Decreased Non-Insulin Dependent Glucose Clearance Contributes to the Rise in FPG in the Non-Diabetic Range.

Diabetes Care Publish Ahead of Print, published online November 13, 2007 Decreased Non-Insulin Dependent Glucose Clearance Contributes to the Rise in FPG in the Non-Diabetic Range. Rucha Jani, M.D., Marjorie Molina, M.D, Masafumi Matsuda, MD, Bogdan Balas, MD, Alberto Chavez, MD, Ralph A. DeFronzo, M.D and Muhammad Abdul-Ghani, M.D., PhD. The Division of Diabetes, University of Texas Health Science Center at San Antonio Running title: Fasting Hyperglycemia in the Non-Diabetic Range Corresponding Author: Muhammad Abdul-Ghani, M.D, PhD. University of Texas Health Science Center at San Antonio Diabetes Division 7703 Floyd Curl Drive San Antonio, TX 78229 albarado@uthscsa.edu Received for publication 13 August 2007 and accepted in revised form 6 November 2007. Copyright American Diabetes Association, Inc., 2007

ABSTRACT Objective: To assess the contribution of decreased glucose clearance to the rise in fasting plasma glucose (FPG) in the non-diabetic range. Research Design and Methods: 120 subjects with normal glucose tolerance received an OGTT and euglycemic insulin clamp with 3-[ 3 H] glucose. The basal and insulin-stimulated rates of glucose appearance, glucose disappearance, glucose clearance, and basal hepatic insulin resistance index were calculated. Simple Pearson correlation was used to assess the relationship between variables. Results: The increase in FPG (range= 75 to 125 mg/dl) correlated (r=0.32, p<0.0001) with the increase in BMI (range = 20-50 kg/m 2 ). The fasting plasma insulin concentration (FPI) also increased progressively with the increase in BMI (r=0.62, p<0.0001). However despite increasing FPI, the basal glucose clearance rate declined and correlated with the increase in BMI (r =-0.56, p<0.0001). Basal hepatic glucose production (HGP) decreased with increasing BMI (r = -0.51, p<0.0001) and correlated inversely with the increase in FPI (r= -0.32, p< 0.0001). The hepatic insulin resistance (basal HGP x FPI) increased with rising BMI (r=0.52, p<0.0001). During the insulin clamp, glucose disposal declined with increasing BMI (r= -0.64, p<0.0001) and correlated with the basal glucose clearance (r=0.39, p<0.0001). Conclusion: These results demonstrate that, in non-diabetic subjects, rising FPG is associated with a decrease (not an increase) in basal hepatic glucose production and is explained by a reduction in glucose clearance.

H yperglycemia is a sine qua-non in type 2 diabetes. The hyperglycemia is manifested both as fasting hyperglycemia and postprandial hyperglycemia. The mechanisms which regulate the plasma glucose concentration during the postabsorptive state are distinct from those which regulate postprandial plasma glucose levels (1-3). Following glucose ingestion approximately two-thirds of the glucose load is taken up by the skeletal muscle and one-third by the liver (4,5), while glucose-stimulated insulin secretion causes the suppression of hepatic glucose production (5). During the postabsorptive state the liver is responsible for the majority of endogenous glucose production, while most of glucose uptake takes place in insulin insensitive (brain and splanchnic) tissues. Only 25% of glucose uptake occurs in insulin sensitive tissues, primarily skeletal muscle, during fasting conditions (6). During the postabsorptive state, tissue glucose uptake is closely matched by hepatic glucose production (HGP) (7,8). The primary determinant of basal HGP is the fasting plasma insulin concentration and small increases in the portal plasma insulin concentration markedly suppress HGP (9,10). Previous studies have demonstrated that the increase in fasting plasma glucose concentration in subjects with type 2 diabetes mellitus primarily is due to an increase in hepatic glucose production which occurs in the presence of fasting hyperinsulinemia, indicating the presence of hepatic insulin resistance (8). Studies examining the relationship between HGP and fasting plasma glucose concentration in type 2 diabetic individuals have demonstrated that basal HGP does not start to increase until the FPG exceeds 140-160 mg/dl (8,11). Since basal HGP remains unchanged with FPG concentrations up to 140-160 mg/dl (8,11), the etiology of the increase in FPG remains unclear. We postulated that a decrease in tissue glucose uptake was responsible for the increase in FPG over this range. The aim of this study was to assess the relationship between tissue glucose clearance during the postabsorptive state and FPG concentration in the nondiabetic range of plasma glucose levels. RESEARCH DESIGN AND METHODS The participants included 120 normal healthy subjects (F/M =67/53; age = 38+ 1 year; BMI = 29.1 + 0.6 kg/m 2 ; FPG =93 + 1 mg/dl; 2h PG = 115 + 2; FPI = 9 + 1 uu/ml, 2h PI = 65 + 01). All subjects were of Mexican American origin and had a normal oral (75 gram) glucose tolerance test (FPG <126 mg/dl and 2h PG <140 mg/dl). All subjects had normal liver, cardiopulmonary, and kidney function as determined by medical history, physical examination, screening blood tests, electrocardiogram, and urinalysis. No subject was taking any medication known to affect glucose tolerance. Body weight was stable (±2 kg) for at least 3 months before study in all subjects. The study protocol was approved by the Institutional Review Board of the University of Texas Health Science Center, San Antonio, Texas and informed written consent was obtained from all subjects before their participation. All studies were performed at the General Clinical Research Center of the University of Texas Health Science Center at 0800 hours following a 10- to 12-h overnight fast. OGTT. Before the start of the OGTT, a small polyethylene catheter was placed into an antecubital vein and blood samples were collected at 30, 15, 0, 30, 60, 90, and 120 min for the measurement of plasma glucose and insulin concentrations. On the day of the OGTT lean body mass was measured with Dual Energy X-ray Absorptiometry (DEXA)

Euglycemic Insulin Clamp. Before the start of the insulin clamp, a catheter was placed into an antecubital vein for the infusion of all test substances. A second catheter was inserted retrogradely into a vein on the dorsum of the hand, and the hand was placed into a thermoregulated box heated to 70 C. At 0800 h, all subjects received a primed (25 µci)- continuous (0.25 µci/min) infusion of 3-[ 3 H] glucose (DuPont NEN Life Science Products, Boston, MA), which was continued for the 4 hour duration of the study. Two hours after the start of tritiated glucose, subjects received a primed-continuous insulin infusion at the rate of 240 pmol (40 mu) min 1 m 2 for 120 min. During the last 30 min of the basal equilibration period (90-120 min), blood samples were taken at 5- to 10-min intervals for the determination of plasma glucose and insulin concentrations and tritiated glucose radioactivity. During the insulin infusion, plasma glucose concentration was measured every 5 min, and a variable infusion of 20% glucose was adjusted, based on the negative feedback principle, to maintain the plasma glucose concentration at each subject s fasting plasma glucose level with a coefficient of variation <5%. Blood samples were collected every 15 min from 120 to 210 min and every 5 10 min from 210 to 240 min for the determination of plasma glucose and insulin concentrations and tritiated glucose radioactivity. Calculations. Following an overnight fast, steady-state conditions prevail, and endogenous (primarily reflects hepatic) glucose production (HGP) was calculated as the tritiated glucose infusion rate (dpm/min) divided by the plasma tritiated glucose specific activity (dpm/mg). During the insulin clamp, non steady-state conditions for tritiated glucose specific activity prevail, and the rate of glucose appearance (Ra) was calculated with Steele s equation (12). The rate of residual HGP during the insulin clamp was calculated by subtracting the rate of exogenous glucose infusion from the tracerderived Ra. The insulin-stimulated rate of total glucose disposal (TGD) was calculated by adding the rate of residual HGP to the exogenous glucose infusion rate. Plasma glucose clearance rate during the fasting state was calculated as the rate of HGP divided by fasting plasma glucose concentration. Hepatic insulin resistance was calculated as the product of HGP and fasting plasma insulin (13). Statistical Analysis. All values are expressed as mean + SEM. ANOVA was used to compare the difference between the means of the 4 quantiles. Simple Pearson correlation was used to assess the relationship between variables. For multivariate regression, FPG was considered dependent variable and BMI, FPI, HGP and glucose clearance were considered independent variables. Non normally distributed variables (i.e., BMI) were log transformed. Statistical significance was considered at P<0.05. RESULTS The increase in FPG over the nondiabetic range of fasting plasma glucose levels (75 to 125 mg/dl) in the present study correlated positively (r=0.32, p<0.0001) with the increase in BMI (range = 20-50 kg/m 2 ) (Figure 1A). However, over this same range, HGP decreased with the increase in BMI (r=- 0.51, p<0.0001) (Figure 1B). We also examined the relationship between HGP, expressed per lean body mass, and BMI. The negative relationship between HGP and BMI remained when HGP was expressed per lean body mass (r=-0.31, p=0.004). To further examine the relationships between obesity, FPG, and HGP, subjects were divided into four quartiles. The metabolic and anthropometric characteristics of subjects in the four quartiles are shown in Table 1. The four groups were comparable in age and gender, but FPG progressively increased from quartile 1 through quartile 4

(P<0.001 with ANOVA). However, HGP, whether expressed per total body weight or lean body mass, progressively decrease (not increased) (p<0.001 with ANOVA). Since fasting plasma insulin (FPI) is the primary regulator of HGP, we examined the relationship between FPI and BMI. As expected, fasting plasma insulin concentration progressively increased from subjects in quartile 1 through 4 (p<0.0001) (Table 1). FPI correlated strongly and positively with BMI (r=0.62, p<0.0001; Figure 1C) and negatively with HGP (r=-0.34, p<0.0001). The product of HGP and FPI, an index of hepatic insulin resistance, progressively increased from quartile 1 through 4 p=0.0002), and correlated closely with BMI (r=0.52, p<0.0001; Figure 2A). However, despite the marked increase in FPI, the glucose clearance rate was decreased with increasing BMI in quartiles 1 through 4 (p<0.0001) and the plasma glucose clearance rate correlated inversely with the BMI (r=-0.56, p<0.0001, Figure 2B). To assess the contribution of obesity to the relationship between FPG and glucose clearance, we compared the correlation coefficient between the two variables in lean (BMI<27, n = 48) and overweight/obese subjects (BMI >27, n=72). The correlation coefficient was -0.43 in lean subjects and - 0.27 in obese subjects. In a multivariate model, using FPG as the dependent variable and BMI, FPI, HGP and glucose clearance as the independent variables, all 4 independent variables significantly correlated with FPG, which was explained by the following equation: FPG= 30.2 + 1.5 log(bmi) + 51.8 HGP - 0.29 FPI - 36.1 Glucose Clearance Collectively, BMI, HGP, FPI, and glucose clearance explained ~60% of the variability in FPG, where more than half of it was explained by glucose clearance and bhgp. When related to FPG, both HGP (r=0.17, p=0.05) and glucose clearance rate (r=-0.42, p<0.0001) displayed a negative correlation (Figures 3A and 3B). A positive correlation between the glucose clearance rate and total body glucose disposal during the insulin clamp (r=0.39, p<0.0001) was observed. DISCUSSION The results of the present study demonstrate that, in nondiabetic subjects, the increase in BMI is associated with an increase in FPG and, paradoxically, with a decrease in HGP. Since HGP is the main contributor to the elevated FPG concentration in type 2 diabetic subjects (8), one might have expected that the increase in FPG observed with increasing BMI would be associated with a rise in HGP. However, the results of this study demonstrate the opposite. The inverse relationship between FPG and HGP is most striking when one compares subjects in the highest BMI quartile with subjects in the lowest BMI quartile (Table 2), and it excludes the possibility that an increase in hepatic glucose production is responsible for the increase in fasting plasma glucose in the nondiabetic range. Since under postabsorptive conditions steady state conditions exist with respect to the fasting plasma glucose concentration, the elevated FPG concentration in obese subjects must be explained by a decrease in tissue glucose clearance. Indeed, when the glucose clearance in obese subjects is compared to that in lean subjects, there is a 34% decrease in glucose clearance rate. Furthermore, the glucose clearance rate correlates negatively with the increase in BMI. These results indicate that the increase in FPG, which accompanies the increase in BMI, primarily results from the decline in glucose clearance and not from excess production of glucose by the liver. Obese subjects, as expected, had a 2.5- fold increase in fasting plasma insulin concentration compared to lean individuals,

and the fasting plasma insulin concentration rose progressively with increasing BMI (Table 1, Figure 1C). The plasma insulin concentration is the main regulator of HGP (9,10). Thus, the rise in FPI with increasing BMI leads to a progressive decrease in HGP from quartile 1 through quartile 4 and HGP was strongly and inversely correlated with the FPI (r=-0.34, p<0.0001). Obesity per se seems has a small effect on FPG. Consistent with this, the inverse relationship between FPG and glucose clearance also was observed in lean subjects (BMI <27). Further the contribution of BMI (multivariate analysis) to the increase in FPG was much smaller compared to the contributions of glucose clearance and hepatic glucose production (see equation 1), indicating that the primary determinants of FPG are the HGP and tissue glucose clearance. Moreover, the impact of obesity to increase the FPG is due primarily to the decrease in tissue glucose clearance. We previously have shown that the basal insulin secretion rate increases with the increase in FPG in the non-diabetic range (14). However, when the insulin secretory rate (ISR) was related to FPG, the ratio of ISR/FPG remained constant across the entire nondiabetic range of FPG levels (14). These results indicate that (i) although glucosestimulated insulin secretion is markedly impaired with increasing FPG, basal insulin secretion is not affected by fasting hyperglycemia, and (ii) fasting hyperinsulinemia is a compensatory beta cell response to fasting hyperglycemia. The resultant fasting hyperinsulinemia which accompanies fasting hyperglycemia inhibits HGP and explains the present observation that HGP declines as FPG increases within the nondiabetic range. Thus, the decrease in HGP associated with the increase in BMI can be viewed as a compensatory physiological response to fasting hyperglycemia, which aims to ameliorate the rise in FPG. However, the increased hepatic insulin resistance in obese subjects, which also strongly correlates with increasing BMI, renders the liver more resistant to the action of insulin and results in an incomplete suppression of HGP. We previously have shown that the increase in insulin secretion rate in response to the rise in FPG peaks at a FPG concentration of ~140 mg/dl and declines thereafter (15). This results in an inverted U-shaped curve relating the fasting plasma insulin and FPG concentrations (15). It is noteworthy that the increase in HGP becomes evident only when FPG exceeds 140-160 mg/dl (8,15). Collectively, these observations indicate that at FPG <140 mg/dl the increase in FPG primarily is due to a decrease in tissue glucose clearance. Once the FPG exceeds ~140-160 mg/dl, the decline in insulin secretion, in the presence of hepatic insulin resistance, results in an increase in HGP resulting in a rise in FPG in type 2 diabetic individuals. A decrease in noninsulin dependent glucose clearance previously has been reported in subjects with type 2 diabetes (16). During the postabsorptive state, ~50% of glucose uptake occurs in the brain, ~ 25% in the splanchnic tissues, and ~25% in skeletal muscle. Glucose uptake by the brain and splanchnic tissues is insulin independent (1). Therefore, it is unlikely that decreased brain or splanchnic glucose uptake can account for the decline in glucose clearance observed with rising FPG levels in the present study. Glucose uptake in skeletal muscle occurs via both insulin dependent (GLUT4) and insulin independent (GLUT1) mechanisms (17). Skeletal muscle is well known to be resistant to the action of insulin (18) and this insulin resistance could contribute, in part to the decline in basal tissue glucose clearance observed in the present study. Similarly, a decrease in GLUT1, the insulin independent glucose transporter, in skeletal muscle, could contribute to the decrease in tissue glucose

clearance observed in normal glucose tolerant obese subjects in the present study. One previous study has reported a decrease in the amount of GLUT1 protein in skeletal muscle in subjects with type 2 diabetes compared to healthy controls (19,20). It is unclear whether the decrease in GLUT1 expression in subjects with type 2 diabetes represents a primary defect or occurs secondary to hyperglycemia. It also is unclear at what fasting plasma glucose level the decrease in GLUT1 expression becomes evident. Nonetheless, a decrease in GLUT1 expression in subjects with elevated FPG concentrations within the nondiabetic range could explain, in part, decrease in glucose clearance observed with rising FPG in the present study. Insulin-stimulated glucose uptake in skeletal muscle correlated well with noninsulin dependent glucose clearance during the fasting state. Thus, it is possible that the same defect responsible for the impairment in insulin-stimulated glucose uptake could explain the decrease in basal glucose clearance since the majority (>80%) of glucose disposal during the euglycemic insulin clamp occurs in muscle (21). In summary, the results of the present study demonstrate that the decrease in noninsulin dependent glucose clearance is the primary factor that contributes to the increase in fasting plasma glucose concentration within the nondiabetic range. The decline in basal HGP observed with rising BMI is explained by the increase in fasting plasma insulin concentration that represents a compensatory response to the obesity-related insulin resistance.

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TABLE 1. Anthropometric, laboratory and metabolic characteristics of lean and obese subjects Quantile 1 Quantile 2 Quantile 3 Quantile 4 ANOVA Number 30 30 30 30 Age (years) 37±2 39 + 2 37 + 2 35±2 NS Gender (M/F) 12/18 13/17 15/15 13/17 NS BMI (kg/m 2 ) 22.9± 0.25 26.6 + 0.4 29.4 + 0.2 35.5± 0.9 <0.0001 FPG (mg/dl) 90±1 92 + 1 95 + 1 97±1 0.003 FPI (uu/ml) 6±1 7+1 8+1 16±2 <0.00001 HGP (mg/kg min) 2.09 ±0.08 2.04 +0.07 1.69 + 0.07 1.51±0.09 <0.0001 HGP 3.21±0.06 3.12+0.09 2.60 + 0.12 2.62±0.08 <0.007 (mg/kg LBM min Glucose Clearance 2.35±0.09 2.21 +0.1 1.79 + 0.07 1.60±0.09 <0.0001 (ml/kg min) Glucose Clearance 3.63±0.07 3.14 + 0.11 2.88 + 0.09 2.70±0.09 <0.0001 (ml/kg LBM min) HGP X FPI 12.3±1.4 15.3+1.5 17.7+1.2 27.7±3.7 <0.0001 rhgp X SSPI 12.6 + 3.5 21.3 + 4.5 26.5 + 5.5 27.6 + 5.6 0.003 FPG = fasting plasma glucose; FPI = fasting plasma insulin; HGP = hepatic glucose production. rhgp= residual hepatic glucose production during insulin clamp; SSPI steady state plasma insulin.

FIGURE LEGENDS Figure 1. Relationship between BMI and fasting plasma glucose concentration (A), hepatic glucose production (B), and fasting plasma insulin concentration (C). Figure 2. Relationship between the hepatic insulin resistance index, measured as the product of fasting plasma insulin concentration (FPI) and hepatic glucose production (HGP) (A) and the rate of glucose clearance (B). Figure 3. Relationship between fasting plasma glucose concentration and glucose clearance rate (A) and hepatic glucose production (B).

FIGURE 1A Fasting plasma Glucose Conc (mg/dl) 120 r = 0.32 p<0.0001 110 100 90 80 20 30 40 50 BMI (kg/m 2 )

FIGURE 1B HGP (mg/kg.min) 3 2 1 r = -0.51 p < 0.0001 0 20 30 40 50 BMI (kg/m 2 )

FIGURE 1C Fasting Plasma Insulin Conc (uu/ml) 50 40 30 20 10 r=0.62 p<0.0001 0 20 30 40 50 BMI (kg/m 2 )

FIGURE 2A 100 80 r=0.52 p<0.0001 FPI X HGP 60 40 20 Col 1 vs Col 3 0 20 30 40 50 BMI (kg/m 2 )

FIGURE 2B 4 r=-0.56 p<0.0001 Glucose Clearance Rate (ml/kg.min) 3 2 1 0 20 30 40 50 BMI (kg/m 2 )

FIGURE 3A Basal HGP (mg/kg.min) 3 2 1 Col 10 vs Col 11 r=-0.17 p=0.05 80 90 100 110 120 Fasting Plasma Glucose (mg/dl)

FIGURE 3B Glucose Clearance (ml/kg.min) 4 3 2 1 r=-0.40 p<0.0001 0 80 90 100 110 120 Fasting Plasma Glucose (mg/dl)