Acute hyperglycemia alters the ability of the normal beta -cell to sense and respond to glucose

Jürgen Meyer1, Jeppe Sturis2, Martin Katschinski1, Rudolf Arnold1, Burkhard Göke3, and Maria M. Byrne1

1 Clinical Research Unit for Gastrointestinal Endocrinology, Department of Internal Medicine, Philipps University, 35033 Marburg, Germany; 2 Novo Nordisk, 2880 Bagsvaerd, Denmark; and 3 Department of Gastroenterology, Ludwig-Maximilians University, 81377 Munich, Germany.


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Impaired glucose tolerance (IGT) and non-insulin-dependent diabetes mellitus (NIDDM) are associated with an impaired ability of the beta -cell to sense and respond to small changes in plasma glucose. The aim of this study was to establish whether acute hyperglycemia per se plays a role in inducing this defect in beta -cell response. Seven healthy volunteers with no family history of NIDDM were studied on two occasions during a 12-h oscillatory glucose infusion with a periodicity of 144 min. Once, low-dose glucose was infused at a mean rate of 6 mg · kg-1 · min-1 and amplitude 33% above and below the mean rate, and, once, high-dose glucose was infused at 12 mg · kg-1 · min-1 and amplitude 16% above and below the mean rate. Mean glucose levels were significantly higher during the high-dose compared with the low-dose glucose infusion [9.5 ± 0.8 vs. 6.8 ± 0.2 mM (P < 0.01)], resulting in increased mean insulin secretion rates [ISRs; 469.1 ± 43.8 vs. 268.4 ± 29 pmol/min (P < 0.001)] and mean insulin levels [213.6 ± 46 vs. 67.9 ± 10.9 pmol/l (P < 0.008)]. Spectral analysis evaluates the regularity of oscillations in glucose, insulin secretion, and insulin at a predetermined frequency. Spectral power for glucose, ISR, and insulin was reduced during the high-dose glucose infusion [11.8 ± 1.4 to 7.0 ± 1.6 (P < 0.02), 7.6 ± 1.5 to 3.2 ± 0.5 (P < 0.04), and 10.5 ± 1.6 to 4.6 ± 0.7 (P < 0.01), respectively]. In conclusion, short-term infusion of high-dose glucose to obtain glucose levels similar to those previously seen in IGT subjects results in reduced spectral power for glucose, ISR, and insulin. The reduction in spectral power previously observed for ISR in IGT or NIDDM subjects may be due partly to hyperglycemia.

insulin secretion; connecting peptide; oscillations; spectral power


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IMPAIRED GLUCOSE TOLERANCE (IGT) is a relatively common condition defined by plasma glucose concentration greater than 7.8 mM and less than 11.1 mM, 2 h after ingestion of a 75-g glucose load (standard oral glucose tolerance test) (33). It is characterized by the presence of insulin resistance (7) and early defects in beta -cell function (1, 3, 18, 19). First-phase insulin secretory responses (ISR) to intravenous glucose are significantly reduced in relation to the degree of insulin resistance (3), and there is an impaired ability of the beta -cell to sense and respond to small changes in plasma glucose concentrations (3, 18). Hyperglycemic progression in IGT subjects has been shown to be associated with a progressive decline in beta -cell function (32), and the restoration of normoglycemia in non-insulin-dependent diabetes mellitus (NIDDM) subjects has been shown to improve beta -cell dysfunction (13).

Chronic hyperglycemia has been shown to have a deleterious effect on both insulin secretion and insulin action, a concept termed "glucose toxicity" (23, 34). The possible mechanisms whereby hyperglycemia may induce defects in glucose-induced insulin secretion include downregulation of several genes including GLUT2 and glucokinase (4, 5, 12, 14, 26), and induction of mitochondrial defects (16, 17). However, to date, very few data come from human studies, because acute or chronic experimental hyperglycemia is difficult to generate, and the nonavailability of pancreatic biopsies is a major impediment that has resulted in limited molecular or biochemical information about hyperglycemia-induced beta -cell defects in humans.

Using an oscillatory glucose infusion protocol, we previously demonstrated that, in IGT subjects, there is an impaired ability of the beta -cell to sense and respond to small changes in plasma glucose compared with controls (3). However, during this protocol, mean plasma glucose levels were significantly higher in IGT subjects compared with controls (9.8 ± 0.6 mM vs. 8.3 ± 0.2 mM). To establish whether short-term hyperglycemia plays a role in inducing this beta -cell defect, we attempted to mimic this degree of hyperglycemia in normal subjects by infusing high-dose glucose in an oscillatory manner. This enabled us to study the pattern of insulin secretion in response to short-term hyperglycemia in normal subjects.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects

Studies were performed in seven healthy male Caucasian volunteers aged 24-35 yr (mean ± SE: 28 ± 1 yr). Subjects were within 10% of ideal body weight (body mass index 23.8 ± 0.7 kg/m2) and had normal fasting glucose (5.0 ± 0.1 mM) and insulin levels (24.1 ± 3.3 pmol/l). None of the participants had a personal or family history of diabetes. Subjects had no other medical illnesses and were not receiving any medications. All subjects were placed on a weight maintenance diet consisting of >= 200 g carbohydrate/day for 2 wk before each study. All studies were performed in the Clinical Research Center of the University of Marburg. The protocol was approved by the Ethics Committee, and all subjects gave written informed consent.

Experimental Protocol

Each subject was studied on two occasions separated by intervals of >= 1 wk. The studies were performed in random order in each subject. All studies were performed after a 12-h overnight fast beginning at 0800, unless otherwise stated, with subjects in the recumbent position. An intravenous catheter was placed in each forearm, one for blood sampling and one for the administration of glucose. In all studies, the arm containing the sampling catheter was maintained in a heating blanket to ensure arterialization of the venous sample.

Administration of Oscillatory Glucose Infusion

It has been previously demonstrated that the peripheral administration of glucose in an oscillatory pattern results in regular oscillations in plasma glucose (25). In normal subjects, the beta -cell is able to detect and respond to repetitive increases and decreases in glucose with parallel changes in insulin secretion (25). This adjustment of the insulin secretory oscillations to the glucose oscillations is termed "entrainment." Lack of entrainment by glucose is an early manifestation of beta -cell dysfunction in individuals with IGT and mild NIDDM (3, 18). To establish whether short-term hyperglycemia per se plays a role in inducing this defect, 20% dextrose was infused on one occasion in an oscillatory pattern for 12 h at a mean rate of 6 mg · kg-1 · min-1 (low dose), with a periodicity of 144 min. The amplitude of the administered oscillations was 33% above and below the mean rate, as previously described (25). On another occasion, 20% dextrose was infused in a similar manner but at a mean rate of 12 mg · kg-1 · min-1 (high dose) and amplitude 16% above and below the mean rate to match the pulse amplitude achieved in the low-dose study. Each study consisted of an initial 2-h period (0800-1000) to allow a steady state to be achieved. This was followed by a subsequent period of 10 h (1000-2000), during which time blood samples were drawn at 15-min intervals for glucose, insulin, and C-peptide.

Assays

Plasma glucose levels were measured by the glucose oxidase technique (YSI, Schlag, Bergisch-Gladbach, Germany). The coefficient of variation of this method is <2%. Plasma insulin was measured by the Abbott IMx Microparticle Enzyme Immunoassay, which shows cross-reactivity with proinsulin of <0.005%. The average intra-assay coefficient of variation was 5%. Plasma C-peptide was measured using a commercially available radioimmunoassay kit (Biermann, Bad Nauheim, Germany) with an average intra-assay coefficient of variation of 4%.

Data Analysis: Determination of ISRs

Standard kinetic parameters for C-peptide clearance adjusted for age, sex, and body surface area were utilized (28). These parameters were used to derive, in each 15-min interval between blood sampling, the ISR from the plasma C-peptide concentrations by deconvolution, as previously described (6, 21).

Ultradian Oscillations in Insulin Secretion

Spectral analysis. Each individual ISR profile from the oscillatory glucose infusion protocol was submitted to spectral analysis to investigate whether the oscillations were entrainable as previously reported (25). Each spectrum was normalized on the assumption of the total variance of each series being 100% and is expressed as the normalized spectral power (SP). Each series was detrended with the first difference filter before spectral estimates were calculated using a Tukey window of 24 data points, as described by Jenkins and Watts (9).

Pulses of insulin secretion. Pulses of glucose and ISR were identified using Ultra, a computer program for pulse detection (27), with a threshold for pulse significance of twice the intra-assay coefficient of variation of the glucose assay, and of three times the intra-assay coefficient of variation of the C-peptide assay. Previous studies have shown that such a detection limit results in a false-positive rate <1% and thus minimizes the impact of any cumulative error resulting from deconvolution. For each significant pulse, the increment was defined as the difference between the level at the peak and the level at the preceding trough and was expressed in absolute concentration units (i.e., absolute increment). The relative amplitude of the pulses was calculated by dividing the absolute amplitude of each individual pulse by the value at the preceeding trough. Pulse amplitudes were based on medians rather than means because of the non-Gaussian nature of pulse distribution.

Statistical Analyses

All results are expressed as means ± SE. Data analysis was performed using the Statistical Analysis System (SAS version 6 Edition for Personal Computers, SAS Institute, Cary, NC). The significance of differences within individuals was determined using paired t-tests. Differences were considered to be significant if P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Volume of Dextrose Infused

Mean volume of 20% dextrose infused during the low-dose infusion was 1,652.3 ± 61.5 ml compared with 3,306.3 ± 123.0 ml during the high-dose infusion (P < 0.0001).

Mean 10-h Levels of Plasma Glucose, Insulin, and ISR

Mean glucose, ISR, and insulin levels were significantly higher during the high-dose compared with the low-dose glucose infusion study. Mean and individual values are shown in Table 1.

                              
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Table 1.   Mean 10-h glucose, ISR, and insulin responses to low- and high-dose glucose infusions

Number and Amplitude of Oscillations

The number of glucose pulses was similar during low- vs. high-dose glucose infusion [5.0 ± 0.7 vs. 5.2 ± 0.5 (P = 0.1)], as was the number of pulses of ISR [4.3 ± 0.4 vs. 4.2 ± 0.2 (P = 0.5)]. The mean absolute amplitude of the glucose pulses did not differ, being 2.44 ± 0.3 during the low-dose glucose infusion and 2.35 ± 0.3 during the high-dose infusion (P = 0.4). The median absolute amplitude of the pulses of ISR was also not significantly different during low- and high-dose glucose infusion [192.4 ± 24.7 vs. 252.8 ± 48.7 (P = 0.3)]. The mean relative amplitude of the glucose pulses was significantly lower during the high-dose infusion, 0.29 ± 0.02, compared with 0.47 ± 0.06 during the low-dose infusion (P < 0.01).

Relationship Between Glucose and ISR During Low- and High-Dose Glucose Infusion

In normal subjects during a low-dose glucose infusion, each pulse of glucose is tightly coupled to a pulse of ISR (25). This is clearly seen from the data from three representative subjects shown in Fig. 1, A, C, and E. During high-dose glucose infusion, there is a reduction in the tight coupling between glucose and ISR (Fig. 1, B, D, and F). To determine whether insulin secretion is entrained by glucose in individual subjects, the temporal profiles of glucose, insulin secretion, and insulin were analyzed by spectral analysis. This method evaluates the regularity of insulin secretory oscillations at a predetermined frequency. Peaks in plasma glucose spectra occurred at 144 min, corresponding to the period of exogenous glucose infusion. There was a reduction in mean SP for glucose during high-dose glucose infusion from 11.8 ± 1.4 to 7.0 ± 1.6 (P < 0.02). This was associated with a reduction in mean SP for ISR from 7.6 ± 1.5 to 3.2 ± 0.5 (P < 0.04) and for insulin from 10.5 ± 1.6 to 4.6 ± 0.7 (P < 0.01) during high-dose glucose infusion. Individual normalized SP values are shown in Fig. 2. The drop in SP for glucose from the low- to high-dose glucose infusion study did not correlate with the drop in SP for ISR (P < 0.8) or insulin (P < 0.7).


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Fig. 1.   Profiles of glucose and insulin secretion rates (ISR) in 3 individual subjects during low-dose oscillatory glucose infusion (A, C, E) and during high-dose glucose infusion (B, D, F). In subject 1 (A and B) spectral power (SP) for glucose and ISR decreased from 15.7 and 6.4 to 5.7 and 5.7, respectively, during the high-dose glucose infusion; in subject 5 (C and D), SP for glucose and ISR decreased from 11.2 and 12.4 to 8.0 and 3.4, respectively; in subject 6 (E and F) SP for glucose and ISR decreased from 13.8 and 10.7 to 10.2 and 1.7, respectively.



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Fig. 2.   Comparison of individual normalized SP for glucose (left), ISR (middle) and insulin (right) during low- and high-dose oscillatory glucose infusion.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Under both basal and stimulated conditions, normal insulin secretion is oscillatory, with periods in the 80- to 150-min range (20). These ultradian oscillations are of importance in the maintenance of normal glucose homeostasis (24). Many of these pulses in insulin and C-peptide are synchronous with pulses of a similar period in glucose, raising the possibility that these oscillations are a product of the insulin-glucose feedback mechanism. In this study, ultradian oscillations in insulin secretion were evaluated during short-term low-dose and high-dose oscillatory glucose infusions with an oscillatory period of 144 min. The study was designed to determine, under short-term experimental conditions, whether the periodicity of the ultradian oscillations could be entrained to the frequency of the exogenous glucose stimulus. If a nonlinear, self-oscillating system is perturbed exogenously with a periodic stimulus, different types of oscillatory behaviors may emerge, one of which is entrainment. When entrainment occurs, the oscillation of the system reacts to the exogenous stimulus by adjusting its period to that stimulus. In fact, consistent with previous studies (25), when low-dose glucose is administered in an oscillatory pattern in this study, entrainment of the glucose and ISR profiles occurs. In contrast, in this study, short-term oscillatory high-dose glucose infusion resulted in entrainment of neither glucose nor ISR.

The lack of entrainment of glucose during acute high-dose glucose infusion (yielding plasma glucose concentrations of 9.5 mM, similar to those previously seen in IGT subjects) in nonobese, insulin-sensitive subjects may be due to a combination of defective insulin secretion and altered peripheral glucose utilization in the presence of acute hyperglycemia (30, 34). If peripheral glucose uptake is not entrained during acute hyperglycemia, this will feed back to the beta -cell. The abnormal insulin secretory pattern to higher glucose levels will also affect peripheral glucose utilization to a larger degree in insulin-sensitive subjects (22) compared with insulin-resistant subjects. In our previous studies, subjects with classic NIDDM and IGT demonstrated a lack of entrainment of ISR; however, entrainment of glucose profiles was seen in these two groups (3, 18). However, in insulin-sensitive subjects with glucokinase mutations, there was also a lack of entrainment of glucose in addition to ISR, with mean plasma glucose concentrations of 13.1 mM (2).

In this study, the absolute amplitude of the glucose oscillations did not differ between the two studies, as this was controlled by the exogenous glucose infusion. However, the relative amplitude of the glucose oscillations was significantly lower during the high-dose glucose infusion. This could potentially influence our results. This reduction in spectral power for glucose seen during the high-dose glucose infusion may also play a role in the reduction in the observed regularity of insulin secretion. However, it is not the whole explanation, because the drop in spectral power for glucose during the high-dose glucose infusion study did not correlate with the drop in spectral power for ISR.

The role of glucotoxicity in humans remains controversial. Flax et al. (8) demonstrated in subjects with normal glucose tolerance that, in response to 2 days of basal hyperglycemia (6.0 mM), both basal and stimulated beta -cell responses to glucose were enhanced. We have shown that a 42-h period of hyperglycemia, 6.9 mM in normal glucose-tolerant subjects and 7.5 mM in IGT subjects, primes the insulin-secretory response to a subsequent glucose stimulus (3), whereas this priming effect was lost in subjects with NIDDM (12.7 mM). Several in vitro and in vivo studies have previously demonstrated that insulin-secretory responses are increased by exposure to glucose (15, 29, 31). Even in subjects with glucokinase mutations with severely reduced ISRs, a 42-h period of hyperglycemia (9.7 mM) resulted in a subsequent 45% increase in ISR at each 1 mM glucose interval (2). Despite preservation of the priming effect of glucose, the entrainment of glucose was lost during an exogenous oscillatory glucose infusion, suggesting that different mechanisms are involved in controlling these two processes.

Exposure to high doses of glucose for prolonged periods, however, may actually induce defects in insulin secretion (12, 13) and insulin action. There is considerable evidence that prolonged exposure of the beta -cell to high glucose may induce defects in insulin secretion in patients with classical NIDDM that are improved by interventions that lower the plasma glucose concentrations (11). An important study in adults with variable fasting glucose levels indicated that glucose-induced insulin secretion is abolished once a level of 115 mg/dl is achieved, and some impairment may occur at lower levels such as 100 mg/dl (1). Studies in various rodent models have also shown that deranged insulin secretion can be found with elevations of glucose levels that are difficult to distinguish from normal (12). beta -Cells exposed to hyperglycemia have been shown to have differential expression of many transporters and enzymes including GLUT2, glucokinase, mitochondrial glycerol phosphate dehydrogenase, pyruvate carboxylase, lactate dehydrogenase, hexokinase, glucose-6-phosphatase, and transcription factors (pancreatic duodenal homeobox protein-1, hepatocyte nuclear factors, Nkx6.1, Pax6) (10, 14, 26, 35).

In conclusion, this study demonstrates that an acute high-dose oscillatory glucose infusion yielding short-term hyperglycemia (9.5 mM) in healthy volunteers results in reduced entrainment of glucose and ISR. This finding suggests that hyperglycemia may be partially responsible for the reduction in entrainment in ISR in IGT subjects.


    ACKNOWLEDGEMENTS

We thank Sabine Jennemann and Elke Birkenstock for excellent technical support.


    FOOTNOTES

This study was supported by the Deutsche Forschungsgemeinschaft, Grant Ar 149/1-2.

Address for reprint requests and other correspondence: M. M. Byrne, Institute of Reproductive Medicine, Univ. of Münster, Domagkstrasse 11, 48129 Munster, Germany (E-mail: byrne{at}uni-muenster.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpendo.00427.2001

Received 24 September 2001; accepted in final form 4 December 2001.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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