Knockdown of the gene encoding Drosophila tribbles homologue 3 (Trib3) improves insulin sensitivity through peroxisome proliferator-activated receptor-γ (PPAR-γ) activation in a rat model of insulin resistance.

AIMS/HYPOTHESIS: Insulin action is purportedly modulated by Drosophila tribbles homologue 3 (TRIB3), which in vitro prevents thymoma viral proto-oncogene (AKT) and peroxisome proliferator-activated receptor-γ (PPAR-γ) activation. However, the physiological impact of TRIB3 action in vivo remains controversial. METHODS: We investigated the role of TRIB3 in rats treated with either a control or Trib3 antisense oligonucleotide (ASO). Tissue-specific insulin sensitivity was assessed in vivo using a euglycaemic-hyperinsulinaemic clamp. A separate group was treated with the PPAR-γ antagonist bisphenol-A-diglycidyl ether (BADGE) to assess the role of PPAR-γ in mediating the response to Trib3 ASO. RESULTS: Trib3 ASO treatment specifically reduced Trib3 expression by 70% to 80% in liver and white adipose tissue. Fasting plasma glucose, insulin concentrations and basal rate of endogenous glucose production were unchanged. However, Trib3 ASO increased insulin-stimulated whole-body glucose uptake by ~50% during the euglycaemic-hyperinsulinaemic clamp. This was attributable to improved skeletal muscle glucose uptake. Despite the reduction of Trib3 expression, AKT2 activity was not increased. Trib3 ASO increased white adipose tissue mass by 70% and expression of Ppar-γ and its key target genes, raising the possibility that Trib3 ASO improves insulin sensitivity primarily in a PPAR-γ-dependent manner. Co-treatment with BADGE blunted the expansion of white adipose tissue and abrogated the insulin-sensitising effects of Trib3 ASO. Finally, Trib3 ASO also increased plasma HDL-cholesterol, a change that persisted with BADGE co-treatment. CONCLUSIONS/INTERPRETATION: These data suggest that TRIB3 inhibition improves insulin sensitivity in vivo primarily in a PPAR-γ-dependent manner and without any change in AKT2 activity.

The ATP/DNA ratio is a better indicator of islet cell viability than the ADP/ATP ratio.

Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio.

Glutamine is the major precursor for GABA synthesis in rat neocortex in vivo following acute GABA-transaminase inhibition.

The objective of the present study was to assess the degree to which astrocytic glutamine provides carbon for net synthesis of GABA in the rat neocortex in vivo. Isotopic labeling of GABA and glutamate from astrocytic glutamine was followed in halothane anesthetized and ventilated rats during an intravenous infusion of [2-(13)C]glucose. A net increase in GABA was achieved by administration of the GABA-transaminase inhibitor, gabaculine to suppress catabolism of GABA and recycling of (13)C label. (13)C Percentage enrichments of GABA, glutamate and glutamine were assessed in tissue extracts using (13)C-edited (1)H nuclear magnetic resonance at 8.4 T. GABA levels increased 2.6 micromol/g at 2 h and 6.1 micromol/g at 5 h after gabaculine, whereas glutamate and glutamine decreased in toto by 5.6 micromol/g at 2 h and 3.1 micromol/g at 5 h. Selective enrichment of glutamine, glutamate, and GABA C3's over other carbon positions was observed consistent with a precursor role for astrocytic glutamine. Between 1 h (control) and 3 h (gabaculine-treated) of [2-(13)C]glucose infusion, (13)C percentage enrichment increased in glutamine C3 (from 3.2+/-0.5 to 7.0+/-0.9%), glutamate C3 (from 1.8+/-0.5 to 3.4+/-0.9%), and GABA C3 (from 2.7+/-1.6 to 4.8+/-0.4%). The measured incremental [3-(13)C]GABA concentration (0.15 micromol/g) was close to the predicted value (0.13 micromol/g) that would be expected if the increase in GABA were produced entirely from glutamine compared to glutamate (0.07 micromol/g) based on the average precursor enrichments between 1 and 3 h. We conclude that glutamine is the major source of GABA carbon in the rat neocortex produced acutely following GABA-T inhibition by gabaculine in vivo.

Effect of triiodothyronine on mitochondrial energy coupling in human skeletal muscle.

The mechanism underlying the regulation of basal metabolic rate by thyroid hormone remains unclear. Although it has been suggested that thyroid hormone might uncouple substrate oxidation from ATP synthesis, there are no data from studies on humans to support this hypothesis. To examine this possibility, we used a novel combined (13)C/(31)P nuclear magnetic resonance (NMR) approach to assess mitochondrial energy coupling in skeletal muscle of seven healthy adults before and after three days of triiodothyronine (T(3)) treatment. Rates of ATP synthesis and tricarboxylic acid (TCA) cycle fluxes were measured by (31)P and (13)C NMR spectroscopy, respectively, and mitochondrial energy coupling was assessed as the ratio. Muscle TCA cycle flux increased by approximately 70% following T(3) treatment. In contrast, the rate of ATP synthesis remained unchanged. Given the disproportionate increase in TCA cycle flux compared with ATP synthesis, these data suggest that T(3) promotes increased thermogenesis in part by promoting mitochondrial energy uncoupling in skeletal muscle.

Effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion on in vivo glucose and lipid metabolism in lean and obese Zucker rats.

Activation of AMP-activated protein kinase (AMPK) with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofurano-side (AICAR) increases glucose transport in skeletal muscle via an insulin-independent pathway. To examine the effects of AMPK activation on skeletal muscle glucose transport activity and whole-body carbohydrate and lipid metabolism in an insulin-resistant rat model, awake obese Zuckerfa/fa rats (n = 26) and their lean (n = 23) littermates were infused for 90 min with AICAR, insulin, or saline. The insulin infusion rate (4 mU.kg(-1).min(-1)) was selected to match the glucose requirements during AICAR (bolus, 100 mg/kg; constant, 10 mg.kg(-1).min(-1)) isoglycemic clamps in the lean rats. The effects of these identical AICAR and insulin infusion rates were then examined in the obese Zucker rats. AICAR infusion increased muscle AMPK activity more than fivefold (P < 0.01 vs. control and insulin) in both lean and obese rats. Plasma triglycerides, fatty acid concentrations, and glycerol turnover, as assessed by [2-13C]glycerol, were all decreased in both lean and obese rats infused with AICAR (P < 0.05 vs. basal), whereas insulin had no effect on these parameters in the obese rats. Endogenous glucose production rates, measured by [U-13C]glucose, were suppressed by >50% during AICAR and insulin infusions in both lean and obese rats (P < 0.05 vs. basal). In lean rats, rates of whole-body glucose disposal increased by more than two-fold (P < 0.05 vs. basal) during both AICAR and insulin infusion; [3H]2-deoxy-D-glucose transport activity increased to a similar extent, by >2.2-fold (both P < 0.05 vs. control), in both soleus and red gastrocnemius muscles of lean rats infused with either AICAR or insulin. In the obese Zucker rats, neither AICAR nor insulin stimulated whole-body glucose disposal or soleus muscle glucose transport activity. However, AICAR increased glucose transport activity by approximately 2.4-fold (P < 0.05 vs. control) in the red gastrocnemius from obese rats, whereas insulin had no effect. In summary, acute infusion of AICAR in an insulin-resistant rat model activates skeletal muscle AMPK and increases glucose transport activity in red gastrocnemius muscle while suppressing endogenous glucose production and lipolysis. Because type 2 diabetes is characterized by diminished rates of insulin-stimulated glucose uptake as well as increased basal rates of endogenous glucose production and lipolysis, these results suggest that AICAR-related compounds may represent a new class of antidiabetic agents.

Stimulating effects of low-dose fructose on insulin-stimulated hepatic glycogen synthesis in humans.

Fructose has been shown to have a catalytic effect on glucokinase activity in vitro; however, its effects on hepatic glycogen metabolism in humans is unknown. To address this question, we used (13)C nuclear magnetic resonance (NMR) spectroscopy to noninvasively assess rates of hepatic glycogen synthesis and glycogenolysis under euglycemic (approximately 5 mmol/l) hyperinsulinemic conditions (approximately 400 pmol/l) with and without a low-dose infusion of fructose (approximately 3.5 micromol. kg(-1). min(-1)). Six healthy overnight-fasted subjects were infused for 4 h with somatostatin (0.1 micromol. kg(-1). min(-1)) and insulin (240 pmol. m(-2). min(-1)). During the initial 120 min, [1-(13)C]glucose was infused to assess glycogen synthase flux followed by an approximately 120-min infusion of unlabeled glucose to assess rates of glycogen phosphorylase flux. Acetaminophen was given to assess the percent contribution of the direct and indirect (gluconeogenic) pathways of glycogen synthesis by the (13)C enrichment of plasma UDP-glucuronide and C-1 of glucose. In the control studies, the flux through glycogen synthase and glycogen phosphorylase was 0.31 +/- 0.06 and 0.17 +/- 0.04 mmol/l per min, respectively, and the rate of net hepatic glycogen synthesis was 0.14 +/- 0.05 mmol/l per min. In the fructose studies, the glycogen synthase flux increased 2.5-fold to 0.79 +/- 0.16 mmol/l per min (P = 0.018 vs. control), whereas glycogen phosphorylase flux remained unchanged (0.24 +/- 0.06; P = 0.16 vs. control). The infusion of fructose resulted in a threefold increase in rates of net hepatic glycogen synthesis (0.54 +/- 0.12 mmol/l per min; P = 0.008 vs. control) without affecting the pathways of hepatic glycogen synthesis (direct pathway approximately 60% in both groups). We conclude that during euglycemic hyperinsulinemia, a low-dose fructose infusion causes a threefold increase in net hepatic glycogen synthesis exclusively through stimulation of glycogen synthase flux. Because net hepatic glycogen synthesis has been shown to be diminished in patients with poorly controlled type 1 and type 2 diabetes, stimulation of hepatic glycogen synthesis by this mechanism may be of potential therapeutic value.

Contribution of net hepatic glycogen synthesis to disposal of an oral glucose load in humans.

The contribution of hepatic glycogen synthesis to whole body glucose disposal after an oral glucose load was examined using (13)C nuclear magnetic resonance (NMR) spectroscopy to measure liver glycogen content in healthy, volunteers after an overnight fast. In group 1 (n = 14), hepatic glycogen synthesis was measured using (13)C-NMR spectroscopy for 240 minutes after ingestion of 98 +/- 1 g glucose. Liver volumes were measured using magnetic resonance imaging (MRI). To assess the direct (glucose --> glucose-6-P --> glucose-1-P --> uridine diphosphate (UDP)-glucose --> glycogen) and indirect (3-carbon units --> --> glycogen) pathways of liver glycogen synthesis, group 2 (n = 6) was studied with an identical glucose load enriched with [1-(13)C]glucose along with acetaminophen to noninvasively assess the (13)C enrichment in hepatic UDP-glucose. The fasting hepatic glycogen content was 305 +/- 17 mmol/L liver, and the liver volume was 1.46 +/- 0.07 L. For the initial 180 minutes after ingestion of glucose, hepatic glycogen concentrations increased linearly (r =.94, P =.0006) achieving a maximum concentration of 390 +/- 7 mmol/L liver and then remained constant until the end of the study. The mean maximum rate of net hepatic glycogen synthesis was 0.48 +/- 0.07 mmol/L liver-minute. Total liver glycogen synthesis could account for 16.7 +/- 3.8 g (17% +/- 4%) of the glucose ingested, and of this, 10.5 +/- 2.4 g (63% +/- 7%) was synthesized by the direct pathway. In conclusion, after ingestion of 98 g of glucose: (1) 16.7 +/- 3.8 g (17% +/- 4%) glucose was stored in the liver as glycogen, and (2) 63% +/- 7% (10.5 +/- 2.4 g) of this glycogen was formed via the direct pathway.

In vivo effects of uncoupling protein-3 gene disruption on mitochondrial energy metabolism.

To clarify the role of uncoupling protein-3 (UCP3) in skeletal muscle, we used NMR and isotopic labeling experiments to evaluate the effect of UCP3 knockout (UCP3KO) in mice on the regulation of energy metabolism in vivo. Whole body energy expenditure was determined from the turnover of doubly labeled body water. Coupling of mitochondrial oxidative phosphorylation in skeletal muscle was evaluated from measurements of rates of ATP synthesis (using (31)P NMR magnetization transfer experiments) and tricarboxylic acid (TCA) cycle flux (calculated from the time course of (13)C enrichment in C-4 and C-2 of glutamate during an infusion of [2-(13)C]acetate). At the whole body level, we observed no change in energy expenditure. However, at the cellular level, skeletal muscle UCP3KO increased the rate of ATP synthesis from P(i) more than 4-fold under fasting conditions (wild type, 2.2 +/- 0.6 versus knockout, 9.1 +/- 1.4 micromol/g of muscle/min, p < 0.001) with no change in TCA cycle flux rate (wild type, 0.74 +/- 0.04 versus knockout, 0.71 +/- 0.03 micromol/g of muscle/min). The increased efficiency of ATP production may account for the significant (p < 0.05) increase in the ratio of ATP to ADP in the muscle of UCP3KO mice (5.9 +/- 0.3) compared with controls (4.5 +/- 0.4). The data presented here provide the first evidence of uncoupling activity by UCP3 in skeletal muscle in vivo.

In vivo (13)C NMR measurement of neurotransmitter glutamate cycling, anaplerosis and TCA cycle flux in rat brain during.

The aims of this study were twofold: (i) to determine quantitatively the contribution of glutamate/glutamine cycling to total astrocyte/neuron substrate trafficking for the replenishment of neurotransmitter glutamate; and (ii) to determine the relative contributions of anaplerotic flux and glutamate/glutamine cycling to total glutamine synthesis. In this work in vivo and in vitro (13)C NMR spectroscopy were used, with a [2-(13)C]glucose or [5-(13)C]glucose infusion, to determine the rates of glutamate/glutamine cycling, de novo glutamine synthesis via anaplerosis, and the neuronal and astrocytic tricarboxylic acid cycles in the rat cerebral cortex. The rate of glutamate/glutamine cycling measured in this study is compared with that determined from re-analysis of (13)C NMR data acquired during a [1-(13)C]glucose infusion. The excellent agreement between these rates supports the hypothesis that glutamate/glutamine cycling is a major metabolic flux ( approximately 0.20 micromol/min/g) in the cerebral cortex of anesthetized rats and the predominant pathway of astrocyte/neuron trafficking of neurotransmitter glutamate precursors. Under normoammonemic conditions anaplerosis was found to comprise 19-26% of the total glutamine synthesis, whilst this fraction increased significantly during hyperammonemia ( approximately 32%). These findings indicate that anaplerotic glutamine synthesis is coupled to nitrogen removal from the brain (ammonia detoxification) under hyperammonemic conditions.

NMR spectroscopy in beta cell engineering and islet transplantation.

Islet transplantation is a promising method for restoring normoglycemia and alleviating the long term complications of diabetes. Widespread application of islet transplantation is hindered by the limited supply of human islets and requires a large increase in the availability of suitable insulin secreting tissue as well as robust quality assessment methodologies that can ensure safety and in vivo efficacy. We explore the application of nuclear magnetic resonance (NMR) spectroscopy in two areas relevant to beta cell engineering and islet transplantation: (1) the effect of genetic alterations on glucose metabolism, and (2) quality assessment of islet preparations prior to transplantation. Results obtained utilizing a variety of NMR techniques demonstrate the following: (1) Transfection of Rat1 cells with the c-myc oncogene (which may be involved in cell proliferation and cell cycle regulation) and overexpression of Bcl-2 (which may protect cells from stresses such as hypoxia and exposure to cytokines) introduce a wide array of alterations in cellular biochemistry, including changes in anaerobic and oxidative glucose metabolism, as assessed by 13C and 31P NMR spectroscopy. (2) Overnight incubation of islets and beta cells in the bottom of centrifuge tubes filled with medium at room temperature, as is sometimes done in islet transportation, exposes them to severe oxygen limitations that may cause cell damage. Such exposure, leading to reversible or irreversible damage, can be observed with NMR-detectable markers using conventional 13C and 31P NMR spectroscopy of extracts. In addition, markers of irreversible damage (as well as markers of hypoxia) can be detected and quantified without cell extraction using high-resolution magic angle spinning 1H NMR spectroscopy. Finally, acute ischemia in a bed of perfused beta cells leads to completely reversible changes that can be followed in real time with 31P NMR spectroscopy.

Mechanism of troglitazone action in type 2 diabetes.

To examine the metabolic pathways by which troglitazone improves insulin responsiveness in patients with type 2 diabetes, the rate of muscle glycogen synthesis was measured by 13C-nuclear magnetic resonance (NMR) spectroscopy. The rate-controlling steps of insulin-stimulated muscle glucose metabolism were assessed using 31P-NMR spectroscopic measurement of intramuscular glucose-6-phosphate (G-6-P) combined with a novel 13C-NMR method to assess intracellular glucose concentrations. Seven healthy nonsmoking subjects with type 2 diabetes were studied before and after completion of 3 months of troglitazone (400 mg/day) therapy. After troglitazone treatment, rates of insulin-stimulated whole-body glucose uptake increased by 58+/-11%, from 629+/-82 to 987+/-156 micromol x m(-2) x min(-1) (P = 0.008), which was associated with an approximately 3-fold increase in rates of insulin-stimulated glucose oxidation (from 119+/-41 to 424+/-70 micromol x m(-2) x min(-1); P = 0.018) and muscle glycogen synthesis (26+/-17 vs. 83+/-35 micromol x l(-1) muscle x min(-1); P = 0.025). After treatment, muscle G-6-P concentrations increased by 0.083+/-0.019 mmol/l (P = 0.008 vs. pretreatment) during the hyperglycemic-hyperinsulinemic clamp, compared with no significant changes in intramuscular G-6-P concentrations in the pretreatment study, reflecting an improvement in glucose transport and/or hexokinase activity. The concentrations of intracellular free glucose did not differ between the pre- and posttreatment studies and remained >50-fold lower in concentration (<0.1 mmol/l) than what would be expected if hexokinase activity was rate-controlling. These results indicate that troglitazone improves insulin responsiveness in skeletal muscle of patients with type 2 diabetes by facilitating glucose transport activity, which thereby leads to increased rates of muscle glycogen synthesis and glucose oxidation.

Regulation of myocardial [(13)C]glucose metabolism in conscious rats.

Administration of supplemental glucose and/or insulin is postulated to improve the outcome from myocardial ischemia by increasing the heart's relative utilization of glucose as an energy substrate. To examine the degree to which circulating glucose and insulin levels actually influence myocardial substrate preference in vivo, we infused conscious, chronically catheterized rats with D-[1-(13)C]glucose and compared steady-state (13)C enrichment of plasma glucose with that of myocardial glycolytic ([3-(13)C]alanine) and oxidative ([4-(13)C]glutamate) intermediary metabolites. In fasting rats, [3-(13)C]alanine-to-[1-(13)C]glucose and [4-(13)C]glutamate-to-[3-(13)C]alanine ratios averaged 0.16 +/- 0.12 and 0.14 +/- 0.03, respectively, indicating that circulating glucose contributed 32% of myocardial glycolytic flux, whereas subsequent flux through pyruvate dehydrogenase contributed 14% of total tricarboxylic acid (TCA) cycle activity. Raising plasma glucose to 11 mmol/l, or insulin to 500 pmol/l, increased these contributions equivalently. At supraphysiological (>6,500 pmol/l) insulin levels, the plasma glucose contribution to glycolysis increased further, and addition of hyperglycemia made it the sole glycolytic substrate, yet [4-(13)C]glutamate-to-[3-(13)C]alanine ratios remained /=40% of myocardial TCA cycle flux.

Persistent changes in myocardial glucose metabolism in vivo during reperfusion of a limited-duration coronary occlusion.

BACKGROUND: Rapid reperfusion of an occluded coronary artery salvages regional mechanical function, but this benefit may not be realized for hours or days because of postischemic stunning. Recovery from stunning is incompletely understood but may involve adaptive changes in heart glucose metabolism. METHODS AND RESULTS: To examine whether reversible coronary occlusion produces sustained changes in regional glucose metabolism in vivo, we performed a 20-minute left coronary artery occlusion followed by 24 hours of open-artery reperfusion in intact rats. Coronary occlusion produced stunning of the anterolateral left ventricle that resolved over 24 hours. When examined at 24 hours, reperfused regions were fully contractile and viable by vital staining and microscopy but demonstrated 25% reduction in blood flow and 50% increased uptake of circulating glucose, as estimated by in vivo [(13)N]NH(3) and [(18)F]fluorodeoxyglucose (FDG) tracer uptake. Reperfused regions had largely inactive glycogen synthase, low rates of glycogen synthesis, and persistent 50% glycogen depletion but increased flux of plasma [1-(13)C]glucose into myocardial [3-(13)C]alanine, indicating preferential shunting of imported glucose away from storage and into glycolysis. CONCLUSIONS: Sustained increases in regional glycolytic consumption of circulating glucose occur during reperfusion of a limited-duration coronary occlusion. This suggests a role for glycolytic ATP in the recovery from postischemic stunning in vivo. Furthermore, [(13)N]NH(3) /FDG regional mismatch may constitute a clinically accessible late metabolic signature of regional myocardial ischemia.

Intense exercise stimulates albumin synthesis in the upright posture.

We tested the hypothesis that an elevation in albumin synthetic rate contributes to increased plasma albumin content during exercise-induced hypervolemia. Albumin synthetic rate was measured in seven healthy subjects at 1-5 and 21-22 h after 72 min of intense (85% peak oxygen consumption rate) intermittent exercise and after 5 h recovery in either upright (Up) or supine (Sup) postures. Deuterated phenylalanine (d(5)-Phe) was administrated by a primed-constant infusion method, and fractional synthetic rate (FSR) and absolute synthetic rate (ASR) of albumin were calculated from the enrichment of d(5)-Phe in plasma albumin, determined by gas chromatography-mass spectrometry. FSR of albumin in Up increased significantly (P < 0.05) from 4.9 +/- 0.9%/day at control to 7.3 +/- 0.9%/day at 22 h of recovery. ASR of albumin increased from 87.9 +/- 17.0 to 141.1 +/- 16.6 mg albumin. kg body wt(-1). day(-1). In contrast, FSR and ASR of albumin were unchanged in Sup (3.9 +/- 0.4 to 4.0 +/- 1.4%/day and 74.2 +/- 8.9 to 85.3 +/- 23.9 mg albumin. kg body wt(-1). day(-1) at control and 22 h of recovery, respectively). Increased albumin synthesis after upright intense exercise contributes to the expansion of greater albumin content and its maintenance. We conclude that stimuli related to posture are critical in modulating the drive for albumin synthesis after intense exercise.

A critical evaluation of mass isotopomer distribution analysis of gluconeogenesis in vivo.

There are conflicting reports concerning the reliability of mass isotopomer distribution analysis (MIDA) for estimating the contribution of gluconeogenesis to total glucose production (f) during [(13)C]glycerol infusion. We have evaluated substrate-induced effects on rate of appearance (R(a)) of glycerol and glucose and f during [2-(13)C]glycerol infusion in vivo. Five groups of mice were fasted for 30 h and then infused with [2-(13)C]glycerol at variable rates and variable (13)C enrichments (group I: 20 micromol. kg(-1). min(-1), 99% (13)C; group II: 60 micromol. kg(-1). min(-1), 60% (13)C; group III: 60 micromol. kg(-1). min(-1), 99% (13)C; group IV: 120 micromol. kg(-1). min(-1), 40% (13)C; or group V: 120 micromol. kg(-1). min(-1), 99% (13)C). The total glycerol R(a) increased from approximately 104 to approximately 157 and to approximately 210 micromol. kg(-1). min(-1) as the infusion of [2-(13)C]glycerol increased from 20 to 60 and to 120 micromol. kg(- 1). min(-1), respectively. As the amount of 99% enriched [2-(13)C]glycerol increased from 20 to 60 and to 120 micromol. kg(-1). min(-1) (groups I, III, and V, respectively), plasma glycerol enrichment increased from approximately 21 to approximately 42 and to approximately 57% and the calculated f increased from approximately 27 to approximately 56 and to approximately 87%, respectively. Similar plasma glycerol enrichments were observed in groups I, II, and IV (i. e., approximately 21-24%), yet f increased from approximately 27 to approximately 57 and to approximately 86% in groups II and IV, respectively. Estimates of absolute gluconeogenesis increased from approximately 14 to approximately 33 and approximately 86 micromol. kg(-1). min(-1) as the infusion of [2-(13)C]glycerol increased from 20 to 60 and 120 micromol. kg(-1). min(-1). Plausible estimates of f were obtained only under conditions that increased total glycerol R(a) approximately 2-fold (P < 0.001) and increased glucose R(a) approximately 1.5-fold (P < 0.01) above basal. We conclude that in 30-h fasted mice, 1) estimates of f by MIDA with low infusion rates of [2-(13)C]glycerol yield erroneous results and 2) reasonable estimates of f are obtained at glycerol infusion rates that perturb glycerol and glucose metabolism.

Impaired glucose transport as a cause of decreased insulin-stimulated muscle glycogen synthesis in type 2 diabetes.

BACKGROUND: Insulin resistance, a major factor in the pathogenesis of type 2 diabetes mellitus, is due mostly to decreased stimulation of glycogen synthesis in muscle by insulin. The primary rate-controlling step responsible for the decrease in muscle glycogen synthesis is not known, although hexokinase activity and glucose transport have been implicated. METHODS: We used a novel nuclear magnetic resonance approach with carbon-13 and phosphorus-31 to measure intramuscular glucose, glucose-6-phosphate, and glycogen concentrations under hyperglycemic conditions (plasma glucose concentration, approximately 180 mg per deciliter [10 mmol per liter]) and hyperinsulinemic conditions in six patients with type 2 diabetes and seven normal subjects. In vivo microdialysis of muscle tissue was used to determine the gradient between plasma and interstitial-fluid glucose concentrations, and open-flow microperfusion was used to determine the concentrations of insulin in interstitial fluid. RESULTS: The time course and concentration of insulin in interstitial fluid were similar in the patients with diabetes and the normal subjects. The rates of whole-body glucose metabolism and muscle glycogen synthesis and the glucose-6-phosphate concentrations in muscle were approximately 80 percent lower in the patients with diabetes than in the normal subjects under conditions of matched plasma insulin concentrations. The mean (+/-SD) intracellular glucose concentration was 2.0+/-8.2 mg per deciliter (0.11+/-0.46 mmol per liter) in the normal subjects. In the patients with diabetes, the intracellular glucose concentration was 4.3+/-4.9 mg per deciliter (0.24+/-0.27 mmol per liter), a value that was 1/25 of what it would be if hexokinase were the rate-controlling enzyme in glucose metabolism. CONCLUSIONS: Impaired insulin-stimulated glucose transport is responsible for the reduced rate of insulin-stimulated muscle glycogen synthesis in patients with type 2 diabetes mellitus.

Free fatty acid-induced insulin resistance is associated with activation of protein kinase C theta and alterations in the insulin signaling cascade.

To examine the mechanism by which free fatty acids (FFAs) induce insulin resistance in vivo, awake chronically catheterized rats underwent a hyperinsulinemic-euglycemic clamp with or without a 5-h preinfusion of lipid/heparin to raise plasma FFA concentrations. Increased plasma FFAs resulted in insulin resistance as reflected by a approximately 35% reduction in the glucose infusion rate (P < 0.05 vs. control). The insulin resistance was associated with a 40-50% reduction in 13C nuclear magnetic resonance (NMR)-determined rates of muscle glycogen synthesis (P < 0.01 vs. control) and muscle glucose oxidation (P < 0.01 vs. control), which in turn could be attributed to a approximately 25% reduction in glucose transport activity as assessed by 2-[1,2-3H]deoxyglucose uptake in vivo (P < 0.05 vs. control). This lipid-induced decrease in insulin-stimulated muscle glucose metabolism was associated with 1) a approximately 50% reduction in insulin-stimulated insulin receptor substrate (IRS)-1-associated phosphatidylinositol (PI) 3-kinase activity (P < 0.05 vs. control), 2) a blunting in insulin-stimulated IRS-1 tyrosine phosphorylation (P < 0.05, lipid-infused versus glycerol-infused), and 3) a four-fold increase in membrane-bound, or active, protein kinase C (PKC) theta (P < 0.05 vs. control). We conclude that acute elevations of plasma FFA levels for 5 h induce skeletal muscle insulin resistance in vivo via a reduction in insulin-stimulated muscle glycogen synthesis and glucose oxidation that can be attributed to reduced glucose transport activity. These changes are associated with abnormalities in the insulin signaling cascade and may be mediated by FFA activation of PKC theta.

Effects of free fatty acids on glucose transport and IRS-1-associated phosphatidylinositol 3-kinase activity.

To examine the mechanism by which free fatty acids (FFA) induce insulin resistance in human skeletal muscle, glycogen, glucose-6-phosphate, and intracellular glucose concentrations were measured using carbon-13 and phosphorous-31 nuclear magnetic resonance spectroscopy in seven healthy subjects before and after a hyperinsulinemic-euglycemic clamp following a five-hour infusion of either lipid/heparin or glycerol/heparin. IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity was also measured in muscle biopsy samples obtained from seven additional subjects before and after an identical protocol. Rates of insulin stimulated whole-body glucose uptake. Glucose oxidation and muscle glycogen synthesis were 50%-60% lower following the lipid infusion compared with the glycerol infusion and were associated with a approximately 90% decrease in the increment in intramuscular glucose-6-phosphate concentration, implying diminished glucose transport or phosphorylation activity. To distinguish between these two possibilities, intracellular glucose concentration was measured and found to be significantly lower in the lipid infusion studies, implying that glucose transport is the rate-controlling step. Insulin stimulation, during the glycerol infusion, resulted in a fourfold increase in PI 3-kinase activity over basal that was abolished during the lipid infusion. Taken together, these data suggest that increased concentrations of plasma FFA induce insulin resistance in humans through inhibition of glucose transport activity; this may be a consequence of decreased IRS-1-associated PI 3-kinase activity.

Differential effects of safflower oil versus fish oil feeding on insulin-stimulated glycogen synthesis, glycolysis, and pyruvate dehydrogenase flux in skeletal muscle: a 13C nuclear magnetic resonance study.

To examine the effects of safflower oil versus fish oil feeding on in vivo intramuscular glucose metabolism and relative pyruvate dehydrogenase (PDH) versus tricarboxylic acid (TCA) cycle flux, rats were pair-fed on diets consisting of 1) 59% safflower oil, 2) 59% menhaden fish oil, or 3) 59% carbohydrate (control) in calories. Rates of glycolysis and glycogen synthesis were assessed by monitoring [1-(13)C]glucose label incorporation into [1-(13)C]glycogen, [3-(13)C]lactate, and [3-(13)C]alanine in the hindlimb of awake rats via 13C nuclear magnetic resonance (NMR) spectroscopy during a euglycemic (approximately 6 mmol/l) hyperinsulinemic (approximately 180 microU/ml) clamp. A steady-state isotopic analysis of lactate, alanine, and glutamate was used to determine the relative PDH versus TCA cycle flux present in muscle under these conditions. The safflower oil-fed rats were insulin resistant compared with control and fish oil-fed rats, as reflected by a markedly reduced glucose infusion rate (Ginf) during the clamp (21.4 +/- 2.3 vs. 31.6 +/- 2.8 and 31.7 +/- 1.9 mg x kg(-1) x min(-1) in safflower oil versus control and fish oil groups, respectively, P < 0.006). This decrease in insulin-stimulated glucose disposal in the safflower oil group was associated with a lower rate of glycolysis (21.7 +/- 2.2 nmol x g(-1) x min(-1)) versus control (62.1 +/- 10.3 nmol x g(-1) x min(-1), P < 0.001) and versus fish oil (45.7 +/- 6.7 nmol x g(-1) x min(-1), P < 0.04), as no change in glycogen synthesis (103 +/- 15, 133 +/- 19, and 125 +/- 14 nmol x g(-1) x min(-1) in safflower oil, fish oil, and control, respectively) was detected. The intramuscular triglyceride (TG) content was increased in the safflower oil group (7.3 +/- 0.8 micromol/g) compared with the control group (5.2 +/- 0.8 micromol/g, P < 0.05) and the fish oil group (3.6 +/- 1.1 micromol/g, P < 0.01). Conversely, the percent PDH versus TCA cycle flux was decreased in the safflower oil (43 +/- 8%) versus the control (73 +/- 8%, P < 0.01) and fish oil (64 +/- 6%, P < 0.05) groups. These data suggest that the reduced insulin-stimulated glucose disposal attributed to safflower oil feeding was a consequence of reduced glycolytic flux associated with an increase in relative free fatty acid/ketone oxidation versus TCA cycle flux, whereas fish oil feeding did not alter glucose metabolism and may in part be protective of insulin-stimulated glucose disposal by limiting intramuscular TG deposition.

Mechanism by which glucose and insulin inhibit net hepatic glycogenolysis in humans.

13C NMR spectroscopy was used to assess flux rates of hepatic glycogen synthase and phosphorylase in overnight-fasted subjects under one of four hypoglucagonemic conditions: protocol I, hyperglycemic (approximately 10 mM) -hypoinsulinemia (approximately 40 pM); protocol II, euglycemic (approximately 5 mM) -hyperinsulinemia (approximately 400 pM); protocol III, hyperglycemic (approximately 10 mM) -hyperinsulinemia (approximately 400 pM); and protocol IV; euglycemic (approximately 5 mM) -hypoinsulinemia (approximately 40 pM). Inhibition of net hepatic glycogenolysis occurred in both protocols I and II compared to protocol IV but via a different mechanism. Inhibition of net hepatic glycogenolysis occurred in protocol I mostly due to decreased glycogen phosphorylase flux, whereas in protocol II inhibition of net hepatic glycogenolysis occurred exclusively through the activation of glycogen synthase flux. Phosphorylase flux was unaltered, resulting in extensive glycogen cycling. Relatively high rates of net hepatic glycogen synthesis were observed in protocol III due to combined stimulation of glycogen synthase flux and inhibition of glycogen phosphorylase flux. In conclusion, under hypoglucagonemic conditions: (a) hyperglycemia, per se, inhibits net hepatic glycogenolysis primarily through inhibition of glycogen phosphorylase flux; (b) hyperinsulinemia, per se, inhibits net hepatic glycogenolysis primarily through stimulation of glycogen synthase flux; (c) inhibition of glycogen phosphorylase and the activation of glycogen synthase are not necessarily coupled and coordinated in a reciprocal fashion; and (d) promotion of hepatic glycogen cycling may be the principal mechanism by which insulin inhibits net hepatic glycogenolysis and endogenous glucose production in humans under euglycemic conditions.