Increased rates of glucose production are strongly correlated with increased fasting plasma glucose concentrations in patients with type 2 diabetes (18
). We found that the rate of glucose production was increased twofold in the diabetic subjects as compared with that in the control subjects, which could be entirely attributed to an approximately threefold increase in the rate of gluconeogenesis as assessed by the 13
C NMR method and mostly to a roughly twofold increase in the rate of gluconeogenesis as assessed by the 2
O method. These results are consistent with our previous study examining the rates of gluconeogenesis in normal and poorly controlled type 2 diabetic subjects, using 13
C NMR (23
). Metformin treatment resulted in a 25–30% reduction in fasting plasma glucose concentrations and glucose production, which is consistent with the results of previous studies (2
). This decrease in glucose production could be entirely accounted for by a reduction in the rates of gluconeogenesis as determined by both methods.
Because the NMR method measures rates of net hepatic glycogenolysis, whereas the 2
O method measures rates of total hepatic glycogenolysis (net hepatic glycogenolysis + hepatic glycogen cycling), estimates of hepatic glycogen cycling were made by subtracting rates of net hepatic glycogenolysis (NMR measured) from the rates of total hepatic glycogenolysis (2
O measured). There was no detectable hepatic glycogen cycling in the control subjects, which is consistent with an earlier study using these two methods (26
). In contrast, there was considerable glycogen cycling in the poorly controlled diabetic subjects, which accounted for ~25% of endogenous glucose production (). Hyperglucagonemia and hyperinsulinemia, which are typically present in patients with type 2 diabetes, have both been shown to promote glycogen cycling (27
). Although the plasma concentrations of these hormones tended to be higher in the diabetic subjects than in the control subjects, the differences were not significant; however, it is likely that differences in portal vein concentrations of these hormones were much greater.
Three different studies have previously examined the effect of metformin on the rates of net hepatic glycogenolysis and gluconeogenesis in patients with type 2 diabetes with conflicting results. Stumvoll et al. (3
) studied 10 obese diabetic individuals before and after 16 weeks of treatment with metformin (2,550 mg/day), using [3-14
C]lactate to estimate the rates of gluconeogenesis. Using this approach, these investigators found that metformin decreased endogenous glucose production through a 37% reduction in rates of gluconeogenesis. However, because of unknown dilution of the 14
C label in the tricarboxylic acid cycle, this approach does not accurately quantify rates of gluconeogenesis. In addition, the diabetic subjects lost on average ~3 kg of body wt during this study, which may have obscured the independent role of metformin in lowering the rates of glucose production.
Cusi et al. (4
) used the same [3-14
C]lactate approach to assess rates of gluconeogenesis in 20 type 2 diabetic subjects before and after 15 weeks of metformin treatment (2,500 mg/day) in a randomized double-blind placebo-controlled trial. Fifteen of the subjects were taking sulfonylureas at the time of enrollment. This medication was continued during the trial, and metformin was added. These investigators reported that metformin treatment significantly decreased glucose production, but they found no change in the contribution of gluconeogenesis from lactate. They therefore concluded that metformin decreased glucose production by inhibiting hepatic glycogenolysis. It is unclear why these investigators arrived at a result opposite to that of Stumvoll et al. (3
) despite using the identical approach.
More recently, Christiansen et al. (5
) examined the mechanism of metformin’s action in diabetic subjects using mass isotopomer distribution analysis to estimate the rates of gluconeogenesis using [2-13
C]glycerol. Five obese subjects (BMI 38 ± 3) with type 2 diabetes were studied before and after 4 weeks of treatment with metformin. In this study, gluconeogenesis contributed only ~24% of overall glucose production in the diabetic subjects. These investigators therefore concluded that an increased rate of glycogenolysis was entirely responsible for the increased rate of glucose production, which is contrary to the current and previous 13
C NMR studies (23
). These investigators also found that metformin treatment led to a 17% reduction in the rate of glucose production, with no change in the absolute rate of gluconeogenesis and concluded that this occurred through a 21% reduction in hepatic glycogenolysis. It is unclear why Christiansen et al. obtained the opposite results from those obtained in the present study. However, recent studies have demonstrated limitations in their approach, resulting in underestimation of gluconeogenic rates (30
The data from the present study are consistent with in vitro studies demonstrating an inhibitory effect of metformin on gluconeogenesis. Radziuk et al. (34
) reported that metformin inhibited gluconeogenesis in perfused liver, primarily through inhibition of hepatic lactate uptake. Argaud et al. (35
) found that metformin decreased ATP concentration in isolated rat hepatocytes. Because ATP is an allosteric inhibitor of pyruvate kinase, these investigators hypothesized that the metformin-induced reduction in glucose production was the result of increased pyruvate kinase flux. However, Large and Beylot (36
) observed no decrease in ATP concentration or in the uptake of gluconeogenic precursors by perfused livers obtained from fasted streptozocin-injected diabetic rats after metformin treatment. They hypothesized that metformin decreased gluconeogenic flux through inhibition of pyruvate carboxylase-phosphoenolpyruvate carboxykinase activity and possibly through increased conversion of pyruvate to alanine. However, it should be noted that all of these in vitro studies used very high doses of metformin (250–350 mg/kg), which are 8- to 12-fold higher than the doses used in the treatment of diabetic patients.
Metformin treatment also lowered the plasma free fatty acid concentrations by 30% in the diabetic subjects, which is consistent with the results of some (37
) but not all previous studies (3
). Plasma free fatty acids have been shown to play an important role in the regulation of hepatic glucose production (41
). It is possible that reduction in plasma free fatty acid concentration after metformin treatment also contributed to the reduced rates of gluconeogenesis.
In summary, we examined the effect of metformin treatment on rates of total and net hepatic glycogenolysis, gluconeogenesis, and hepatic glycogen cycling in poorly controlled type 2 diabetic patients who were matched to healthy control subjects using 13C NMR and 2H2O techniques. We found the following: 1) Poorly controlled diabetic subjects had a twofold increase in rates of glucose production and extensive hepatic glycogen cycling accounting for ~25% of their glucose production; 2) the increased rate of glucose production in the diabetic subjects could be attributed to an increased rate of gluconeogenesis; and 3) metformin treatment in the diabetic subjects decreased rates of glucose production through a reduction in the rate of gluconeogenesis.