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 13 C nuclear magnetic resonance spectroscopy, were combined with estimates of contributions to glucose production of gluconeogenesis and glycogenolysis, measured by labeling of blood glucose by 2 H from ingested 2 H 2 O. Glucose production was measured using [6,6-2 H 2 ]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 · m -2 · min -1 , P < 0.0001). Metformin reduced that rate by 24% (to 0.53 ± 0.03 mmol · m -2 · 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 · m -2 · min -1 ) and metformin reduced that rate by 36% (to 0.38 ± 0.03 mmol · m -2 · min -1 , P = 0.01). By the 2 H 2 O method, there was a twofold increase in rates of gluconeogenesis in diabetic subjects (0.42 ± 0.04 mmol · m -2 · min -1 ), which decreased by 33% after metformin treatment (0.28 ± 0.03 mmol · m -2 · 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. A lthough it is generally agreed that metformin reduces fasting plasma glucose concentrations by reducing rates of hepatic glucose production (1,2), its effect on the relative contributions of hepatic glycogenolysis and gluconeogenesis remains controversial. Some studies conclude that metformin works mostly by reducing rates of gluconeogenesis (3); others, that it works by reducing rates of hepatic glycogenolysis (4,5).Because of limitations of the methods used in the previous studies to assess gluconeogenesis and glycogenolysis, we used two independent and complementary methods to assess these processes in patients with poorly controlled type 2 diabetes before and after 3 months of metformin therapy. 13C nuclear magnetic resonance (NMR) spectroscopy was used to directly measure rates of net hepatic glycogenolysis, in combination with [6,6-2 H 2 ]glucose administration, to calculate the rates of endogenous glucose production (6). Rates of gluconeogenesis were estimated by subtracting the rates of net hepatic glycogenolysis from the rates of endogenous glucose production. In addition...

Healthy subjects ingested 2 H 2 O and after 14, 22, and 42 h of fasting the enrichments of deuterium in the hydrogens bound to carbons 2, 5, and 6 of blood glucose and in body water were determined. The hydrogens bound to the carbons were isolated in formaldehyde which was converted to hexamethylenetetramine for assay. Enrichment of the deuterium bound to carbon 5 of glucose to that in water or to carbon 2 directly equals the fraction of glucose formed by gluconeogenesis. The contribution of gluconeogenesis to glucose production was 47 Ϯ 4% after 14 h, 67 Ϯ 4% after 22 h, and 93 Ϯ 2% after 42 h of fasting. Glycerol's conversion to glucose is included in estimates using the enrichment at carbon 5, but not carbon 6. Equilibrations with water of the hydrogens bound to carbon 3 of pyruvate that become those bound to carbon 6 of glucose and of the hydrogen at carbon 2 of glucose produced via glycogenolysis are estimated from the enrichments to be ‫ف‬ 80% complete. Thus, rates of gluconeogenesis can be determined without corrections required in other tracer methodologies. After an overnight fast gluconeogenesis accounts for ‫ف‬ 50% and after 42 h of fasting for almost all of glucose production in healthy subjects. ( J. Clin. Invest. 1996. 98:378-385.)

Glucagon-like peptide 1 (GLP-1) is potentially a very attractive agent for treating type 2 diabetes. We explored the effect of short-term (1 week) treatment with a GLP-1 derivative, liraglutide (NN2211), on 24-h dynamics in glycemia and circulating free fatty acids, islet cell hormone profiles, and gastric emptying during meals using acetaminophen. Furthermore, fasting endogenous glucose release and gluconeogenesis (3-3 Hglucose infusion and 2 H 2 O ingestion, respectively) were determined, and aspects of pancreatic islet cell function were elucidated on the subsequent day using homeostasis model assessment and first-and second-phase insulin response during a hyperglycemic clamp (plasma glucose ϳ16 mmol/l), and, finally, on top of hyperglycemia, an arginine stimulation test was performed. For accomplishing this, 13 patients with type 2 diabetes were examined in a double-blind, placebo-controlled crossover design. Liraglutide (6 g/kg) was administered subcutaneously once daily. Liraglutide significantly reduced the 24-h area under the curve for glucose (P ‫؍‬ 0.01) and glucagon (P ‫؍‬ 0.04), whereas the area under the curve for circulating free fatty acids was unaltered. Twenty-four-hour insulin secretion rates as assessed by deconvolution of serum C-peptide concentrations were unchanged, indicating a relative increase. Gastric emptying was not influenced at the dose of liraglutide used. Fasting endogenous glucose release was decreased (P ‫؍‬ 0.04) as a result of a reduced glycogenolysis (P ‫؍‬ 0.01), whereas gluconeogenesis was unaltered. First-phase insulin response and the insulin response to an arginine stimulation test with the presence of hyperglycemia were markedly increased (P < 0.001), whereas the proinsulin/insulin ratio fell (P ‫؍‬ 0.001). The disposition index (peak insulin concentration after intravenous bolus of glucose multiplied by insulin sensitivity as assessed by homeostasis model assessment) almost doubled during liraglutide treatment (P < 0.01). Both during hyperglycemia per se and after arginine exposure, the glucagon responses were reduced during liraglutide administration (P < 0.01 and P ‫؍‬ 0.01). Thus, 1 week's treatment with a single daily dose of the GLP-1 derivative liraglutide, operating through several different mechanisms including an ameliorated pancreatic islet cell function in individuals with type 2 diabetes, improves glycemic control throughout 24 h of daily living, i.e., prandial and nocturnal periods. This study further emphasizes GLP-1 and its derivatives as a promising novel concept for treatment of type 2 diabetes.

A method is introduced for estimating the contribution of gluconeogenesis to glucose production. 2H20 is administered orally to achieve 0.5% deuterium enrichment in body water. Enrichments are determined in the hydrogens bound to carbons 2 and 6 of blood glucose and in urinary water. Enrichment at carbon 6 of glucose is assayed in hexamethylenetetramine, formed from formaldehyde produced by periodate oxidation of the glucose. Enrichment at carbon 2 is assayed in lactate formed by enzymatic transfer of the hydrogen from glucose via sorbitol to pyruvate. The fraction gluconeogenesis contributes to glucose production equals the ratio of the enrichment at carbon 6 to that at carbon 2 or in urinary water. Applying the method, the contribution of gluconeogenesis in healthy subjects was 23-42% after fasting 14 h, increasing to 59-84% after fasting 42 h. Enrichment at carbon 2 to that in urinary water was 1.12±0.13. Therefore, the assumption that hydrogen equilibrated during hexose-6-P isomerization was fulfilled. The 3H/14C ratio in glucose formed from [3-3H,3-'4C]lactate given to healthy subjects was 0.1 to 0.2 of that in the lactate. Therefore equilibration during gluconeogenesis of the hydrogen bound to carbon 6 with that in body water was 80-90% complete, so that gluconeogenesis is underestimated by 10-20%. Glycerol's contribution to gluconeogenesis is not included in these estimates. The method is applicable to studies in humans of gluconeogenesis at safe doses of 21H20. (J. Clin. Invest. 1995. 95:172-178.)

. Plasma glucose concentrations decreased by ~10% (P < 0.01), whereas plasma insulin increased by ~47% (P = 0.02) after 9 h of lipid infusion. EGP declined from 9.3 ± 0.5 (lipid) and 9.0 ± 0.8 µmol · kg -1 · min -1 (glycerol) to 8.4 ± 0.5 and 8.2 ± 0.7 µmol · kg -1 · min -1 , respectively (P < 0.01). Contribution of GNG similarly rose (P < 0.01) from 46 ± 4 and 52 ± 3% to 65 ± 8 and 78 ± 7%. To exclude interaction of FFAs with insulin secretion, the study was repeated at fasting plasma insulin (~35 pmol/l) and glucagon (~90 ng/ml) concentrations using somatostatin-insulin-glucagon clamps. Plasma glucose increased by ~50% (P < 0.005) during lipid but decreased by ~12% during glycerol infusion (P < 0.005). EGP remained unchanged over the 9-h period (9.9 ± 1.2 vs. 9.0 ± 1.1 µmol · kg -1 · min -1 ). GNG accounted for 62 ± 5 (lipid) and 60 ± 6% (glycerol) of EGP at time 0 and rose to 74 ± 3% during lipid infusion only (P < 0.05 vs. glycerol: 64 ± 4%). In conclusion, high plasma FFA concentrations increase the percent contribution of GNG to EGP and may contribute to increased rates of GNG in patients with type 2 diabetes. Diabetes 49:701-707, 2000 E levation of plasma free fatty acid (FFA) concentrations is often associated with obesity (1) and type 2 diabetes (2). The close correlation between whole-body glucose uptake and fasting plasma FFA concentrations in lean normoglycemic offspring of type 2 diabetic parents (3) indicates that FFAs might play a pivotal role in the early events leading to insulin resistance (4,5).Plasma FFA elevation induced by lipid/heparin infusions during hyperinsulinemic clamps has repeatedly been shown to decrease insulin-dependent whole-body glucose disposal (5-8). Reports on a correlation between plasma FFAs and hepatic insulin sensitivity are more controversial. Fasting plasma FFAs correlate with the magnitude of hyperglycemia and endogenous glucose production (EGP) (9), which has been attributed to increased lipid oxidation in type 2 diabetes (10). Under hyperinsulinemic conditions, lipid/heparin infusion either increased (6,11) or had no effect on (12-14) EGP. At postabsorptive plasma insulin concentrations, plasma FFA elevation caused marked increases in EGP during somatostatin-insulin clamps (12,15), but not after an overnight fast (15,16). Similarly, inhibition of lipolysis by nicotinic acid or its derivative, acipimox, decreased basal EGP in some (17,18) but not other (19,20) studies. These apparent discrepancies could result from FFA-induced insulin secretion counterbalancing the stimulatory effect of FFAs on EGP (15) or from hepatic autoregulation preventing an increase in EGP under conditions that might favor hepatic gluconeogenesis (GNG) (16). Of note, increased GNG was documented in type 2 diabetes from a variety of precursors (21,22), whereas contradictory effects of FFAs on the contribution of GNG to EGP have been reported during lipid/ heparin infusion or acipimox studies (16,18,20,23).In most studies, glycerol was not infused during control experiments to match the lipid-i...

The use of2H2O in estimating gluconeogenesis’ contribution to glucose production (%GNG) was examined during progressive fasting in three groups of healthy subjects. One group ( n = 3) ingested2H2O to a body water enrichment of ≈0.35% 5 h into the fast. %GNG was determined at 2-h intervals from the ratio of the enrichments of the hydrogens at C-5 and C-2 of blood glucose, assayed in hexamethylenetetramine. %GNG increased from 40 ± 8% at 10 h to 93 ± 6% at 42 h. Another group ingested2H2O over 2.25 h, beginning at 11 h ( n = 7) and 19 h ( n = 7) to achieve ≈0.5% water enrichment. Enrichment in plasma water and at C-2 reached steady state ≈1 h after completion of2H2O ingestion. The C-5-to-C-2 ratio reached steady state by the completion of 2H2O ingestion. %GNG was 54 ± 2% at 14 h and 64 ± 2% at 22 h. A 3-h [6,6-2H2]glucose infusion was also begun to estimate glucose production from enrichments at C-6, again in hexamethylenetetramine. Glucose produced by gluconeogenesis was 0.99 ± 0.06 mg ⋅ kg−1 ⋅ min−1at both 14 and 22 h. In a third group ( n = 3) %GNG reached steady state ≈2 h after2H2O ingestion to only ≈0.25% enrichment. In conclusion, %GNG by 2 h after2H2O ingestion and glucose production using [6,6-2H2]glucose infusion, begun together, can be determined from hydrogen enrichments at blood glucose C-2, C-5, and C-6. %GNG increases gradually from the postabsorptive state to 42 h of fasting, without apparent change in the quantity of glucose produced by gluconeogenesis at 14 and 22 h.

Based on our earlier work, a 2.5-fold increase in insulin secretion should completely inhibit hepatic glucose production through the hormone's direct effect on hepatic glycogen metabolism. The aim of the present study was to test the accuracy of this prediction and to confirm that gluconeogenic flux, as measured by three independent techniques, was unaffected by the increase in insulin. A 40-min basal period was followed by a 180-min experimental period in which an increase in insulin was induced, with euglycemia maintained by peripheral glucose infusion. Arterial and hepatic sinusoidal insulin levels increased from 10 ؎ 2 to 19 ؎ 3 and 20 ؎ 4 to 45 ؎ 5 U/ml, respectively. Net hepatic glucose output decreased rapidly from 1.90 ؎ 0.13 to 0.23 ؎ 0.16 mg ⅐ kg ؊1 ⅐ min ؊1 . Three methods of measuring gluconeogenesis and glycogenolysis were used: 1) the hepatic arteriovenous difference technique (n ‫؍‬ 8), 2) the [ (1) showed that hepatic glucose production (HGP) can be inhibited by selective increases in the arterial or portal vein insulin concentration. In response to a 14-U/ml increase in arterial insulin (no change in portal insulin), a Ͼ50% reduction in net hepatic glucose output (NHGO) was observed. Likewise, a 14-U/ml increase in portal insulin (no change in arterial insulin) also resulted in a Ͼ50% reduction in NHGO. In addition, the above studies showed that insulin acted directly on the liver, with a rise in hepatic sinusoidal insulin quickly inhibiting HGP by reducing net hepatic glycogenolysis. The indirect effect of insulin on HGP, on the other hand, resulted from a decrease in gluconeogenic flux rate caused by a reduction in the flow of gluconeogenic amino acids and glycerol to the liver and diversion of carbon derived from glycogenolysis to lactate rather than glucose. The reduction in HGP in this group was also, in part, the result of a decrease in net hepatic glycogenolysis, which occurred as a result of a slight rise in the hepatic sinusoidal insulin level, which, in turn, occurred as a result of the rise in hepatic artery insulin. It took 1 h to detect a significant indirect effect of insulin on HGP.Sindelar et al.(1) created selective changes in the arterial or portal insulin level by infusing somatostatin to inhibit insulin secretion and replacing insulin by infusion through a peripheral and/or portal catheter. Stimulation of pancreatic insulin secretion, on the other hand, results in an increase in both portal and arterial levels of the hormone. Therefore, in the present study, our aim was to determine if a two-to threefold increase in insulin, occurring simultaneously in portal and peripheral blood, would inhibit HGP primarily through an effect on glycogen metabolism. Although Sindelar et al. (1) reported that portally delivered insulin did not affect gluconeogenic flux, their estimate of the latter relied solely on the measurement of the net hepatic uptake (arteriovenous [AV] difference) of gluconeogenic precursors. In the present study, we combined the hepatic AV difference technique, along...

Contributions of renal glucose production to whole-body glucose turnover were determined in healthy individuals by using the arteriovenous balance technique across the kidneys and the splanchnic area combined with intravenous infusion of [U-13C6]glucose, [3-(3)H]glucose, or [6-(3)H]glucose. In the postabsorptive state, the rate of glucose appearance was 11.5 +/- 0.6 micromol x kg(-1) x min(-1). Hepatic glucose production, calculated as the sum of net glucose output (9.8 +/- 0.8 micromol x kg(-1) x min(-1)) and splanchnic glucose uptake (2.2 +/- 0.3 micromol x kg(-1) x min(-1)) accounted for the entire rate of glucose appearance. There was no net exchange of glucose across the kidney and no significant renal extraction of labeled glucose. The renal contribution to total glucose production calculated from the arterial, hepatic, and renal venous 13C-enrichments (glucose M+6) was 5 +/- 2%. In the 60-h fasted state, the rate of glucose appearance was 8.2 +/- 0.3 micromol x kg(-1) x min(-1). Hepatic glucose production, estimated as net splanchnic output (5.8 +/- 0.7 micromol x kg(-1) x min(-1)) plus splanchnic uptake (0.6 +/- 0.3 micromol x kg(-1) x min(-1)) accounted for 79% of the rate of glucose appearance. There was a significant net renal output of glucose (0.9 +/- 0.3 micromol x kg(-1) x min(-1)), but no significant extraction of labeled glucose across the kidney. The renal contribution to whole-body glucose turnover calculated from the 13C-enrichments was 24 +/- 3%. We concluded that 1) glucose production by the human kidney in the postabsorptive state, in contrast to recent reports, makes at most only a minor contribution (approximately 5%) to blood glucose homeostasis, but that 2) after 60-h of fasting, renal glucose production may account for 20-25% of whole-body glucose turnover.