Both fasting glucose production and disappearance are abnormal in people with "mild" and "severe" type 2 diabetes

Rita Basu, W. Frederick Schwenk, and Robert A. Rizza

Division of Endocrinology, Mayo Clinic, Rochester, Minnesota 55905

Submitted 4 December 2003 ; accepted in final form 16 February 2004


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To determine whether regulation of fasting endogenous glucose production (EGP) and glucose disappearance (Rd) are both abnormal in people with type 2 diabetes, EGP and Rd were measured in 7 "severe" (SD), 9 "mild" (MD), and 12 nondiabetic (ND) subjects (12.7 ± 0.6 vs. 8.1 ± 0.4 vs. 5.1 ± 0.4 mmol/l) after an overnight fast and during a hyperglycemic pancreatic clamp. Fasting insulin was higher in both the SD and MD than ND subjects, whereas fasting glucagon only was increased (P < 0.05) in SD. Fasting EGP, glycogenolysis, gluconeogenesis, and Rd all were increased (P < 0.05) in SD but did not differ in MD or ND. On the other hand, when glucose (~11 mmol/l), insulin (~72 pmol/l), and glucagon (~140 pg/ml) concentrations were raised to values similar to those observed in the severe diabetic subjects, EGP was higher (P < 0.001) and Rd lower (P < 0.01) in both SD and MD than in ND. The higher EGP in the SD and MD than ND during the clamp was the result of increased (P < 0.05) rates of glycogenolysis (4.2 ± 1.7 vs. 3.5 ± 1.0 vs. 0.0 ± 0.8 µmol·kg–1·min–1), since gluconeogenesis did not differ among groups. We conclude that neither glucose production nor disappearance is appropriate for the prevailing glucose and insulin concentrations in people with mild or severe diabetes. Both increased rates of gluconeogenesis (likely because of higher glucagon concentrations) and lack of suppression of glycogenolysis contribute to excessive glucose production in type 2 diabetics.

liver; insulin resistance; fasting hyperglycemia


FASTING HYPERGLYCEMIA is commonly observed in untreated type 2 diabetes. The relative contribution of excessive endogenous glucose production (EGP) and defective glucose uptake has been debated, particularly in individuals who have only a modest elevation in their fasting glucose concentration (7, 9, 10, 15, 18, 19, 21, 23, 29, 37, 40, 42, 43). Most (7, 9, 10, 15, 18, 19, 21, 23, 37, 40, 42, 43), but not all (28, 29, 46), investigators have reported that EGP is increased in people with "severe" (e.g., fasting glucose >9 mmol/l) hyperglycemia. On the other hand, fasting EGP has generally been reported not to differ from nondiabetic rates when glucose concentrations are only mildly elevated (e.g., 7–9 mmol/l; see Refs. 7, 15, and 42) . In contrast, the latter individuals have been consistently noted to have rates of fasting glucose uptake that are not appropriate for their elevated glucose concentration. This has led to the concept that defects in glucose uptake are involved in the pathogenesis of type 2 diabetes, whereas fasting excessive EGP is a late secondary event (9, 15, 29).

Discrepant reports as to whether fasting glucose production is increased in type 2 diabetes may in part be explained by technical problems that in the past have hindered accurate measurement of glucose production in the diabetic subjects. As demonstrated by Hother-Nielsen and Beck-Nielsen (28), inadequate tracer priming in hyperglycemic diabetic subjects delays the achievement of steady state. This can cause a falsely low tracer-to-tracee ratio that in turn can lead to an overestimation of glucose production in the diabetic subjects (28). This error can be avoided by proportionately adjusting the priming dose based on the fasting glucose concentration (28, 46). However, lack of adequate priming does not appear to be the sole explanation for the discrepant results, since glucose production in people with severe type 2 diabetes has been reported to be either increased (7, 21, 22) or not increased (28, 46) despite the use of the same proportionate priming method.

Small increases in either insulin or glucose result in rapid and substantial suppression of EGP in nondiabetic individuals (26, 39, 41, 47, 55). Because both glucose and insulin concentrations generally are elevated in people with mild type 2 diabetes, it could be argued that even "normal" (i.e., not different from nondiabetic rates) rates of EGP are excessive (31). If so, then alterations in the regulation of EGP may also be present in individuals with "mild" and severe hyperglycemia and, therefore, may contribute to rather than merely be a consequence of fasting hyperglycemia.

The present experiments sought to address this question by measuring EGP and glucose disappearance in people with either mild (defined as fasting glucose 7–9 mmol/l) or severe (>9 mmol/l) diabetes in the fasting state and comparing results with those observed in nondiabetic subjects. Care was taken to ensure that the plasma tracer-to-tracee ratio was at steady state, thereby enabling accurate measurement of glucose turnover. In addition, EGP and glucose disappearance were measured after an overnight fast, when glucose, insulin, and glucagon concentrations differed, and during a hyperglycemic pancreatic clamp, when glucose, insulin, and glucagon in the nondiabetic and mild diabetic subjects were raised to values similar to those observed in the severe diabetic subjects, thereby facilitating comparison. Rates of glycogenolysis and gluconeogenesis also were measured to determine whether the cause of excessive glucose production (if present) differs with the severity of diabetes.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects. After approval from the Mayo Institutional Review Board, 7 subjects with severe diabetes (prospectively defined as a fasting plasma glucose concentration >9 mmol/l), 9 subjects with mild diabetes (prospectively defined as a fasting glucose concentration 7–9 mmol/l), and 12 nondiabetic subjects gave informed written consent to participate in the study. Subjects were matched for age, gender, and body mass index (Table 1). All subjects were in good health, at a stable weight, and did not engage in regular vigorous physical exercise. The diabetic subjects had no evidence of clinically significant diabetic complications. At the time of screening, three subjects in the severe diabetic group were on insulin, one on metformin, and three on both metformin and a sulfonylurea. In the mild group, one was on insulin, one on a sulfonylurea, and three on metformin, and four were treated with lifestyle modification alone. Oral hypoglycemic agents were discontinued 3 wk before study, and long-acting insulin was switched to regular insulin beginning with the midday meal 2 days before study. The four subjects previously on long-acting insulin injected ~4–8 units of regular insulin before each meal depending on their plasma glucose concentrations. The last injection of regular insulin was taken with the midday meal on the day before study. At the time of study, subjects were on no medications other than thyroxin and estrogen replacement therapy. All subjects were instructed to follow a weight-maintenance diet containing 55% carbohydrate, 30% fat, and 15% protein for at least 3 days before the study date.


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Table 1. Subject characteristics

 
Experimental design. Subjects were admitted to the General Clinical Research Center at 1700 the evening before the study and ate a standard 10 kcal/kg meal (55% carbohydrate, 30% fat, and 15% protein) at 1800. The subjects then remained fasting until the end of the study. After sampling blood for baseline enrichment, 1.67 mg·kg–1·body–1 2H2O was given in three divided doses at 1800, 2000, and 2200. Small sips of water (containing 2H2O) were permitted upon request. At 0600, an 18-gauge cannula was inserted in a forearm vein for tracer and hormone infusions. Another 18-gauge cannula was inserted in a retrograde fashion in a dorsal hand vein of the opposite arm, and the hand was placed in a heated box (~55°C) to enable sampling of arterialized venous blood. A primed (fasting glucose in mmol/l divided by 5.5 mmol/l times 12 µCi) continuous (0.12 µCi/min) infusion of [3-3H]glucose (New England Nuclear, Boston, MA) was started at 0700 and continued until the end of the study. A constant infusion containing insulin (0.2 mU·kg–1·min–1), somatostatin (60 ng·kg–1·min–1), glucagon (0.65 ng·kg–1·min–1), and growth hormone (3 ng·kg–1·min–1) was started 15 h after the last meal at 0900 (i.e., time 0) and continued at the same rate until the end of the study. If necessary, exogenous glucose (containing [3-3H]glucose) was infused in amounts sufficient to raise and maintain plasma glucose at ~11 mmol/l. In addition, to mimic the anticipated decrease in EGP, the basal infusion of [3-3H]glucose was reduced by 50% in the diabetic and 25% in the nondiabetic subjects at 1 min and kept constant thereafter (3). Arterialized venous blood samples were collected at regular intervals for measurement of tracer and hormone concentrations.

Analytical techniques. Plasma samples were placed on ice, centrifuged at 4°C, separated, and stored at –20°C until assay. Glucose concentrations were 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 were measured by RIA using reagents supplied by Linco Research (St. Louis, MO). Free fatty acid (FFA) concentrations were measured using a colorimetric assay (COBAS; Roche Diagnostics, Indianapolis, IN). HbA1C was measured by affinity chromatography (normal range 4–6.3%; Gly-Affin, Akron, OH). Body composition was measured using dual-energy X-ray absorptiometry (DPX scanner; Hologic, Waltham, MA). Plasma [3-3H]glucose specific activity was measured as previously described (2, 45). Plasma 2H2O enrichment and enrichment of 2H2O on the 5th carbon of glucose were measured as previously described (1) using the method of Landau et al. (34) and Schumann et al. (50). C-5 glucose enrichment and, therefore, gluconeogenesis and glycogenolysis could not be measured after HPLC purification in one nondiabetic and one subject with mild diabetes because of lack of sufficient blood for analysis.

Calculations. Concentrations and rates from –30 to 0 (basal) and 210–240 (clamp) min were meaned for the purposes of analysis. All rates are expressed per kilogram lean body mass. Glucose appearance and disappearance were calculated using the steady-state equations of Steele et al. (53). EGP was calculated by subtracting the exogenous glucose infusion rate from the glucose appearance. Rates of gluconeogenesis were calculated by multiplying the ratio of plasma C-5 glucose to 2H2O enrichment times glucose appearance, which in the basal state equals EGP and during the clamps equals the sum of EGP and the exogenous glucose infusion rate (1, 34, 50). Previous experiments have shown that multiplying the plasma C-5 glucose-to-2H2O ratio times glucose appearance provides an estimate of gluconeogenesis that is equal to that obtained by multiplying the plasma C-5 glucose-to-plasma C-2 glucose ratio times EGP both in the fasting state (7, 11) and during exogenous glucose infusion at rates equivalent to those employed in the present experiments (1). Glycogenolysis was calculated by subtracting the rate of gluconeogenesis from EGP (1, 7, 21, 22). This approach measures the contribution of gluconeogenesis and glycogenolysis to EGP rather than the contribution of these processes to the intrahepatic glucose 6-phosphate pool.

Concentrations and rates were compared among groups using ANOVA followed where appropriate by Student's two-tailed nonpaired test. A P value <0.05 was considered as statistically significant.


    RESULTS
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 RESULTS
 DISCUSSION
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Plasma glucose, insulin, and C-peptide concentrations. By design, fasting plasma glucose concentrations (Fig. 1, top) were higher (P < 0.001) in the severe diabetic than mild diabetic subjects, which in turn were higher (P < 0.001) than those of the nondiabetic subjects (12.7 ± 0.6 vs. 8.1 ± 0.4 vs. 5.1 ± 0.4). Glucose concentrations were increased by means of an exogenous glucose infusion in the mild and nondiabetic subjects to values that did not differ from 120 min onward. Glucose concentrations averaged 11.4 ± 0.3 and 11.2 ± 0.5 mmol/l, respectively, in the mild diabetic and nondiabetic subjects during the final hour of the clamp. Glucose concentrations in the severe diabetic subjects remained elevated during the pancreatic clamp. This resulted in glucose concentrations during the final 30 min of the clamp (13.7 ± 0.8 mmol/l) that were higher (P < 0.05) than those present over the same interval in the mild diabetic or nondiabetic subjects.



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Fig. 1. Glucose (top), insulin (middle), and C-peptide (bottom) concentrations observed in nondiabetic ({blacktriangleup}) and mild ({blacksquare}) and severe ({bullet}) diabetic subjects before and during a hyperglycemic pancreatic clamp. Somatostatin, insulin, and replacement infusions of glucagon and growth hormone were started at time 0 (dotted line).

 
Fasting plasma insulin concentrations (Fig. 1, middle) were higher in both the severe (P = 0.05) and mild (P < 0.01) diabetic subjects than in the nondiabetic subjects (46 ± 6 vs. 58 ± 9 vs. 32 ± 4 pmol/l). In contrast, fasting insulin concentrations did not differ in the severe and mild diabetic subjects. Plasma insulin concentrations during the final 30 min of the clamp did not differ among the severe, mild, and nondiabetic subjects (70 ± 5 vs. 76 ± 5 vs. 73 ± 4 pmol/l).

Fasting C-peptide concentrations (Fig. 1, bottom) were higher (P < 0.05) in the mild diabetic than nondiabetic subjects (0.77 ± 0.14 vs. 0.47 ± 0.30 nmol/l) but did not differ from those in the severe diabetic subjects (0.48 ± 0.07 nmol/l). The somatostatin infusion, started at time 0, resulted in comparable (P = 0.11) and near-complete suppression of C-peptide concentrations in the severe diabetic, mild diabetic, and nondiabetic subjects (0.06 ± 0.01 vs. 0.15 ± 0.04 vs. 0.13 ± 0.02 nmol/l).

Glucagon and growth hormone concentrations. Fasting plasma glucagon concentrations (Fig. 2, top) were higher (P < 0.05) in the severe diabetic subjects than both the mild diabetic and nondiabetic subjects (170 ± 11 vs. 139 ± 9 vs. 139 ± 6 pg/ml). Fasting plasma glucagon concentrations did not differ in the mild diabetic and nondiabetic subjects. Plasma glucagon fell during the clamp in the severe diabetic subjects to values that did not differ from those present in the mild and nondiabetic subjects (151 ± 6 vs. 131 ± 7 vs. 139 ± 7 pg/ml, respectively).



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Fig. 2. Plasma glucagon (top) and growth hormone (bottom) concentrations observed in nondiabetic ({blacktriangleup}) and mild ({blacksquare}) and severe ({bullet}) diabetic subjects before and during a hyperglycemic pancreatic clamp. Somatostatin, insulin, and replacement infusions of glucagon and growth hormone were started at time 0 (dotted line).

 
Growth hormone concentrations did not differ either before (0.8 ± 0.3 vs. 0.6 ± 0.3 vs. 0.6 ± 0.3 ng/ml) or during (0.7 ± 0.1 vs. 0.8 ± 0.1 vs. 0.9 ± 0.1 ng/ml) the clamp among the severe diabetic, mild diabetic, and nondiabetic subjects (Fig. 2, bottom).

Glucose infusion rate and [3-3H]glucose specific activity. The glucose infusion rate required to maintain target glucose concentration during the final 30 min of the clamp (Fig. 3, top) was greater (P < 0.01) in the nondiabetic subjects than in either the severe or mild diabetic subjects (5.7 ± 0.8 vs. 0.4 ± 0.4 vs. 1.0 ± 0.5 µmol·kg–1·min–1). On the other hand, the glucose infusion rates did not differ in the severe and mild diabetic subjects.



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Fig. 3. The iv glucose infusion rate required to maintain target glucose concentrations (top) and plasma [3-3H]glucose specific activity (bottom) observed in nondiabetic ({blacktriangleup}) and mild ({blacksquare}) and severe ({bullet}) diabetic subjects. Somatostatin, insulin, and replacement infusions of glucagon and growth hormone were started at time 0 (dotted line).

 
Plasma [3-3H]glucose specific activity remained within 10% of basal values throughout the study in all groups, enabling accurate measurement of glucose production (Fig. 3, bottom).

EGP and rates of glucose disappearance. EGP before the clamp (Fig. 4, top) was higher (P < 0.002) in the severe diabetic than in the mild diabetic and nondiabetic subjects (24.1 ± 1.2 vs. 18.0 ± 0.9 vs. 18.0 ± 1.0 µmol·kg–1·min–1). Basal EGP did not differ in the mild diabetic and nondiabetic subjects. In contrast, EGP during the final 30 min of the clamp was higher (P < 0.01) in both the severe and mild diabetic subjects than in the nondiabetic subjects (14.1 ± 1.3 vs. 14.0 ± 0.7 vs. 8.6 ± 1.0 µmol·kg–1·min–1). EGP during the clamp did not differ in the severe and mild diabetic subjects.



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Fig. 4. Rates of endogenous glucose production (top) and glucose disappearance (bottom) observed in the nondiabetic and mild and severe diabetic subjects at baseline and during the final 30 min of hyperglycemic pancreatic clamp. *P < 0.01 vs. nondiabetic subjects. {dagger}P < 0.01 vs. mild diabetic subjects.

 
Glucose disappearance before the clamp (Fig. 4, bottom) was higher (P < 0.002) in the severe diabetic than in the mild diabetic and nondiabetic subjects (24.1 ± 1.2 vs. 18.0 ± 0.9 vs. 18.0 ± 1.0 µmol·kg–1·min–1). As with EGP, glucose disappearance did not differ in the mild diabetic and nondiabetic subjects before the clamp. However, glucose disappearance during the final 30 min of the clamp was lower (P < 0.05) in both the severe and mild diabetic subjects than the nondiabetic subjects (16.6 ± 1.8 vs. 16.5 ± 0.7 vs. 38.2 ± 4.2 µmol·kg–1·min–1). Glucose disappearance did not differ in the severe and mild diabetic subjects during the clamp.

Contribution of glycogenolysis and gluconeogenesis to EGP. Glycogenolysis before the clamp (Fig. 5, top) was higher (P < 0.02) in the 7 severe diabetic than in the 8 mild diabetic and 11 nondiabetic subjects (8.4 ± 1.1 vs. 5.0 ± 0.6 vs. 5.0 ± 0.8 µmol·kg–1·min–1). Basal glycogenolysis did not differ in the mild diabetic and nondiabetic subjects. As with EGP, glycogenolysis during the final 30 min of the clamp was higher (P < 0.05) in both the severe and mild diabetic subjects than in the nondiabetic subjects (4.2 ± 1.7 vs. 3.5 ± 1.0 vs. 0.0 ± 0.8 µmol·kg–1·min–1) but did not differ in the severe and mild diabetic subjects.



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Fig. 5. Rates of glycogenolysis (top) and gluconeogenesis (bottom) observed in nondiabetic and mild and severe diabetic subjects at baseline and during the final 30 min of a hyperglycemic pancreatic clamp. *P < 0.05 vs. nondiabetic subjects. {dagger}P < 0.05 vs. mild diabetic subjects.

 
The percent contribution of gluconeogenesis to EGP did not differ among groups before the clamps (Table 2). However, because of higher rates of EGP, gluconeogenesis (Fig. 5, bottom) was higher (P < 0.05) in the severe diabetic than in the mild diabetic and nondiabetic subjects (15.7 ± 1.3 vs. 11.9 ± 1.0 vs. 12.7 ± 0.7 µmol·kg–1·min–1). Basal gluconeogenesis did not differ in the mild diabetic and nondiabetic subjects. Gluconeogenesis during the clamp did not differ in the severe diabetic, mild diabetic, and nondiabetic subjects (9.9 ± 0.9 vs. 9.1 ± 0.8 vs. 8.2 ± 0.9 µmol·kg–1·min–1).


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Table 2. Enrichment of C-5 glucose, plasma water, glucose appearance, gluconeogenesis, and glycogenolysis

 
FFA concentrations. Plasma FFAs (Fig. 6) did not differ (ANOVA, P = 0.06) among the three groups before the clamp. However, post hoc analysis indicated that FFA were slightly higher (P < 0.02) in the severe diabetic than nondiabetic subjects (0.54 ± 0.06 vs. 0.38 ± 0.04 mmol/l) but did not differ from those in the mild diabetic group (0.50 ± 0.06 mmol/l). Plasma FFAs fell in all groups during the clamp to values that were higher (P < 0.001) in both the severe and mild diabetic subjects than in the nondiabetic subjects (0.34 ± 0.07 vs. 0.30 ± 0.06 vs. 0.10 ± 0.03 mmol/l). Plasma FFAs did not differ in the severe and mild diabetic subjects during the clamp.



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Fig. 6. Free fatty acid concentrations observed in nondiabetic and mild and severe diabetic subjects before and during the final 30 min of study with a hyperglycemic pancreatic clamp. *P < 0.05 vs. nondiabetic subjects.

 
Correlations. EGP before the clamp was correlated with both plasma glucose (r = 0.69; P < 0.001) and FFA concentrations (r = 0.52; P < 0.01). EGP during the last 30 min of the clamp also was correlated (r = 0.52; P < 0.01) with the plasma FFA concentrations present over the same interval. On the other hand, there was an inverse relationship between plasma FFA and glucose disappearance both before (r = 0.51; P < 0.001) and during (r = 0.43; P < 0.05) the clamps. Neither glycogenolysis nor gluconeogenesis correlated with plasma glucose or FFA either before or during the clamp.


    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
There has been debate as to whether type 2 diabetes is associated with increased EGP (7, 9, 10, 15, 18, 19, 2123, 28, 29, 31, 37, 40, 42, 43, 46). The present experiments provide insight into the origins of the debate. They indicate that, when measured in the presence of plasma tracer and tracee steady state, EGP is elevated in individuals whose fasting plasma glucose is in excess of 10 mmol/l (7, 9, 10, 15, 18, 19, 2123, 31, 37, 40, 42, 43) but is not different from nondiabetic rates in diabetic subjects whose fasting glucose is <9 mmol/l (7, 15, 29, 31, 42). Such data have been interpreted in the past as indicating that excessive EGP only occurs in individuals with severe fasting hyperglycemia. However, consistent with the recent review of Radzuik and Pye (44), the present studies indicate that the allegedly normal rates of EGP in subjects with mild diabetes occurred in the presence of increased glucose and insulin concentrations, indicating that the rates in fact were elevated inappropriately (31, 43). This was clearly evident in the present experiments when glucose and insulin concentrations in the nondiabetic and mild diabetic subjects were raised to levels approximating those present in the severe diabetic subjects. Under these circumstances, EGP clearly was excessive in the subjects with mild and more severe type 2 diabetes. The increase in EGP in the severe diabetic subjects in the basal state was the result of an increase in glycogenolysis and gluconeogenesis. On the other hand, when glucose, insulin, and glucagon concentrations were raised in the nondiabetic and mild diabetic subjects to levels approximating those present in the severe diabetic subjects, excessive EGP was entirely accounted for by increased rates of glycogenolysis in both the severe and mild diabetic subjects.

Insulin and glucose suppress and glucagon stimulates EGP (12, 17, 39, 41, 47, 49, 55). Glucose concentrations will rise when glucose appearance exceeds disappearance. The rise in glucose causes an increase glucose disappearance and a decrease in glucose production in part because of the effects of glucose per se and in part because of an associated increase in insulin secretion (12, 16, 17, 20, 32, 39, 43, 49, 55). Glucose concentrations will plateau when the rates of glucose production again equal glucose disappearance. In the present experiments, this equilibration occurred in the mild diabetic subjects at a glucose concentration of ~8 mmol/l and at an insulin concentration that was approximately two times as high as that present in the nondiabetic subjects. Although the resultant rates of glucose production and disappearance did not differ numerically from those present in the nondiabetic subjects, they clearly were not appropriate for the higher glucose and insulin concentrations, as was evident when glucose and insulin concentrations were matched during the clamp. In addition, insulin secretion obviously was impaired, since insulin secretion was not sufficient to restore the fasting glucose concentration to nondiabetic levels.

A similar situation pertained to the subjects with severe diabetes; however, glucose production and disappearance equilibrated at a higher glucose concentration. Insulin and C-peptide concentrations did not differ in the severe and mild diabetic subjects, indicating that a greater glycemic stimuli was required in the former to achieve the same circulating insulin and C-peptide concentrations as the latter. However, in contrast to the pattern observed in the mild diabetic subjects, glucose production and disappearance in the severe diabetic subjects equilibrated at rates that were significantly higher than those in the nondiabetic subjects. Taken together, these data suggest that inappropriately elevated rates of glucose production and inappropriately decreased rates of glucose disappearance combined with defects in insulin secretion and excessive glucagon release all contribute to fasting hyperglycemia in people with type 2 diabetes.

The present experiments provide insight into the cause of the excessive rates of glucose production in the diabetic subjects. Glucagon and FFA both can modulate the effects of insulin and glucose on glucose production (5, 14, 17, 25, 35, 36, 38, 41, 49, 51, 52). An acute increase in glucagon results in an increase in glycogenolysis, whereas a chronic increase also enhances gluconeogenesis (13). Elevated FFA increase gluconeogenesis and impair suppression of glucose production at least in part by inhibiting insulin-induced suppression of glycogenolysis (6, 8, 48). In the present experiments, both glucagon and FFA were elevated in the severe diabetic subjects in the fasting state and therefore likely contributed to the increased rates of glycogenolysis and gluconeogenesis observed in these individuals.

Infusion of exogenous insulin and inhibition of endogenous hormone secretion during clamp resulted in comparable insulin concentrations in all three groups. Assuming an approximate 2:1 portal venous-to-peripheral venous insulin gradient (27, 54), the modest increase in peripheral insulin concentrations likely resulted in little if any change in portal insulin concentrations. However, there was only a minimal decrease in FFA in the mild and severe diabetic subjects. This observation is consistent with previous studies that have shown that diabetes impairs suppression of lipolysis (24). Peripheral glucagon concentrations fell in the severe diabetic subjects but remained unchanged in the mild diabetic and nondiabetic subjects during the clamp because of infusion of somatostatin and exogenous glucagon. However, because in the fasting state portal glucagon concentrations are 40–50% higher than peripheral glucagon concentrations (4, 30), the reduction in glucagon secretion during the somatostatin infusion likely resulted in portal glucagon concentrations that were equal to or lower than systemic glucagon concentrations in all groups. This fall in portal glucagon likely contributed to the concurrent decrease in glucose production and gluconeogenesis that also was observed in all groups. In addition, the concurrent increase in plasma glucose concentrations resulted in essentially complete suppression of glycogenolysis in the nondiabetic subjects, consistent with previous reports that hyperglycemia markedly inhibits glycogenolsysis in both nondiabetic animals (14, 49) and humans (41). In contrast, the approximately threefold higher FFA concentrations observed in diabetic subjects during the clamps (Fig. 6) was accompanied by higher rates of glucose production and glycogenolysis in both the "severe" and "mild" diabetic subjects. Taken together, these data suggest that elevated glucagon and FFA contribute to the altered regulation of glycogenolysis and gluconeogenesis in people with diabetes mellitus.

We are unaware of previous studies that have compared glycogenolysis in diabetic and nondiabetic subjects in the presence of low insulin concentrations and matched but elevated glucose concentrations. Several studies using the deuterated water method have reported no difference in fasting rates of glycogenolysis between diabetic and nondiabetic subjects (7, 21). Furthermore, Magnusson et al. (37) have reported that net glycogenolysis (the net balance between glycogen synthesis and degradation) measured with NMR spectroscopy was lower in diabetic than nondiabetic subjects. However, in all of the above studies, glucose concentrations in the diabetic subjects were considerably higher than those present in the nondiabetic subjects. Because a lesser degree of hyperglycemia resulted in near-complete suppression of glycogenolysis in nondiabetic subjects in the current and previous studies (41), ongoing glycogenolysis in the diabetic subjects (whether equal to or lower than that observed in euglycemic nondiabetic subjects) was not appropriate for the prevailing glucose concentration.

The present experiments suffer from several limitations. The C-5 of glucose is labeled with 2H2O during gluconeogenesis, whereas the C-2 of glucose is labeled during equilibration of glucose 6-phosphate derived from gluconeogenesis, glycogenolysis, and phosphorylation of extracellular glucose with fructose 6-phosphate. Therefore, although the plasma ratio of C-5-to-C-2 deuterated glucose provides an index of gluconeogenesis, C-5-labeled glucose 6-phosphate could have been incorporated into glycogen followed by subsequent release into plasma. If so, the increased rates of glycogenolysis observed in the diabetic patients could have resulted in an overestimate of gluconeogenesis. The report by Landau (33) that this so-called hepatic glycogen cycling is minimal in nondiabetic subjects but increased with diabetes supports such a possibility. Use of the deuterated water method also assumes that exchange of 2H2O at the level of the transaldolase reaction and flux through the pentose shunt are equal in the groups being compared (50). Presently, it is not known whether these assumptions are true in nondiabetic subjects and diabetic subjects with varying degrees of hyperglycemia. Future studies will be required to address these important questions.

We assume that differences between the mild and severe groups are the result of differences in their metabolic milieu at the time of study rather than their chronic level of glycemic control, since their HbA1c concentrations did not differ at the time of screening. In addition, because all oral antihyperglycemic drugs were discontinued 3 wk before study and intermediate-acting insulin was withdrawn 48 h before study, we believe it is improbable that the results were influenced by residual effects of their antecedent therapy. Fasting insulin concentrations were higher in the diabetic than nondiabetic subjects, whereas the insulin concentrations did not differ during the clamps among groups. This suggests that the increment in portal insulin concentrations likely was smaller in the diabetic than nondiabetic subjects. It is possible that a smaller increment in portal insulin led to lesser suppression of glucose production in the diabetic subjects. On the other hand, the rates of glucose production and glycogenolysis remained higher in both the mild and severe diabetic subjects despite several hours of matched glucose and insulin concentrations. Therefore, the present experiments cannot determine whether the response to a change in glucose and insulin concentrations is abnormal in people with type 2 diabetes. However, they do demonstrate that the rates of glucose production and glycogenolysis were not appropriate for the prevailing glucose and insulin concentrations. Glucose concentrations were matched by means of an exogenous glucose infusion in the mild diabetic and nondiabetic subjects. However, glucose concentrations present during the clamp in the individuals with severe diabetes remained higher than those in the other two groups. Therefore, EGP may have been even higher in the severe diabetic subjects if their glucose concentrations had been lowered to match those present in the mild diabetic and nondiabetic subjects. Comparable glucose concentrations potentially could have been achieved in all groups if we had used a higher basal insulin infusion rate. However, we chose not to do so, since we were concerned that higher insulin concentrations would result in near-maximal suppression of EGP in both the mild and nondiabetic groups, thereby obscuring potential differences. We also hesitated to clamp glucose concentrations in the mild diabetic and nondiabetic subjects at higher levels since we were concerned the greater glycemic stimulus would overcome the ability of somatostatin to inhibit insulin secretion. We therefore do not know whether regulation of hepatic glucose release was equally abnormal in the severe and mild diabetic subjects. We merely know that EGP was inappropriately elevated in both groups.

As anticipated, glucose disappearance increased in the nondiabetic subjects during the clamp, presumably because of the increase in glucose and peripheral insulin concentrations. On the other hand, glucose disappearance during the clamp remained unchanged in the mild diabetic subjects and decreased slightly in the severe diabetic subjects. Although insulin- and glucose-induced stimulation of glucose uptake is impaired in type 2 diabetes, it is not absent (10, 19, 20, 26, 32, 39). We therefore would have anticipated either a slight increase or no change in glucose disappearance in the severe diabetic subjects. It is possible that portal insulin concentrations decreased rather than increased in the diabetic subjects, resulting in a decrease in hepatic glucose uptake. Alternatively, non-insulin-dependent sites of glucose disposal (e.g., urinary glucose loss) may have contributed to the fall in glucose disappearance. Gluconeogenesis was measured with the deuterated water method (11, 34, 50). This technique assumes the presence of steady state. Steady state likely was approached in both the basal state and during the clamps, since 2H2O was given the evening before study and since the exogenous glucose infusion rate remained essentially constant in all groups during the final 3 h of the clamps. In addition, as is evident from Table 2, the plasma 2H2O enrichment did not change during the 4.5 h of study. Because this method measures the enrichment of 2H2O on the fifth carbon of plasma glucose, it is possible that a portion of the C-5 glucose first passed through glycogen. If so, glycogenolysis will be underestimated and gluconeogenesis overestimated. Therefore, the lack of suppression of glycogenolysis in the diabetic subjects during the clamps may have led to an overestimate of gluconeogenesis. Future studies will be required to address this possibility.

In summary, the present studies indicate that, when considered in light of the prevailing insulin and glucose concentrations, EGP is inappropriately increased and glucose disappearance inappropriately decreased in people with mild and severe type 2 diabetes mellitus. Although hyperglucagonemia may contribute to increased rates of gluconeogenesis in people with severe type 2 diabetes, glycogenolysis is inappropriately elevated in people with mild and severe type 2 diabetes, even when glucose, insulin, and glucagon concentrations are matched. Therefore, treatment strategies that normalize regulation of glycogenolysis, gluconeogenesis, or ideally both are likely to improve glycemic control in people with type 2 diabetes. Because FFAs were elevated in the severe diabetic subjects before and in both diabetic groups during the clamp, these data also lend further support to the concept that alterations in fat metabolism contribute to inappropriately elevated rates of glucose production in people with type 2 diabetes mellitus.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by National Institutes of Health (NIH) Grants DK-29953 and RR-00585, a Novo Nordisk research infrastructure grant, and the Mayo Foundation. R. Basu was supported by an American Diabetes Association Mentor based fellowship.


    ACKNOWLEDGMENTS
 
We thank B.Dicke, L. Heins, R. Rood, C. Nordyke, M. Persson, and C. Ford for technical assistance; J. Feehan, B. Norby, and the staff of the Mayo General Clinical Research Center for assistance in performing the studies; and M. Davis for assistance in preparation of the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Rizza, Mayo Clinic, 200 1st St. SW, Rm 5-194 Joseph, Rochester, MN 55905 (E-mail: rizza.robert{at}mayo.edu).

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.


    REFERENCES
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