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.)

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.