1Department of Internal Medicine, Division of Endocrinology, Metabolism and Nutrition, and 2Department of Anesthesiology, Division of Research, Mayo Clinic and Foundation, Rochester, Minnesota 55905
Submitted 4 December 2002 ; accepted in final form 6 March 2003
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ABSTRACT |
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glucose effectiveness; glucose turnover
The kinetic response of EGP to a change in glucose concentration is less well defined. In the presence of constant insulin concentrations, clamping glucose at a progressively higher concentration results in a progressive decrease in steady-state rates of EGP (2, 20, 22). Furthermore, EGP decreases when glucose concentration is increased in a pattern mimicking that commonly observed following ingestion of a carbohydrate-containing meal (5). However, in the latter experiments, EGP remained below basal rates when glucose concentrations fell back toward euglycemic concentrations, suggesting a sustained effect of antecedent hyperglycemia. On the other hand, those experiments did not include control studies in which glucose concentrations were maintained at euglycemic levels over the same interval of time. Therefore, the lower rates of EGP at the end of the experiments may have been merely due to a fall resulting from with the extended period of fasting.
The kinetic response of EGP to glucose has both theoretical and practical implications. The "cold" minimal model has been extensively used to assess insulin action and glucose effectiveness (6). This model assumes that a change in plasma glucose results in a proportionate change in EGP. Sequential hyperglycemic clamps have also been used to measure the effects of glucose on glucose production (13, 17, 20). Because rates of EGP observed after an increase in glucose concentration to a new plateau are compared with those observed before the increase, this method assumes that the effects of glucose on EGP are sustained and not time dependent. In addition, due to fenestrations in the portal vein, equilibration between hepatic venous and hepatic sinusoidal glucose concentrations is believed to be rapid (8). If so and if modulation of hepatic metabolism by glucose is also rapid, then a change in the plasma glucose concentration should result in a prompt and reciprocal change in EGP. On the other hand, if equilibration and/or intrahepatic signaling are slow, then a change in EGP will lag behind a change in plasma glucose concentration.
The present study therefore was undertaken to determine whether, in the presence of constant "basal" insulin concentrations, a change in plasma glucose concentration would result in a prompt and reciprocal change in EGP. To do so, we measured EGP when glucose concentrations were either clamped at 5 mmol/l for 6 h, rapidly raised to
7 mmol/l (the threshold for diagnosing diabetes) and maintained at that level for 6 h, or varied so that glucose peaked at
11.1 mmol/l at 1 h and returned to basal levels by 4 h (a pattern commonly observed in people with impaired glucose tolerance). To ensure comparable but constant hepatic insulin concentrations on all occasions, endogenous hormone secretion was inhibited with somatostatin and exogenous insulin infused at rates resulting in a plasma concentration of
65 pmol/l. We report that, under these conditions, sustained hyperglycemia results in prompt and sustained suppression of EGP, whereas a continuous change in plasma glucose concentration is accompanied by a rapid and reciprocal change in EGP.
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SUBJECTS, MATERIALS, AND METHODS |
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Experimental design. Each subject was studied on one occasion. Subjects were admitted to the Mayo Clinic General Clinical Research Center at 1800 the evening before the study. After ingestion of a standard 10-kcal/kg meal (55% carbohydrate, 30% fat, and 15% protein), subjects fasted until the end of the study. On the morning of the study at 0600 (-180 min), an 18-gauge cannula was inserted into the dominant forearm, and a primed continuous infusion of [3-3H]glucose (12 µCi bolus, 0.12 µCi/min continuous; New England Nuclear, Boston, MA) was started and continued until the end of the study.
At 0730 (-90 min), a 3.5-cm double-lumen Teflon arterial catheter was placed in the nondominant brachial artery with aseptic technique under local 1% lidocaine anesthesia. The catheter was connected to a pressure transducer and flushed with normal saline at 2 ml/h to maintain patency. One port was used for blood draws and arterial pressure monitoring, and the second port was used for local infusion of acetylcholine and sodium nitroprusside as part of a separate study examining the effect of hyperglycemia on endothelial function.
Intravenous infusions of somatostatin (60 ng·kg-1·min-1), human growth hormone (3 ng · kg-1 · min-1), glucagon (0.65 ng · kg-1 · min-1), and insulin (0.2 mU · kg-1 · min-1) were started at -90 min and continued at the same rate thoroughout the study. Glucose (containing [3-3H]glucose) was infused intravenously in amounts sufficient to maintain the plasma glucose concentration at 5 mmol/l until time 0. On the euglycemia study day, glucose was clamped at this level throughout the remainder of the study. On the sustained-hyperglycemia study day, plasma glucose was rapidly raised to
7.0 mmol/l and maintained at that level for the duration of the study. On the profile study day, the glucose infusion rate was adjusted so as to increase plasma glucose to
11.1 mmol/l by 60 min, followed by a decrease to
7 mmol/l by 240 min, followed by a further decrease to
5 mmol/l by 300 min. This pattern was chosen because it mimics the glucose profile commonly observed in people with impaired glucose tolerance following ingestion of 75 g of glucose and because it resulted in a glucose area above basal equal to that on the sustained-hyperglycemia study day.
The constant intravenous [3-3H]glucose infusion rate used to measure baseline (i.e., before initiation of the various glucose clamps) EGP was progressively altered beginning at time 0 in a pattern anticipated to approximate the pattern of change of EGP (0180 min: 50% basal rate; 181300 min: 25%; 301420 min: 15%; 010 min: 90%; 1020 min: 80%; 2030 min: 65%; 3040 min: 55%; 4050 min: 45%; 5075 min: 35%; 75120 min: 40%; 120300 min: 45%; 300360 min: 50%). This was done in an effort to minimize the change in plasma glucose specific activity, thereby enabling accurate measurement of glucose turnover (3, 15). This resulted in a mean plasma glucose specific activity that averaged -3.2 ± 11.2, 4.7 ± 7.0, and 15.1 ± 4.7% of basal on the euglycemia, sustained-hyperglycemia, and profile study days, respectively.
Analytical techniques. Arterial plasma samples were placed in ice, centrifuged at 4°C, separated, and stored at -20°C until assay. Plasma glucose concentration was measured using a glucose oxidase method (Yellow Springs Instrument, Yellow Springs, OH). Plasma insulin and growth hormone concentrations were measured using a chemiluminescence assay with reagents obtained from Beckman (Access Assay; Beckman, Chaska, MN). Plasma glucagon and C-peptide concentrations were measured by radioimmunoassay (Linco Research, St. Charles, MO). Body composition was measured using dual-energy X-ray absorptiometry (DEXA scanner; Hologic, Waltham, MA). Plasma [3-3H]glucose specific activity was measured using liquid scintillation counting as previously described (26).
Calculations. Glucose specific activity was smoothed using the method of Bradley et al. (9). Total glucose appearance was calculated with the non-steady-state equations of Steele et al. (30) using the actual tracer infusion rate for each interval and an assumed volume of distribution of 200 ml/kg and the pool correction factor to equal 0.65. EGP was calculated by subtracting the glucose infusion rate for each interval from the tracer-determined rate of glucose appearance.
Statistical analysis. Data in the text and figures are expressed as means ± SE. All rates of turnover are expressed as micromoles per kilogram of lean body mass per minute. The mean of the glucose production rates from -30 to 0 min was considered as basal, and the mean of values from 330 to 360 min was considered as study end. Area above or below basal was calculated using the trapezoidal rule. ANOVA was used to test differences among groups. A P value of <0.05 was considered to be statistically significant.
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RESULTS |
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Plasma glucose concentrations and EGP. Plasma glucose concentrations did not differ on the euglycemia, sustained-hyperglycemia, and profile study days either before the start of the somatostatin infusion (4.9 ± 0.1 vs. 4.8 ± 0.1 vs. 5.0 ± 0.1 mmol/l, respectively) or during the basal period from -30 to 0 min (5.4 ± 0.1 vs. 5.5 ± 0.1 vs. 5.5 ± 0.1 mmol/l, respectively). Plasma glucose concentrations from time 0 onward averaged 5.3 ± 0.1 mmol/l on the euglycemia study day (Fig. 2). Plasma glucose concentration on the sustained-hyperglycemia study day increased to 7.1 ± 0.3 mmol/l within 30 min and was maintained at an average of 7.2 ± 0.1 mmol/l. Plasma glucose concentrations on the profile study day increased to a peak of 11.2 ± 0.5 mmol/l by 60 min and then decreased to 9.8 ± 0.4 mmol/l by 120 min to 7.4 ± 0.3 mmol/l by 180 min and to 5.1 ± 0.1 mmol/l by the end of the study. This resulted in a glucose area above basal that did not differ between the sustained-hyperglycemia and profile study days (2,823 ± 27 vs. 2,942 ± 56 mmol/6 h).
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Basal rates of EGP did not differ on the euglycemia, sustained-hyperglycemia, and profile study days (24.4 ± 2.1 vs. 20.3 ± 1.7 vs. 27.8 ± 4.6 µmol · kg-1 · min-1, respectively). On the euglycemia study day, EGP fell over the 1st h of the experiment, rose slightly during the next 2 h, and then fell gradually over the final 3 h of the experiment to rates that averaged 13.1 ± 3.7 µmol · kg-1 · min-1 by study end (P < 0.01 vs. basal). The increase in glucose concentrations on the sustained-hyperglycemia study day resulted in prompt and sustained suppression of EGP to rates at study end (6.7 ± 1.2 µmol · kg-1 · min-1) that remained lower (P < 0.01) than those present on the euglycemia study day. The increase in glucose concentration on the profile study day also resulted in suppression of EGP to a nadir of 5.2 ± 3.2 µmol · kg-1 · min-1 at 60 min (i.e., coincident with the peak plasma glucose concentration), which was lower (P < 0.05) than that present at the same time on the sustained-hyperglycemia study day (11.3 ± 1.0 µmol · kg-1 · min-1). EGP on the profile study day then rose as glucose concentrations fell. EGP on the profile study day rose to rates by 180 min (11.1 ± 1.5 µmol · kg-1 · min-1) that did not differ from those on the sustained-hyperglycemia study day (11.7 ± 1.7 µmol · kg-1 · min-1, P = 0.8) and by 360 min (12.2 ± 2.2 µmol · kg-1 · min-1) that did not differ from those present on the euglycemia study day (12.6 ± 3.4 µmol · kg-1 · min-1). Of note, as with glucose concentration, although the pattern of change of EGP differed on the sustained-hyperglycemia and profile study days, the degree of suppression of EGP below basal did not differ on the two occasions (-1,952 ± 204 vs. -1,922 ± 246 mmol · kg-1 · 6 h-1).
Relationship between plasma glucose concentration and EGP. To gain greater insight into the relationship between glucose and EGP, the mean plasma glucose concentration at each time point on each study day was plotted against the mean EGP present at the same time point. As is evident in Fig. 3, regardless of the pattern of change of glucose, there was an inverse relationship between glucose concentration and EGP (r = 0.63; P < 0.001), further supporting the conclusion that the prevailing glucose concentration per se was the primary determinant of EGP.
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DISCUSSION |
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Insulin and glucose both regulate EGP. Because of the presence of fenestrations in the portal venous system (8), equilibration between plasma and hepatic sinusoidal insulin and glucose concentrations is believed to be rapid. Insulin initiates its intracellular signaling cascade by binding to its extracellular insulin receptor (33). In contrast, the means by which glucose regulates liver glucose metabolism is less clear. Uptake via hepatic GLUT2 transporters and phosphorylation by glucokinase appear to be important, because glucokinase deficiency decreases (14, 32) and overexpression increases (23) hepatic glycogen synthesis. We (22) as well as other investigators (2, 20) have shown that, in the presence of basal insulin concentrations, an increase in glucose concentrations within the physiological range results in linear suppression of EGP measured after 23 h of sustained hyperglycemia. The present data extend these observations by showing that the hepatic response to glucose is rapid both when glucose concentration is rising and when it is falling.
The rapid fall in EGP is consistent with previous reports by Ader et al. (2) and Nielsen et al. (22) that EGP, measured using the so-called "hot Ginf" (i.e., glucose infusion) method (15), is suppressed immediately following the initiation of a hyperglycemic clamp. It is also consistent with the pattern of suppression of EGP that we observed when glucose concentrations varied in a manner resembling the glucose profile study day of the present experiments (5). However, in those studies, EGP remained below baseline rates at the termination of the experiment. This was reminiscent of the delay in resumption of EGP observed when elevated insulin concentrations were acutely lowered back to basal level, indicating that the half-life of insulin's effect on the liver is longer that its plasma half-life (10). However, in our earlier glucose profile studies (5), glucose concentrations at the end of the study were still above baseline values. In addition, a euglycemic control group was not included in those experiments. We therefore could not determine whether the apparent persistent suppression of EGP was due to an effect of antecedent hyperglycemia or to a time-dependent fall in EGP due to prolonged fasting. The equivalent rates of EGP in the present experiments from 5 h onward (i.e., when glucose concentrations were the same) on the euglycemia and profile study days shows that the current rather than the antecedent glucose concentration is the primary determinant of EGP. The fall of EGP on the euglycemia day also emphasizes the need for a time-dependent control when the effect of a change in glucose concentration on EGP is being evaluated. Comparison of rates of EGP at the end of the experiment to baseline rates would have led to the erroneous conclusion that EGP was suppressed on the profile study day and would have overestimated the degree of suppression on the sustained-hyperglycemia study day.
The ability of glucose to suppress glucose production is dependent on the prevailing insulin and glucagon concentrations (16, 28). Glucose-induced suppression of EGP is severely impaired in insulin-deficient type 1 diabetic patients (24), indicating that "permissive" amounts of insulin are required for glucose to exert its effects. Peripheral insulin concentrations were 30 pmol/l before the start of the somatostatin infusion and were maintained at
65 pmol/l on all three study days. Somatostatin resulted in essentially complete suppression of endogenous insulin secretion on all three occasions. Assuming a portal venous-to-peripheral insulin gradient of
1.62.0 (18, 31), this suggests that portal insulin concentrations either remained the same or increased slightly. On the other hand, plasma glucagon concentrations were the same before and after the somatostatin-glucagon infusion, suggesting a drop in portal glucagon concentrations (7). However, because the somatostatin, insulin, and glucagon infusions all were started 90 min before time 0, portal venous hormone concentrations were constant and equal on all occasions at the start of the experiments. Nevertheless, it is likely that both basal rates of EGP and the magnitude of response of EGP to a change in glucose concentration would differ in the presence of differing insulin and glucagon concentrations. However, the pattern of response (i.e., EGP falls as glucose rises and rises as glucose falls) presumably would be the same.
Of note, glucose concentrations were not perfectly clamped on the euglycemic study day. Glucose concentrations tended to rise during the first 4560 min, fall during the next 2 h reaching a nadir at 180 min, rise reaching a second plateau at
300 min, and then remain more or less stable thereafter. These changes in glucose concentrations were accompanied by a relatively rapid fall in EGP during the first 60 min, a rise in EGP during the next 2 h that reached a peak at
180 min, a fall in EGP until
300 min, and then a more gradual decrease thereafter. Although these changes in glucose and EGP on the euglycemic study day make calculation of absolute differences among euglycemia, hyperglycemia, and profile study days difficult, they further highlight the reciprocal relationship between small changes in glucose concentration and EGP. In addition, we have measured only EGP. Previous studies in both animals and humans have shown that hyperglycemia suppresses EGP primarily by inhibiting glycogenolysis and by stimulating intrahepatic glycogen cycling (21, 25, 27). We therefore presume that the resumption of EGP as glucose concentrations returned to basal values on the profile day was mediated primarily by an increase in glycogenolysis. Measurement of the relative contributions of glycogenolysis and gluconeogenesis to EGP in future studies will be required to address this point specifically. Finally, net hepatic glucose uptake was not measured in this study. Because glucose stimulates its own uptake as well as suppressing its own release, it likely that hepatic glucose uptake also changed as glucose concentrations changed. Future studies will be required to determine whether comparable increases in integrated glycemic exposure achieved by temporally different glucose profiles result in equivalent increases in hepatic glucose uptake.
In summary, these studies lend further support to the importance of glucose in the regulation of EGP. They indicate that, in the presence of basal insulin concentrations, a change in glucose concentration results in a rapid and reciprocal change in EGP. EGP falls as glucose concentration rises and rises as glucose concentration falls. The response is proportionate to the prevailing glucose concentration and does not appear to be influenced by the antecedent pattern of change in glucose. These data emphasize that alterations in EGP in various disease states such as diabetes need to be interpreted in light of the prevailing glucose concentrations, and suppression of EGP by glucose can compensate, in part or in whole, for a defect in insulin availability or action (4, 13, 19, 20, 22, 29).
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ACKNOWLEDGMENTS |
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The National Institutes of Health, US Public Health Service (DK-29953, AR-08610, HL-63328, and RR-00585), and the Mayo Foundation supported this study. P. Shah was supported by a Novo-Nordisk research fellowship, R. Basu by an American Diabetes Association mentor-based fellowship, and A. Reed by a Falk fellowship grant.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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