Time-Dependent Effects of Free Fatty Acids on Glucose Effectiveness in Type 2 Diabetes
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糖尿病学杂志 2006年第6期
Division of Endocrinology and Diabetes Research and Training Center, Albert Einstein College of Medicine, Bronx, New York
EGP, endogenous glucose production, FFA, free fatty acid
ABSTRACT
Impaired effectiveness of glucose to suppress endogenous glucose production (EGP) is an important cause of worsening hyperglycemia in type 2 diabetes. Elevated free fatty acids (FFAs) may impair glucose effectiveness via several mechanisms, including rapid changes in metabolic fluxes and/or more gradual changes in gene expression of key enzymes or other proteins. Thus, we examined the magnitude and time course of effects of FFAs on glucose effectiveness in type 2 diabetes and whether glucose effectiveness can be restored by lowering FFAs. Glucose fluxes ([3-3H]-glucose) were measured during 6-h pancreatic clamp studies, at euglycemia (5 mmol/l glucose, t = 0eC240 min), and hyperglycemia (10 mmol/l, t = 240eC360 min). We studied 19 poorly controlled subjects with type 2 diabetes (HbA1c 10.9 ± 0.4%, age 50 ± 3 years, BMI 30 ± 2 kg/m2) on at least two occasions with saline (NAeC group) or nicotinic acid (NA group) infusions for 3, 6, or 16 h (NA3h, NA6h, and NA16h groups, respectively) to lower FFAs to nondiabetic levels. As a reference group, glucose effectiveness was also assessed in 15 nondiabetic subjects. There was rapid improvement in hepatic glucose effectiveness following only 3 h of NA infusion (NA3h = 31 ± 6% suppression of EGP with hyperglycemia vs. NAeC = 8 ± 7%; P < 0.01) and complete restoration of glucose effectiveness after 6 h of NA (NA6h = 41 ± 8% suppression of EGP; P = NS vs. nondiabetic subjects). Importantly, the loss of hepatic glucose effectiveness in type 2 diabetes is completely reversible upon correcting the increased FFA concentrations. A longer duration of FFA lowering may be required to overcome the chronic effects of increased FFAs on hepatic glucose effectiveness.
Increased endogenous glucose production (EGP) may be the principal cause of fasting hyperglycemia in type 2 diabetes (1eC3). Several mechanisms have been proposed to explain this observation, including defective overnight insulin secretion and relative insulin deficiency, enhanced delivery of gluconeogenic substrates, hepatic insulin resistance, and impairment of the effectiveness of glucose per se to regulate its own production (1). Indeed, in addition to the suppressive effects of insulin on EGP (4,5), we and others (6eC10) have reported direct inhibitory effects of hyperglycemia on EGP in nondiabetic individuals, independent of other hormonal or metabolic signals. These studies all demonstrated that hyperglycemia per se inhibits EGP in nondiabetic individuals, and this phenomenon is referred to as "glucose effectiveness." This glucose effectiveness is markedly blunted in people with type 2 diabetes (11eC13) and contributes substantially to worsening hyperglycemia. Importantly, we have demonstrated that glucose effectiveness is completely restored in individuals with type 2 diabetes by normalizing the chronic metabolic defects (12).
In addition to chronic hyperglycemia, the metabolic milieu in less than optimally controlled type 2 diabetes is characterized by sustained elevations in free fatty acid (FFA) levels (14), which rise in proportion to worsening glycemic control (12). Of note, we have reported that 72 h of normoglycemia with intensive insulinization in subjects with poorly controlled type 2 diabetes restored glucose effectiveness together with normalization of plasma FFA levels (12). We recently demonstrated that elevating circulating FFA levels in nondiabetic subjects antagonized the regulation of EGP by hyperglycemia, while conversely, normalizing FFA levels for 6 h markedly improved this regulation in subjects with type 2 diabetes. Furthermore, in type 2 diabetic subjects with good glycemic control (HbA1c [A1C] <7%), both FFA levels and glucose effectiveness were comparable with nondiabetic subjects (15). Thus, the inability of hepatic glucose fluxes to respond to hyperglycemia in poorly controlled type 2 diabetic subjects may be, at least in part, due to sustained increases in plasma FFAs. Loss of insulin’s inhibitory effects on lipolysis probably contributes to the FFA elevations in type 2 diabetes, thus ultimately impairing glucose effectiveness. Since long-term normalization of FFA levels remains an elusive goal, delineating the mechanism(s) whereby FFAs impact glucose effectiveness could be therapeutically advantageous.
An increase in de novo synthesis of glucose via gluconeogenesis is the major process responsible for increased EGP in type 2 diabetes. Increased FFAs are known to potently stimulate gluconeogenesis (16,17). Additionally, increased FFA availability has been shown to affect the relative flux through the key hepatic enzymes, glucokinase and glucose-6-phosphatase, by altering both gene expression and enzyme activity (18eC23).
Thus, we studied the time-dependent effects of increased FFAs on glucose effectiveness and the extent to which these effects are reversible in type 2 diabetes. We thereby aimed to establish the magnitude, time course, and reversibility of the impact of FFAs on glucose effectiveness in type 2 diabetes. Indeed, we observed significant improvement in glucose effectiveness in poorly controlled type 2 diabetic individuals following only 3 h of FFA lowering and a maximal effect after 6 h. The complete restoration of glucose effectiveness with FFA lowering underscores the importance of FFA levels to this regulation and the potential therapeutic benefit of lowering FFAs in type 2 diabetes.
RESEARCH DESIGN AND METHODS
Subjects aged 18eC65 years were recruited, and informed written consent was obtained in accordance with the policies of the institutional committee on clinical investigations (Table 1). Subjects with type 2 diabetes (n = 19) were in poor glycemic control (A1C 8%). Except for diabetes, all type 2 diabetic subjects were in general good health and did not have proliferative retinopathy, significant diabetic renal disease (urine microalbumin <100 mg/dl), or symptomatic neuropathy. Insulin was discontinued 24 h prior, sulfonylureas and metformin 3 days prior, and thiazolidinediones at least 8 days prior to the day of the clamp studies. The nondiabetic volunteers (n = 15) were taking no medications and had no family history of type 2 diabetes. A 2-h oral glucose tolerance test was performed to ensure normal glucose tolerance.
Nondiabetic subjects were admitted to the General Clinical Research Center on the morning of the study. To ensure that initial plasma glucose levels were <140 mg/dl in all subjects on the morning of study, subjects with type 2 diabetes were admitted the evening before the study for low-dose overnight insulin infusion, and an 18-gauge catheter was inserted in an antecubital vein for infusions. Beginning at 10:00 P.M. on the night before the study, a variable intravenous infusion of insulin was started, and the rate was adjusted according to an algorithm based on hourly blood glucose measurement. As previously described, overnight correction of glucose did not affect glucose effectiveness in these poorly controlled type 2 diabetic subjects (24).
Euglycemic-hyperglycemic pancreatic clamp studies.
At 7 A.M. on the morning of the study, an 18-gauge catheter was inserted in an antecubital vein for infusions and a contralateral hand vein was cannulated in a retrograde fashion for arterialized venous blood sampling. To obtain arterialized venous blood, this hand was kept in a warming Plexiglas box maintained at 55°C. The experimental protocols lasted 6 h and consisted of an initial 4-h euglycemic period followed by a 2-h hyperglycemic period as previously described (12,15).
At t = 0 min, a primed-continuous infusion of high-performance liquid chromatographyeCpurified [3-3H]-glucose (New England Nuclear, Boston, MA) was started (prime infusion 22 e藽i) and then continued at 0.15 e藽i/min for 6 h (Fig. 1). Infusions containing somatostatin, growth hormone, glucagon, and insulin were also initiated at t = 0 min (12,15). Plasma glucose concentrations were measured at 10- to 15-min intervals during the initial 240 min of the study and maintained at normal fasting concentrations (90 mg/dl) by frequently adjusting the infusion rate of insulin during the first 120 min and maintaining these individualized insulin infusion rates for the duration of the studies. At 240 min, plasma glucose concentrations were acutely increased to 180 mg/dl and then clamped at this level with variable 20% glucose infusions. [3-3H]-glucose (0.1 e藽i/ml) was added to the "cold" 20% glucose to maintain constant glucose specific activity (15). From 0 to 360 min, blood samples were obtained to measure plasma glucose, insulin, glucagon, C-peptide, FFAs, glycerol, lactate, and 3-3H-glucose specific activity. All infusions were stopped at 360 min.
To determine the impact of FFAs on glucose effectiveness, glucose fluxes were compared between euglycemia and hyperglycemia in the nondiabetic group and type 2 diabetic subjects under the various conditions outlined below:
Nondiabetic subjects underwent a control pancreatic clamp study as described above with saline infusion only. A subgroup of these subjects have been previously described (15).
All type 2 diabetic subjects were studied under paired conditions, with a control (saline infusion [NAeC) study and an active infusion (nicotinic acid [NA]) study, at least 1 month apart under the following conditions: a) saline control studies (NAeC) paired with one of the following study types: b) NA3h (n = 9): with infusion of nicotinic acid (0.017 mg · kgeC1 · mineC1) for the final 3 h of the clamp study or c) NA6h (n = 8): with infusion of nicotinic acid (USP grade; Sigma, St. Louis, MO) at 0.015 mg · kgeC1 · mineC1 throughout the 6-h clamp studies. This rate was selected to decrease FFA levels to those observed in nondiabetic subjects (33,34). The NA was pyrogen free, pH adjusted to 7.0 with NaOH, and filter sterilized through a 0.2-micron filter before use. d) NA16h (n = 8): infusion of nicotinic acid (0.008 mg · kgeC1 · mineC1) starting at 10 P.M. and continuing for the duration of the clamp study at a rate of 0.015 mg · kgeC1 · mineC1.
These rates of NA infusion were chosen based on the literature and pilot studies to match FFA levels in the final hour in all groups.
Analytical procedures.
Plasma glucose was measured with a Beckman glucose analyzer (Fullerton, CA) by use of the glucose oxidase method. Plasma [3-3H]-glucose and tritiated watereCspecific activities were measured as previously described (4,11). Plasma insulin, C-peptide, glucagon FFAs, glycerol and lactate, and A1C were measured as previously described (15).
Calculations.
Rates of glucose appearance (Ra) and glucose uptake (Rd) were calculated using Steele’s steady-state equation (26). Rates of EGP were calculated as previously described (26). Data for glucose turnover, plasma hormones, and substrate concentrations represent the mean values during the final 60 min of the euglycemic period (t = 180eC240 min) and the final 60 min of the hyperglycemic period (t = 300eC360 min).
Statistical analysis.
Analysis of the data were performed using SPSS Version 11.5. For averaged data, Student’s t tests were utilized, using paired t tests for comparisons of euglycemic and hyperglycemic intervals in each group and nonpaired t tests to compare the nondiabetic with the type 2 diabetic group. ANOVA was used to compare the various study conditions within the type 2 diabetic group. All data are presented as means ± SE, unless otherwise specified. A P value <0.05 was considered significant.
RESULTS
Baseline (fasting) patient characteristics.
The rate of overnight insulin infusion required to maintain plasma glucose in the target range of 90eC120 mg/dl in the type 2 diabetic subjects averaged 2.67 ± 0.20 units/h. There was no difference in overnight insulin infusion rates for the different study conditions. In the nondiabetic subjects, fasting plasma glucose levels averaged 99 ± 1 mg/dl and plasma FFA levels were 443 ± 57 e蘒/ml. In type 2 diabetic subjects, basal (t = 0) plasma glucose was 124 ± 3 mg/dl and plasma FFA levels were 349 ± 44 e蘒/ml (averaged for all studies after overnight insulin infusion). There was no difference in fasting plasma glucose and fasting FFAs for the three NA groups (Tables 1 and 2).
General clamp study conditions.
During the clamps, plasma glucose levels were similar in type 2 diabetic and in nondiabetic subjects (during the euglycemic study period, 98 ± 1 mg/dl for type 2 diabetic and 96 ± 1 mg/dl for nondiabetic subjects and during the hyperglycemic study period, 185 ± 1 mg/dl for type 2 diabetic and 183 ± 1 mg/dl for nondiabetic subjects, respectively). Plasma glucose levels during both euglycemia and hyperglycemia were similar between pairs of studies (NA versus NAeC). Glucose specific activity was constant following tracer equilibration during both euglycemia and hyperglycemia in each group (Fig. 2).
Although there was a trend toward higher average insulin infusion rates required to maintain euglycemia in type 2 diabetic (0.23 ± 0.02 e蘒 · kgeC1 · mineC1) when compared with nondiabetic (0.18 ± 0.01 e蘒 · kgeC1 · mineC1) subjects, this did not reach statistical significance (P = 0.056). Importantly, plasma insulin levels did not differ between euglycemic and hyperglycemic study periods or between pairs of the NA studies (Table 2). The former comparison is particularly important since the principal comparison is between euglycemia and hyperglycemia in the same study. Plasma glucagon levels also remained stable in all groups. C-peptide levels were suppressed by somatostatin infusion in all studies and did not differ between pairs of studies in either basal, euglycemic, or hyperglycemic periods. Although there was a trend toward increased C-peptide with the onset of hyperglycemia in nondiabetic subjects, the levels remained suppressed at far-lower-than-baseline levels (Table 2). Plasma lactate levels were not different between paired studies.
Saline control studies (NAeC and nondiabetic subjects).
In the tables, all study results will be compared with the paired saline control studies in the same subjects, e.g., NA3h versus NAeC. There was a marked decrease in glucose effectiveness in the type 2 diabetic subjects when compared with nondiabetic subjects. The average rate of glucose infusion required to maintain the target hyperglycemic plateau during the last 60 min of the hyperglycemic period was decreased by more than half in the type 2 diabetic compared with the nondiabetic subjects (1.00 ± 0.19 mg · kgeC1 · mineC1 in type 2 diabetes vs. 3.10 ± 0.20 mg · kgeC1 · mineC1 in nondiabetic subjects; P < 0.001). EGP was suppressed by 48 ± 3% with hyperglycemia in nondiabetic subjects but failed to suppress with hyperglycemia in type 2 diabetic subjects (9 ± 3%, P < 0.001 vs. nondiabetic subjects; Fig. 3). Also, while the percent increase in glucose uptake between the euglycemic and hyperglycemic study periods was 127 ± 72% for nondiabetic subjects, there was a more modest increase of 34 ± 8% in type 2 diabetic subjects (P < 0.001 vs. nondiabetic group). Importantly, plasma FFA concentrations were higher in type 2 diabetic than in nondiabetic subjects during both euglycemia (type 2 diabetes = 412 ± 99 e蘭ol/l vs. no diabetes = 227 ± 31 e蘭ol/l; P < 0.001) and hyperglycemia (type 2 diabetes = 435 ± 81 e蘭ol/l vs. no diabetes = 190 ± 29 e蘭ol/l; P < 0.001) (Fig. 4). Thus, while there were time-dependent decreases in FFA values with the onset of hyperglycemia in nondiabetic subjects (P < 0.001), FFA levels remained elevated in the type 2 diabetic group throughout the entire 6 h of NAeC studies. This is consistent with our previous studies (15).
NA studies
FFA and glycerol levels.
As shown in Fig. 4, FFA values during the final hour of all NA+ studies were comparable with the saline control studies in the nondiabetic subjects. Significant lowering in FFA levels was attained after 1 h of NA infusion; hence, significant lowering of FFAs was observed for the final 2 h of the NA3h studies and during the final 5 h of the NA6h studies. In the NA16h studies, FFA levels were lower at the beginning of the clamp when compared with NAeC (P < 0.01) and remained reduced throughout. Of note, the different NA infusion rates selected for each duration attained comparable FFA reduction in all three groups during the final hour (P = NS by ANOVA).
There were no significant changes in glycerol levels during the NA studies (NA3h = 85 ± 6 vs. NAeC = 84 ± 7, NA6h = 52 ± 3 vs. NAeC = 76 ± 4, and NA16h = 92 ± 15 vs. NAeC = 108 ± 9 e蘭ol/l; P = NS for all). These findings differ from two reports (27,28) in which nicotinic acid reduced glycerol levels in fasting individuals. However, a recent study (29) using nicotinic acid in the presence of high physiologic insulin was consistent with our findings, since nicotinic acid lowered FFAs but had no effect on plasma glycerol levels. Of note, the former pair of studies were performed under basal fasting conditions, while the latter pair of studies were in the presence of hyperinsulinemia and hyperglycemia, respectively. Both conditions inhibit gluconeogenesis, and hence there would be less consumption of glycerol via gluconeogenesis. This is likely to balance the reduced production of glycerol via lipolysis, particularly since lipolysis of a triacyl-glycerol molecule would yield only one-third as much glycerol as fatty acids when expressed on a molar basis.
Glucose fluxes.
To raise blood glucose quickly to 180 mg/dl at 240 min, glucose infusion rates at the onset of hyperglycemia were calculated based on each subject’s weight and were similar to the paired NAeC studies. Glucose infusion rates became significantly different by 315 min in the NA6h group and 330 min in the NA3h and NA16h groups compared with the paired NAeC studies. The average rate of glucose infusion required to maintain hyperglycemia during the last 60 min in the NA studies (NA3h = 2.22 ± 0.56, NA6h = 2.29 ± 0.33, and NA16h = 2.56 ± 0.46 mg · kgeC1 · mineC1) was increased more than twofold above the rates required in the paired NAeC studies (1.11 ± 0.40, 0.66 ± 0.21, and 0.74 ± 0.18 mg · kgeC1 · mineC1, respectively). Thus, normalizing plasma FFA levels for 3 h significantly increased glucose effectiveness in the type 2 diabetic subjects. The degree of suppression of EGP with hyperglycemia in the NA studies (NA3h = 31 ± 6%, NA6h = 41 ± 8%, and NA16h = 47 ± 4%) was significantly greater than in the NAeC studies in the same individuals (NAeC 8 ± 7%, 7 ± 6%, and 14 ± 4%, respectively, P = 0.001; Figs. 3 and 5). The suppression of EGP with NA for 6 and 16 h was comparable with the degree of suppression observed in the nondiabetic subjects, while there was less suppression of EGP in the NA3h studies (P = 0.02). This indicates that significant improvements were seen at 3 h and complete normalization observed after 6 h of FFA lowering.
Also, the percent increase in glucose uptake (Figs. 3 and 5) between the euglycemic and hyperglycemic study periods was 63 ± 10% for the NA6h vs. 26 ± 10% in the NAeC (P = 0.01) and 64 ± 20% for the NA16h vs. 30 ± 14% change in the NAeC studies (P = 0.04). However, this stimulation of glucose uptake by hyperglycemia was substantially less than the 127% increase observed in nondiabetic subjects. While there was a trend toward increased glucose uptake after only 3 h of FFA lowering in type 2 diabetic subjects (NA3h 70 ± 14% vs. NAeC 43 ± 17%), it did not reach statistical significance (P = 0.09). Thus, there were improvements in peripheral glucose effectiveness following 6 h of FFA lowering, but normalization was not achieved even after 16 h.
Of note, we previously performed studies with coinfusion of both nicotinic acid and liposyn in type 2 diabetic subjects (n = 5) to prevent the nicotinic acideCinduced decline in FFA levels (15). The percent change in EGP and glucose uptake in the coinfusion studies were eC9 ± 10 and 37 ± 17%, respectively, and thus were not significantly different from the current NAeC studies. Thus, the effects of nicotinic acid on glucose effectiveness are likely due to FFA lowering and not to nonspecific effects of nicotinic acid.
DISCUSSION
Although the effects of increased FFA levels on hepatic insulin action are well established (30), the effects of FFAs on hepatic glucose effectiveness are underrecognized. Indeed, a careful review of the literature suggests that this is an important phenomenon. Previous studies (31) have suggested that glucose-induced reductions in FFA levels account for the majority of suppression of EGP by glucose in nondiabetic individuals. In support of this idea, we previously reported that in type 2 diabetic individuals with optimal glycemic control, EGP displays normal suppression of FFA levels by hyperglycemia and thus normal glucose effectiveness (12). Moreover, 72 h of intensive glycemic control with insulin in poorly controlled type 2 diabetic subjects restored glucose effectiveness in concert with normalization of plasma FFA levels (12). Shah et al. (33) reported that elevation of plasma FFA levels in healthy women blunted the suppression of EGP by both hyperinsulinemia and hyperglycemia (35), although glucose effectiveness was not specifically examined. When plasma FFA levels were increased to >2 mmol/l for 9 h under pancreatic clamp conditions in healthy subjects, the resulting 50% rise in plasma glucose concentrations failed to suppress EGP (33).
The present experiments examined the impact of altering FFA levels for variable durations on glucose effectiveness in type 2 diabetic individuals. Euglycemic-hyperglycemic pancreatic clamp techniques were used to study the ability of hyperglycemia per se to regulate EGP and glucose uptake under fixed hormonal conditions. In these subjects with poorly controlled type 2 diabetes, there was a rapid improvement in hepatic glucose effectiveness following only 3 h of nicotinic acid infusion (2 h of FFA lowering) and a complete restoration of glucose effectiveness with 6-h infusion (5 h of FFA normalization). This time course of FFA-induced changes in glucose effectiveness is consistent with rapid changes in flux through gluconeogenesis and/or rapid changes in the activity of key hepatic enzymes. The magnitude of the effect we report suggests that the inhibitory effects of FFAs on glucose effectiveness contribute importantly to worsening hyperglycemia in individuals with type 2 diabetes.
Increased circulating FFAs have been shown to alter both the gene expression and the activity of hepatic enzymes responsible for regulating EGP. Specifically, FFAs promote hepatic glucose-6-phosphatase gene expression (18,19) and decrease hepatic glucokinase expression (20). Studies of the roles of these enzymes in regulating glucose fluxes are consistent with their importance in determining glucose effectiveness. Dual defects in these key regulatory enzymes could impair glucose effectiveness, since defective glucokinase activity would impair the liver’s ability to "sense" rising glucose levels (34,35), while increased glucose-6-phosphatase would stimulate EGP by both glycogenolysis and gluconeogenesis. The fact that 6 h of FFA lowering was required to fully normalize glucose effectiveness in the current studies suggests that adequate time was required to reverse chronic effects of FFAs on hepatic gene expression.
Increased FFAs have also been shown to increase gluconeogenesis both in vivo and in vitro (18,36). Indeed, type 2 diabetes is characterized by augmented rates of gluconeogenesis, which contribute to the inappropriate elevations in EGP in these individuals (37). The current studies were designed to explore potential mechanisms whereby increased FFA levels impact glucose effectiveness in humans. The rapid effects of lowering FFAs observed in these studies could be consistent with rapid changes in gluconeogenesis. This will be the focus of further investigation.
It is important to note that our studies were performed under basal insulin conditions, since they were designed to study glucose effectiveness and not insulin sensitivity. Of note, there was no effect of lowering FFA levels on rates of EGP during euglycemia (i.e., NA16h or NA6h versus their respective controls). This demonstrates that sensitivity to basal insulin was not affected by the modest lowering in FFA levels. This is consistent with the observation of Whiteall et al. (38) that lowering triglycerides and FFAs with gemfibrozil did not alter insulin sensitivity during low-dose insulin infusion. We also found that there was no change in insulin sensitivity (as assessed by the insulin infusion rates required to lower blood glucose) overnight despite lower FFA levels in the NA16h studies. This confirms that the improved suppression of EGP observed during hyperglycemia with FFA lowering was due to improved glucose effectiveness rather than a change in insulin sensitivity.
It is intriguing that nicotinic acid acutely improved glucose effectiveness in these poorly controlled type 2 diabetic subjects, despite concerns about its chronic use in type 2 diabetes (39). Use of niaspan in 125 subjects with type 2 diabetes for up to 65 weeks had very modest increases in plasma glucose (<10 mg/dl) and no change in A1C (40), although FFA levels were not assessed systematically. Of note, rebound elevations in FFA levels occur following administration of nicotinic acid, generally rising well above the pretreatment FFA levels. Indeed, 7-day treatment with nicotinic acid in nondiabetic subjects resulted in insulin resistance that was proportional to the increases in fasting FFAs (41). It is therefore likely that repeated rebound rises in FFA levels may have mild detrimental effects on glucose control with chronic administration of nicotinic acid. Use of extended-release nicotinic acid was recently shown to reduce the degree of FFA rebound. Despite a nonsignificant trend of more type 2 diabetic patients needing adjustment of diabetes medication, there was no change in A1C (42).
Lowering FFA levels also improved peripheral glucose effectiveness, i.e., the ability of hyperglycemia per se to stimulate whole-body glucose uptake. Previous studies have demonstrated that elevated FFAs inhibited glucose-mediated glucose uptake under conditions of physiologic insulin levels, similar to the present studies (43). Of note, FFAs have been shown to directly impact both the expression (44) and translocation (45) of glucose transporters in skeletal muscle. In contrast to the complete restoration of hepatic glucose effectiveness, FFA lowering for up to 16 h only partially restored peripheral glucose effectiveness. However, we previously reported that correction of glucose and FFA levels for 72 h improved peripheral glucose effectiveness to the level of nondiabetic subjects (12). Therefore it is possible that a longer duration of FFA lowering would have completely restored glucose effectiveness. Additionally, since peripheral insulin resistance is not rapidly restored by correcting metabolic defects (46), the permissive effects of insulin on peripheral glucose effectiveness (47) are still likely to be impaired in these poorly controlled type 2 diabetic subjects.
To conclude, our data suggest that elevated FFA levels contribute to the lack of hepatic and peripheral glucose effectiveness in individuals with type 2 diabetes. Of note, FFA levels are chronically elevated in type 2 diabetes, and attaining long-term FFA lowering in these individuals is currently not a specific therapeutic goal in type 2 diabetes. Since elevated FFA levels appear to significantly contribute to the loss of glucose effectiveness in type 2 diabetes and thus overall glycemic control, understanding the pathogenesis of this important defect could have considerable therapeutic benefit.
ACKNOWLEDGMENTS
This work was supported in part by grants from the American Diabetes Association (to M.H. and J.T.) and the National Insitutes of Health (Einstein General Clinical Research Center [GCRC] RR12248, K12 RR17672 [to P.K.], P01-AG021654, and DK20541 [Diabetes Research and Training Center {DRTC}]). Dr. Hawkins was a Clinical Associate Physician of the GCRC (3 M01 RR 12248eC03S1) and is a recipient of a Beeson Award from the American Federation of Aging Research.
The authors thank Angela Stangarone for coordinating studies, Robin Sgueglia for determinations performed in the DRTC Hormone Assay Core, Dr. Hillel Cohen for biostatistical assistance, and the staff of the Albert Einstein College of Medicine GCRC for their outstanding patient care. We also acknowledge Drs. Luciano Rossetti, Daniel Stein; and Harry Shamoon for many helpful discussions.
FOOTNOTES
DOI: 10.2337/db05-0988
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
DeFronzo R: The triumvirate: -cell, muscle and liver: a collusion responsible for type 2 diabetes. Diabetes 37:667eC687, 1988
Bogardus C, Lillioja S, Howard B, Reaven G, Mott D: Relationships between insulin secretion, insulin action, and fasting plasma glucose concentration in nondiabetic and noninsulin-dependent diabetic subjects. J Clin Invest 74:1238eC1246, 1984
Campbell P, Mandarino L, Gerich J: Quantification of the relative impairment in actions of insulin on hepatic glucose production and peripheral glucose uptake in non-insulin-dependent diabetes mellitus. Metabolism 37:15eC22, 1988
Mittelman SD, Fu YY, Rebrin K, Steil G, Bergman RN: Indirect effect of insulin to suppress endogenous glucose production is dominant, even with hyperglucagonemia. J Clin Invest 100:3121eC3130, 1997
Fisher SJ, Kahn CR: Insulin signaling is required for insulin’s direct and indirect action on hepatic glucose production. J Clin Invest 111:463eC468, 2003
Bergman R: Interaction of insulin and glucose in the control of hepatic glucose balance. Am J Physiol 227:1314eC1322, 1974
Liljenquist JE, Mueller GL, Cherrington AD, Perry JM, Rabinowitz D: Hyperglycemia per se (insulin and glucagon withdrawn) can inhibit hepatic glucose production in man. J Clin Endocrinol Metab 48:171eC175, 1979
Sacca L, Hendler R, Sherwin RS: Hyperglycemia inhibits glucose production in man independent of changes in glucoregulatory hormone. J Clin Endocrinol Metab 47:1160eC1163, 1978
DeFronzo RA, Ferrannini E, Hendler R, Felig P, Wahren J: Regulation of splanchic and peripheral glucose uptake by insulin and hyperglycemia in man. Diabetes 32:35eC45, 1983
Bell PM, Firth RG, Rizza RA: Effect of hyperglycemia on glucose production and utilization in humans. Diabetes 35:642eC648, 1986
Mevorach M, Giacca A, Aharon Y, Hawkins M, Shamoon H, Rossetti L: Regulation of endogenous glucose production by glucose per se is impaired in type 2 diabetes mellitus. J Clin Invest 102:744eC753, 1998
Hawkins M, Gabriely I, Wozniak R, Mevorach M, Rossetti L, Shamoon H: The effect of glycemic control on hepatic and peripheral glucose effectiveness in type 2 diabetes mellitus. Diabetes 51:2179eC2189, 2002
Nagasaka S, Tokuyama K, Kusaka I, Hayashi H, Rokkaku K, Nakamura T, Kawakami A, Higashiyama M, Ishikawa S, Saito T: Endogenous glucose production and glucose effectiveness in type 2 diabetic subjects derived from stable-labeled minimal model approach. Diabetes 48:1054eC1060, 1990
Reaven GM, Hollenbeck C, Jeng CY, Wu MS, Chen YD: Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes 37:1020eC1024, 1988
Hawkins M, Tonelli J, Kishore P, Stein D, Ragucci E, Gitig A, Reddy K: Contribution of elevated free fatty acid levels to the lack of glucose effectiveness in type 2 diabetes. Diabetes 52:2748eC2758, 2003
Williamson JR, Kreisberg RA, Felts PW: Mechanism for the stimulation of gluconeogenesis by fatty acids in perfused rat liver. Proc Natl Acad Sci U S A 56:247eC254, 1966
Antras-Ferry J, Le Bigot G, Robin P, Robin D, Forest C: Stimulation of phosphoenolpyruvate carboxykinase gene expression by fatty acids. Biochem Biophys Res Commun 203:385eC391, 1994
Lam TK, Carpentier A, Lewis GF, van de Werve G, Fantus IG, Giacca A: Mechanisms of the free fatty acid-induced increase in hepatic glucose production. Am J Physiol Endocrinol Metab 284:E863eCE873, 2003
Massillon D, Barzilai N, Hawkins M, Prus-Wertheimer D, Rossetti L: Induction of hepatic glucose-6-phosphatase gene expression by lipid infusion. Diabetes 46:153eC157, 1997
van de Werve G, Lange A, Newgard C, Mechin MC, Li Y, Berteloot A: New lessons in the regulation of glucose metabolism taught by the glucose 6-phosphatase system. Eur J Biochem 267:1533eC1549, 2000
Jump DB, Clarke SD, Thelen A, Liimatta M: Coordinate regulation of glycolytic and lipogenic gene expression by polyunsaturated fatty acids. J Lipid Res 35:1076eC1084, 1994
Lam TK, van de Werve G, Giacca A: Free fatty acids increase basal hepatic glucose production and induce hepatic insulin resistance at different sites. Am J Physiol Endocrinol Metab 284:E281eCE290, 2003
Oakes N, Cooney G, Camilleri S, Chisholm D, Kraegen E: Mechanisms of liver and muscle insulin resistance induced by chronic high-fat feeding. Diabetes 46:1768eC1774, 1997
Basu R, Basu A, Nielsen M, Shah P, Rizza RA: Effect of overnight restoration of overnight euglycemia on glucose effectiveness in type 2 diabetes mellitus. J Clin Endocrinol Metab 84:2314eC2319, 1999
Steele R: Influences of glucose loading and of injected insulin on hepatic glucose output. Ann N Y Acad Sci 82:420eC430, 1959
Rossetti L, Giaccari A: Relative contribution of glycogen synthesis and glycolysis to insulin-mediated glucose uptake: a dose-response euglycemic clamp study in normal and diabetic rats. J Clin Invest 85:1785eC1792, 1990
Wang W, Basinger A, Neese R, Christiansen M, Hellerstein M: Effects of nicotinic acid on fatty acid kinetics, fuel selection, and pathways of glucose production in women. Am J Physiol Endocrinol Metab 279:50eC59, 2000
Boden Chen X, Capulong E, Mozzoli M: Effects of free fatty acids on gluconeogenesis and autoregulation of glucose production in type 2 diabetes. Diabetes 50:810eC816, 2001
Carpentier AC, Frisch F, Cyr D, Genereux P, Patterson BW, Giguere R, Baillargeon JP: On the suppression of plasma nonesterified fatty acids by insulin during enhanced intravascular lipolysis in humans. Am J Physiol Endocrinol Metab 289:E849eCR856, 2005
Service FJ: Hypoglycemic disorders. N Engl J Med 332:1144eC1152, 1995
Shah P, Basu A, Rizza R: Fat-induced liver insulin resistance. Curr Diab Rep 3:214eC218, 2003
Best JD, Kahn SE, Ader M, Watanabe RM, Ni TC, Bergman RN: Role of glucose effectiveness in the determination of glucose tolerance. Diabetes Care 19:1018eC1030, 1996
Shah P, Vella A, Basu A, Basu R, Adkins A, Schwenk WF, Johnson CM, Nair KS, Jensen MD, Rizza RA: Elevated free fatty acids impair glucose metabolism in women: decreased stimulation of muscle glucose uptake and suppression of splanchnic glucose production during combined hyperinsulinemia and hyperglycemia. Diabetes 52:38eC42, 2003
Roden M, Stingl H, Chandramouli V, Schumann WC, Hofer A, Landau BR, Nowotny P, Waldhausl W, Shulman GI: Effects of free fatty acid elevation on postabsorptive endogenous glucose production and gluconeogenesis in humans. Diabetes 49:701eC707, 2000
Hawkins M, Gabriely I, Wozniak R, Vilcu C, Shamoon H, Rossetti L: Fructose improves the ability of hyperglycemia per se to regulate glucose production in type 2 diabetes. Diabetes 51:606eC614, 2002
Basu A, Basu R, Shah P, Vella A, Johnson CM, Jensen M, Nair KS, Schwenk WF, Rizza RA: Type 2 diabetes impairs splanchnic uptake of glucose but does not alter intestinal glucose absorption during enteral glucose feeding: additional evidence for a defect in hepatic glucokinase activity. Diabetes 50:1351eC1362, 2001
Boden G: Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes 46:3eC10, 1997
Boden G, Chen X, Stein TP: Gluconeogenesis in moderately and severely hyperglycemic patients with type 2 diabetes mellitus. Am J Physiol Endocrinol Metab 280:E23eCE30, 2001
Whitelaw DC, Smith JM, Nattrass M: Effects of gemfibrozil on insulin resistance to fat metabolism in subjects with type 2 diabetes and hypertriglyceridaemia. Diabetes Obes Metab 3:187eC194, 2002
American Diabetes Association: Management of dyslipidemia in adults with diabetes. Diabetes Care 21:179eC182, 1998
Elam MB, Hunninghake DB, Davis KB, Garg R, Johnson C, Egan D, Kostis JB, Sheps DS, Brinton EA, the ADMIT Investigators: Effect of niacin on lipid and lipoprotein levels and glycemic control in patients with diabetes and peripheral arterial disease: the ADMIT Study: a randomized trial. JAMA 284:1263eC1270, 2000
Poynten AM, Gan SK, Kriketos AD, O’Sullivan A, Kelly JJ, Ellis BA, Chisholm DJ, Campbell LV: Nicotinic acid-induced insulin resistance is related to increased circulating fatty acids and fat oxidation but not muscle lipid content. Metabolism 52:699eC704, 2003
Vega GL, Cater NB, Meguro S, Grundy SM: Influence of extended-release nicotinic acid on nonesterified fatty acid flux in the metabolic syndrome with atherogenic dyslipidemia. Am J Cardiol 95:1309eC1313, 2005
Baron AD, Brechtel G, Edelman SV: Effects of free fatty acids and ketone bodies on in vivo non-insulin-mediated glucose utilization and production in humans. Metabolism 38:1056eC1061, 1989
Long SD, Pekala PH: Regulation of GLUT4 gene expression by arachidonic acid: evidence for multiple pathways, one of which requires oxidation to prostaglandin E2. J Biol Chem 271:1138eC1144, 1996
Zierath JR, Housenecht KL, Gnudi L, Kahn BB: High-fat feeding impairs insulin-stimulated GLUT4 recruitment via an early insulin-signaling defect. Diabetes 46:215eC223, 1997
Wise SD, Nielsen MF, Cryer PE, Rizza RA: Overnight normalization of glucose concentrations improves hepatic but not extrahepatic insulin action in subjects with type 2 diabetes mellitus. J Clin Endocrinol Metab 83:2461eC2469, 1998
Edelman SV, Laakso M, Wallace P, Brechtel G, Olefsky EM, Baron AD: Kinetics of insulin-mediated and non-insulin-mediated glucose uptake in humans. Diabetes 39:955eC964, 1990(Preeti Kishore, Julia Ton)
EGP, endogenous glucose production, FFA, free fatty acid
ABSTRACT
Impaired effectiveness of glucose to suppress endogenous glucose production (EGP) is an important cause of worsening hyperglycemia in type 2 diabetes. Elevated free fatty acids (FFAs) may impair glucose effectiveness via several mechanisms, including rapid changes in metabolic fluxes and/or more gradual changes in gene expression of key enzymes or other proteins. Thus, we examined the magnitude and time course of effects of FFAs on glucose effectiveness in type 2 diabetes and whether glucose effectiveness can be restored by lowering FFAs. Glucose fluxes ([3-3H]-glucose) were measured during 6-h pancreatic clamp studies, at euglycemia (5 mmol/l glucose, t = 0eC240 min), and hyperglycemia (10 mmol/l, t = 240eC360 min). We studied 19 poorly controlled subjects with type 2 diabetes (HbA1c 10.9 ± 0.4%, age 50 ± 3 years, BMI 30 ± 2 kg/m2) on at least two occasions with saline (NAeC group) or nicotinic acid (NA group) infusions for 3, 6, or 16 h (NA3h, NA6h, and NA16h groups, respectively) to lower FFAs to nondiabetic levels. As a reference group, glucose effectiveness was also assessed in 15 nondiabetic subjects. There was rapid improvement in hepatic glucose effectiveness following only 3 h of NA infusion (NA3h = 31 ± 6% suppression of EGP with hyperglycemia vs. NAeC = 8 ± 7%; P < 0.01) and complete restoration of glucose effectiveness after 6 h of NA (NA6h = 41 ± 8% suppression of EGP; P = NS vs. nondiabetic subjects). Importantly, the loss of hepatic glucose effectiveness in type 2 diabetes is completely reversible upon correcting the increased FFA concentrations. A longer duration of FFA lowering may be required to overcome the chronic effects of increased FFAs on hepatic glucose effectiveness.
Increased endogenous glucose production (EGP) may be the principal cause of fasting hyperglycemia in type 2 diabetes (1eC3). Several mechanisms have been proposed to explain this observation, including defective overnight insulin secretion and relative insulin deficiency, enhanced delivery of gluconeogenic substrates, hepatic insulin resistance, and impairment of the effectiveness of glucose per se to regulate its own production (1). Indeed, in addition to the suppressive effects of insulin on EGP (4,5), we and others (6eC10) have reported direct inhibitory effects of hyperglycemia on EGP in nondiabetic individuals, independent of other hormonal or metabolic signals. These studies all demonstrated that hyperglycemia per se inhibits EGP in nondiabetic individuals, and this phenomenon is referred to as "glucose effectiveness." This glucose effectiveness is markedly blunted in people with type 2 diabetes (11eC13) and contributes substantially to worsening hyperglycemia. Importantly, we have demonstrated that glucose effectiveness is completely restored in individuals with type 2 diabetes by normalizing the chronic metabolic defects (12).
In addition to chronic hyperglycemia, the metabolic milieu in less than optimally controlled type 2 diabetes is characterized by sustained elevations in free fatty acid (FFA) levels (14), which rise in proportion to worsening glycemic control (12). Of note, we have reported that 72 h of normoglycemia with intensive insulinization in subjects with poorly controlled type 2 diabetes restored glucose effectiveness together with normalization of plasma FFA levels (12). We recently demonstrated that elevating circulating FFA levels in nondiabetic subjects antagonized the regulation of EGP by hyperglycemia, while conversely, normalizing FFA levels for 6 h markedly improved this regulation in subjects with type 2 diabetes. Furthermore, in type 2 diabetic subjects with good glycemic control (HbA1c [A1C] <7%), both FFA levels and glucose effectiveness were comparable with nondiabetic subjects (15). Thus, the inability of hepatic glucose fluxes to respond to hyperglycemia in poorly controlled type 2 diabetic subjects may be, at least in part, due to sustained increases in plasma FFAs. Loss of insulin’s inhibitory effects on lipolysis probably contributes to the FFA elevations in type 2 diabetes, thus ultimately impairing glucose effectiveness. Since long-term normalization of FFA levels remains an elusive goal, delineating the mechanism(s) whereby FFAs impact glucose effectiveness could be therapeutically advantageous.
An increase in de novo synthesis of glucose via gluconeogenesis is the major process responsible for increased EGP in type 2 diabetes. Increased FFAs are known to potently stimulate gluconeogenesis (16,17). Additionally, increased FFA availability has been shown to affect the relative flux through the key hepatic enzymes, glucokinase and glucose-6-phosphatase, by altering both gene expression and enzyme activity (18eC23).
Thus, we studied the time-dependent effects of increased FFAs on glucose effectiveness and the extent to which these effects are reversible in type 2 diabetes. We thereby aimed to establish the magnitude, time course, and reversibility of the impact of FFAs on glucose effectiveness in type 2 diabetes. Indeed, we observed significant improvement in glucose effectiveness in poorly controlled type 2 diabetic individuals following only 3 h of FFA lowering and a maximal effect after 6 h. The complete restoration of glucose effectiveness with FFA lowering underscores the importance of FFA levels to this regulation and the potential therapeutic benefit of lowering FFAs in type 2 diabetes.
RESEARCH DESIGN AND METHODS
Subjects aged 18eC65 years were recruited, and informed written consent was obtained in accordance with the policies of the institutional committee on clinical investigations (Table 1). Subjects with type 2 diabetes (n = 19) were in poor glycemic control (A1C 8%). Except for diabetes, all type 2 diabetic subjects were in general good health and did not have proliferative retinopathy, significant diabetic renal disease (urine microalbumin <100 mg/dl), or symptomatic neuropathy. Insulin was discontinued 24 h prior, sulfonylureas and metformin 3 days prior, and thiazolidinediones at least 8 days prior to the day of the clamp studies. The nondiabetic volunteers (n = 15) were taking no medications and had no family history of type 2 diabetes. A 2-h oral glucose tolerance test was performed to ensure normal glucose tolerance.
Nondiabetic subjects were admitted to the General Clinical Research Center on the morning of the study. To ensure that initial plasma glucose levels were <140 mg/dl in all subjects on the morning of study, subjects with type 2 diabetes were admitted the evening before the study for low-dose overnight insulin infusion, and an 18-gauge catheter was inserted in an antecubital vein for infusions. Beginning at 10:00 P.M. on the night before the study, a variable intravenous infusion of insulin was started, and the rate was adjusted according to an algorithm based on hourly blood glucose measurement. As previously described, overnight correction of glucose did not affect glucose effectiveness in these poorly controlled type 2 diabetic subjects (24).
Euglycemic-hyperglycemic pancreatic clamp studies.
At 7 A.M. on the morning of the study, an 18-gauge catheter was inserted in an antecubital vein for infusions and a contralateral hand vein was cannulated in a retrograde fashion for arterialized venous blood sampling. To obtain arterialized venous blood, this hand was kept in a warming Plexiglas box maintained at 55°C. The experimental protocols lasted 6 h and consisted of an initial 4-h euglycemic period followed by a 2-h hyperglycemic period as previously described (12,15).
At t = 0 min, a primed-continuous infusion of high-performance liquid chromatographyeCpurified [3-3H]-glucose (New England Nuclear, Boston, MA) was started (prime infusion 22 e藽i) and then continued at 0.15 e藽i/min for 6 h (Fig. 1). Infusions containing somatostatin, growth hormone, glucagon, and insulin were also initiated at t = 0 min (12,15). Plasma glucose concentrations were measured at 10- to 15-min intervals during the initial 240 min of the study and maintained at normal fasting concentrations (90 mg/dl) by frequently adjusting the infusion rate of insulin during the first 120 min and maintaining these individualized insulin infusion rates for the duration of the studies. At 240 min, plasma glucose concentrations were acutely increased to 180 mg/dl and then clamped at this level with variable 20% glucose infusions. [3-3H]-glucose (0.1 e藽i/ml) was added to the "cold" 20% glucose to maintain constant glucose specific activity (15). From 0 to 360 min, blood samples were obtained to measure plasma glucose, insulin, glucagon, C-peptide, FFAs, glycerol, lactate, and 3-3H-glucose specific activity. All infusions were stopped at 360 min.
To determine the impact of FFAs on glucose effectiveness, glucose fluxes were compared between euglycemia and hyperglycemia in the nondiabetic group and type 2 diabetic subjects under the various conditions outlined below:
Nondiabetic subjects underwent a control pancreatic clamp study as described above with saline infusion only. A subgroup of these subjects have been previously described (15).
All type 2 diabetic subjects were studied under paired conditions, with a control (saline infusion [NAeC) study and an active infusion (nicotinic acid [NA]) study, at least 1 month apart under the following conditions: a) saline control studies (NAeC) paired with one of the following study types: b) NA3h (n = 9): with infusion of nicotinic acid (0.017 mg · kgeC1 · mineC1) for the final 3 h of the clamp study or c) NA6h (n = 8): with infusion of nicotinic acid (USP grade; Sigma, St. Louis, MO) at 0.015 mg · kgeC1 · mineC1 throughout the 6-h clamp studies. This rate was selected to decrease FFA levels to those observed in nondiabetic subjects (33,34). The NA was pyrogen free, pH adjusted to 7.0 with NaOH, and filter sterilized through a 0.2-micron filter before use. d) NA16h (n = 8): infusion of nicotinic acid (0.008 mg · kgeC1 · mineC1) starting at 10 P.M. and continuing for the duration of the clamp study at a rate of 0.015 mg · kgeC1 · mineC1.
These rates of NA infusion were chosen based on the literature and pilot studies to match FFA levels in the final hour in all groups.
Analytical procedures.
Plasma glucose was measured with a Beckman glucose analyzer (Fullerton, CA) by use of the glucose oxidase method. Plasma [3-3H]-glucose and tritiated watereCspecific activities were measured as previously described (4,11). Plasma insulin, C-peptide, glucagon FFAs, glycerol and lactate, and A1C were measured as previously described (15).
Calculations.
Rates of glucose appearance (Ra) and glucose uptake (Rd) were calculated using Steele’s steady-state equation (26). Rates of EGP were calculated as previously described (26). Data for glucose turnover, plasma hormones, and substrate concentrations represent the mean values during the final 60 min of the euglycemic period (t = 180eC240 min) and the final 60 min of the hyperglycemic period (t = 300eC360 min).
Statistical analysis.
Analysis of the data were performed using SPSS Version 11.5. For averaged data, Student’s t tests were utilized, using paired t tests for comparisons of euglycemic and hyperglycemic intervals in each group and nonpaired t tests to compare the nondiabetic with the type 2 diabetic group. ANOVA was used to compare the various study conditions within the type 2 diabetic group. All data are presented as means ± SE, unless otherwise specified. A P value <0.05 was considered significant.
RESULTS
Baseline (fasting) patient characteristics.
The rate of overnight insulin infusion required to maintain plasma glucose in the target range of 90eC120 mg/dl in the type 2 diabetic subjects averaged 2.67 ± 0.20 units/h. There was no difference in overnight insulin infusion rates for the different study conditions. In the nondiabetic subjects, fasting plasma glucose levels averaged 99 ± 1 mg/dl and plasma FFA levels were 443 ± 57 e蘒/ml. In type 2 diabetic subjects, basal (t = 0) plasma glucose was 124 ± 3 mg/dl and plasma FFA levels were 349 ± 44 e蘒/ml (averaged for all studies after overnight insulin infusion). There was no difference in fasting plasma glucose and fasting FFAs for the three NA groups (Tables 1 and 2).
General clamp study conditions.
During the clamps, plasma glucose levels were similar in type 2 diabetic and in nondiabetic subjects (during the euglycemic study period, 98 ± 1 mg/dl for type 2 diabetic and 96 ± 1 mg/dl for nondiabetic subjects and during the hyperglycemic study period, 185 ± 1 mg/dl for type 2 diabetic and 183 ± 1 mg/dl for nondiabetic subjects, respectively). Plasma glucose levels during both euglycemia and hyperglycemia were similar between pairs of studies (NA versus NAeC). Glucose specific activity was constant following tracer equilibration during both euglycemia and hyperglycemia in each group (Fig. 2).
Although there was a trend toward higher average insulin infusion rates required to maintain euglycemia in type 2 diabetic (0.23 ± 0.02 e蘒 · kgeC1 · mineC1) when compared with nondiabetic (0.18 ± 0.01 e蘒 · kgeC1 · mineC1) subjects, this did not reach statistical significance (P = 0.056). Importantly, plasma insulin levels did not differ between euglycemic and hyperglycemic study periods or between pairs of the NA studies (Table 2). The former comparison is particularly important since the principal comparison is between euglycemia and hyperglycemia in the same study. Plasma glucagon levels also remained stable in all groups. C-peptide levels were suppressed by somatostatin infusion in all studies and did not differ between pairs of studies in either basal, euglycemic, or hyperglycemic periods. Although there was a trend toward increased C-peptide with the onset of hyperglycemia in nondiabetic subjects, the levels remained suppressed at far-lower-than-baseline levels (Table 2). Plasma lactate levels were not different between paired studies.
Saline control studies (NAeC and nondiabetic subjects).
In the tables, all study results will be compared with the paired saline control studies in the same subjects, e.g., NA3h versus NAeC. There was a marked decrease in glucose effectiveness in the type 2 diabetic subjects when compared with nondiabetic subjects. The average rate of glucose infusion required to maintain the target hyperglycemic plateau during the last 60 min of the hyperglycemic period was decreased by more than half in the type 2 diabetic compared with the nondiabetic subjects (1.00 ± 0.19 mg · kgeC1 · mineC1 in type 2 diabetes vs. 3.10 ± 0.20 mg · kgeC1 · mineC1 in nondiabetic subjects; P < 0.001). EGP was suppressed by 48 ± 3% with hyperglycemia in nondiabetic subjects but failed to suppress with hyperglycemia in type 2 diabetic subjects (9 ± 3%, P < 0.001 vs. nondiabetic subjects; Fig. 3). Also, while the percent increase in glucose uptake between the euglycemic and hyperglycemic study periods was 127 ± 72% for nondiabetic subjects, there was a more modest increase of 34 ± 8% in type 2 diabetic subjects (P < 0.001 vs. nondiabetic group). Importantly, plasma FFA concentrations were higher in type 2 diabetic than in nondiabetic subjects during both euglycemia (type 2 diabetes = 412 ± 99 e蘭ol/l vs. no diabetes = 227 ± 31 e蘭ol/l; P < 0.001) and hyperglycemia (type 2 diabetes = 435 ± 81 e蘭ol/l vs. no diabetes = 190 ± 29 e蘭ol/l; P < 0.001) (Fig. 4). Thus, while there were time-dependent decreases in FFA values with the onset of hyperglycemia in nondiabetic subjects (P < 0.001), FFA levels remained elevated in the type 2 diabetic group throughout the entire 6 h of NAeC studies. This is consistent with our previous studies (15).
NA studies
FFA and glycerol levels.
As shown in Fig. 4, FFA values during the final hour of all NA+ studies were comparable with the saline control studies in the nondiabetic subjects. Significant lowering in FFA levels was attained after 1 h of NA infusion; hence, significant lowering of FFAs was observed for the final 2 h of the NA3h studies and during the final 5 h of the NA6h studies. In the NA16h studies, FFA levels were lower at the beginning of the clamp when compared with NAeC (P < 0.01) and remained reduced throughout. Of note, the different NA infusion rates selected for each duration attained comparable FFA reduction in all three groups during the final hour (P = NS by ANOVA).
There were no significant changes in glycerol levels during the NA studies (NA3h = 85 ± 6 vs. NAeC = 84 ± 7, NA6h = 52 ± 3 vs. NAeC = 76 ± 4, and NA16h = 92 ± 15 vs. NAeC = 108 ± 9 e蘭ol/l; P = NS for all). These findings differ from two reports (27,28) in which nicotinic acid reduced glycerol levels in fasting individuals. However, a recent study (29) using nicotinic acid in the presence of high physiologic insulin was consistent with our findings, since nicotinic acid lowered FFAs but had no effect on plasma glycerol levels. Of note, the former pair of studies were performed under basal fasting conditions, while the latter pair of studies were in the presence of hyperinsulinemia and hyperglycemia, respectively. Both conditions inhibit gluconeogenesis, and hence there would be less consumption of glycerol via gluconeogenesis. This is likely to balance the reduced production of glycerol via lipolysis, particularly since lipolysis of a triacyl-glycerol molecule would yield only one-third as much glycerol as fatty acids when expressed on a molar basis.
Glucose fluxes.
To raise blood glucose quickly to 180 mg/dl at 240 min, glucose infusion rates at the onset of hyperglycemia were calculated based on each subject’s weight and were similar to the paired NAeC studies. Glucose infusion rates became significantly different by 315 min in the NA6h group and 330 min in the NA3h and NA16h groups compared with the paired NAeC studies. The average rate of glucose infusion required to maintain hyperglycemia during the last 60 min in the NA studies (NA3h = 2.22 ± 0.56, NA6h = 2.29 ± 0.33, and NA16h = 2.56 ± 0.46 mg · kgeC1 · mineC1) was increased more than twofold above the rates required in the paired NAeC studies (1.11 ± 0.40, 0.66 ± 0.21, and 0.74 ± 0.18 mg · kgeC1 · mineC1, respectively). Thus, normalizing plasma FFA levels for 3 h significantly increased glucose effectiveness in the type 2 diabetic subjects. The degree of suppression of EGP with hyperglycemia in the NA studies (NA3h = 31 ± 6%, NA6h = 41 ± 8%, and NA16h = 47 ± 4%) was significantly greater than in the NAeC studies in the same individuals (NAeC 8 ± 7%, 7 ± 6%, and 14 ± 4%, respectively, P = 0.001; Figs. 3 and 5). The suppression of EGP with NA for 6 and 16 h was comparable with the degree of suppression observed in the nondiabetic subjects, while there was less suppression of EGP in the NA3h studies (P = 0.02). This indicates that significant improvements were seen at 3 h and complete normalization observed after 6 h of FFA lowering.
Also, the percent increase in glucose uptake (Figs. 3 and 5) between the euglycemic and hyperglycemic study periods was 63 ± 10% for the NA6h vs. 26 ± 10% in the NAeC (P = 0.01) and 64 ± 20% for the NA16h vs. 30 ± 14% change in the NAeC studies (P = 0.04). However, this stimulation of glucose uptake by hyperglycemia was substantially less than the 127% increase observed in nondiabetic subjects. While there was a trend toward increased glucose uptake after only 3 h of FFA lowering in type 2 diabetic subjects (NA3h 70 ± 14% vs. NAeC 43 ± 17%), it did not reach statistical significance (P = 0.09). Thus, there were improvements in peripheral glucose effectiveness following 6 h of FFA lowering, but normalization was not achieved even after 16 h.
Of note, we previously performed studies with coinfusion of both nicotinic acid and liposyn in type 2 diabetic subjects (n = 5) to prevent the nicotinic acideCinduced decline in FFA levels (15). The percent change in EGP and glucose uptake in the coinfusion studies were eC9 ± 10 and 37 ± 17%, respectively, and thus were not significantly different from the current NAeC studies. Thus, the effects of nicotinic acid on glucose effectiveness are likely due to FFA lowering and not to nonspecific effects of nicotinic acid.
DISCUSSION
Although the effects of increased FFA levels on hepatic insulin action are well established (30), the effects of FFAs on hepatic glucose effectiveness are underrecognized. Indeed, a careful review of the literature suggests that this is an important phenomenon. Previous studies (31) have suggested that glucose-induced reductions in FFA levels account for the majority of suppression of EGP by glucose in nondiabetic individuals. In support of this idea, we previously reported that in type 2 diabetic individuals with optimal glycemic control, EGP displays normal suppression of FFA levels by hyperglycemia and thus normal glucose effectiveness (12). Moreover, 72 h of intensive glycemic control with insulin in poorly controlled type 2 diabetic subjects restored glucose effectiveness in concert with normalization of plasma FFA levels (12). Shah et al. (33) reported that elevation of plasma FFA levels in healthy women blunted the suppression of EGP by both hyperinsulinemia and hyperglycemia (35), although glucose effectiveness was not specifically examined. When plasma FFA levels were increased to >2 mmol/l for 9 h under pancreatic clamp conditions in healthy subjects, the resulting 50% rise in plasma glucose concentrations failed to suppress EGP (33).
The present experiments examined the impact of altering FFA levels for variable durations on glucose effectiveness in type 2 diabetic individuals. Euglycemic-hyperglycemic pancreatic clamp techniques were used to study the ability of hyperglycemia per se to regulate EGP and glucose uptake under fixed hormonal conditions. In these subjects with poorly controlled type 2 diabetes, there was a rapid improvement in hepatic glucose effectiveness following only 3 h of nicotinic acid infusion (2 h of FFA lowering) and a complete restoration of glucose effectiveness with 6-h infusion (5 h of FFA normalization). This time course of FFA-induced changes in glucose effectiveness is consistent with rapid changes in flux through gluconeogenesis and/or rapid changes in the activity of key hepatic enzymes. The magnitude of the effect we report suggests that the inhibitory effects of FFAs on glucose effectiveness contribute importantly to worsening hyperglycemia in individuals with type 2 diabetes.
Increased circulating FFAs have been shown to alter both the gene expression and the activity of hepatic enzymes responsible for regulating EGP. Specifically, FFAs promote hepatic glucose-6-phosphatase gene expression (18,19) and decrease hepatic glucokinase expression (20). Studies of the roles of these enzymes in regulating glucose fluxes are consistent with their importance in determining glucose effectiveness. Dual defects in these key regulatory enzymes could impair glucose effectiveness, since defective glucokinase activity would impair the liver’s ability to "sense" rising glucose levels (34,35), while increased glucose-6-phosphatase would stimulate EGP by both glycogenolysis and gluconeogenesis. The fact that 6 h of FFA lowering was required to fully normalize glucose effectiveness in the current studies suggests that adequate time was required to reverse chronic effects of FFAs on hepatic gene expression.
Increased FFAs have also been shown to increase gluconeogenesis both in vivo and in vitro (18,36). Indeed, type 2 diabetes is characterized by augmented rates of gluconeogenesis, which contribute to the inappropriate elevations in EGP in these individuals (37). The current studies were designed to explore potential mechanisms whereby increased FFA levels impact glucose effectiveness in humans. The rapid effects of lowering FFAs observed in these studies could be consistent with rapid changes in gluconeogenesis. This will be the focus of further investigation.
It is important to note that our studies were performed under basal insulin conditions, since they were designed to study glucose effectiveness and not insulin sensitivity. Of note, there was no effect of lowering FFA levels on rates of EGP during euglycemia (i.e., NA16h or NA6h versus their respective controls). This demonstrates that sensitivity to basal insulin was not affected by the modest lowering in FFA levels. This is consistent with the observation of Whiteall et al. (38) that lowering triglycerides and FFAs with gemfibrozil did not alter insulin sensitivity during low-dose insulin infusion. We also found that there was no change in insulin sensitivity (as assessed by the insulin infusion rates required to lower blood glucose) overnight despite lower FFA levels in the NA16h studies. This confirms that the improved suppression of EGP observed during hyperglycemia with FFA lowering was due to improved glucose effectiveness rather than a change in insulin sensitivity.
It is intriguing that nicotinic acid acutely improved glucose effectiveness in these poorly controlled type 2 diabetic subjects, despite concerns about its chronic use in type 2 diabetes (39). Use of niaspan in 125 subjects with type 2 diabetes for up to 65 weeks had very modest increases in plasma glucose (<10 mg/dl) and no change in A1C (40), although FFA levels were not assessed systematically. Of note, rebound elevations in FFA levels occur following administration of nicotinic acid, generally rising well above the pretreatment FFA levels. Indeed, 7-day treatment with nicotinic acid in nondiabetic subjects resulted in insulin resistance that was proportional to the increases in fasting FFAs (41). It is therefore likely that repeated rebound rises in FFA levels may have mild detrimental effects on glucose control with chronic administration of nicotinic acid. Use of extended-release nicotinic acid was recently shown to reduce the degree of FFA rebound. Despite a nonsignificant trend of more type 2 diabetic patients needing adjustment of diabetes medication, there was no change in A1C (42).
Lowering FFA levels also improved peripheral glucose effectiveness, i.e., the ability of hyperglycemia per se to stimulate whole-body glucose uptake. Previous studies have demonstrated that elevated FFAs inhibited glucose-mediated glucose uptake under conditions of physiologic insulin levels, similar to the present studies (43). Of note, FFAs have been shown to directly impact both the expression (44) and translocation (45) of glucose transporters in skeletal muscle. In contrast to the complete restoration of hepatic glucose effectiveness, FFA lowering for up to 16 h only partially restored peripheral glucose effectiveness. However, we previously reported that correction of glucose and FFA levels for 72 h improved peripheral glucose effectiveness to the level of nondiabetic subjects (12). Therefore it is possible that a longer duration of FFA lowering would have completely restored glucose effectiveness. Additionally, since peripheral insulin resistance is not rapidly restored by correcting metabolic defects (46), the permissive effects of insulin on peripheral glucose effectiveness (47) are still likely to be impaired in these poorly controlled type 2 diabetic subjects.
To conclude, our data suggest that elevated FFA levels contribute to the lack of hepatic and peripheral glucose effectiveness in individuals with type 2 diabetes. Of note, FFA levels are chronically elevated in type 2 diabetes, and attaining long-term FFA lowering in these individuals is currently not a specific therapeutic goal in type 2 diabetes. Since elevated FFA levels appear to significantly contribute to the loss of glucose effectiveness in type 2 diabetes and thus overall glycemic control, understanding the pathogenesis of this important defect could have considerable therapeutic benefit.
ACKNOWLEDGMENTS
This work was supported in part by grants from the American Diabetes Association (to M.H. and J.T.) and the National Insitutes of Health (Einstein General Clinical Research Center [GCRC] RR12248, K12 RR17672 [to P.K.], P01-AG021654, and DK20541 [Diabetes Research and Training Center {DRTC}]). Dr. Hawkins was a Clinical Associate Physician of the GCRC (3 M01 RR 12248eC03S1) and is a recipient of a Beeson Award from the American Federation of Aging Research.
The authors thank Angela Stangarone for coordinating studies, Robin Sgueglia for determinations performed in the DRTC Hormone Assay Core, Dr. Hillel Cohen for biostatistical assistance, and the staff of the Albert Einstein College of Medicine GCRC for their outstanding patient care. We also acknowledge Drs. Luciano Rossetti, Daniel Stein; and Harry Shamoon for many helpful discussions.
FOOTNOTES
DOI: 10.2337/db05-0988
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
DeFronzo R: The triumvirate: -cell, muscle and liver: a collusion responsible for type 2 diabetes. Diabetes 37:667eC687, 1988
Bogardus C, Lillioja S, Howard B, Reaven G, Mott D: Relationships between insulin secretion, insulin action, and fasting plasma glucose concentration in nondiabetic and noninsulin-dependent diabetic subjects. J Clin Invest 74:1238eC1246, 1984
Campbell P, Mandarino L, Gerich J: Quantification of the relative impairment in actions of insulin on hepatic glucose production and peripheral glucose uptake in non-insulin-dependent diabetes mellitus. Metabolism 37:15eC22, 1988
Mittelman SD, Fu YY, Rebrin K, Steil G, Bergman RN: Indirect effect of insulin to suppress endogenous glucose production is dominant, even with hyperglucagonemia. J Clin Invest 100:3121eC3130, 1997
Fisher SJ, Kahn CR: Insulin signaling is required for insulin’s direct and indirect action on hepatic glucose production. J Clin Invest 111:463eC468, 2003
Bergman R: Interaction of insulin and glucose in the control of hepatic glucose balance. Am J Physiol 227:1314eC1322, 1974
Liljenquist JE, Mueller GL, Cherrington AD, Perry JM, Rabinowitz D: Hyperglycemia per se (insulin and glucagon withdrawn) can inhibit hepatic glucose production in man. J Clin Endocrinol Metab 48:171eC175, 1979
Sacca L, Hendler R, Sherwin RS: Hyperglycemia inhibits glucose production in man independent of changes in glucoregulatory hormone. J Clin Endocrinol Metab 47:1160eC1163, 1978
DeFronzo RA, Ferrannini E, Hendler R, Felig P, Wahren J: Regulation of splanchic and peripheral glucose uptake by insulin and hyperglycemia in man. Diabetes 32:35eC45, 1983
Bell PM, Firth RG, Rizza RA: Effect of hyperglycemia on glucose production and utilization in humans. Diabetes 35:642eC648, 1986
Mevorach M, Giacca A, Aharon Y, Hawkins M, Shamoon H, Rossetti L: Regulation of endogenous glucose production by glucose per se is impaired in type 2 diabetes mellitus. J Clin Invest 102:744eC753, 1998
Hawkins M, Gabriely I, Wozniak R, Mevorach M, Rossetti L, Shamoon H: The effect of glycemic control on hepatic and peripheral glucose effectiveness in type 2 diabetes mellitus. Diabetes 51:2179eC2189, 2002
Nagasaka S, Tokuyama K, Kusaka I, Hayashi H, Rokkaku K, Nakamura T, Kawakami A, Higashiyama M, Ishikawa S, Saito T: Endogenous glucose production and glucose effectiveness in type 2 diabetic subjects derived from stable-labeled minimal model approach. Diabetes 48:1054eC1060, 1990
Reaven GM, Hollenbeck C, Jeng CY, Wu MS, Chen YD: Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes 37:1020eC1024, 1988
Hawkins M, Tonelli J, Kishore P, Stein D, Ragucci E, Gitig A, Reddy K: Contribution of elevated free fatty acid levels to the lack of glucose effectiveness in type 2 diabetes. Diabetes 52:2748eC2758, 2003
Williamson JR, Kreisberg RA, Felts PW: Mechanism for the stimulation of gluconeogenesis by fatty acids in perfused rat liver. Proc Natl Acad Sci U S A 56:247eC254, 1966
Antras-Ferry J, Le Bigot G, Robin P, Robin D, Forest C: Stimulation of phosphoenolpyruvate carboxykinase gene expression by fatty acids. Biochem Biophys Res Commun 203:385eC391, 1994
Lam TK, Carpentier A, Lewis GF, van de Werve G, Fantus IG, Giacca A: Mechanisms of the free fatty acid-induced increase in hepatic glucose production. Am J Physiol Endocrinol Metab 284:E863eCE873, 2003
Massillon D, Barzilai N, Hawkins M, Prus-Wertheimer D, Rossetti L: Induction of hepatic glucose-6-phosphatase gene expression by lipid infusion. Diabetes 46:153eC157, 1997
van de Werve G, Lange A, Newgard C, Mechin MC, Li Y, Berteloot A: New lessons in the regulation of glucose metabolism taught by the glucose 6-phosphatase system. Eur J Biochem 267:1533eC1549, 2000
Jump DB, Clarke SD, Thelen A, Liimatta M: Coordinate regulation of glycolytic and lipogenic gene expression by polyunsaturated fatty acids. J Lipid Res 35:1076eC1084, 1994
Lam TK, van de Werve G, Giacca A: Free fatty acids increase basal hepatic glucose production and induce hepatic insulin resistance at different sites. Am J Physiol Endocrinol Metab 284:E281eCE290, 2003
Oakes N, Cooney G, Camilleri S, Chisholm D, Kraegen E: Mechanisms of liver and muscle insulin resistance induced by chronic high-fat feeding. Diabetes 46:1768eC1774, 1997
Basu R, Basu A, Nielsen M, Shah P, Rizza RA: Effect of overnight restoration of overnight euglycemia on glucose effectiveness in type 2 diabetes mellitus. J Clin Endocrinol Metab 84:2314eC2319, 1999
Steele R: Influences of glucose loading and of injected insulin on hepatic glucose output. Ann N Y Acad Sci 82:420eC430, 1959
Rossetti L, Giaccari A: Relative contribution of glycogen synthesis and glycolysis to insulin-mediated glucose uptake: a dose-response euglycemic clamp study in normal and diabetic rats. J Clin Invest 85:1785eC1792, 1990
Wang W, Basinger A, Neese R, Christiansen M, Hellerstein M: Effects of nicotinic acid on fatty acid kinetics, fuel selection, and pathways of glucose production in women. Am J Physiol Endocrinol Metab 279:50eC59, 2000
Boden Chen X, Capulong E, Mozzoli M: Effects of free fatty acids on gluconeogenesis and autoregulation of glucose production in type 2 diabetes. Diabetes 50:810eC816, 2001
Carpentier AC, Frisch F, Cyr D, Genereux P, Patterson BW, Giguere R, Baillargeon JP: On the suppression of plasma nonesterified fatty acids by insulin during enhanced intravascular lipolysis in humans. Am J Physiol Endocrinol Metab 289:E849eCR856, 2005
Service FJ: Hypoglycemic disorders. N Engl J Med 332:1144eC1152, 1995
Shah P, Basu A, Rizza R: Fat-induced liver insulin resistance. Curr Diab Rep 3:214eC218, 2003
Best JD, Kahn SE, Ader M, Watanabe RM, Ni TC, Bergman RN: Role of glucose effectiveness in the determination of glucose tolerance. Diabetes Care 19:1018eC1030, 1996
Shah P, Vella A, Basu A, Basu R, Adkins A, Schwenk WF, Johnson CM, Nair KS, Jensen MD, Rizza RA: Elevated free fatty acids impair glucose metabolism in women: decreased stimulation of muscle glucose uptake and suppression of splanchnic glucose production during combined hyperinsulinemia and hyperglycemia. Diabetes 52:38eC42, 2003
Roden M, Stingl H, Chandramouli V, Schumann WC, Hofer A, Landau BR, Nowotny P, Waldhausl W, Shulman GI: Effects of free fatty acid elevation on postabsorptive endogenous glucose production and gluconeogenesis in humans. Diabetes 49:701eC707, 2000
Hawkins M, Gabriely I, Wozniak R, Vilcu C, Shamoon H, Rossetti L: Fructose improves the ability of hyperglycemia per se to regulate glucose production in type 2 diabetes. Diabetes 51:606eC614, 2002
Basu A, Basu R, Shah P, Vella A, Johnson CM, Jensen M, Nair KS, Schwenk WF, Rizza RA: Type 2 diabetes impairs splanchnic uptake of glucose but does not alter intestinal glucose absorption during enteral glucose feeding: additional evidence for a defect in hepatic glucokinase activity. Diabetes 50:1351eC1362, 2001
Boden G: Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes 46:3eC10, 1997
Boden G, Chen X, Stein TP: Gluconeogenesis in moderately and severely hyperglycemic patients with type 2 diabetes mellitus. Am J Physiol Endocrinol Metab 280:E23eCE30, 2001
Whitelaw DC, Smith JM, Nattrass M: Effects of gemfibrozil on insulin resistance to fat metabolism in subjects with type 2 diabetes and hypertriglyceridaemia. Diabetes Obes Metab 3:187eC194, 2002
American Diabetes Association: Management of dyslipidemia in adults with diabetes. Diabetes Care 21:179eC182, 1998
Elam MB, Hunninghake DB, Davis KB, Garg R, Johnson C, Egan D, Kostis JB, Sheps DS, Brinton EA, the ADMIT Investigators: Effect of niacin on lipid and lipoprotein levels and glycemic control in patients with diabetes and peripheral arterial disease: the ADMIT Study: a randomized trial. JAMA 284:1263eC1270, 2000
Poynten AM, Gan SK, Kriketos AD, O’Sullivan A, Kelly JJ, Ellis BA, Chisholm DJ, Campbell LV: Nicotinic acid-induced insulin resistance is related to increased circulating fatty acids and fat oxidation but not muscle lipid content. Metabolism 52:699eC704, 2003
Vega GL, Cater NB, Meguro S, Grundy SM: Influence of extended-release nicotinic acid on nonesterified fatty acid flux in the metabolic syndrome with atherogenic dyslipidemia. Am J Cardiol 95:1309eC1313, 2005
Baron AD, Brechtel G, Edelman SV: Effects of free fatty acids and ketone bodies on in vivo non-insulin-mediated glucose utilization and production in humans. Metabolism 38:1056eC1061, 1989
Long SD, Pekala PH: Regulation of GLUT4 gene expression by arachidonic acid: evidence for multiple pathways, one of which requires oxidation to prostaglandin E2. J Biol Chem 271:1138eC1144, 1996
Zierath JR, Housenecht KL, Gnudi L, Kahn BB: High-fat feeding impairs insulin-stimulated GLUT4 recruitment via an early insulin-signaling defect. Diabetes 46:215eC223, 1997
Wise SD, Nielsen MF, Cryer PE, Rizza RA: Overnight normalization of glucose concentrations improves hepatic but not extrahepatic insulin action in subjects with type 2 diabetes mellitus. J Clin Endocrinol Metab 83:2461eC2469, 1998
Edelman SV, Laakso M, Wallace P, Brechtel G, Olefsky EM, Baron AD: Kinetics of insulin-mediated and non-insulin-mediated glucose uptake in humans. Diabetes 39:955eC964, 1990(Preeti Kishore, Julia Ton)