The aim of this study was to establish the contribution of insulin resistance to the morning (a.m.) versus afternoon (p.m.) lower glucose tolerance of people with type 2 diabetes (T2D). Eleven subjects with T2D (mean [SD] diabetes duration 0.79 [0.23] years, BMI 28.3 [1.8] kg/m2, A1C 6.6% [0.26%] [48.9 (2.9) mmol/mol]), treatment lifestyle modification only) and 11 matched control subjects without diabetes were monitored between 5:00 and 8:00 a.m. and p.m. (in random order) on one occasion (study 1), and on a subsequent occasion, they underwent an isoglycemic clamp (a.m. and p.m., both between 5:00 and 8:00, insulin infusion rate 10 mU/m2/min) (study 2). In study 1, plasma glucose, insulin, C-peptide, and glucagon were higher and insulin clearance lower in subjects with T2D a.m. versus p.m. and versus control subjects (P < 0.05), whereas free fatty acid, glycerol, and β-hydroxybutyrate were lower a.m. versus p.m. However, in study 2 at identical hyperinsulinemia a.m. and p.m. (∼150 pmol/L), glucose Ra and glycerol Ra were both less suppressed a.m. versus p.m. (P < 0.05) in subjects with T2D. In contrast, in control subjects, glucose Ra was more suppressed a.m. versus p.m. Leucine turnover was no different a.m. versus p.m. In conclusion, in subjects with T2D, insulin sensitivity for glucose (liver) and lipid metabolism has diurnal cycles (nadir a.m.) opposite that of control subjects without diabetes already at an early stage, suggesting a marker of T2D.

Article Highlights

  • In people with type 2 diabetes (T2D), fasting hyperglycemia is greater in the morning (a.m.) versus the afternoon (p.m.), and insulin sensitivity for glucose and lipid metabolism is lower a.m. versus p.m.

  • This pattern is the reverse of the physiological diurnal cycle of people without diabetes who are more insulin sensitive a.m. versus p.m.

  • These new findings have been observed in the present study in people without obesity but with recent-onset T2D, with good glycemic control, and in the absence of confounding pharmacological treatment.

  • It is likely that the findings represent a specific marker of T2D, possibly present even in prediabetes before biochemical and clinical manifestations.

Diurnal cycles in metabolism have long been reported in rodents, mammals, and humans as part of the complex circadian system, which anticipates recurring 24-h anabolic feeding and catabolic fasting to increase metabolic efficiency (1,2). In humans, several studies in healthy subjects without diabetes have observed greater glucose tolerance (311) as a result of greater insulin sensitivity (79) and greater β-cell responsiveness (48) in the morning (a.m.) compared with the evening (p.m.) hours. These observations may suggest that the early morning hours are more suitable for feeding, whereas the evening hours are more suitable for fasting (2). Misalignment and/or disruption of this rhythmic cycle induced by mistimed light exposure, food intake, or sleep (12), among other factors, adversely affect metabolic health in humans and may be involved in the pathogenesis of metabolic diseases like type 2 diabetes (T2D) (2).

People with T2D appear to have an inversion of the 24-h physiological cycle of greater glucose tolerance a.m. versus p.m., as first reported by Hatlehol (13) in 1924, and confirmed by more contemporary studies (1418). This characteristic of T2D is known as the dawn phenomenon (19,20).

In 1996, Boden et al. (17) reported that in people with T2D studied with the hyperglycemic clamp during 3 days of fasting, insulin sensitivity exhibits rhythmic diurnal cycles that are consistent day to day. Interestingly, the a.m. nadir of insulin sensitivity coincided with the peak of endogenous glucose production (EGP) (17). Shortly after, our group reported that in people with T2D, the nocturnal rise of EGP is driven primarily by gluconeogenesis and accounts for fasting hyperglycemia along with impaired responsiveness of the β-cell (21). These results have been subsequently confirmed and expanded by the elegant study of Radziuk and Pye (22). Taken together, these studies suggest that the diurnal cycles of glucose tolerance in T2D in the fasting state are associated with intraday changes of insulin sensitivity at the level of the liver. However, no study so far has directly examined EGP a.m. and p.m. in T2D with the glucose clamp at insulin concentrations suitable to specifically explore EGP and lipid metabolism (23).

The aim of the current study was to establish diurnal cycles of insulin sensitivity in people with T2D compared with people without diabetes using a glucose clamp a.m. and p.m. at insulin concentrations designed to establish glucose metabolism (primarily in the liver), and lipid and protein metabolism. To avoid confounders, such as variable diabetes duration, diabetic complications, obesity, glucotoxicity (24), and effects of glucose-lowering drugs, only people without obesity but with T2D of very recent clinical onset and with good glycemic control in the absence of pharmacological treatment were studied.

Subjects

The study was carried out according to the tenets of the Declaration of Helsinki after obtaining informed written consent from all volunteers and approval by the local ethics committee of University of Perugia, Italy. People with T2D and negative for GADA antibodies were considered eligible for the study if they 1) had a known duration of diabetes of ≤1 year; 2) were treated for diabetes only with lifestyle intervention for the 3 months before the study in the absence of any glucose-lowering drug and had good glycemic control (A1C <53 mmol/mol [7.0%]); and 3) had no other diseases apart from diabetes. Age-, sex-, and BMI-matched control subjects without diabetes, as indicated by fasting plasma glucose (PG) and oral glucose tolerance test; with no family history of diabetes and no other diseases; and not taking any medications were studied.

Study Design

Control subjects and subjects with T2D were studied on two occasions (study 1 and study 2) at a 2-month interval (Supplementary Fig. 1). Study 2 was always performed after study 1. Study 1 was observational, i.e., metabolic monitoring in the fasting 5:00–08:00 a.m. on one occasion and a week later at 5:00–8:00 p.m. (random order), with no intervention. Study 2 was a hyperinsulinemic-isoglycemic clamp performed at the same time as observational study 1 (between 5:00 and 8:00 a.m. and p.m. in random order). PG was clamped in control subjects at the individual PG values observed at time 0 h (T0 h) in study 1, a.m. and p.m., respectively. In subjects with T2D, the clamp was made isoglycemic by allowing PG to decrease after initiation of intravenous insulin to the mean value observed in control subjects at T2 h in study 1 and by maintaining it until T3 h.

In study 1 and study 2, subjects were admitted to the Clinical Research Unit of the Section of Endocrinology and Metabolism, Department of Medicine and Surgery, University of Perugia, at 6:00 p.m. on the evening before the a.m. study on the next day (i.e., 11 h before T0 h at 5:00 a.m.) and in case of the p.m. study, at 6:00 a.m. on the morning of the same day (i.e., T0 h at 5:00 p.m.). To ensure the same duration of fasting prior to the a.m. and p.m. studies, control subjects and subjects with T2D had a standardized meal (10 kcal/kg, 52% carbohydrates, 28% fat, and 20% protein) served at 6:30 p.m. and at 6:30 a.m. to be eaten in 30 min on the occasion of the a.m. and p.m. study, respectively. Thereafter, subjects were placed in bed maintained in a supine position, and a forearm vein was cannulated with an 18-gauge catheter needle kept patent with NaCl 0.9% infusion. Subjects were invited to sleep until 30 min before T0 h of the a.m. and p.m. studies. Thirty minutes before T0 h, an 18-gauge needle was inserted in a retrograde fashion in a dorsal vein of one hand maintained in a thermoregulated plexiglass box at 65°C to arterialize venous blood (25) for sampling for glucose, hormones, and metabolites at frequent intervals. In study 2, 2 h prior to T0 h, a primed, sterile, pyrogen-free constant infusion (prime 300 mg, infusion rate 3.3 mg/min) of [6,6-2H2]glucose, along with [1,1,2,3,3-2H5]glycerol (prime 4.5 μmol/L/kg; infusion rate 0.3 μmol/L/kg/min) and 13C-leucine (prime 0.7 mg/kg; infusion rate 0.7 mg/kg/h) (Cambridge Isotopes Laboratories, Cambridge, MA) were started and maintained throughout the studies to determine glucose, glycerol, and leucine kinetics, respectively (Supplementary Fig. 1). At T0 h, an intravenous infusion of human regular insulin (Eli Lilly Italia SpA), diluted to 1 unit/mL in 100 mL of 0.9% NaCl solution containing 2 mL of the subject’s blood, was initiated and continued at the rate of 10 mU/m2/min until 180 min. An infusion of 20% glucose enriched with 2% [6,6-2H2]glucose at a variable rate was used to maintain PG at the target according to the original principle of the manual clamp technique (25,26), while avoiding non–steady-state errors in measurement of glucose turnover (27).

Analytical Methods

Bedside PG was measured in triplicate every 5–15 min using the YSI 2300 STAT glucose analyzer (Yellow Springs Instruments, Yellow Springs, OH). Plasma C-peptide and glucagon were measured by a commercial radioimmunoassay kit (DRG Instruments GmbH, Marburg, Germany). The serum insulin concentration was also measured by a commercial radioimmunoassay kit specific for human insulin (DRG Instruments GmbH), with a range of detection of 3.125–200 μU/mL. A1C was determined by high-performance liquid chromatography using an Hi-AUTO A1C, TM HA 8121 apparatus (DIC; Daiichi, Kogaku Co., Ltd., Kyoto, Japan), Diabetes Control and Complications Trial (DCCT) aligned (upper limit in control subjects without diabetes <6.1%). Free fatty acids (FFAs), blood glycerol, β-hydroxybutyrate, alanine, and lactate were analyzed by previously described fluorimetric methods (25,26). To determine glucose, protein, and lipid kinetics in study 2, arterialized venous blood samples were taken every 10 min from −20 to 0 min and 150–180 min to measure the plasma concentration and enrichment of leucine, α-ketoisocaproic acid, glucose, and glycerol.

Calculations and Statistical Analysis

The insulin secretory rate (ISR) (28), the estimated portal venous insulin concentration (29), and the prehepatic insulin-to-glucagon (I/Glg) molar ratio (30) were calculated as previously described. The endogenous metabolic clearance rate of insulin (eMCRI) was calculated as the ratio of total insulin secretion (as reconstructed from C-peptide concentrations [28]) to the serum insulin area under the curve (AUC) for corresponding T0 h–T3 h of a.m. and p.m. of observational study 1 as previously described (30). The index of fasting β-cell function was calculated as the ratio AUC − ISR/AUC − PG in observational study 1. The HOMA of insulin resistance was calculated from data of observational study 1 (T3 h), and the indices of insulin resistance at the tissue level (hepatic, adipose, and lipoprotein) were calculated from data of clamp study 2, as previously reported (31). The linear trapezoidal rule was used to calculate the AUC for all parameters. Clamp quality was expressed as the coefficient of variation of the PG measurements from T2.5 h through T3 h. Glucose fluxes were calculated based on a non–steady-state assumption, and the total Ra and Rd were calculated using a modified form of the Steele equation to account for the addition of stable labeled tracer to the exogenous glucose infusate (27). EGP (primarily hepatic) was obtained as the difference between Ra and the exogenous glucose infusion rate (GIR) during the clamp. Glucose utilization was obtained by adding the GIR to the EGP. When the EGP yielded a negative number, it was assumed to be 0, and the corresponding GIR was assumed as glucose utilization. Glucose clearance was calculated by dividing the rate of glucose utilization by the prevailing PG concentration (32).

The statistical analysis was based on repeated-measures ANOVA followed by Tukey-Kramer multiple comparison test. Sex was not considered as a factor in the statistical analysis. Data that did not follow a normal distribution according to the Shapiro-Wilk test were analyzed on log-transformed data. A total sample size of 11 subjects with T2D was calculated to achieve 90% power to detect a mean of paired differences in blood glucose (a.m. vs. p.m.) of 20% with an SD of differences of 20% and a significance level (α) of 0.05 using a two-sided paired z test. A total sample of 11 subjects without diabetes was also enrolled as the control group. Continuous variables are expressed as mean (SD) in the tables and text, and as mean (SE) in the figures. Statistical analysis was usually performed using NCSS 21/PASS 11 software (NCSS, Kaysville, UT).

Data and Resource Availability

The data sets generated during the current study are available from the corresponding author upon reasonable request.

Subjects

A total of 22 matched subjects were studied (11 control subjects without diabetes and 11 with T2D) (Table 1). Subjects with T2D had a short known duration of diabetes with modest increase in fasting PG and A1C and were all treated with lifestyle intervention only in the absence of glucose-lowering drugs.

Table 1

Subject demographic and clinical characteristics

Control subjects (n = 11)Subjects with T2D (n = 11)P
Sex, n    
 Male  
 Female  
Age (years) 52.8 (5.5) 53.3 (5.7) 0.822 
Ethnicity, n    
 Caucasian 11 11  
 Other  
Diabetes duration (years) — 0.79 (0.23)  
BMI (kg/m228.1 (2.1) 28.3 (1.8) 0.832 
eGFR (mL/min/1.73 m295 (8.9) 96 (7.6) 0.761 
A1C    
 % 5.31 (0.31) 6.6 (0.26) <0.000 
 mmol/mol 34.6 (3.5) 48.9 (2.9) <0.000 
Fasting PG (mg/dL) 88 (3.1) 132 (4.1) <0.000 
Fasting serum insulin (pmol/L) 64 (14) 88 (10) 0.001 
HOMA-IR^ 1.9 (0.3) 4.9 (0.6) <0.001 
Fasting plasma C-peptide (nmol/L) 1.29 (0.49) 0.92 (0.38) 0.048 
Fasting plasma glucagon (pmol/L) 18.5 (3.6) 29.4 (6.9) <0.000 
Treatment of diabetes — Lifestyle only  
Control subjects (n = 11)Subjects with T2D (n = 11)P
Sex, n    
 Male  
 Female  
Age (years) 52.8 (5.5) 53.3 (5.7) 0.822 
Ethnicity, n    
 Caucasian 11 11  
 Other  
Diabetes duration (years) — 0.79 (0.23)  
BMI (kg/m228.1 (2.1) 28.3 (1.8) 0.832 
eGFR (mL/min/1.73 m295 (8.9) 96 (7.6) 0.761 
A1C    
 % 5.31 (0.31) 6.6 (0.26) <0.000 
 mmol/mol 34.6 (3.5) 48.9 (2.9) <0.000 
Fasting PG (mg/dL) 88 (3.1) 132 (4.1) <0.000 
Fasting serum insulin (pmol/L) 64 (14) 88 (10) 0.001 
HOMA-IR^ 1.9 (0.3) 4.9 (0.6) <0.001 
Fasting plasma C-peptide (nmol/L) 1.29 (0.49) 0.92 (0.38) 0.048 
Fasting plasma glucagon (pmol/L) 18.5 (3.6) 29.4 (6.9) <0.000 
Treatment of diabetes — Lifestyle only  

Data are mean (SD) unless otherwise indicated. eGFR, estimated glomerular filtration rate; HOMA-IR, HOMA of insulin resistance.

^HOMA-IR = (fasting plasma insulin [mU/mL] × fasting PG [mg/dL]) / 405.

Study 1: Monitoring a.m. and p.m.

PG, Serum Insulin, Plasma C-Peptide, and ISR

Plasma glucose in subjects with T2D was greater a.m. than p.m., reaching a mean peak of 140 (10) mg/dL and 100 (14) mg/dL (P < 0.001), respectively, at a median T1 h (Fig. 1 and Table 2). In addition, PG in subjects with T2D was greater than in control subjects (P < 0.001). In the latter, PG was no different a.m. versus p.m.

Figure 1

PG, serum insulin, and plasma C-peptide concentrations and ISRs in control subjects and subjects with T2D in study 1 (monitoring) between 5:00 and 8:00 a.m. and p.m. Data are mean (SE). *P < 0.05 after repeated-measures ANOVA with Tukey-Kramer multiple comparison test.

Figure 1

PG, serum insulin, and plasma C-peptide concentrations and ISRs in control subjects and subjects with T2D in study 1 (monitoring) between 5:00 and 8:00 a.m. and p.m. Data are mean (SE). *P < 0.05 after repeated-measures ANOVA with Tukey-Kramer multiple comparison test.

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Table 2

T0 h–T3 h AUCs of parameters measured between 5:00 and 8:00 a.m. and p.m., in study 1 and study 2*

Control subjectsSubjects with T2D
a.m.p.m.Pa.m.p.m.PP#
Study 1        
 PG (mg ⋅ h/dL) 253 (16) 256 (16) 0.908 406 (31) 283 (34) <0.001 <0.001 
 Insulin (pmol ⋅ h/mL) 186 (25) 193 (29) 0.681 254 (17) 186 (29) <0.001 <0.001 
 C-peptide (nmol ⋅ h/L) 2.3 (0.8) 2.3 (0.7) 0.961 2.4 (1.0) 1.9 (0.7) 0.003 0.001 
 ISR (pmol ⋅ m2 ⋅ h/L) 298 (104) 299 (100) 0.946 325 (131) 264 (95) 0.003 <0.001 
 IRG (pmol ⋅ h/L) 55 (7.1) 56 (11.0) 0.883 76 (8.1) 56 (4.6) <0.001 <0.001 
 eMCRI (L/m2 ⋅ h) 4.9 (1.6) 4.7 (1.3) 0.945 3.6 (1.4) 4.4 (1.7) 0.012 0.014 
 I/Glg molar ratio 21 (6.7) 21 (6.7) 0.971 16 (3.7) 18 (3.8) 0.016 0.041 
 FFA (mmol ⋅ h/L) 0.42 (0.12) 0.46 (0.16) 0.841 0.97 (0.22) 2.09 (0.4) <0.001 <0.001 
 Glycerol (mmol ⋅ h/L) 0.21 (0.04) 0.20 (0.03) 0.961 0.27 (0.07) 0.30 (0.06) <0.001 0.001 
 β-Hydroxybutyrate (mmol ⋅ h/L) 0.78 (0.29) 0.75 (0.20) 0.994 1.45 (0.45) 2.84 (0.51) <0.001 <0.001 
Study 2        
 PG (mg ⋅ h/dL) (AUC T0 h–T3 h252 (17) 256 (17) 0.762 316 (19) 274 (24) <0.001 <0.001 
 PG (mg ⋅ h/dL) (AUC T2.5 h–T3 h42.4 (2.5) 43.1 (2.8) 0.891 42.8 (3.1) 43.1 (2.5) 0.912 0.474 
 Insulin (pmol ⋅ h/mL) 389 (38) 392 (45) 0.763 428 (29) 443 (20) 0.524 0.571 
 GIR (mg/kg) 261 (22) 172 (27) 0.039 41 (37) 142 (59) <0.001 <0.001 
Control subjectsSubjects with T2D
a.m.p.m.Pa.m.p.m.PP#
Study 1        
 PG (mg ⋅ h/dL) 253 (16) 256 (16) 0.908 406 (31) 283 (34) <0.001 <0.001 
 Insulin (pmol ⋅ h/mL) 186 (25) 193 (29) 0.681 254 (17) 186 (29) <0.001 <0.001 
 C-peptide (nmol ⋅ h/L) 2.3 (0.8) 2.3 (0.7) 0.961 2.4 (1.0) 1.9 (0.7) 0.003 0.001 
 ISR (pmol ⋅ m2 ⋅ h/L) 298 (104) 299 (100) 0.946 325 (131) 264 (95) 0.003 <0.001 
 IRG (pmol ⋅ h/L) 55 (7.1) 56 (11.0) 0.883 76 (8.1) 56 (4.6) <0.001 <0.001 
 eMCRI (L/m2 ⋅ h) 4.9 (1.6) 4.7 (1.3) 0.945 3.6 (1.4) 4.4 (1.7) 0.012 0.014 
 I/Glg molar ratio 21 (6.7) 21 (6.7) 0.971 16 (3.7) 18 (3.8) 0.016 0.041 
 FFA (mmol ⋅ h/L) 0.42 (0.12) 0.46 (0.16) 0.841 0.97 (0.22) 2.09 (0.4) <0.001 <0.001 
 Glycerol (mmol ⋅ h/L) 0.21 (0.04) 0.20 (0.03) 0.961 0.27 (0.07) 0.30 (0.06) <0.001 0.001 
 β-Hydroxybutyrate (mmol ⋅ h/L) 0.78 (0.29) 0.75 (0.20) 0.994 1.45 (0.45) 2.84 (0.51) <0.001 <0.001 
Study 2        
 PG (mg ⋅ h/dL) (AUC T0 h–T3 h252 (17) 256 (17) 0.762 316 (19) 274 (24) <0.001 <0.001 
 PG (mg ⋅ h/dL) (AUC T2.5 h–T3 h42.4 (2.5) 43.1 (2.8) 0.891 42.8 (3.1) 43.1 (2.5) 0.912 0.474 
 Insulin (pmol ⋅ h/mL) 389 (38) 392 (45) 0.763 428 (29) 443 (20) 0.524 0.571 
 GIR (mg/kg) 261 (22) 172 (27) 0.039 41 (37) 142 (59) <0.001 <0.001 

Data are mean (SD). IRG, immunoreactive glucagon.

*

And as reported in Figs. 14.

#

From repeated-measures ANOVA (interaction term).

Serum insulin concentration was greater in subjects with T2D a.m. versus control subjects and decreased p.m. to values no longer different from control subjects at that time of day (Table 2). In control subjects, serum insulin was no different a.m. versus p.m. (Table 2 and Fig. 1). The a.m. plasma C-peptide in subjects with T2D was similar to that of control subjects (Table 2) and decreased significantly p.m., like insulin. The ISR in the subjects with T2D was greater a.m. than p.m. (Table 2), although it was not different from control subjects. The p.m. ISR in subjects with T2D was lower than in control subjects a.m. (P = 0.018) and p.m. (P = 0.008), respectively.

Plasma Glucagon, eMCRI, and Prehepatic I/Glg Molar Ratio

The a.m. plasma glucagon was higher in subjects with T2D versus control subjects but decreased p.m. to values no longer different from control subjects (Fig. 2 and Table 2). The eMCRI was lower a.m. than p.m. in the subjects with T2D and was also lower than that of control subjects (Table 2). The prehepatic I/Glg molar ratio a.m. was lower in subjects with T2D versus control subjects both a.m. and p.m. However, in T2D, the ratio increased p.m. versus a.m. (Table 2).

Figure 2

Plasma glucagon concentration, eMCRI, and prehepatic I/Glg molar ratios in control subjects and subjects with T2D in study 1 (monitoring) between 5:00 and 8:00 a.m. and p.m. Data are mean (SE). *P < 0.05 after repeated-measures ANOVA with Tukey-Kramer multiple comparison test.

Figure 2

Plasma glucagon concentration, eMCRI, and prehepatic I/Glg molar ratios in control subjects and subjects with T2D in study 1 (monitoring) between 5:00 and 8:00 a.m. and p.m. Data are mean (SE). *P < 0.05 after repeated-measures ANOVA with Tukey-Kramer multiple comparison test.

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FFAs, Blood Glycerol, and β-Hydroxybutyrate

In control subjects, serum FFAs, blood glycerol, and β-hydroxybutyrate did not differ a.m. versus p.m. In subjects with T2D, FFAs were higher versus control subjects both a.m. and p.m. (Fig. 3 and Table 2). However, in contrast to the pattern a.m. to p.m. of PG, FFAs were higher p.m. versus a.m. Blood glycerol and β-hydroxybutyrate, similar to FFAs, were higher in subjects with T2D versus control subjects both a.m. and p.m. (Table 2), and both were higher p.m. versus a.m.

Figure 3

Serum FFAs, blood glycerol, and β-hydroxybutyrate concentrations in control subjects and subjects with T2D in study 1 (monitoring) between 5:00 and 8:00 a.m. and p.m. Data are mean (SE). *P < 0.05 after repeated-measures ANOVA with Tukey-Kramer multiple comparison test.

Figure 3

Serum FFAs, blood glycerol, and β-hydroxybutyrate concentrations in control subjects and subjects with T2D in study 1 (monitoring) between 5:00 and 8:00 a.m. and p.m. Data are mean (SE). *P < 0.05 after repeated-measures ANOVA with Tukey-Kramer multiple comparison test.

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Study 2: Clamp Studies

PG, Serum Insulin Concentration, and GIRs

In control subjects, the mean PG concentration at T0 ha.m. was 84 (5.6) mg/dL and 86 (6) mg/dL at T0 hp.m. (Fig. 4 and Table 2). These values were superimposable to those of study 1, and PG was clamped at this target. Mean PG was 84 (5.6) mg/dL and 85.5 (5.8) mg/dL during the 3-h a.m. and p.m. study 2, respectively (isoglycemic clamp), with a coefficient of variation of PG a.m. and p.m. of <7%. In subjects with T2D, in the a.m. study, the mean PG at T0 ha.m. was 118 (16) mg/dL, and PG was allowed to decrease during insulin infusion until it reached the target of 85 mg/dL by 120 min and then clamped at 85 mg/dL. Mean PG was 85.5 (1.0) mg/dL over the last 30 min of the a.m. study (coefficient of variation 2%). In the p.m. study, subjects with T2D had a mean PG at T0 h of 93 (11) mg/dL, and then it was allowed to decrease to 85 mg/dL as in the a.m. study. Mean PG was 85.6 (4.8) mg/dL over the last 30 min of the p.m. study (coefficient of variation 5.6%).

Figure 4

PG and serum insulin concentrations and GIRs in control subjects and subjects with T2D in study 2 before and during infusion of insulin at a fixed rate (0.25 mU/kg/min) and glucose at a variable rate to maintain PG at 85 mg/dL between 5:00 and 8:00 a.m. and p.m. Data are mean (SE). *P < 0.05 after repeated-measures ANOVA with Tukey-Kramer multiple comparison test.

Figure 4

PG and serum insulin concentrations and GIRs in control subjects and subjects with T2D in study 2 before and during infusion of insulin at a fixed rate (0.25 mU/kg/min) and glucose at a variable rate to maintain PG at 85 mg/dL between 5:00 and 8:00 a.m. and p.m. Data are mean (SE). *P < 0.05 after repeated-measures ANOVA with Tukey-Kramer multiple comparison test.

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Serum insulin concentrations increased to similar values in the a.m. and p.m. clamp studies in control subjects and subjects with T2D (Table 2). GIR increased more slowly and to lower values by the end of studies in subjects with T2D versus control subjects both a.m. and p.m. However, whereas GIR was higher a.m. versus p.m. in control subjects, subjects with T2D, GIR had an opposite pattern, with lower values a.m. versus p.m. (Table 2).

Glucose and Glycerol Turnover

Mean EGP was higher a.m. in subjects with T2D versus control subjects at T0 h (2.2 [0.2] vs. 1.8 [0.12] mg/kg/min, P = 0.001, respectively), despite higher serum insulin, and was less suppressed by hyperinsulinemia at the end of the clamp. However, EGP decreased p.m. in subjects with T2D, despite lower serum insulin at T0 h, and was more suppressed during the hyperinsulinemic clamp versus a.m. (Fig. 5 and Table 3). This pattern was not evident in control subjects in whom EGP instead tended to be more suppressed a.m. versus p.m. Glucose utilization increased in response to hyperinsulinemia in control subjects a.m., but not p.m. In subjects with T2D, glucose utilization did not increase in response to insulin either a.m. or p.m. (Table 3). Similarly, glucose clearance increased significantly only in control subjects a.m. versus p.m. (Table 3).

Figure 5

Rates of EGP and glycerol Ra in control subjects and subjects with T2D in study 2 before (T0 h) and at the end of the clamp (T3 h, which is the mean 150–180-min value) during infusion of insulin (0.25 mU/kg/min) between 5:00 and 8:00 a.m. and p.m. Data are mean (SE). *P < 0.05 after repeated-measures ANOVA with Tukey-Kramer multiple comparison test.

Figure 5

Rates of EGP and glycerol Ra in control subjects and subjects with T2D in study 2 before (T0 h) and at the end of the clamp (T3 h, which is the mean 150–180-min value) during infusion of insulin (0.25 mU/kg/min) between 5:00 and 8:00 a.m. and p.m. Data are mean (SE). *P < 0.05 after repeated-measures ANOVA with Tukey-Kramer multiple comparison test.

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Table 3

Hormonal and metabolic parameters of carbohydrate, lipid, and protein metabolism in study 2 (clamp) at baseline (T0 h) and over the last 30 min of clamp (T3 h) a.m. and p.m. in control subjects and subjects with T2D

Control subjectsSubjects with T2D
a.m.p.m.Pa.m.p.m.PP#
Glucagon (pmol/L)        
 T0 h 18.3 (3.3) 18.9 (3.6)  25.4 (4.7) 22.3 (3.3)   
 T3 h 16.6 (2.6) 17.2 (2.3)  22.8 (4.5) 19 (3.1)   
 Mean difference (T3 h – T0 h−1.7 (1.4) −1.7 (1.5) 0.994 −2.7 (1.2) −3.3 (2.4) 0.838 0.609 
C-peptide (nmol/L)        
 T0 h 0.79 (0.16) 0.83 (0.13  0.74 (0.43) 0.57 (0.40)   
 T3 h 0.46 (0.12) 0.51 (0.13  0.38 (0.31) 0.38 (0.35)   
 Mean difference (T3 h – T0 h−0.33 (0.08) −0.32 (0.12) 0.987 −0.36 (0.15) −0.19 (0.08) <0.001 <0.001 
ISR (pmol/min/m2       
 T0 h 101 (20.1) 106 (17.4)  102 (45.8) 85 (69.8)   
 T3 h 76 (15.2) 81 (13.2)  62 (20.4) 64 (18.0)   
 Mean difference (T3 h – T0 h−25.2 (5.1) −25.5 (4.2) 0.996 −39.9 (26.3) −20.8 (61.0) 0.245 0.368 
EGP (mg/kg/min)        
 T0 h 1.81 (0.12) 1.88 (0.11)  2.22 (0.20) 1.92 (0.14)   
 T3 h 0.14 (0.15) 0.54 (0.37)  1.42 (0.42) 0.56 (0.43)   
 Mean difference (T3 h – T0 h−1.66 (0.19) −1.35 (0.34) 0.091 −0.79 (0.43) −1.36 (0.48) 0.001 <0.001 
Glucose utilization (mg/kg/min)        
 T0 h 1.81 (0.11) 1.88 (0.11)  2.22 (0.17) 1.95 (0.14)   
 T3 h 2.37 (0.19) 2.02 (0.37)  2.03 (0.17) 1.94 (0.25)   
 Mean difference (T3 h – T0 h0.56 (0.27) 0.14 (0.38) 0.008 −0.20 (0.27) −0.01 (0.21) 0.391 0.001 
Glucose clearance (ml/kg/min)        
 T0 h 2.16 (0.19) 2.19 (0.17)  1.90 (0.14) 2.11 (0.12)   
 T3 h 2.81 (0.27) 2.38 (0.46)  2.37 (0.29) 2.27 (0.17)   
 Mean difference (T3 h – T0 h0.65 (0.33) 0.19 (0.45) 0.005 0.47 (0.29) 0.17 (0.37) 0.116 0.305 
FFA (mmol/L)        
 T0 h 0.143 (0.037) 0.166 (0.034)  0.399 (0.143) 0.830 (0.181)   
 T3 h 0.070 (0.022) 0.076 (0.018)  0.175 (0.045) 0.145 (0.030)   
 Mean difference (T3 h – T0 h−0.073 (0.018) −0.091 (0.019) 0.942 −0.223 (0.123) −0.685 (0.161) <0.001 <0.001 
β-Hydroxybutyrate (mmol/L)        
 T0 h 0.263 (0.077) 0.272 (0.060)  0.396 (0.282) 0.888 (0.240)   
 T3 h 0.141 (0.028) 0.145 (0.034)  0.202 (0.060) 0.149 (0.081)   
 Mean difference (T3 h – T0 h−0.122 (0.068) −0.127 (0.061) 0.981 −0.194 (0.268) −0.742 (0.234) <0.001 <0.001 
Glycerol (mmol/L)        
 T0 h 0.070 (0.007) 0.072 (0.008)  0.092 (0.015) 0.102 (0.014)   
 T3 h 0.034 (0.005) 0.036 (0.004)  0.057 (0.010) 0.044 (0.007)   
 Mean difference (T3 h – T0 h−0.036 (0.003) −0.036 (0.004) 0. 972 −0.035 (0.005) −0.058 (0.009) <0.001 <0.001 
Glycerol Ra (μmol/kg/min)        
 T0 h 1.51 (0.2) 1.53 (0.2)  2.38 (0.3) 2.73 (0.3)   
 T3 h 0.54 (0.1) 0.55 (0.1)  1.82 (0.2) 1.22 (0.1)   
 Mean difference (T3 h – T0 h−0.96 (0.12) −0.98 (0.12) 0.944 −0.56 (0.08) −1.51 (0.17) <0.001 <0.001 
Leucine Ra (μmol/kg/min)        
 T0 h 1.59 (0.13) 1.60 (0.16)  1.58 (0.17) 1.58 (0.13)   
 T3 h 1.32 (0.11) 1.2 (0.14)  1.33 (0.18) 1.32 (0.12)   
 Mean difference (T3 h – T0 h−0.27 (0.15) −0.39 (0.13) 0.968 −0.24 (0.18) −0.26 (0.13) 0.971 0.906 
Control subjectsSubjects with T2D
a.m.p.m.Pa.m.p.m.PP#
Glucagon (pmol/L)        
 T0 h 18.3 (3.3) 18.9 (3.6)  25.4 (4.7) 22.3 (3.3)   
 T3 h 16.6 (2.6) 17.2 (2.3)  22.8 (4.5) 19 (3.1)   
 Mean difference (T3 h – T0 h−1.7 (1.4) −1.7 (1.5) 0.994 −2.7 (1.2) −3.3 (2.4) 0.838 0.609 
C-peptide (nmol/L)        
 T0 h 0.79 (0.16) 0.83 (0.13  0.74 (0.43) 0.57 (0.40)   
 T3 h 0.46 (0.12) 0.51 (0.13  0.38 (0.31) 0.38 (0.35)   
 Mean difference (T3 h – T0 h−0.33 (0.08) −0.32 (0.12) 0.987 −0.36 (0.15) −0.19 (0.08) <0.001 <0.001 
ISR (pmol/min/m2       
 T0 h 101 (20.1) 106 (17.4)  102 (45.8) 85 (69.8)   
 T3 h 76 (15.2) 81 (13.2)  62 (20.4) 64 (18.0)   
 Mean difference (T3 h – T0 h−25.2 (5.1) −25.5 (4.2) 0.996 −39.9 (26.3) −20.8 (61.0) 0.245 0.368 
EGP (mg/kg/min)        
 T0 h 1.81 (0.12) 1.88 (0.11)  2.22 (0.20) 1.92 (0.14)   
 T3 h 0.14 (0.15) 0.54 (0.37)  1.42 (0.42) 0.56 (0.43)   
 Mean difference (T3 h – T0 h−1.66 (0.19) −1.35 (0.34) 0.091 −0.79 (0.43) −1.36 (0.48) 0.001 <0.001 
Glucose utilization (mg/kg/min)        
 T0 h 1.81 (0.11) 1.88 (0.11)  2.22 (0.17) 1.95 (0.14)   
 T3 h 2.37 (0.19) 2.02 (0.37)  2.03 (0.17) 1.94 (0.25)   
 Mean difference (T3 h – T0 h0.56 (0.27) 0.14 (0.38) 0.008 −0.20 (0.27) −0.01 (0.21) 0.391 0.001 
Glucose clearance (ml/kg/min)        
 T0 h 2.16 (0.19) 2.19 (0.17)  1.90 (0.14) 2.11 (0.12)   
 T3 h 2.81 (0.27) 2.38 (0.46)  2.37 (0.29) 2.27 (0.17)   
 Mean difference (T3 h – T0 h0.65 (0.33) 0.19 (0.45) 0.005 0.47 (0.29) 0.17 (0.37) 0.116 0.305 
FFA (mmol/L)        
 T0 h 0.143 (0.037) 0.166 (0.034)  0.399 (0.143) 0.830 (0.181)   
 T3 h 0.070 (0.022) 0.076 (0.018)  0.175 (0.045) 0.145 (0.030)   
 Mean difference (T3 h – T0 h−0.073 (0.018) −0.091 (0.019) 0.942 −0.223 (0.123) −0.685 (0.161) <0.001 <0.001 
β-Hydroxybutyrate (mmol/L)        
 T0 h 0.263 (0.077) 0.272 (0.060)  0.396 (0.282) 0.888 (0.240)   
 T3 h 0.141 (0.028) 0.145 (0.034)  0.202 (0.060) 0.149 (0.081)   
 Mean difference (T3 h – T0 h−0.122 (0.068) −0.127 (0.061) 0.981 −0.194 (0.268) −0.742 (0.234) <0.001 <0.001 
Glycerol (mmol/L)        
 T0 h 0.070 (0.007) 0.072 (0.008)  0.092 (0.015) 0.102 (0.014)   
 T3 h 0.034 (0.005) 0.036 (0.004)  0.057 (0.010) 0.044 (0.007)   
 Mean difference (T3 h – T0 h−0.036 (0.003) −0.036 (0.004) 0. 972 −0.035 (0.005) −0.058 (0.009) <0.001 <0.001 
Glycerol Ra (μmol/kg/min)        
 T0 h 1.51 (0.2) 1.53 (0.2)  2.38 (0.3) 2.73 (0.3)   
 T3 h 0.54 (0.1) 0.55 (0.1)  1.82 (0.2) 1.22 (0.1)   
 Mean difference (T3 h – T0 h−0.96 (0.12) −0.98 (0.12) 0.944 −0.56 (0.08) −1.51 (0.17) <0.001 <0.001 
Leucine Ra (μmol/kg/min)        
 T0 h 1.59 (0.13) 1.60 (0.16)  1.58 (0.17) 1.58 (0.13)   
 T3 h 1.32 (0.11) 1.2 (0.14)  1.33 (0.18) 1.32 (0.12)   
 Mean difference (T3 h – T0 h−0.27 (0.15) −0.39 (0.13) 0.968 −0.24 (0.18) −0.26 (0.13) 0.971 0.906 

Data are mean (SD).

#

From repeated-measures ANOVA (interaction term).

Glycerol Ra in control subjects was similar at T0 ha.m. and p.m. and was similarly suppressed by hyperinsulinemia at the end of clamp. In subjects with T2D, glycerol Ra was higher at T0 h and was less suppressed by hyperinsulinemia versus control subjects both a.m. and p.m. However, the suppression of Ra in subjects with T2D was greater p.m. versus a.m. (Table 3).

Plasma Glucagon, C-Peptide, ISR, and Lipid Metabolism

Plasma glucagon was higher a.m. at T0 h in subjects with T2D versus control subjects. Glucagon suppression to clamped hyperinsulinemia was similar in control subjects and subjects with T2D (Table 3). C-peptide values and ISR in control subjects and subjects with T2D were similar at T0 ha.m. and were similarly suppressed by hyperinsulinemia at T3 h, but more so in a.m. in subjects with T2DM, likely because PG was decreased at the same time compared with values at T0 h.

Serum FFAs, blood β-hydroxybutyrate, and glycerol at T0 h of study 2 reproduced the pattern already observed in study 1. In response to hyperinsulinemia, FFAs, β-hydroxybutyrate, and glycerol were similarly suppressed a.m. versus p.m. in control subjects. In subjects with T2D, the values at the end of clamp remained greater than those of control subjects. Leucine turnover was similar in subjects with T2D and control subjects, with no differences between a.m. and p.m. (Table 3).

Indices of β-Cell Function and Insulin Resistance

β-Cell function (explored in the fasting state as ratio ISR/PG) was impaired in subjects with T2D compared with control subjects both a.m. and p.m. and more so a.m. versus p.m. (Supplementary Table 1). Subjects with T2D were insulin resistant compared with control subjects a.m. but not p.m. Insulin resistance was present at the liver and adipose tissue levels a.m. and only at the adipose tissue level p.m. (Supplementary Table 1).

The current study demonstrates deviations of the diurnal cycling of glucose and lipid homeostasis in people with T2D compared with individuals without diabetes. In people with T2D, fasting hyperglycemia is greater a.m. versus p.m. and is paralleled by lower insulin sensitivity for glucose and lipid but not protein metabolism. This pattern is the reverse of the physiological diurnal cycle of people without diabetes, who are more insulin sensitive a.m. versus p.m. These new findings have been observed in the current study in people without obesity but with recent-onset T2D, with good glycemic control, and in the absence of confounding pharmacological treatment. It is likely that they represent a specific marker of T2D that is possibly present even in prediabetes before biochemical and clinical manifestations.

Reduced insulin sensitivity for glucose metabolism a.m. versus p.m. in people with T2D was suggested by observational study 1 and then confirmed by the hyperinsulinemic-isoglycemic clamp of study 2. However, the interpretation of lipid metabolism is less straightforward than glucose metabolism given the lower FFAs observed a.m. versus p.m. (Fig. 3), a result opposite to that observed for PG. The higher serum insulin concentrations a.m., likely driven by higher PG (Fig. 1), might have suppressed FFAs more a.m. versus p.m., given the greater insulin sensitivity of adipose tissue versus liver and muscle glucose metabolism (3336). In fact, when serum insulin concentrations were made comparable a.m. and p.m. in the clamp study 2, lipolysis was less suppressed a.m. versus p.m. (glycerol Ra) (Table 3). Thus, we conclude that in T2D, the inversion of physiological diurnal cycling of insulin sensitivity involves both glucose and lipid metabolism, but not protein metabolism (Table 3). The latter finding is in line with previous observations made in the morning hours (37,38).

Comments

The higher serum insulin in T2D a.m. versus p.m. are expected to be the result of a higher ISR likely driven by higher PG. However, C-peptide values and ISR a.m. were still inappropriately low for the prevailing fasting PG (Table 3), suggesting impaired β-cell function. However, the lower eMCRI observed in T2D a.m. versus p.m. might likely contribute to the higher serum insulin concentrations a.m. A lower eMCRI has long been reported in T2D (39) and interpreted as a compensatory mechanism contributing to hyperinsulinemia to counteract insulin resistance and mitigate hyperglycemia (40).

The current study explored insulin sensitivity primarily at the liver and adipose tissue levels with an hyperinsulinemic clamp at a low rate of insulin infusion (10 mU/m2/min) known to produce serum insulin concentrations close to the estimated insulin concentration which produces a half-maximal effect (EC50) for suppression of EGP (23,33,34) and lipolysis (3336). The current study did not establish the insulin-mediated glucose uptake (primarily muscle), which is stimulated at plasma insulin concentrations in the range of 400–600 pmol/L of the postprandial state (23,34). However, even with the modest increase of serum insulin to ∼130–150 pmol/L of the present study 2, there was an increase in glucose utilization and clearance a.m. in control subjects, but not in the subjects with T2D (Table 3), indirectly suggesting muscle insulin resistance. This would explain the typically greater hyperglycemia postbreakfast versus the afternoon in T2D (20,41).

In T2D, the greater insulin resistance a.m. was associated with increased glucagon and lower I/Glg molar ratio, a key factor in the regulation of EGP (30,42). Hyperglucagonemia a.m. occurred despite greater intraislet ISR and hyperinsulinemia by exogenous insulin (Table 3). Conversely, the decrease of glucagon p.m., with increased I/Glg molar ratio, contributed to the lower EGP p.m. versus a.m.

Although in the current study the diurnal cycling of insulin sensitivity exhibited an a.m. nadir common to glucose and lipid metabolism in T2D, there were differences in keeping with physiology of hierarchical responses to insulin (42). In healthy subjects, the EC50 of insulin antilipolytic effects (estimated from suppression of FFA turnover or glycerol Ra) lies between 51 and 106 pmol/L (3335), similarly to EC50 for suppression of circulating FFAs (91 pmol/L) (36). Previous studies in T2D have shown that the insulin EC50 for suppression of FFAs is increased versus control subjects (104 vs. 62 pmol/L, respectively), but it remains lower than the insulin EC50 for suppression of EGP (180 and 97 pmol/L in subjects with T2D and control subjects, respectively) (34), suggesting that the physiological difference between glucose and lipid metabolism is maintained also in the insulin resistance state of T2D (34). In the present investigation, a full dose-response study was not done. However, it is tempting to speculate on the patterns shown in Supplementary Fig. 2, where the dose-response curves were obtained from the fitting of data of study 2. In control subjects, there is a right shift of EC50p.m. versus a.m. (both for EGP and glycerol Ra), suggesting lower sensitivity p.m. versus a.m. On the contrary, in subjects with T2D, the EC50 values were right-shifted versus those of control subjects, indicating insulin resistance, and were higher a.m. versus p.m., suggesting that insulin resistance is greater a.m. (Supplementary Fig. 2).

Interpretation of the Findings and Relevance to T2D

The dysregulation of the diurnal cycling of insulin sensitivity in T2D has long been recognized, but the mechanisms have not been established. A role for the morning increase in cortisol has been suggested (17). Radziuk and Pye (22) postulated a dysfunction of the suprachiasmatic nucleus leading to a dysregulation of neural control of whole-glucose metabolism and found a correlation with melatonin levels. Increased activity of the autonomic sympathetic nervous system might contribute, but neural activity appears reduced, not increased, a.m. (22). In type 1 diabetes, a role for nocturnal surges of growth hormone has been proposed (43). Regardless of the mechanisms, the findings of the current study of a.m. versus p.m. difference in hepatic and lipid insulin sensitivity may be relevant to the interpretation of metabolic data in clinical and experimental research at different times of the day. To avoid the confounder of a.m. versus p.m. cycling of insulin sensitivity, different people or the same people examined on different days should be studied always at the same time. Also, when two experiments are repeated in sequence on the same day with a time interval of hours and compared with each other, such as the glucose loads to investigate the Staub-Traugott effect (44), the possible role of variable hepatic insulin sensitivity between the two tests should be considered.

The current study emphasizes the early defect of T2D, i.e., reduced insulin sensitivity a.m. with exaggerated EGP (primarily gluconeogenesis [21,42]). This, in combination with reduced β-cell responsiveness, results in day-long hyperglycemia, and supports the need for timely therapeutic intervention. Metformin (21,45) given at night, long-acting insulin analogs (basal insulin) (4648), and long-acting glucagon-like peptide 1 receptor agonists (49), which do not induce hypoglycemia p.m. when insulin sensitivity increases, along with a low-carbohydrate load a.m., appear to be the most appropriate treatment for this goal.

Study Limitations

The study examined a small number of individuals of the large and heterogeneous population with T2D (50). Insulin sensitivity was examined primarily at the liver and adipose tissue levels, not at the muscle level. β-Cell function has been studied only in the fasting steady-state condition and not in response to dynamic stimuli (oral and/or intravenous glucose or meal load). Additional studies are required to fill these gaps. Finally, the sequence of study 1 and study 2 was not randomized, but such an approach was pivotal to successfully clamp PG in study 2 in subjects with T2D at values matched for those of control subjects (isoglycemic clamp).

Conclusion

The inversion of the diurnal cycling of both glucose and lipid metabolism in recent-onset T2D represents an early metabolic marker of the disease. The finding calls for timely therapeutic intervention of the disease mechanism to counteract the excess lipolysis and EGP a.m. while reducing muscle IR. Care should be taken in using glucose-lowering drugs not to increase the risk of hypoglycemia p.m. when insulin sensitivity increases.

This article contains supplementary material online at https://doi.org/10.2337/figshare.23646849.

Acknowledgments. The authors are deeply grateful to the subjects who volunteered for the study. The experiments of this study were done by Dr. Walkyria Pimenta, fellow from the Department of Clinical Medicine Faculdade de Medicina Botucatu, University of São Paulo State, São Paulo, Brazil. The authors also thank Dr. Cristina Cordoni and Dr. Mauro Lepore, Univeristy of Perugia, Italy, for helpful contributions to the clamp studies.

Funding. This study was supported by research funds provided by the University of Perugia.

Duality of Interest. G.P. has received research grants from GlaxoSmithKline and Novo Nordisk. F.P. has received honoraria for lectures and consultations from AstraZeneca, Sanofi, Eli Lilly, Mundipharma, and Medtronic. G.B.B. has received honoraria for lectures and consultations from Sanofi and Menarini. C.G.F. has received honoraria for lecturing and consultations from Sanofi and travel grants from Menarini. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. P.L. enrolled patients, collaborated in performing clamps, analyzed data, and reviewed and edited the manuscript. G.P. and S.P. collaborated in the study design and in performing clamps, analyzed data, and reviewed and edited the manuscript. F.P. collaborated in the study design and in performing clamps, analyzed data, contributed to the interpretation of results, and reviewed and edited the manuscript. M.D.F. contributed to the interpretation of results and the final text of the manuscript. A.T. calculated the ISR and eMCRI, contributed to the interpretation of results, and reviewed and edited the manuscript. G.B.B. provided the study concept and design, supervised the protocol development and the research, and wrote the manuscript. C.G.F. analyzed data, performed the statistical analysis, and wrote the manuscript. C.G.F. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in an abstract poster presentation at the 58th Scientific Sessions of the American Diabetes Association, Chicago, IL, 13–16 June 1998; 35th Annual Meeting of the European Association for the Study of Diabetes, Brussels, Belgium, 28 September–2 October 1999; 60th Scientific Sessions of the American Diabetes Association, San Antonio, TX, 10–13 June 2000; and the 61st Scientific Sessions of the American Diabetes Association, Philadelphia, PA, 22–26 June 2001.

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