Abstract
Context
A better understanding of nocturnal regulation of glucose homeostasis will provide the framework for designing rational therapeutic strategies to improve the management of overnight glucose in patients with type 2 diabetes (T2D).
Objective
To establish the nocturnal pattern and regulation of glucose production (EGP) in humans and to determine whether the pattern is dysregulated in people with T2D.
Design
Subjects were infused with [3-3H] glucose overnight. Arterial blood samples were drawn for hormones and analytes to estimate EGP throughout the night. Deuterium-labeled water was provided to measure gluconeogenesis (GNG) using the hexamethylenetetramine method of Landau.
Setting
Mayo Clinic Clinical Research Trials Unit, Rochester, MN, USA.
Participants and Interventions
A total of 43 subjects [23 subjects with T2D and 20 nondiabetic (ND) subjects comparable for age and body mass index] were included in this study.
Main Outcome(s) Measure(s)
Glucose and EGP.
Results
Plasma glucose, C-peptide, and glucagon concentrations were higher throughout the night, whereas insulin concentrations were higher in subjects with T2D vs ND subjects at 1:00 and 4:00 am but similar at 7:00 am. EGP was higher in the subjects with T2D than in the ND subjects throughout the night (P < 0.001). Glycogenolysis (GGL) fell and GNG rose, resulting in significantly higher (P < 0.001) rates of GNG at 4:00 and 7:00 am and significantly (P < 0.001) higher rates of GGL at 1:00, 4:00, and 7:00 am in T2D as compared with ND.
Conclusions
These data imply that optimal therapies for T2D for nocturnal/fasting glucose control should target not only the absolute rates of EGP but also the contributing pathways of GGL and GNG sequentially.
Nocturnal glucose production was measured using state-of-the-art techniques to understand overnight glucose control in T2D. Drugs targeting late-night GNG may optimize morning glucose control in T2D.
Individuals with type 2 diabetes (T2D) have fasting hyperglycemia because of increased rates of endogenous glucose production (EGP) measured in the morning (1–3) attributable to increased rates of gluconeogenesis (GNG) and glycogenolysis (GGL) (2, 3). However, it is currently unknown if the higher glucose concentrations observed in the morning in individuals with T2D are a result of glucose levels rising throughout the night or rising only during the latter part of the night. It is unknown if the so-called “dawn phenomenon” frequently observed in type 1 diabetes occurs in T2D. Because studies have hinted at the critical role of circadian rhythm and the crosstalk between clock and metabolic transcription networks in glucose homeostasis (4–7), we studied nocturnal pattern and regulation of glucose production and to ascertain whether the pattern is dysregulated in T2D. A better understanding of nocturnal regulation of glucose homeostasis will provide the framework for future studies to design rational therapeutic strategies aimed at restoring overnight glucose turnover and therefore glucose concentrations to normal. Prior studies have established that fasting hyperglycemia in T2D in the morning is due to increased rates of EGP (2, 7, 8) measured with the isotope dilution technique (9). Elegant studies conducted by Chen et al. (10) in a small cohort of healthy subjects (n = 6) and subjects with T2D (n = 8) indicated that glucose disposal rates were higher overnight in those with T2D. However, they did not quantify GNG or GGL in the overnight period, and subjects in the healthy cohort were leaner, which may have confounded data interpretation. Magnusson et al. (11) quantified net hepatic GGL using 13C nuclear magnetic resonance (NMR) to show that the lower glycogen content observed in the diabetic liver indirectly accounted for the higher GNG and could explain the higher EGP observed in T2D.
The purpose of our study was to determine if the temporal pattern of nocturnal glucose turnover differed in subjects with T2D compared with nondiabetic (ND) subjects. We are not aware of prior studies where a cohort of comparable healthy subjects and individuals with T2D were studied to quantify overnight glucose production. In addition, we sought to determine if there were temporal differences in the contribution of GNG and GGL to nocturnal EGP in people with T2D when compared with ND subjects. To do so, we concomitantly used the isotope dilution technique and the deuterated water method of Landau (12) to measure nocturnal EGP and the contribution of GNG and GGL. We report that rates of EGP were higher throughout the night in subjects with T2D than in ND subjects. In addition, whereas the contribution of GGL and GNG to EGP remained essentially constant throughout the night in the ND subjects, the contribution of GGL to EGP progressively fell and the contribution of GNG progressively rose during the night in subjects with T2D, indicating a dynamic and altered regulation of these processes.
Materials and Methods
Study design
Subjects reported to the Mayo Clinic Clinical Research Trials Unit (CRTU) for a screening visit the morning after an overnight fast. Subjects provided written informed consent. A history and physical examination were performed, and the subject’s height and weight were recorded. A dietary history was obtained by a dietician to ensure adherence to a weight-maintaining diet between screening and study visits. Body composition was measured by Lunar iDXA software v. 15.0 (GE Health Care Technologies, Chicago, IL). Subjects were screened for sleep-related disorders using the Sleep Questionnaire. At the time of the screening, 14 subjects with T2D were on metformin, seven were on combination of metformin and sulfonylurea, and two were on sulfonylurea only. Subjects with T2D were asked to stop using metformin and/or sulfonylureas for 2 weeks prior to the study visit and to monitor their finger stick glucose values regularly during this period. If three successive finger stick glucose values exceeded 300 mg/dL, subjects were asked to resume their medication and were withdrawn from the study. Subjects were asked to refrain from unaccustomed physical exercise and to maintain their weight within 2% between screening and study visits.
Subjects who met the enrollment criteria were admitted to the CRTU at 4:00 pm and were provided a standard 10 kcal/kg meal (50% carbohydrate, 20% protein, 30% fat) at 5:00 pm. In the evening, a pulse oximeter was placed to track oxygen saturation levels throughout the night to rule out sleep apnea. The purpose of this first night was to acclimatize the subject to the CRTU environment and to minimize sleep disruptions during the study period on the second night.
Subjects were awakened at 6:00 am the next day and received three standardized caffeine-free meals (50% carbohydrate, 20% protein, and 30% fat) at 7:00 am and at 12:00 and 5:00 pm prepared by the CRTU metabolic kitchen. Each meal consisted of 33% of the subject’s total estimated calorie intake based on the Harris Benedict + 20% calorie requirements. Physical activity was monitored by an accelerometer, and subjects were asked to walk on a treadmill periodically at a predetermined slow speed to mimic the activities of daily living (13–15). The subjects remained fasting after the evening meal until the end of the study. Subjects were provided deuterated water (2H2O; 5 g/kg total body water), which was 55% and 45% of body weight in men and women, respectively, at 6:00, 8:00, and 10:00 pm in split doses (∼1.7 g/kg total body water) for estimation of GNG as previously described (2). All water consumed overnight contained 2H2O to maintain steady state.
At 8:00 pm, an arterial line was inserted by respiratory therapists (under local anesthesia and aseptic precautions) into a radial artery to obtain arterial samples periodically throughout the night. Arterial catheterizations minimized sleep disruptions and discomfort associated with the retrograde intravenous hand vein technique. Arterial catheters could not be placed in four subjects in whom we inserted a retrograde intravenous catheter into a hand vein for blood draws and kept heated within plexiglass box (maintained at 55°C) to enable drawing of arterialized venous blood (2). An intravenous cannula was inserted into a vein on the contralateral forearm for tracer infusion. In the evening, a pulse oximeter was placed as on day 1. Primed continuous infusion of [3-3H] glucose (0.06 µCi/min), with priming dose based on the fasting glucose, was started at 10:00 pm and continued until the end of the study to measure glucose turnover. Similarly, [6,6-2H2] glucose was started at 1:00 am and [1-13C] glucose was started at 4:00 am and continued until the end of the study as part of separate experiments to estimate tracer recycling.
At 7:00 am the following morning, all infusions and blood draws were stopped, and all cannulae were removed. The subjects were provided breakfast and discharged from the CRTU with instructions on care for the arterial and venipuncture sites.
Analytical techniques
Plasma samples were placed on ice, centrifuged at 4°C, separated, and stored at −20°C until assay. Plasma glucose concentrations were measured using a glucose oxidase method (YSI, Inc., Yellow Springs, OH). Plasma [3-3H] glucose specific activity was measured by liquid scintillation (16). Levels of [5-2H] glucose and [2-2H] glucose were estimated by the hexamethylenetetramine (HMT) method (2, 12). Insulin was analyzed using the DxI 800 (Beckman Instruments, Chaska, MN). C-peptide and glucagon were analyzed using a radioimmunoassay (Roche Diagnostics, Indianapolis, IN and Millipore Corporation, Billerica, MA, respectively). Cortisol was measured with the competitive binding immunoenzymatic assay on the DxI 800 (Beckman Instruments).
Methods
After approval by the Radioactive Drug Research Committee and FDA for use of radioisotope and the Institutional Review Board, 20 ND individuals and 23 individuals with T2D provided written informed consent to participate in the study. ND subjects were healthy with normal fasting glucose and normal HbA1c and did not have a history of diabetes in first-degree family members. Subjects with T2D did not have microvascular or macrovascular complications apart from mild background retinopathy and had an HbA1c <9%.
All subjects had normal kidney (creatinine ≤1.4 mg/dL in women and ≤1.5 mg/dL in men) and liver function, and none of the subjects had evidence of cardiac, renal, pulmonary, or hepatic disorders. Subjects on medications that could affect glucose metabolism (e.g., corticosteroids, tricyclic-antidepressants, benzodiazepines, and opiates) or with a history of cerebrovascular disease, anemia, alcoholism, or substance abuse were excluded. All subjects were required to be at a stable weight for minimum 2 weeks prior to the study.
Calculations
Rates of EGP were estimated throughout the night, but emphasis was on 1:00, 4:00, and 7:00 am (9). Because the tracer to tracee ratio was constant (Supplemental Fig. 1), steady-state equations were applied. The rates of EGP from 12:00 to 7:00 am were measured with [3-3H] glucose. GNG rate was calculated by multiplying the plasma C5 glucose to C2 glucose ratio by EGP. GGL rate was calculated by subtracting the rate of GNG from the rate of EGP (3).
Statistics
Data are described as means and SD or frequency and percentage for sample characteristics unless otherwise indicated. EGP was estimated as the mean of the sample obtained 10 minutes prior and at the top of the hour at 1:00, 4:00, and 7:00 am and is expressed per kilogram fat free mass per minute. A repeated measures model was estimated using a mixed model framework to allow for missing observations (one observation at 4:00 am, two observations at 7:00 am) at the time of sample collection. The mixed model included fixed effects for study group (ND vs T2D), tracer, and their interaction. EGP was calculated at 1:00, 4:00, and 7:00 am using [3-3H] glucose specific activity. Post hoc comparisons of the means within a group were computed.
To test for differences in profiles of hormones overnight, a linear mixed model framework was used. Although the hormones were measured more frequently during the night, these analyses were focused on the primary assessment times of 10:00 pm and 1:00, 4:00, and 7:00 am.
The P values reported for comparisons of means are two-sided and have not been inflated to reflect correction for multiple testing. Data analysis was conducted using the SAS System version 9.4 (SAS, Cary, NC).
Results
Subject characteristics are presented in Table 1. Twenty ND subjects [12 men; age, 56 ± 11 years; body mass index (BMI), 31 ± 3 kg/m2] and 23 subjects with T2D (11 men; age, 53 ± 11 years; BMI, 33 ± 4 kg/m2; HbA1c, 7.9 ± 1.0%; duration of T2D, 9.3 ± 4.7 years) completed the study.
Table 1.
Baseline Characteristics of the Subjects
|
ND (n = 20)
|
T2D (n = 23)
|
P Value | |||
|---|---|---|---|---|---|
| Mean | SD | Mean | SD | ||
| Age, y | 55.9 | 10.7 | 52.6 | 11.4 | 0.35 |
| Sex, M/F | 12/8 | 11/12 | – | ||
| Diabetes duration, y | – | – | 9.3 | 4.7 | – |
| Weight, kg | 92.7 | 10.0 | 95.8 | 11.9 | 0.36 |
| BMI, kg/m2 | 31.0 | 2.6 | 32.6 | 4.0 | 0.12 |
| Fat free mass, kg | 55.7 | 8.6 | 56.9 | 10.2 | 0.69 |
| Body fat, % | 40.5 | 6.0 | 41.2 | 7.6 | 0.74 |
| Fasting plasma glucose, mM | 4.9 | 0.4 | 9.5 | 2.4 | <0.001 |
| HbA1c, %/mmol/mol | 5.4/36.0 | 0.6/8.0 | 7.9/62.6 | 1.0/10.8 | <0.001 |
| Hemoglobin, g/dL | 14.4 | 1.1 | 14.2 | 1.1 | 0.54 |
| Resting energy expenditure, kcal/d | 1594.4 | 238.3 | 1760.1 | 246.1 | 0.03 |
Concentrations of plasma glucose, insulin, C-peptide, glucagon, and cortisol
Plasma glucose concentrations were higher (P < 0.0001) in the subjects with T2D (∼12 mM) at each time point than in the ND subjects (∼5 mM) during the night (Fig. 1). Plasma insulin concentrations, however, showed differences in the profiles overnight (P = 0.0041 for the group by sampling period interaction). Insulin concentrations were higher (P < 0.0001) in the subjects with T2D than in the ND subjects during the initial part of night and decreased to the point that the concentrations were no longer statistically significant at 7:00 am (P = 0.45). Insulin concentrations in the ND subjects did not change overnight.
Figure 1.
Mean (SD) concentrations of plasma (A) glucose, (B) insulin, (C) c-peptide, and (D) glucagon between 10:00 pm and 7:00 am observed for ND subjects and subjects with T2D.
Plasma C-peptide concentrations followed the pattern observed with plasma insulin throughout the night in both groups, although in this case, the concentrations remained statistically higher in T2D at each time point (P < 0.03 for the four time points analyzed statistically). Glucagon concentrations were higher (P < 0.05) in subjects with T2D than in ND subjects throughout the night, comparably falling in both groups from 10:00 pm to 7:00 am (P = 0.47).
Plasma cortisol (graph not shown; data provided in Table 2) demonstrated a comparable gradual increase through the night in subjects with T2D (5.8 vs 9.7 vs 12.9 µg/dL at 1:00, 4:00, and 7:00 am, respectively) and ND subjects (5.8 vs 8.7 vs 12.9 µg/dL at 1:00, 4:00, and 7:00 am, respectively).
Table 2.
Plasma Concentrations of Glucose and Hormones Overnight
|
ND
|
T2D
|
P Value b | |||||
|---|---|---|---|---|---|---|---|
| n | Mean | SD | n | Mean | SD | ||
| Glucose | |||||||
| 10:00 pm | 20 | 5.13 | 0.48 | 23 | 12.03 | 3.98 | <0.0001 |
| 1:00 am | 20 | 5.20 | 0.33 | 23 | 11.43 | 3.79 | <0.0001 |
| 4:00 am | 20 | 5.22 | 0.37 | 20 | 11.74 | 4.44 | <0.0001 |
| 7:00 am | 20 | 5.34 | 0.47 | 21 | 11.90 | 3.65 | <0.0001 |
| Estimated change (95% CI)a | 0.21 (−0.21 to 0.63) | 0.03 (−0.37 to 0.44) | 0.56 | ||||
| Study group | – | – | – | – | – | – | <0.0001 |
| Sample collection time | – | – | – | – | – | – | 0.0669 |
| Group × time interaction | – | – | – | – | – | – | 0.14 |
| Insulin | |||||||
| 10:00 pm | 20 | 44.37 | 23.82 | 23 | 70.90 | 39.23 | 0.0014 |
| 1:00 am | 20 | 42.36 | 23.05 | 23 | 56.82 | 32.43 | 0.0770 |
| 4:00 am | 20 | 40.83 | 13.99 | 22 | 48.52 | 25.19 | 0.2640 |
| 7:00 am | 20 | 41.85 | 19.31 | 21 | 47.31 | 22.52 | 0.4507 |
| Estimated change (95% CI)a | −2.52 (−10.95 to 5.91) | −22.87 (−30.99 to −14.76) | 0.0008 | ||||
| Study group | – | – | – | – | – | – | 0.0557 |
| Sample collection time | – | – | – | – | – | – | <0.0001 |
| Group × time interaction | – | – | – | – | – | – | 0.0041 |
| C-peptide | |||||||
| 10:00 pm | 20 | 1.18 | 0.40 | 23 | 1.68 | 0.54 | <0.0001 |
| 1:00 am | 20 | 0.88 | 0.27 | 23 | 1.29 | 0.40 | 0.0003 |
| 4:00 am | 20 | 0.86 | 0.24 | 22 | 1.16 | 0.34 | 0.0059 |
| 7:00 am | 20 | 0.84 | 0.23 | 21 | 1.08 | 0.34 | 0.0321 |
| Estimated change (95% CI)a | −0.35 (−0.45 to −0.25) | −0.60 (−0.70 to −0.51) | 0.0003 | ||||
| Study group | – | – | – | – | – | – | 0.0005 |
| Sample collection time | – | – | – | – | – | – | <0.0001 |
| Group × time interaction | – | – | – | – | – | – | 0.0018 |
| Glucagon | |||||||
| 10:00 pm | 20 | 97.20 | 18.21 | 23 | 119.57 | 32.04 | 0.0048 |
| 1:00 am | 20 | 85.25 | 18.36 | 23 | 106.35 | 30.94 | 0.0078 |
| 4:00 am | 20 | 82.00 | 21.75 | 20 | 100.35 | 29.04 | 0.0167 |
| 7:00 am | 20 | 81.75 | 22.27 | 21 | 101.76 | 26.56 | 0.0145 |
| Estimated change (95% CI)a | −15.45 (−21.28 to −9.62) | −18.40 (−24.02 to −12.77) | 0.47 | ||||
| Study group | – | – | – | – | – | – | 0.0065 |
| Sample collection time | – | – | – | – | – | – | <0.0001 |
| Group × time interaction | – | – | – | – | – | – | 0.84 |
| Cortisol | |||||||
| 10:00 pm | 20 | 4.31 | 3.09 | 23 | 7.10 | 4.24 | 0.0405 |
| 1:00 am | 20 | 6.22 | 4.77 | 23 | 5.83 | 6.04 | 0.77 |
| 4:00 am | 20 | 9.40 | 4.07 | 20 | 8.69 | 5.52 | 0.70 |
| 7:00 am | 20 | 12.60 | 2.79 | 21 | 12.94 | 3.45 | 0.74 |
| Estimated change (95% CI)a | 8.29 (5.73 to 10.85) | 5.95 (3.50 to 8.40) | 0.19 | ||||
| Study group | – | – | – | – | – | – | 0.4779 |
| Sample collection time | – | – | – | – | – | – | <0.0001 |
| Group × time interaction | – | – | – | – | – | – | 0.22 |
Estimated change is a model-based estimate of the change in hormone from 10:00 pm to 7:00 am. The P value reported for this row is for the test of equal changes between study groups.
P values, which are unadjusted for multiple comparisons, result from mixed model consisting of fixed effects for study group, sample collection time, and their interaction. A random subject effect was included in the model.
Enrichments of C5 and C2 glucose for estimation of GNG
Plasma C5 glucose, C2 glucose enrichment, and ratio of C5/C2 glucose are presented in Table 3.
Table 3.
Plasma C5 Glucose and C2 Glucose Enrichment
| 1:00 am | 4:00 am | 7:00 am | |
|---|---|---|---|
| C5 glucose | |||
| ND | 0.27 ± 0.04 | 0.29 ± 0.04 | 0.31 ± 0.05 |
| T2D | 0.19 ± 0.04a | 0.25 ± 0.06a | 0.30 ± 0.05 |
| C2 glucose | |||
| ND | 0.54 ± 0.05 | 0.54 ± 0.05 | 0.53 ± 0.05 |
| T2D | 0.54 ± 0.06 | 0.53 ± 0.06 | 0.53 ± 0.06 |
| C5/C2 glucose ratio | |||
| ND | 0.51 ± 0.07 | 0.55 ± 0.07 | 0.58 ± 0.09 |
| T2D | 0.37 ± 0.07a | 0.47 ± 0.09a | 0.57 ± 0.07 |
Data are mean ± SD.
P < 0.05, ND vs T2D.
Plasma C5 concentrations were significantly higher in ND subjects than in subjects with T2D at 1:00 and 4:00 am (P < 0.05) but were not different at 7:00 am (P = 0.76), and C2 glucose concentrations were not different between ND subjects and subjects with T2D throughout the night (P = 0.6). This resulted in C5/C2 glucose concentrations that were significantly different between ND subjects and subjects with T2D at 1:00 and 4:00 am (P < 0.05) but not different at 7:00 am (P = 0.9).
Rates of EGP
The [3-3H] glucose tracer/tracee ratio (specific activity) remained constant (Supplemental Fig. 1) in subjects with T2D and ND subjects throughout the night, permitting the use of Steele steady state equations for calculation of glucose turnover.
The time course of EGP shows higher EGP in the subjects with T2D than in the ND subjects throughout the night (Fig. 2, upper panel). EGP was higher in the subjects with T2D than in the ND subjects at 1:00 am (18.5 ± 3.9 vs 13.6 ± 2.0 µmol/kg/min; P < 0.001), at 4:00 am (16.9 ± 3.8 vs 12.0 ± 1.7 µmol/kg/min; P < 0.001), and at 7:00 am (15.8 ± 3.1 vs 12.0 ± 1.8 µmol/kg/min; P < 0.001).
Figure 2.
Time course of EGP observed in ND subjects (diamonds) and subjects with T2D (squares). An infusion of [3-3H] glucose was started at 10:00 pm (upper panel). Rates of EGP observed in the ND control (gray bars) and subjects with T2D (black bars) at 1:00, 4:00, and 7:00 am (lower panel). *P < 0.001 vs T2D. FFM, fat free mass.
Rates of GNG and GGL
Fractional rates of GNG were measured using the deuterated water method of Landau et al. (12) (Fig. 3). Rates of GNG progressively increased throughout the night in subjects with T2D (6.91 vs 8.02 vs 9.01 µmol/kg/min at 1:00, 4:00, and 7:00 am, respectively) but not in ND subjects (6.96 vs 6.6 vs 6.8 µmol/kg/min at 1:00, 4:00, and 7:00 am, respectively). In contrast, rates of GGL in the subjects with T2D progressively decreased throughout the night (11.4 vs 8.6 vs 6.8 µmol/kg/min at 1:00, 4:00, and 7:00 am, respectively) but remained essentially unchanged in the ND subjects (6.8 vs 5.5 vs 4.8 µmol/kg/min at 1:00, 4:00, and 7:00 am, respectively). This resulted in significantly higher (P < 0.001) rates of GNG in subjects with T2D than in ND subjects at 4:00 and 7:00 am and significantly (P < 0.001) higher rates of GGL in subjects with T2D than in the ND subjects at 1:00, 4:00, and 7:00 am.
Figure 3.
Rates of GNG (upper panel) and GGL (lower panel) observed in ND subjects (gray bars) and patients with T2D (black bars) at 1:00, 4:00, and 7:00 am. *P < 0.001 vs T2D. FFM, fat free mass.
Discussion
The present data indicate that glucose concentrations are higher in subjects with T2D than ND individuals throughout the night without any evidence of a point in time when glucose concentrations diverge between the groups. In fact, although glucose concentrations and EGP were higher in the subjects with T2D, they were parallel to those observed in the ND subjects throughout the night. Furthermore, in contrast to prior reports (17), there was no evidence of an early-morning increase in glucose concentration or EGP in the subjects with T2D or the ND subjects. On the other hand, whereas the contribution of GGL and GNG remained essentially constant in the ND subjects throughout the night, the contribution of GGL to EGP progressively fell and that of GNG progressively rose during the night in the subjects with T2D. These data indicated that (1) higher rates of EGP account for the higher nocturnal glucose concentrations in people with T2D and (2) the nocturnal regulation of GGL and GNG is altered in T2D.
Tracer recycling due to concurrent glycogen synthesis and breakdown has been a topic of interest among investigators using stable and radioisotopes for research (18, 19). Glucose tracers can be incorporated into glycogen via glycogen synthesis, with subsequent release via GGL into the glucose pool. In the current study, we used sequential tracer infusions to determine if tracer recycling was sufficient to affect the measurement of EGP. The observation that, under the current experimental conditions (i.e., during an overnight fast), tracers infused for 9, 6, or 3 hours provided comparable estimates of EGP in subjects with T2D and ND subjects provides strong data against tracer recycling (20). However, this approach provides no information regarding rate and impact of glucose/glucose-6-phosphate cycling on total glucose release. This contrasts with the estimation of EGP, which represents the difference between total glucose release and glucose that re-enters the glucose-6-phosphate pool.
People with T2D have hepatic insulin resistance, as indicated by impaired insulin-induced suppression of EGP, GNG, and GGL (2, 21). The higher nocturnal EGP despite higher insulin and C-peptide concentrations observed in the present experiments are consistent with nocturnal hepatic insulin resistance in individuals with T2D. Glucagon concentrations remained higher in subjects with T2D than in ND subjects throughout the night. The higher glucagon concentrations, by counteracting the effects of insulin on the liver, could have contributed to the higher nocturnal rates of EGP. On the other hand, whereas cortisol concentrations increased during the night, nocturnal cortisol concentrations did not differ significantly in the two groups, and the magnitude of the nocturnal increase in cortisol was comparable in subjects with T2D and ND subjects, making it unlikely that differences in cortisol were the primary cause of the persistently higher rates of EGP.
Krssak et al. (22), using 13C NMR methodology, have shown defective glycogen metabolism in the fed state and under combined hyperglycemia/hyperinsulinemia. However, our experimental conditions and methodology used were very different, which could explain the relative differences in results obtained. We (2, 16, 23–27) and others have reported that fasting rates of GNG measured in the morning are increased in people with T2D. The present studies confirm this observation. However, neither the rate nor the percent contribution of GNG to EGP differed in subjects with T2D and ND subjects at 1:00 am when EGP already was elevated. In contrast, GGL was higher in subjects with T2D than in ND subjects at 1:00 am, accounting for the higher rates of EGP. GGL (whether calculated as an absolute rate or as a percentage of EGP) then fell, and GNG (whether measured as an absolute rate or as a percentage of EGP) then increased during the night in subjects with T2D. In contrast, GNG remained essentially constant during the night in ND subjects, whereas GGL decreased slightly.
The cause of the difference in the temporal pattern of regulation of GGL and GNG in the two groups is not known. EGP reflects the net flux through the glucose-6 phosphatase and glucokinase pathways. Both in vitro and animal data indicate that flux through the glucose-6-phosphatase pathway is increased in T2D. In contrast, flux through the glucokinase pathway is decreased (23), resulting in a decrease in postprandial hepatic glycogen synthesis (28) in subjects with T2D. Despite this decrease, rates of GGL are higher (3, 29) and insulin-induced suppression of GGL is lower in subjects with subjects with T2D and ND subjects (2). It is therefore interesting to speculate that the higher rates of GGL observed in subjects with T2D during the first part of the night waned as hepatic glycogen became depleted more rapidly by the ongoing fast in the subjects with T2D than in ND subjects (28). This could have resulted in a decrease in the intrahepatic glucose-6-phosphate concentration, which in turn (by a mechanism still to be defined) caused a compensatory increase in GNG, which repleted the glucose-6-phosphate pool and, in the presence of a continued increase in glucose-6-phosphatase activity, resulted in a persistent and unchanged rate of EGP. Figure 3 shows that a similar but small inverse change in GGL and GNG occurred in the ND subjects during the night, which may have become more evident if the duration of the fast had been extended. Free fatty acid concentrations (data not shown) decreased in ND subjects throughout the night but not in subjects with T2D, resulting in higher (P < 0.001) plasma free fatty acid concentrations in the subjects with T2D than in the ND subjects at 4:00 and 7:00 am. Further studies are required to examine whether other substrates (alanine, glutamate) were abnormally high in T2D.
These data indicate that, under the current experimental conditions, EGP is greater in subjects with T2D than in ND subjects, signifying that T2D is associated with elevated nocturnal as well as fasting rates of EGP. It is possible that the magnitude of EGP could be dependent on the duration of diabetes and/or the degree of glycemic control (i.e., HbA1c). A prior study (30) in individuals with poorly controlled T2D (HbA1c >14%; n = 7), applying serial NMR technique to measure net hepatic GGL combined with the deuterated water method, showed higher rates of overnight GGL resulting in higher rates of EGP and GNG estimated in the morning in subjects with T2D than in ND subjects. However, this study did not measure EGP throughout the night to determine the temporal pattern of the contributions of GGL and GNG in subjects with T2D.
Our study has some limitations. We studied subjects with T2D who had their oral antidiabetic agents withdrawn prior to the study. Further studies are needed to determine the extent to which different antidiabetic therapies restore nocturnal EGP and/or the nocturnal temporal pattern of changes in rates of GGL and GNG. We studied subjects in the control group who were comparable to the patients with T2D to avoid confounding effects of differences in obesity and/or leanness. We anticipate the differences observed would have been magnified had we studied lean ND subjects because interpretation of the data would have been further confounded by BMI–induced differences in insulin sensitivity. Although we directly measured rates of EGP with the isotope dilution method and measured GNG by the widely accepted HMT technique, we did not directly quantify rates of GGL by NMR studies. However, given the robustness of the isotope dilution and the HMT methodologies, we are confident of the accuracy of our calculated rates of GGL.
Contrary to what we have previously observed in type 1 diabetes (31), we did not find evidence of the putative “dawn” phenomenon in the subjects with T2D. Rather than increasing, plasma glucose, insulin, and C-peptide concentrations and rates of EGP remained constant or fell from 4:00 to 7:00 am. It is possible that an early-morning increase in glucose and/or EGP may occur in patients with T2D of longer duration and/or in patients with limited residual endogenous insulin secretion.
In summary, the present data indicate that EGP is higher in people with T2D than in ND individuals throughout the night rather than merely in the morning. Rates of EGP are higher despite the presence of higher nocturnal glucose and insulin concentration, implying that hepatic insulin resistance contributes, at least in part, to this abnormality. The cause of the increase in EGP in the subjects with T2D differed during the night, being solely due to increased GGL in the first part of the night. Despite no change in EGP, GGL progressively fell and GNG progressively increased in subjects with T2D during the night, resulting in both GGL and GNG contributing to increased EGP by the following morning. These observations have potentially important theoretical as well as practical implications. From a theoretical viewpoint, the inverse changes in GGL and GNG in the presence of unchanged rates of EGP imply that, although elevated, flux through the glucose-phosphate pool to plasma glucose appears to be tightly regulated in people with T2D. From a practical viewpoint, the current data suggest that, to normalize nocturnal glucose metabolism in people with T2D, therapeutic agents will need to regulate GGL and GNG in a coordinated and temporally appropriate pattern. For example, therapeutic strategies that suppress GGL (e.g., glucagon receptor antagonists) could be effective in reducing early nocturnal hyperglycemia, whereas agents that suppress GNG (e.g., metformin, thiazolidinediones, PEPCK inhibitors, and glucose-6-phosphatase inhibitors) could be effective in reducing late nocturnal hyperglycemia.
Supplementary Material
Acknowledgments
We thank the research participants; Barbara Norby, Kelly Dunagan, and Cheryl Shonkwiler (Endocrine Research Unit, Mayo Clinic, Rochester, NY) for conducting the studies; the staff of the Mayo Clinic Center for Translational Science Activities (CTSA) Clinical Research Trials Unit (CRTU), the Metabolomics Core Laboratory, and the Immunochemical Core Laboratory; and Pamela Reich, Michael Slama, Brent McConahey, Benjamin Gran, and Prestin Schwichtenberg (Endocrine Research Unit, Mayo Clinic, Rochester, NY) for sample analyses and assistance with graphics. R.B. is the guarantor of this work, had full access to all the data, and takes full responsibility for the integrity of data and the accuracy of data analysis.
Financial Support: This work was supported by National Institutes of Health Grants R01 DK029953 (to R.B.) and R01 DK085516 (to A.B.) and by Grant UL1 TR000135 from the National Center for Advancing Translational Science, a component of the National Institutes of Health.
Disclosure Summary: The authors have nothing to disclose.
Glossary
Abbreviations:
- BMI
body mass index
- CRTU
Mayo Clinic Clinical Research Trials Unit
- EGP
endogenous glucose production
- GGL
glycogenolysis
- GNG
gluconeogenesis
- HMT
hexamethylenetetramine
- ND
nondiabetic
- NMR
nuclear magnetic resonance
- T2D
type 2 diabetes
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