflammatory effect than EPA in these cells. In conclusion, besides exerting anti-inflammatory effects, DHA and EPA directly regulated glucose and fat metabolism in skeletal muscle cells, although DHA was more effective in doing so than EPA. Thus, by directly enhancing glucose and fat oxidation, DHA may increase glucose disposal and reduce intramyocellular lipid accumulation.

Heterogeneity in Insulin‐Stimulated Glucose Uptake Among Different Muscle Groups in People

Insulin‐stimulated glucose uptake in skeletal muscles is the major mechanism for postprandial glucose disposal. Insulin‐stimulated glucose uptake is greater in type I than in type II muscles in rats. The cellular capacity for glucose uptake and disposal (insulin receptor, GLUT4, hexokinase, and glycogen synthase expression) is also greater in isolated human type I compared with type II fibers. The goal of the present study was to test the hypothesis that insulin‐stimulated glucose uptake rate varies among different muscle groups and is greater in trunk (erector spinae, rectus abdominis, obliques) than leg (quadriceps, hamstrings) muscles and greater in the hamstrings than the quadriceps because trunk muscles generally have a greater percentage of type I fibers than leg muscles and the hamstrings have a greater percentage of type I fibers than quadriceps muscles. We determined abdominal and thigh muscle glucose uptake rates in 10 healthy lean women (age: 43±13 years, BMI: 23±2 kg/m2; mean±SD) by using a hyperinsulinemic‐euglycemic clamp procedure in conjunction with dynamic positron emission tomography imaging after bolus injection of 18F‐fluorodeoxyglucose. In addition, [6,6‐2H2]glucose was infused to determine whole‐body glucose disposal rate per kg fat‐free mass, which is commonly used as an index of muscle glucose uptake, and a muscle biopsy from the vastus lateralis was obtained to determine myofiber composition. Glucose uptake rate in the erector spinae (128±30 μmol/kg/min) was nearly twice as great as in the rectus abdominis (66±14 μmol/kg/min; P&lt;0.01) and approximately 50% greater than in the obliques (88±23 μmol/kg/min; P&lt;0.05). Quadriceps and hamstring glucose uptake rates were not different from each other (85±42 and 91±37 μmol/kg/min, respectively; P&gt;0.05) and not different from glucose uptake rates in the rectus abdominis and obliques but less than in the erector spinae (P&lt;0.05). No significant correlation between fiber type composition and muscle glucose uptake rate in the quadriceps was detected. Whole body insulin stimulated glucose disposal rate was 93±28 μmol/kg fat‐free mass/min. We conclude that: i) insulin‐stimulated muscle glucose uptake is heterogeneous among muscle groups, ii) it is unlikely that differences in fiber type composition among muscles are responsible for the heterogeneity in glucose uptake rates, and iii) whole‐body glucose disposal rate per kg fat‐free mass does not accurately reflect glucose uptake rates in individual muscles.Support or Funding InformationNIH DK115400, DK56341 (Nutrition Obesity Research Center), DK020579 (Diabetes Research Center), and UL1TR000448 (Clinical Translational Science Award), and ADA 1‐18‐ICTS‐119.

Effects of the adiponectin mimetic compound ALY688 on glucose and fat metabolism in visceral and subcutaneous rat adipocytes

ABSTRACT Adiponectin regulates white adipose tissue (WAT) metabolism and promotes insulin-sensitizing and anti-atherosclerotic effects in vivo. In this context, small molecule adiponectin receptor agonists have become of great therapeutic value for the treatment of metabolic diseases. Here, we investigated the effects of the adiponectin mimetic compound ALY688 on WAT metabolism. To accomplish this, rat epididymal (Epid) and subcutaneous inguinal (Sc Ing) adipocytes were isolated and incubated with ALY688. Subsequently, several parameters of glucose and fat metabolism were assessed. ALY688 promoted AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation, enhanced glucose oxidation, and suppressed fat oxidation in adipocytes from both fat depots. ALY688 did not affect basal and insulin-stimulated rates of glucose uptake, glucose incorporation into lipids, and AKTSer473 and p38 mitogen-activated protein kinase (MAPK) phosphorylations in either Epid or Sc Ing adipocytes. ALY688 did not alter basal lipolysis in Epid and Sc Ing adipocytes, but it enhanced isoproterenol-induced lipolysis in Epid adipocytes. Adiponectin receptor 2 (AdipoR2) mRNA was the prevalent isoform expressed in all adipocytes, and Epid adipocytes displayed significantly higher AdipoR2 mRNA expression than Sc Ing adipocytes. In conclusion, ALY688 can regulate adiposity and affect glycaemic control by altering substrate portioning in the WAT in a fat depot-specific manner.

Different Predictors of Right and Left Ventricular Metabolism in Healthy Middle-Aged Men.

Dysfunction of the right ventricle (RV) plays a crucial role in the outcome of various cardiovascular diseases. Previous studies on RV metabolism are sparse although evidence implies it may differ from left ventricular (LV) metabolism. Therefore, the aims of this study were (1) to determine predictors of RV glucose uptake (GU) and free fatty acid uptake (FFAU) and (2) to compare them to predictors of LV metabolism in healthy middle-aged men. Altogether 28 healthy, sedentary, middle-aged (40–55 years) men were studied. Insulin-stimulated GU and fasting FFAU were measured by positron emission tomography and RV and LV structural and functional parameters by cardiac magnetic resonance. Several parameters related to whole-body health were also measured. Predictors of RV and LV metabolism were determined by pairwise correlation analysis, lasso regression models, and variable clustering using heatmap. RVGU was most strongly predicted by age and moderately by RV ejection fraction (EF). The strongest determinants of RVFFAU were exercise capacity (peak oxygen uptake), resting heart rate, LVEF, and whole-body insulin-stimulated glucose uptake rate. When considering LV metabolism, age and RVEF were associated also with LVGU. In addition, LVGU was strongly, and negatively, influenced by whole-body insulin-stimulated glucose uptake rate. LVFFAU was predicted only by LVEF. This study shows that while RV and LV metabolism have shared characteristics, they also have unique properties. Age of the subject should be taken into account when measuring myocardial glucose utilization. Ejection fraction is related to myocardial metabolism, and even so that RVEF may be more closely related to GU of both ventricles and LVEF to FFAU of both ventricles, a finding supporting the ventricular interdependence. However, only RV fatty acid utilization associates with exercise capacity so that better physical fitness in a relatively sedentary population is related with decreased RV fat metabolism. To conclude, this study highlights the need for further study designed specifically on less-known RV, as the results on LV metabolism and physiology may not be directly applicable to the RV.

Effect of aging on muscle mitochondrial substrate utilization in humans

Previous studies have implicated age-associated reductions in mitochondrial oxidative phosphorylation activity in skeletal muscle as a predisposing factor for intramyocellular lipid (IMCL) accumulation and muscle insulin resistance (IR) in the elderly. To further investigate potential alterations in muscle mitochondrial function associated with aging, we assessed basal and insulin-stimulated rates of muscle pyruvate dehydrogenase (VPDH) flux relative to citrate synthase flux (VCS) in healthy lean, elderly subjects and healthy young body mass index- and activity-matched subjects. VPDH/VCS flux was assessed from the (13)C incorporation from of infused [1-13C] glucose into glutamate [4-13C] relative to alanine [3-13C] assessed by LC-tandem MS in muscle biopsies. Insulin-stimulated rates of muscle glucose uptake were reduced by 25% (P<0.01) in the elderly subjects and were associated with ∼70% (P<0.04) increase in IMCL, assessed by 1H magnetic resonance spectroscopy. Basal VPDH/VCS fluxes were similar between the groups (young: 0.20±0.03; elderly: 0.14±0.03) and increased approximately threefold in the young subjects following insulin stimulation. However, this increase was severely blunted in the elderly subjects (young: 0.55±0.04; elderly: 0.18±0.02, P=0.0002) and was associated with an ∼40% (P=0.004) reduction in insulin activation of Akt. These results provide new insights into acquired mitochondrial abnormalities associated with aging and demonstrate that age-associated reductions in muscle mitochondrial function and increased IMCL are associated with a marked inability of mitochondria to switch from lipid to glucose oxidation during insulin stimulation.

The cholesterol-lowering agent methyl-β-cyclodextrin promotes glucose uptake via GLUT4 in adult muscle fibers and reduces insulin resistance in obese mice.

Insulin stimulates glucose uptake in adult skeletal muscle by promoting the translocation of GLUT4 glucose transporters to the transverse tubule (T-tubule) membranes, which have particularly high cholesterol levels. We investigated whether T-tubule cholesterol content affects insulin-induced glucose transport. Feeding mice a high-fat diet (HFD) for 8 wk increased by 30% the T-tubule cholesterol content of triad-enriched vesicular fractions from muscle tissue compared with triads from control mice. Additionally, isolated muscle fibers (flexor digitorum brevis) from HFD-fed mice showed a 40% decrease in insulin-stimulated glucose uptake rates compared with fibers from control mice. In HFD-fed mice, four subcutaneous injections of MβCD, an agent reported to extract membrane cholesterol, improved their defective glucose tolerance test and normalized their high fasting glucose levels. The preincubation of isolated muscle fibers with relatively low concentrations of MβCD increased both basal and insulin-induced glucose uptake in fibers from controls or HFD-fed mice and decreased Akt phosphorylation without altering AMPK-mediated signaling. In fibers from HFD-fed mice, MβCD improved insulin sensitivity even after Akt or CaMK II inhibition and increased membrane GLUT4 content. Indinavir, a GLUT4 antagonist, prevented the stimulatory effects of MβCD on glucose uptake. Addition of MβCD elicited ryanodine receptor-mediated calcium signals in isolated fibers, which were essential for glucose uptake. Our findings suggest that T-tubule cholesterol content exerts a critical regulatory role on insulin-stimulated GLUT4 translocation and glucose transport and that partial cholesterol removal from muscle fibers may represent a useful strategy to counteract insulin resistance.

Muscle-specific activation of Ca2+/calmodulin-dependent protein kinase IV increases whole-body insulin action in mice

Aerobic exercise increases muscle glucose and improves insulin action through numerous pathways, including activation of Ca(2+)/calmodulin-dependent protein kinases (CAMKs) and peroxisome proliferator γ coactivator 1α (PGC-1α). While overexpression of PGC-1α increases muscle mitochondrial content and oxidative type I fibres, it does not improve insulin action. Activation of CAMK4 also increases the content of type I muscle fibres, PGC-1α level and mitochondrial content. However, it remains unknown whether CAMK4 activation improves insulin action on glucose metabolism in vivo. The effects of CAMK4 activation on skeletal muscle insulin action were quantified using transgenic mice with a truncated and constitutively active form of CAMK4 (CAMK4([Symbol: see text])) in skeletal muscle. Tissue-specific insulin sensitivity was assessed in vivo using a hyperinsulinaemic-euglycaemic clamp and isotopic measurements of glucose metabolism. The rate of insulin-stimulated whole-body glucose uptake was increased by ∼25% in CAMK4([Symbol: see text]) mice. This was largely attributed to an increase of ∼60% in insulin-stimulated glucose uptake in the quadriceps, the largest hindlimb muscle. These changes were associated with improvements in insulin signalling, as reflected by increased phosphorylation of Akt and its substrates and an increase in the level of GLUT4 protein. In addition, there were extramuscular effects: CAMK4([Symbol: see text]) mice had improved hepatic and adipose insulin action. These pleiotropic effects were associated with increased levels of PGC-1α-related myokines in CAMK4([Symbol: see text]) skeletal muscle. Activation of CAMK4 enhances mitochondrial biogenesis in skeletal muscle while also coordinating improvements in whole-body insulin-mediated glucose.

Mitochondrial fragmentation impairs insulin-dependent glucose uptake by modulating Akt activity through mitochondrial Ca2+ uptake

Insulin is a major regulator of glucose metabolism, stimulating its mitochondrial oxidation in skeletal muscle cells. Mitochondria are dynamic organelles that can undergo structural remodeling in order to cope with these ever-changing metabolic demands. However, the process by which mitochondrial morphology impacts insulin signaling in the skeletal muscle cells remains uncertain. To address this question, we silenced the mitochondrial fusion proteins Mfn2 and Opa1 and assessed insulin-dependent responses in L6 rat skeletal muscle cells. We found that mitochondrial fragmentation attenuates insulin-stimulated Akt phosphorylation, glucose uptake and cell respiratory rate. Importantly, we found that insulin induces a transient rise in mitochondrial Ca(2+) uptake, which was attenuated by silencing Opa1 or Mfn2. Moreover, treatment with Ruthenium red, an inhibitor of mitochondrial Ca(2+) uptake, impairs Akt signaling without affecting mitochondrial dynamics. All together, these results suggest that control of mitochondrial Ca(2+) uptake by mitochondrial morphology is a key event for insulin-induced glucose uptake.

A Modified Protocol to Maximize Differentiation of Human Preadipocytes and Improve Metabolic Phenotypes

Adipose stromal cells proliferate and differentiate into adipocytes, providing a valuable model system for studies of adipocyte biology. We compared differentiation protocols for human preadipocytes and report on their metabolic phenotypes. By simply prolonging the adipogenic induction period from the first 3 days to 7 days, the proportion of cells (passage 5–6) acquiring adipocyte morphology increased from 30–70% to over 80% in human subcutaneous preadipocytes. These morphological changes were accompanied by increases in the adipogenic marker expression and improved adipocyte metabolic phenotypes: enhanced responses to beta-adrenergically-stimulated lipolysis and to insulin-stimulated glucose metabolism into triglyceride. Confirming previous studies, FBS dose-dependently inhibited adipogenesis. However, in subcutaneous preadipocytes that differentiate well (donor-dependant high capacity and subcultured fewer than 2 times), the use of 7d-induction protocols in both 3% FBS and serum-free conditions allowed >80% differentiation. Responsiveness to β-adrenergically stimulated lipolysis was lower in 3% FBS. Rates of insulin-stimulated glucose uptake were higher in adipocytes differentiated with 3% FBS, while the sensitivity to insulin was almost identical between the two groups. In summary, extending the length of the induction period in adipogenic cocktail improves the degree of differentiation and responses to key metabolic hormones. This protocol permits functional analysis of metabolic phenotypes in valuable primary human adipocyte cultures through multiple passages.

Walnut leaves against diabetes mellitus? Potential mechanism and active principle

Walnut (Juglans regia – JR) is one of the medicinal plants used against diabetes mellitus in Austrian folk medicine. The anti-diabetic effect of Folium Juglandis has also been scientifically proven by several in vivo studies in mouse models. The present study is designed to scrutinize the active anti-diabetic principle of JR leaves and to achieve first hints of its physiological mechanism of action. The chlorophyll-free methanol extract of JR leaves (JR wC) enhanced the basal glucose uptake in C2C12 myocytes by 1.5 times at concentrations of 25µg/mL compared to untreated cells. Compared with the solvent DMSO, this extract elicted more than doubled insulin-stimulated cellular glucose uptake rate. These effects could partly be explained due toby the inhibition of protein tyrosine phosphatase 1B (PTP 1B). JR wC inhibited PTP 1B by about 80% at a concentration of 25µg/mL. Further separation of this fraction resulted in decrease and dispartment of activity, suggesting synergy between the individual components of JR wC. LC-MS analyses of the active JR wC extract led to the identification of chlorogenic acid, cumaroylquinic acid and naphthoquinone-hexoside, as well as eight flavonoids. Two of the flavonoids were identified as hyperoside and avicularin by comparison with reference compounds. The sugar moiety of three further flavonoids was determined by trimethylsilyl-derivatisation and GC-MS analyses leading to quercetin-L-arabinoside, quercetin-L-rhamnoside and kaempferol-L-arabinoside.

Mitochondrial function, but not glucose uptake, is disrupted in skeletal muscle from Bmal1−/− mice

Molecular clocks regulate circadian rhythms including muscle function and metabolism. Mice lacking Bmal1, a circadian clock transcription factor, showed a decreased in mitochondrial density in skeletal muscle. We hypothesize that mitochondrial function and glucose uptake are compromised in skeletal muscle of Bmal1−/− mice. O2 consumption was measured by polarography in mitochondria from 18 week‐old gastrocnemius muscles. Content of respiratory complexes, UCP3 and VDAC1 was measured in isolated mitochondria by western blot. Glucose uptake was measured with [3H] glucose upon insulin stimulation in the soleus muscles of 13 and 36 week‐old mice. Blood glucose was measured using a commercial glucose meter in 7, 13 and 36 week‐old mice. Serum insulin was measured with an Elisa kit in 13 and 36 week‐old mice. Respiration states 3 (ADP‐driven) and 5 (uncoupled) were 47±6% and 50±8% lower in mitochondria isolated from the gastrocnemius muscles of Bmal1−/− vs. control mice. There were no differences in the content of respiratory complexes, UCP3 and VDAC1. Blood glucose was 61% higher in 7 week‐old Bmal1−/− mice but there was no difference at other ages. Serum insulin was significantly lower in 36 week‐old Bmal1−/− mice. Basal and insulin‐stimulated glucose uptake rates were not different in soleus muscles at any age. We suggest that the molecular clock regulates mitochondrial function but not glucose uptake in mice.

Generation of a Dominant‐Negative Glycogen Targeting Subunit for Protein Phosphatase‐1

Modulation of the expression of the protein phosphatase-1 (PP1) glycogen-targeting subunit PTG exerts profound effects on cellular glycogen metabolism in vitro and in vivo. PTG contains three distinct binding domains for glycogen, PP1, and a common site for glycogen synthase and phosphorylase. The impact of disrupting the PP1-binding domain on PTG function was examined in 3T3-L1 adipocytes. A full-length PTG mutant was generated as an adenoviral construct in which the valine and phenylalanine residues in the conserved PP1-binding domain were mutated to alanine (PTG-VF). Infection of fully differentiated 3T3-L1 adipocytes with the PTG-VF adenovirus reduced glycogen stores by over 50%. In vitro, PTG-VF competitively interfered with wild-type PTG action, suggesting that the mutant construct acted as a dominant-negative molecule. The reduction in cellular glycogen storage was due to a significantly increased rate of glycogen turnover. Interestingly, acute basal and insulin-stimulated glucose uptake and glycogen synthesis rates were enhanced in PTG-VF expressing cells vs. control 3T3-L1 adipocytes, likely as a compensatory response to the loss of glycogen stores. These results indicate that the mutation of the PP1-binding domain on PTG resulted in the generation of a dominant-negative molecule that impeded endogenous PTG action and reduced cellular glycogen levels, through enhancement of glycogenolysis rather than impairment of glycogen synthesis.

A robust assay measuring GLUT4 translocation in rat myoblasts overexpressing GLUT4-myc and AS160_v2

Muscle and fat cells translocate GLUT4 (glucose transporter 4) to the plasma membrane when stimulated by insulin. Usually, this event is measured in differentiated adipocytes, myotubes, or cell lines overexpressing tagged GLUT4 by immunostaining. However, measurement of the translocation in differentiated adipocytes or myotubes or GLUT4 overexpressing cell lines is difficult because of high assay variability caused by either the differentiation protocol or low assay sensitivity. We recently reported the identification of a novel splice variant of AS160 (substrate of 160kDa), namely AS160_v2, and showed that its coexpression with GLUT4 in L6 myoblasts increased the insulin-stimulated glucose uptake rate due to an increased amount of GLUT4 on the cell surface. L6 cells, which coexpress myc-tagged GLUT4 and AS160_v2, can be efficiently used to generate an assay useful for identifying compounds that affect cellular responses to insulin. We compared the EC50 values for radioactive glucose uptake and GLUT4 translocation of different insulins and several small molecules to validate the assay. The use of L6 cells overexpressing AS160_v2 can be considered as a novel tool for the characterization of molecules modulating insulin signaling and GLUT4 translocation, and an image-based assay increases our confidence in the mode of action of the compounds identified.

Mildronate decreases carnitine availability and up-regulates glucose uptake and related gene expression in the mouse heart

Aims l-carnitine has been shown to play a central role in both fat and carbohydrate metabolisms. This study investigated whether acute and long-term treatments with an l-carnitine biosynthesis inhibitor, mildronate (3-(2,2,2-trimethylhydrazinium) propionate), modulate glucose uptake. Main methods The effects of acute and long-term administration of mildronate at a dose of 200 mg/kg (i.p. daily for 20 days) were tested in mouse blood plasma and heart. Key findings Acute administration of mildronate in vivo, or in vitro administration with perfusion buffer in isolated heart experiments, did not induce any effects on glucose blood concentration and uptake in the heart. Mildronate long-term treatment significantly decreased carnitine concentration in plasma and heart tissues, as well as increased the rate of insulin-stimulated glucose uptake by 35% and the expression of glucose transporter 4, hexokinase II, and insulin receptor proteins in mouse hearts. In addition, expression of both carnitine palmitoyltransferases IA and IB were significantly increased. Mildronate long-term treatment statistically significantly decreased fed state blood glucose from 6 ± 0.2 to 5 ± 0.1 mM, but did not affect plasma insulin and C-peptide levels. Significance Our experiments demonstrate for the first time that long-term mildronate treatment decreases carnitine content in the mouse heart and leads to increased glucose uptake and glucose metabolism-related gene expression.

Insulin-Stimulated Rates of Glucose Uptake in Muscle in Hyperthyroidism: The Importance of Blood Flow

In hyperthyroidism, although hepatic insulin resistance is well established, information on the effects of insulin on glucose uptake in skeletal muscle is variable. To investigate this, a meal was given to nine hyperthyroid (HR) and seven euthyroid (EU) subjects. Blood was withdrawn for 360 min from a forearm deep vein and the radial artery for measurements of insulin and glucose. Forearm blood flow (BF) was measured with strain-gauge plethysmography. Glucose flux was calculated as arteriovenous difference multiplied by BF and fractional glucose extraction as arteriovenous difference divided by arterial glucose concentrations. Both groups displayed comparable postprandial glucose levels, with the HR having higher insulin levels than the EU. In the forearm of HR vs. EU: 1) glucose flux was similar [area under the curve (AUC)(0-360) 673 +/- 143 vs. 826 +/- 157 micromol per 100 ml tissue]; 2) BF was increased (AUC(0-360) 3076 +/- 338 vs. 1745 +/- 145 ml per 100 ml tissue, P = 0.005); and 3) fractional glucose extraction was decreased (AUC(0-360) 14.5 +/- 3 vs. 32 +/- 5%min, P = 0.03). These results suggest that, in hyperthyroidism, insulin-stimulated glucose uptake in muscle is impaired; this defect is corrected, at least in part, by the increases in BF.

Visceral Obesity is Associated with High Levels of Serum Squalene

To investigate the impact of visceral obesity on cholesterol metabolism in normoglycemic offspring of patients with type 2 diabetes. The proportion of intra-abdominal fat (IAF) was measured by abdominal computer tomography, and serum cholesterol synthesis and absorption markers were determined by gas-liquid chromatography in 109 normoglycemic offspring of patients with type 2 diabetes. Insulin action was measured with the hyperinsulinemic euglycemic clamp. The gene encoding squalene synthase (farnesyl-diphosphate farnesyltransferase 1) was screened with the single-strand conformation polymorphism analysis and direct sequencing. Cholesterol synthesis markers correlated positively with IAF (r = 0.213 to 0.309, p < or = 0.027) and negatively with the rates of insulin-stimulated whole-body glucose uptake (r = -0.372 to -0.248, p < or = 0.010). However, serum squalene, the first measured precursor of cholesterol synthesis, showed a positive correlation with IAF (r = 0.309, p = 0.001) without any association with subcutaneous fat or insulin sensitivity. Variation in the farnesyl-diphosphate farnesyltransferase 1 gene did not explain elevated serum squalene levels in viscerally obese subjects. From the cholesterol absorption markers, cholestanol was associated negatively with IAF and positively with whole-body glucose uptake (p < 0.05). High serum squalene levels are associated with visceral obesity but not with subcutaneous obesity. Whether this finding is causally connected to visceral obesity remains to be established.

Liver steatosis coexists with myocardial insulin resistance and coronary dysfunction in patients with type 2 diabetes

Nonalcoholic fatty liver (NAFL) is a common comorbidity in patients with type 2 diabetes and links to the risk of coronary syndromes. The aim was to determine the manifestations of metabolic syndrome in different organs in patients with liver steatosis. We studied 55 type 2 diabetic patients with coronary artery disease using positron emission tomography. Myocardial perfusion was measured with [15O]H2O and myocardial and skeletal muscle glucose uptake with 2-deoxy-2-[18F]fluoro-D-glucose during hyperinsulinemic euglycemia. Liver fat content was determined by magnetic resonance proton spectroscopy. Patients were divided on the basis of their median (8%) into two groups with low (4.6 +/- 2.0%) and high (17.4 +/- 8.0%) liver fat content. The groups were well matched for age, BMI, and fasting plasma glucose. In addition to insulin resistance at the whole body level (P = 0.012) and muscle (P = 0.002), the high liver fat group had lower insulin-stimulated myocardial glucose uptake (P = 0.040) and glucose extraction rate (P = 0.0006) compared with the low liver fat group. In multiple regression analysis, liver fat content was the most significant explanatory variable for myocardial insulin resistance. In addition, the high liver fat group had increased concentrations of high sensitivity C-reactive protein, soluble forms of E-selectin, vascular adhesion protein-1, and intercellular adhesion molecule-1 (P < 0.05) and lower coronary flow reserve (P = 0.02) compared with the low liver fat group. In conclusion, in patients with type 2 diabetes and coronary artery disease, liver fat content is a novel independent indicator of myocardial insulin resistance and reduced coronary functional capacity. Further studies will reveal the effect of hepatic fat reduction on myocardial metabolism and coronary function.

Reduced mitochondrial density and increased IRS-1 serine phosphorylation in muscle of insulin-resistant offspring of type 2 diabetic parents

To further explore the nature of the mitochondrial dysfunction and insulin resistance that occur in the muscle of young, lean, normoglycemic, insulin-resistant offspring of parents with type 2 diabetes (IR offspring), we measured mitochondrial content by electron microscopy and insulin signaling in muscle biopsy samples obtained from these individuals before and during a hyperinsulinemic-euglycemic clamp. The rate of insulin-stimulated muscle glucose uptake was approximately 60% lower in the IR offspring than the control subjects and was associated with an approximately 60% increase in the intramyocellular lipid content as assessed by H magnetic resonance spectroscopy. Muscle mitochondrial density was 38% lower in the IR offspring. These changes were associated with a 50% increase in IRS-1 Ser312 and IRS-1 Ser636 phosphorylation and an approximately 60% reduction in insulin-stimulated Akt activation in the IR offspring. These data provide new insights into the earliest defects that may be responsible for the development of type 2 diabetes and support the hypothesis that reductions in mitochondrial content result in decreased mitochondrial function, which predisposes IR offspring to intramyocellular lipid accumulation, which in turn activates a serine kinase cascade that leads to defects in insulin signaling and action in muscle.