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.

Mechanism of impaired insulin-stimulated muscle glucose metabolism in subjects with insulin-dependent diabetes mellitus.

To determine the mechanism of impaired insulin-stimulated muscle glycogen metabolism in patients with poorly controlled insulin-dependent diabetes mellitus (IDDM), we used 13C-NMR spectroscopy to monitor the peak intensity of the C1 resonance of the glucosyl units in muscle glycogen during a 6-h hyperglycemic-hyperinsulinemic clamp using [1-(13)C]glucose-enriched infusate followed by nonenriched glucose. Under similar steady state (t = 3-6 h) plasma glucose (approximately 9.0 mM) and insulin concentrations (approximately 400 pM), nonoxidative glucose metabolism was significantly less in the IDDM subjects compared with age-weight-matched control subjects (37+/-6 vs. 73+/-11 micromol/kg of body wt per minute, P < 0.05), which could be attributed to an approximately 45% reduction in the net rate of muscle glycogen synthesis in the IDDM subjects compared with the control subjects (108+/-16 vs. 195+/-6 micromol/liter of muscle per minute, P < 0.001). Muscle glycogen turnover in the IDDM subjects was significantly less than that of the controls (16+/-4 vs. 33+/-5%, P < 0.05), indicating that a marked reduction in flux through glycogen synthase was responsible for the reduced rate of net glycogen synthesis in the IDDM subjects. 31P-NMR spectroscopy was used to determine the intramuscular concentration of glucose-6-phosphate (G-6-P) under the same hyperglycemic-hyperinsulinemic conditions. Basal G-6-P concentration was similar between the two groups (approximately 0.10 mmol/kg of muscle) but the increment in G-6-P concentration in response to the glucose-insulin infusion was approximately 50% less in the IDDM subjects compared with the control subjects (0.07+/-0.02 vs. 0.13+/-0.02 mmol/kg of muscle, P < 0.05). When nonoxidative glucose metabolic rates in the control subjects were matched to the IDDM subjects, the increment in the G-6-P concentration (0.06+/-0.02 mmol/kg of muscle) was no different than that in the IDDM subjects. Together, these data indicate that defective glucose transport/phosphorylation is the major factor responsible for the lower rate of muscle glycogen synthesis in the poorly controlled insulin-dependent diabetic subjects.

Mechanism of free fatty acid-induced insulin resistance in humans.

To examine the mechanism by which lipids cause insulin resistance in humans, skeletal muscle glycogen and glucose-6-phosphate concentrations were measured every 15 min by simultaneous 13C and 31P nuclear magnetic resonance spectroscopy in nine healthy subjects in the presence of low (0.18 +/- 0.02 mM [mean +/- SEM]; control) or high (1.93 +/- 0.04 mM; lipid infusion) plasma free fatty acid levels under euglycemic (approximately 5.2 mM) hyperinsulinemic (approximately 400 pM) clamp conditions for 6 h. During the initial 3.5 h of the clamp the rate of whole-body glucose uptake was not affected by lipid infusion, but it then decreased continuously to be approximately 46% of control values after 6 h (P < 0.00001). Augmented lipid oxidation was accompanied by a approximately 40% reduction of oxidative glucose metabolism starting during the third hour of lipid infusion (P < 0.05). Rates of muscle glycogen synthesis were similar during the first 3 h of lipid and control infusion, but thereafter decreased to approximately 50% of control values (4.0 +/- 1.0 vs. 9.3 +/- 1.6 mumol/[kg.min], P < 0.05). Reduction of muscle glycogen synthesis by elevated plasma free fatty acids was preceded by a fall of muscle glucose-6-phosphate concentrations starting at approximately 1.5 h (195 +/- 25 vs. control: 237 +/- 26 mM; P < 0.01). Therefore in contrast to the originally postulated mechanism in which free fatty acids were thought to inhibit insulin-stimulated glucose uptake in muscle through initial inhibition of pyruvate dehydrogenase these results demonstrate that free fatty acids induce insulin resistance in humans by initial inhibition of glucose transport/phosphorylation which is then followed by an approximately 50% reduction in both the rate of muscle glycogen synthesis and glucose oxidation.

Impaired net hepatic glycogen synthesis in insulin-dependent diabetic subjects during mixed meal ingestion. A 13C nuclear magnetic resonance spectroscopy study.

Hepatic glycogen concentration was measured in six subjects with insulin-dependent diabetes mellitus (IDDM) and nine weight-matched control subjects using 13C nuclear magnetic resonance spectroscopy during a day in which three isocaloric mixed meals were ingested. The relative fluxes of the direct and indirect (3 carbon units-->-->glycogen) pathways of hepatic glycogen synthesis were also assessed using [1-13C]glucose in combination with acetaminophen to noninvasively sample the hepatic UDP-glucose pool. Mean fasting hepatic glycogen content was similar in the two groups. After each meal, hepatic glycogen content increased, peaking 4-5 h after the meal in both groups. By 11:00 p.m. the IDDM subjects had synthesized only 30% of the glycogen that was synthesized by the control group [IDDM subjects, net increment = 44 +/- 20 (mean +/- SE) mM; control subjects, net increment = 144 +/- 14 mM; P < 0.05]. After breakfast the flux through the gluconeogenic pathway relative to the direct pathway of hepatic glycogen synthesis was 1.7-fold greater in the IDDM subjects (59 +/- 4%) than in the control subjects (35 +/- 4%, P < 0.0003). In conclusion, under mixed meal conditions, subjects with poorly controlled IDDM have a major defect in net hepatic glycogen synthesis and augmented hepatic gluconeogenesis. The former abnormality may result in an impaired glycemic response to counterregulatory hormones, whereas both abnormalities may contribute to postprandial hyperglycemia.

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.

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.

Impaired hepatic glycogen synthesis in glucokinase-deficient (MODY-2) subjects.

All glucokinase gene mutations identified to date have been localized to exons that are common to the pancreatic and hepatic isoforms of the enzyme. While impaired insulin secretion has been observed in glucokinase-deficient subjects the consequences of this mutation on hepatic glucose metabolism remain unknown. To examine this question hepatic glycogen concentration was measured in seven glucokinase-deficient subjects with normal glycosylated hemoglobin and 12 control subjects using 13C nuclear magnetic spectroscopy during a day in which three isocaloric mixed meals were ingested. The relative fluxes of the direct and indirect pathways of hepatic glycogen synthesis were also assessed using [1-13C]glucose in combination with acetaminophen to noninvasively sample the hepatic UDP-glucose pool. Average fasting hepatic glycogen content was similar in glucokinase-deficient and control subjects (279+/-20 vs 284+/-14 mM; mean+/-SEM), and increased in both groups after the meals with a continuous pattern throughout the day. However, the net increment in hepatic glycogen content after each meal was 30-60% lower in glucokinase-deficient than in the control subjects (breakfast, 46% lower, P < 0.02; lunch, 62% lower, P = 0.002; dinner; 30% lower, P = 0.04). The net increment over basal values 4 h after dinner was 105 +/-18 mM in glucokinase-deficient and 148+/-11 mM in control subjects (P = 0.04). In the 4 h after breakfast, flux through the gluconeogenic pathway relative to the direct pathway of hepatic glycogen synthesis was higher in glucokinase-deficient than in control subjects (50+/-2% vs 34+/-5%; P = 0.038). In conclusion glucokinase-deficient subjects have decreased net accumulation of hepatic glycogen and relatively augmented hepatic gluconeogenesis after meals. These results suggest that in addition to the altered beta cell function, abnormalities in liver glycogen metabolism play an important role in the pathogenesis of hyperglycemia in patients with glucokinase-deficient maturity onset diabetes of young.

The roles of insulin and glucagon in the regulation of hepatic glycogen synthesis and turnover in humans.

To determine the respective roles of insulin and glucagon for hepatic glycogen synthesis and turnover, hyperglycemic clamps were performed with somatostatin [0.1 micrograms/(kg.min)] in healthy young men under conditions of: (I) basal fasting) portal vein insulinemia-hypoglucagonemia, (II) basal portal vein insulinemia-basal glucagonemia, and (III) basal peripheral insulinemia-hypoglucagonemia. Synthetic rates, pathway (direct versus indirect) contributions, and percent turnover of hepatic glycogen were assessed by in vivo 13C nuclear magnetic resonance spectroscopy during [1-13C]glucose infusion followed by a natural abundance glucose chase in conjunction with acetaminophen to noninvasively sample the hepatic UDP-glucose pool. In the presence of hyperglycemia (10.4 +/- 0.1 mM) and basal portal vein insulinemia (192 +/- 6 pM), suppression of glucagon secretion (plasma glucagon, I:31 +/- 4, II: 63 +/- 8 pg/ml) doubled the hepatic accumulation of glycogen (Vsyn) compared with conditions of basal glucagonemia [I: 0.40 +/- 0.06, II: 0.19 +/- 0.03 mumol/(liter.min): P < 0.0025]. Glycogen turnover was markedly reduced (I: 19 +/- 7%, II: 69 +/- 12%; P < 0.005), so that net rate of glycogen synthesis increased approximately fivefold (P < 0.001) by inhibition of glucagon secretion. The relative contribution of gluconeogenesis (indirect pathway) to glycogen synthesis was lower during hypoglucagonemia (42 +/- 6%) than during basal glucagonemia (54 +/- 5%; P < 0.005). Under conditions of basal peripheral insulinemia (54 +/- 2 pM) and hypoglucagonemia (III) there was negligible hepatic glycogen synthesis and turnover. In conclusion, small changes in portal vein concentrations of insulin and glucagon independently affect hepatic glycogen synthesis and turnover. Inhibition of glucagon secretion under conditions of hyperglycemia and basal concentrations of insulin results in: (a) twofold increase in rate of hepatic glycogen synthesis, (b) reduction of glycogen turnover by approximately 73%, and (c) augmented percent contribution of the direct pathway to glycogen synthesis compared with conditions of basal glucagonemia.

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.

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.

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.

13C/31P NMR studies on the mechanism of insulin resistance in obesity.

The mechanism of insulin resistance in obesity was examined in ten obese (BMI 33 +/- 1 kg/m2) and nine lean (BMI 22 +/- 1 kg/m2) Caucasian women during a hyperglycemic-hyperinsulinemic clamp using 13C and 31P nuclear magnetic resonance (NMR) spectroscopy to measure rates of muscle glycogen synthesis and intramuscular glucose-6-phosphate (G-6-P) concentrations. Under similar steady-state plasma concentrations of glucose (approximately 11 mmol/l) and insulin (approximately 340 pmol/l), rates of muscle glycogen synthesis were reduced approximately 70% in the obese subjects (52 +/- 8 micromol/[l muscle-min]) as compared with the rates in the lean subjects (176 +/- 22 micromol/[l muscle-min]; P < 0.0001). Basal concentrations of intramuscular G-6-P were similar in the obese and lean subjects; but during the clamp, G-6-P failed to increase in the obese group (deltaG-6-P obese 0.044 +/- 0.011 vs. lean 0.117 +/- 0.011 mmol/l muscle; P < 0.001), reflecting decreased muscle glucose transport and/or phosphorylation activity. We conclude that insulin resistance in obesity can be mostly attributed to impairment of insulin-stimulated muscle glycogen synthesis due to a defect in glucose transport and/or phosphorylation activity.

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.

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.

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.

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.

Triiodothyronine treatment increases substrate cycling between pyruvate carboxylase and malic enzyme in perfused rat liver.

The relative roles of pyruvate kinase and malic enzyme in substrate cycling between pyruvate and oxaloacetate were examined in perfused livers of 24-hour-fasted normal and triiodothyronine (T3)-treated rats using an inhibitor of malic enzyme (hydroxymalonate). Livers were perfused for 60 minutes in a recirculating system with [3-13C]alanine (10 mmol/L, 99% 13C-enriched). The combined flux through pyruvate kinase plus malic enzyme relative to pyruvate carboxylase flux was assessed by the 13C-enrichment ratio of alanine C2 to glucose C5 in the perfusate, determined with 13C and 1H nuclear magnetic resonance (NMR) spectroscopy. In normal rat livers, the relative carbon flux through pyruvate kinase plus malic enzyme to pyruvate carboxylase was 0.18 +/- 0.04, and increased to 0.44 +/- 0.08 (P < .05) in the T3-treated group. After addition of hydroxymalonate, this relative carbon flux was unchanged in normal rat livers, but decreased to 0.15 +/- 0.04 (P < .01) in the T3-treated group, suggesting that the increased carbon flux in T3-treated livers was caused by increased flux through malic enzyme. Malic enzyme activity increased from 0.36 +/- 0.05 U/g liver in normal livers to 2.51 +/- 0.50 U/g liver (P < .05) in the T3-treated group, whereas there was no effect of T3 treatment on pyruvate kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.