Impact of gain-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) on glucose and lipid homeostasis.

LRP5 loss-of-function mutations have been shown to cause profound osteoporosis and have been associated with impaired insulin sensitivity and dysregulated lipid metabolism. We hypothesized that gain-of-function mutations in LRP5 would also affect these parameters. We therefore studied individuals with LRP5 gain-of-function mutations exhibiting high bone mass (HBM) phenotypes and found that while there was no detected change in insulin sensitivity, there was a significant reduction in serum LDL. INTRODUCTION: Wnt signaling through LRP5 represents a newly appreciated metabolic pathway, which potentially represents a target for drug discovery in type 2 diabetes and hyperlipidemia. Studies in animal models suggest a physiologic link between LRP5 and glucose and lipid homeostasis; however, whether it plays a similar role in humans is unclear. As current literature links loss-of-function LRP5 to impaired glucose and lipid metabolism, we hypothesized that individuals with an HBM-causing mutation in LRP5 would exhibit improved glucose and lipid homeostasis. Since studies in animal models have suggested that Wnt signaling augments insulin secretion, we also examined the effect of Wnt signaling on glucose-stimulated insulin secretion on human pancreatic islets. METHODS: This was a matched case-control study. We used several methods to assess glucose and lipid metabolism in 11 individuals with HBM-causing mutations in LRP5. Affected study participants were recruited from previously identified kindreds with HBM-causing LRP5 mutations and included 9 males and 2 females. Two subjects that were being treated with insulin for type 2 diabetes were excluded from our analysis, as this would have obscured our ability to determine the impact of gain-of-function LRP5 mutations on glucose metabolism. The mean age of the evaluated study subjects was 55 ± 7 with a mean BMI of 27.2 ± 2.0. Control subjects were matched and recruited from the general community at an equivalent ratio, with 18 males and 4 females (mean age 56 ± 4; mean BMI 27.2 ± 1.0). Study testing was conducted at an academic medical center. RESULTS: There were no statistically significant differences between affected and matched control populations for HbA1c (p = 0.06), eAG (p = 0.06), insulin (p = 0.82), HOMA-B (p = 0.34), or HOMA-IR (p = 0.66). The mean Insulin Sensitivity Index (ISI) was also similar between control and affected individuals. Total cholesterol (p = 0.43), triglycerides (TG) (p = 0.56), and HDL (p = 0.32) were not different between the same two groups. In a small subset of studied subjects, intramyocellular and hepatic lipid content were similar in the affected individuals and controls when quantified by proton magnetic resonance spectroscopy (MRS). However, the mean value for serum LDL was significantly lower (p = 0.04) in affected individuals. In primary human islets, there were no differences between control and Wnt treatment groups for insulin secretion measured as area under the curve (AUC) for first phase (p = 0.17) or second phase (p = 0.33) insulin secretion. CONCLUSIONS: Although our sample size was small, our data do not support the hypothesis that HBM-causing LRP5 mutations, associated with increased Wnt signaling, improve glucose metabolism in humans. However, it does appear that LRP5 variants may affect LDL metabolism, a major risk factor for coronary artery disease. The molecular mechanisms underpinning this effect warrant further study.

High prevalence of diabetes and pre-diabetes in adults with Williams syndrome.

A standard oral glucose tolerance test (OGTT) was administered to 28 adults with Williams syndrome (WS). Three quarters of the WS subjects showed abnormal glucose curves, meeting diagnostic criteria for either diabetes or the pre-diabetic state of impaired glucose tolerance. Fasting mean glucose and median insulin levels did not differ significantly in the total WS cohort versus age-gender-BMI matched controls, though the glucose area under the curve was greater in the WS subjects. HbA1c levels were not as reliable as the OGTT in diagnosing the presence of diabetes. Given the high prevalence of impaired glucose regulation, adults with WS should be screened for diabetes, and when present should be treated in accordance with standard medical practice. Hemizygosity for a gene mapping to the Williams syndrome chromosome region (WSCR) is likely the major factor responsible for the high frequency of diabetes in WS. Syntaxin-1A is a prime candidate gene based on its location in the WSCR, its role in insulin release, and the presence of abnormal glucose metabolism in mouse models with aberrantly expressed Stx-1a.

13C/31P NMR studies on the role of glucose transport/phosphorylation in human glycogen supercompensation.

This study measured muscle glycogen during a 7-day carbohydrate loading protocol. Twenty healthy subjects (12 male, 8 female) performed 1 hr treadmill/toe-raise exercise immediately before a 3-day low carbohydrate (LoCHO) diet (20 % carbohydrate, 60 % fat, 20 % protein). On day 3 they repeated the exercise and began a 4-day high carbohydrate (HiCHO) diet (90 % carbohydrate, 2 % fat, 8 % protein). The order of administration of the diet was reversed in a subpopulation (n = 3). Interleaved natural abundance 13C/ 31P NMR spectra were obtained before and immediately after exercise, and each day during the controlled diets in order to determine concentrations of glycogen (GLY), glucose-6-phosphate (G6P), and muscle pH. Following exercise, muscle GLY and pH were reduced (p < 0.001) while muscle G6P was elevated (p

Effects of a novel glucagon receptor antagonist (Bay 27-9955) on glucagon-stimulated glucose production in humans.

AIMS/HYPOTHESIS: To study the effects of a specific glucagon receptor antagonist (Bay 27-9955), on plasma glucose concentrations and rates of glucose production in response to hyperglucagonaemia in humans. METHODS: The study was conducted as a two-dose [Low Dose Bay 27-9955 70 mg, (n = 6), High Dose Bay 27-9955 200 mg, (n = 8)], double blind, placebo controlled, crossover study. Basal glucose production was measured after an overnight fast with [6,6-2H]. At 0 min Bay 27-9955 or placebo was administered and at 120 min an infusion of somatostatin [0.1 microg x (kg x min)(-1)], insulin [24 pmol x (m2 x min)(-1)] and glucagon [3 ng x (kg x min)(-1)] was initiated. RESULTS: Basal plasma glucose concentrations were about 5 mmol/l and basal rates of glucose production were about 13 micromol x (kg x min)(-1). During the hyperglucagonaemic period, plasma glucagon concentrations doubled to 100 pg/ml, plasma glucose concentration increased by 75 % to a peak of about 10 mmol/l and glucose production doubled to about 23 micromol x (kg x min)(-1) (p < 0.0001 vs basal). In the High Dose Group these effects of glucagon were markedly blunted, plasma glucose concentrations were 7.6 +/- 1.1 mmol/l (p = 0.012 vs placebo) and rates of glucose production increased minimally to 15.3 +/- 1.9 micromol x (kg-min)(-1) (p < 0.0003 vs placebo]. In the Low Dose Group, there was a proportional decrease in the effects of Bay 27-9955 on these parameters. CONCLUSION/INTERPRETATION: Bay 27-9955 is an effective and safe glucagon antagonist in humans. Given the potentially important role of glucagon in increasing glucose production and gluconeogenesis in patients with Type II (non-insulin-dependent) diabetes mellitus this agent could represent an innovative class of therapeutic agents for the disease.

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.

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.

Nuclear magnetic resonance studies of hepatic glucose metabolism in humans.

Nuclear magnetic resonance (NMR) spectroscopy has made noninvasive and repetitive measurements of human hepatic glycogen concentrations possible. Monitoring of liver glycogen in real-time mode has demonstrated that glycogen concentrations decrease linearly and that net hepatic glycogenolysis contributes only about 50 percent to glucose production during the early period of a fast. Following a mixed meal, hepatic glycogen represents approximately 20 percent of the ingested carbohydrates, while only about 10 percent of an intravenous glucose load is retained by the liver as glycogen. During mixed-meal ingestion, poorly controlled type 1 diabetic patients synthesize only about 30 percent of the glycogen stored in livers of nondiabetic humans studied under similar conditions. Reduced net glycogen synthesis can be improved but not normalized by short-term, intensified insulin treatment. A decreased increment in liver glycogen content following meals was also found in patients with maturity-onset diabetes of the young due to glucokinase mutations (MODY-2). In patients with poorly controlled type 2 diabetes, fasting hyperglycemia can be attributed mainly to increased rates of endogenous glucose production, which was found by 13C NMR to be due to increased rates of gluconeogenesis. Metformin treatment improved fasting hyperglycemia in these patients through a reduction in hepatic glucose production, which could be attributed to a decrease in gluconeogenesis. In conclusion, NMR spectroscopy has provided new insights into the pathogenesis of hyperglycemia in type 1, type 2, and MODY diabetes and offers the potential of providing new insights into the mechanism of action of novel antidabetic therapies.

Mechanism by which metformin reduces glucose production in type 2 diabetes.

To examine the mechanism by which metformin lowers endogenous glucose production in type 2 diabetic patients, we studied seven type 2 diabetic subjects, with fasting hyperglycemia (15.5 +/- 1.3 mmol/l), before and after 3 months of metformin treatment. Seven healthy subjects, matched for sex, age, and BMI, served as control subjects. Rates of net hepatic glycogenolysis, estimated by 13C nuclear magnetic resonance spectroscopy, were combined with estimates of contributions to glucose production of gluconeogenesis and glycogenolysis, measured by labeling of blood glucose by 2H from ingested 2H2O. Glucose production was measured using [6,6-2H2]glucose. The rate of glucose production was twice as high in the diabetic subjects as in control subjects (0.70 +/- 0.05 vs. 0.36 +/- 0.03 mmol x m(-2) min(-1), P < 0.0001). Metformin reduced that rate by 24% (to 0.53 +/- 0.03 mmol x m(-2) x min(-1), P = 0.0009) and fasting plasma glucose concentration by 30% (to 10.8 +/- 0.9 mmol/l, P = 0.0002). The rate of gluconeogenesis was three times higher in the diabetic subjects than in the control subjects (0.59 +/- 0.03 vs. 0.18 +/- 0.03 mmol x m(-2) min(-1) and metformin reduced that rate by 36% (to 0.38 +/- 0.03 mmol x m(-2) x min(-1), P = 0.01). By the 2H2O method, there was a twofold increase in rates of gluconeogenesis in diabetic subjects (0.42 +/- 0.04 mmol m(-2) x min(-1), which decreased by 33% after metformin treatment (0.28 +/- 0.03 mmol x m(-2) x min(-1), P = 0.0002). There was no glycogen cycling in the control subjects, but in the diabetic subjects, glycogen cycling contributed to 25% of glucose production and explains the differences between the two methods used. In conclusion, patients with poorly controlled type 2 diabetes have increased rates of endogenous glucose production, which can be attributed to increased rates of gluconeogenesis. Metformin lowered the rate of glucose production in these patients through a reduction in gluconeogenesis.

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.

Effects of caffeine on muscle glycogen utilization and the neuroendocrine axis during exercise.

To examine the effect of caffeine ingestion on muscle glycogen utilization and the neuroendocrine axis during exercise, we studied 20 muscle glycogen-loaded subjects who were given placebo or caffeine (6 mg/kg) in a double blinded fashion 90 min before cycling for 2 h at 65% of their maximal oxygen consumption. Exercise-induced glycogen depletion in the thigh muscle was noninvasively measured by means of 13C nuclear magnetic resonance spectroscopy (NMR) spectroscopy, and plasma concentrations of substrates and neuroendocrine hormones, including beta-endorphins, were also assessed. Muscle glycogen content was increased 140% above normal values on the caffeine trial day (P < 0.001). After cycling for 2 h, caffeine ingestion was associated with a greater increase in plasma lactate (caffeine: +1.0 +/- 0.2 mmol/L; placebo, +0.1 +/- 0.2 mmol/L; P < 0.005), epinephrine (caffeine, +223 +/- 82 pg/mL; placebo, +56 +/- 26 pg/mL; P < 0.05), and cortisol (caffeine, +12 +/- 3 mg/mL; placebo, +2 +/- 2 mg/mL; P < 0.001) levels. However, plasma free fatty acid concentrations increased (caffeine, +814 +/- 133 mmol/L; placebo, +785 +/- 85 mmol/L; P = NS), and muscle glycogen content decreased (caffeine, -57 +/- 6 mmol/L muscle; placebo, -53 +/- 5 mmol/L muscle; P = NS) to the same extent in both groups. At the same time, plasma beta-endorphin levels almost doubled (from 30 +/- 5 to 53 +/- 13 pg/mL; P < 0.05) in the caffeine-treated group, whereas no change occurred in the placebo group. We conclude that caffeine ingestion 90 min before prolonged exercise does not exert a muscle glycogen-sparing effect in athletes with high muscle glycogen content. However, these data suggest that caffeine lowers the threshold for exercise-induced beta-endorphin and cortisol release, which may contribute to the reported benefits of caffeine on exercise endurance.

Mechanism of muscle glycogen autoregulation in humans.

To examine the mechanism by which muscle glycogen limits its own synthesis, muscle glycogen and glucose 6-phosphate (G-6-P) concentrations were measured in seven healthy volunteers during a euglycemic ( approximately 5.5 mM)-hyperinsulinemic ( approximately 450 pM) clamp using (13)C/(31)P nuclear magnetic resonance spectroscopy before and after a muscle glycogen loading protocol. Rates of glycogen synthase (V(syn)) and phosphorylase (V(phos)) flux were estimated during a [1-(13)C]glucose (pulse)-unlabeled glucose (chase) infusion. The muscle glycogen loading protocol resulted in a 65% increase in muscle glycogen content that was associated with a twofold increase in fasting plasma lactate concentrations (P < 0.05 vs. basal) and an approximately 30% decrease in plasma free fatty acid concentrations (P < 0.001 vs. basal). Muscle glycogen loading resulted in an approximately 30% decrease in the insulin-stimulated rate of net muscle glycogen synthesis (P < 0.05 vs. basal), which was associated with a twofold increase in intramuscular G-6-P concentration (P < 0.05 vs. basal). Muscle glycogen loading also resulted in an approximately 30% increase in whole body glucose oxidation rates (P < 0.05 vs. basal), whereas there was no effect on insulin-stimulated rates of whole body glucose uptake ( approximately 10.5 mg. kg body wt(-1). min(-1) for both clamps) or glycogen turnover (V(syn)/V(phos) was approximately 23% for both clamps). In conclusion, these data are consistent with the hypothesis that glycogen limits its own synthesis through feedback inhibition of glycogen synthase activity, as reflected by an accumulation of intramuscular G-6-P, which is then shunted into aerobic and anaerobic glycolysis.

Intramuscular glycogen and intramyocellular lipid utilization during prolonged exercise and recovery in man: a 13C and 1H nuclear magnetic resonance spectroscopy study.

Depletion of muscle glycogen is considered a limiting performance factor during prolonged exercise, whereas the role of the intramyocellular lipid (IMCL) pool is not yet fully understood. We examined 1) intramyocellular glycogen and lipid utilization during prolonged exercise, 2) resynthesis of muscle glycogen and lipids during recovery, and 3) changes in glycogen content between nonexercising and exercising muscles during recovery. Subjects ran on a treadmill at submaximal intensity until exhaustion. Glycogen concentrations were assessed in thigh, calf, and nonexercising forearm muscle, and IMCL content was measured in soleus muscle using magnetic resonance spectroscopy techniques. At the time of exhaustion, glycogen depletion was 2-fold greater in calf than in thigh muscles, but a significant amount of glycogen was left in both leg muscles. The glycogen concentration in nonexercising forearm muscle decreased during the initial 5 h of recovery to 73% of the baseline value. Duringthe exercise, the IMCL content decreased to 67% and subsequently during recovery increased to 83% of the baseline value. In summary, we found during prolonged running 1) significantly greater muscle glycogen utilization in the calf muscle group than in the thigh muscle group, 2) significant utilization of IMCL in the soleus muscle, and 3) a decrease in glycogen content in nonexercising muscle and an increase in glycogen content in recovering muscles during the postexercise phase. These latter data are consistent with the hypothesis that there is transfer of glycogen by the glucose-lactate and the glucose-->alanine cycle from the resting muscle (forearm) to recovering muscles (thigh and calf) after running exercise.

Glycogen loading alters muscle glycogen resynthesis after exercise.

This study compared muscle glycogen recovery after depletion of approximately 50 mmol/l (DeltaGly) from normal (Nor) resting levels (63.2 +/- 2.8 mmol/l) with recovery after depletion of approximately 50 mmol/l from a glycogen-loaded (GL) state (99.3 +/- 4.0 mmol/l) in 12 healthy, untrained subjects (5 men, 7 women). To glycogen load, a 7-day carbohydrate-loading protocol increased muscle glycogen 1.6 +/- 0.2-fold (P < or = 0.01). GL subjects then performed plantar flexion (single-leg toe raises) at 50 +/- 3% of maximum voluntary contraction (MVC) to yield DeltaGly = 48.0 +/- 1.3 mmol/l. The Nor trial, performed on a separate occasion, yielded DeltaGly = 47.5 +/- 4.5 mmol/l. Interleaved natural abundance (13)C-(31)P-NMR spectra were acquired and quantified before exercise and during 5 h of recovery immediately after exercise. During the initial 15 min after exercise, glycogen recovery in the GL trial was rapid (32.9 +/- 8.9 mmol. l(-1). h(-1)) compared with the Nor trial (15.9 +/- 6.9 mmol. l(-1). h(-1)). During the next 45 min, GL glycogen synthesis was not as rapid as in the Nor trial (0.9 +/- 2.5 mmol. l(-1). h(-1) for GL; 14.7 +/- 3.0 mmol. l(-1). h(-1) for Nor; P < or = 0.005) despite similar glucose 6-phosphate levels. During extended recovery (60-300 min), reduced GL recovery rates continued (1.3 +/- 0.5 mmol. l(-1). h(-1) for GL; 3.9 +/- 0.3 mmol. l(-1). h(-1) for Nor; P < or = 0.001). We conclude that glycogen recovery from heavy exercise is controlled primarily by the remaining postexercise glycogen concentration, with only a transient synthesis period when glycogen levels are not severely reduced.

Determination of the rate of the glutamate/glutamine cycle in the human brain by in vivo 13C NMR.

Recent 13C NMR studies in rat models have shown that the glutamate/glutamine cycle is highly active in the cerebral cortex and is coupled to incremental glucose oxidation in an approximately 1:1 stoichiometry. To determine whether a high level of glutamatergic activity is present in human cortex, the rates of the tricarboxylic acid cycle, glutamine synthesis, and the glutamate/glutamine cycle were determined in the human occipital/parietal lobe at rest. During an infusion of [1-13C]-glucose, in vivo 13C NMR spectra were obtained of the time courses of label incorporation into [4-13C]-glutamate and [4-13C]-glutamine. Using a metabolic model we have validated in the rat, we calculated a total tricarboxylic acid cycle rate of 0.77 +/- 0.07 micromol/min/g (mean +/- SD, n = 6), a glucose oxidation rate of 0.39 +/- 0.04 micromol/min/g, and a glutamate/glutamine cycle rate of 0.32 +/- 0.05 micromol/min/g (mean +/- SD, n = 6). In agreement with studies in rat cerebral cortex, the glutamate/glutamine cycle is a major metabolic flux in the resting human brain with a rate approximately 80% of glucose oxidation.

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.

Preliminary evidence of low cortical GABA levels in localized 1H-MR spectra of alcohol-dependent and hepatic encephalopathy patients.

OBJECTIVE: The aim of the study was to compare levels of neuroactive amino acids in the cerebral cortex of healthy subjects, recently detoxified alcohol-dependent patients, and patients with hepatic encephalopathy. METHOD: Metabolite levels were measured in the occipital cortex by using spatially localized 1H-MRS. Five recently detoxified alcohol-dependent and five hepatic encephalopathy patients with alcohol and non-alcohol-related disease were compared with 10 healthy subjects. RESULTS: The combined level of gamma-aminobutyric acid (GABA) plus homocarnosine was lower in the alcohol-dependent and hepatic encephalopathy patients than in the healthy subjects. CONCLUSIONS: The findings suggest that GABA-ergic systems are altered in both alcohol-dependent and hepatic encephalopathy patients.

Contributions of net hepatic glycogenolysis and gluconeogenesis to glucose production in cirrhosis.

Net hepatic glycogenolysis and gluconeogenesis were examined in normal (n = 4) and cirrhotic (n = 8) subjects using two independent methods [13C nuclear magnetic resonance spectroscopy (NMR) and a 2H2O method]. Rates of net hepatic glycogenolysis were calculated by the change in hepatic glycogen content before ( approximately 11:00 PM) and after ( approximately 7:00 AM) an overnight fast using 13C NMR and magnetic resonance imaging. Gluconeogenesis was calculated as the difference between the rates of glucose production determined with an infusion of [6,6-2H2]glucose and net hepatic glycogenolysis. In addition, the contribution of gluconeogenesis to glucose production was determined by the 2H enrichment in C-5/C-2 of blood glucose after intake of 2H2O (5 ml/kg body water). Plasma levels of total and free insulin-like growth factor I (IGF-I) and IGF-I binding proteins-1 and -3 were significantly decreased in the cirrhotic subjects (P < 0.01 vs. controls). Postprandial hepatic glycogen concentrations were 34% lower in the cirrhotic subjects (P = 0.007). Rates of glucose production were similar between the cirrhotic and healthy subjects [9.0 +/- 0.9 and 10.0 +/- 0.8 micromol. kg body wt-1. min-1, respectively]. Net hepatic glycogenolysis was 3.5-fold lower in the cirrhotic subjects (P = 0.01) and accounted for only 13 +/- 6% of glucose production compared with 40 +/- 10% (P = 0.03) in the control subjects. Gluconeogenesis was markedly increased in the cirrhotic subjects and accounted for 87 +/- 6% of glucose production vs. controls: 60 +/- 10% (P = 0.03). Gluconeogenesis in the cirrhotic subjects, as determined from the 2H enrichment in glucose C-5/C-2, was also increased and accounted for 68 +/- 3% of glucose production compared with 54 +/- 2% (P = 0.02) in the control subjects. In conclusion, cirrhotic subjects have increased rates of gluconeogenesis and decreased rates of net hepatic glycogenolysis compared with control subjects. These alterations are likely important contributing factors to their altered carbohydrate metabolism.

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.