1 Division of Endocrinology and Diabetes, Keck School of Medicine, University of Southern California, Los Angeles, California 90089; 2 Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota 55905; 3 Department of Endocrinology, University of Aarhus, 8000-C Aarhus, Denmark; and 4 Division of Endocrinology and Metabolism, University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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Impaired
insulin secretion in type 2 diabetes is characterized by decreased
first-phase insulin secretion, an increased proinsulin-to-insulin molar
ratio in plasma, abnormal pulsatile insulin release, and heightened
disorderliness of insulin concentration profiles. In the present study,
we tested the hypothesis that these abnormalities are at least partly
reversed by a period of overnight suspension of -cell secretory
activity achieved by somatostatin infusion. Eleven patients with type 2 diabetes were studied twice after a randomly ordered overnight infusion
of either somatostatin or saline with the plasma glucose concentration
clamped at ~8 mmol/l. Controls were studied twice after overnight
saline infusions and then at a plasma glucose concentration of either 4 or 8 mmol/l. We report that in patients with type 2 diabetes,
1) as in nondiabetic humans, insulin is secreted in discrete
insulin secretory bursts; 2) the frequency of pulsatile
insulin secretion is normal; 3) the insulin pulse mass is
diminished, leading to decreased insulin secretion, but this defect can
be overcome acutely by
-cell rest with somatostatin; 4)
the reported loss of orderliness of insulin secretion, attenuated
first-phase insulin secretion, and elevated proinsulin-to-insulin molar
ratio also respond favorably to overnight inhibition by somatostatin.
The results of these clinical experiments suggest the conclusion that
multiple parameters of abnormal insulin secretion in patients with type
2 diabetes mechanistically reflect cellular depletion of immediately
secretable insulin that can be overcome by
-cell rest.
pulsatile insulin secretion; hyperglycemia; orderliness; C-peptide; somatostatin; proinsulin
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INTRODUCTION |
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HUMANS WITH TYPE 2 DIABETES exhibit defective insulin secretion, which is most obviously manifest under conditions of insulin resistance (12, 15, 23, 24, 41, 42). Impaired insulin secretion in type 2 diabetes is often characterized by a decreased first-phase insulin response (41), abnormal pulsatile insulin release (14, 21, 23), an increased ratio of proinsulin to insulin in the systemic circulation (30, 36, 43), and reduced orderliness of plasma insulin patterns (31). Many, but not all, of these parameters of insulin secretion were measured in patients with type 2 diabetes during hyperglycemia and compared with control subjects during normoglycemia. Under these conditions, the overnight fasting plasma insulin concentration (12) and measured insulin secretion rate (23) in patients with type 2 diabetes and nondiabetic controls are comparable, which is presumably due to the offsetting effects of hyperglycemia and impaired insulin secretion.
Chronic hyperglycemia per se may have deleterious effects on
insulin secretion (5, 10, 17,
18, 29), which can be partly ameliorated by
decreasing -cell demand through either weight loss (10,
34), insulin treatment (5, 6,
37, 38), or inhibition of insulin secretion
by diazoxide (9, 19). Based on these
observations, one proposition is that chronic
-cell stimulation is
important in the pathogenesis of impaired insulin secretion in type 2 diabetes (15, 24, 37,
42). In humans with type 2 diabetes, there appears to be a
deficiency of
-cell mass compared with appropriately matched
(comparably obese) nondiabetic controls (13). In patients
with type 2 diabetes, therefore, a decreased number of
-cells are
exposed to an environment of chronic hyperglycemia and insulin
resistance, which could establish the pathophysiological basis for
chronically increased
-cell stimulation.
The absent and/or defective insulin pulses previously reported
in patients with type 2 diabetes could be caused either by a primary
defect in the pacemaker and/or islet coordination system or by a defect
at the level of the islet. We hypothesized that the latter is true,
such that a normally generated impulse (healthy pacemaker) is delivered
in an appropriately synchronized manner (healthy coordination) to
islets but that these fail to respond appropriately to this
stimulus. We further hypothesized that such a deficient response may be
due to deficient immediately releasable insulin stores. A primary
purpose of the present study was to determine whether a period of
-cell rest imposed by overnight somatostatin at physiological
hyperglycemia (a strategy designed to enhance immediately secretable
insulin stores) restores pulsatile insulin release. If a defect in
pacemaker or islet coordination is responsible for the abnormal pattern
of pulsatile insulin release in patients with diabetes, we would not
anticipate restoration of pulsatile insulin release by a period of
somatostatin-imposed
-cell rest. To meet this primary objective,
therefore, the crucial comparison in the present studies is between the
pattern of insulin release in patients with type 2 diabetes after
either somatostatin or saline treatment.
Thus, in the present study, we sought to address the hypothesis that,
in patients with type 2 diabetes, the pattern of impaired insulin
secretion is at least partly reversible after a period of -cell rest
imposed via somatostatin (which inhibits insulin secretion but not
synthesis). This circumstance would argue against the presence of
specific
-cell defects leading to a loss of first-phase insulin
secretion, pulsatile insulin secretion, disordered insulin secretion,
or decreased conversion of proinsulin to insulin, and rather imply that
these defects are at least in part a consequence of chronically
increased
-cell stimulation in the setting of an inadequate number
of
-cells.
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METHODS |
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Study Subjects
Eleven patients with type 2 diabetes (yr since diagnosis 2.4 ± 0.6, range 0.5-6 yr), and six nondiabetic control subjects were studied in the General Clinical Research Center (GCRC) of the Mayo Clinic. The studies were approved by the Mayo Clinic Institutional Review Board (IRB), and all volunteers provided written informed consent. Inclusion criteria for patients with type 2 diabetes were documented fasting hyperglycemia (>7.8 mmol/l) on at least two occasions, a positive family history of type 2 diabetes, onset of diabetes after age 35, and the absence of diabetic ketoacidosis. The inclusion criterion for controls was a normal fasting blood glucose value of <6.1 mmol/l. Exclusion criteria for both type 2 diabetes and control subjects were pregnancy, abnormal liver or renal function tests or a history of hepatic or renal disease, a history of pancreatitis, anemia, symptomatic ischemic heart disease or cardiac failure, and treatment with glucocorticoid or anticonvulsants. Patients with type 2 diabetes and controls were matched for age, gender, and body mass index (Table 1). The diabetic patients were managed with diet (n = 4), sulfonylurea (n = 3), or insulin (n = 4). Sulfonylurea treatment was discontinued 48 h before each study, and NPH or Lente insulin was discontinued 24 h before each study. During those 48 h, patients were monitored frequently to avoid fasting hyperglycemia (>16 mmol/l) and were treated with soluble insulin if required.
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Study Design
Patients with type 2 diabetes were studied twice in the Mayo Clinic GCRC, once after overnight hyperglycemia (SAL) to reproduce the typical metabolic environment observed by theStudy Protocol
Diabetic patients.
All 11 patients with type 2 diabetes were studied twice, after either
an overnight SAL or an overnight SMS. On each occasion, the study
protocol can be conveniently divided into the overnight period
(2000-0700), when subjects were prepared for study
(SAL vs. SMS), and the study period per se
(0700-0832), when the dynamics of insulin secretion
were measured. On both occasions, volunteers were admitted to the GCRC
at 1500, fed a standard meal at 1600, and had two forearm peripheral
intravenous catheters placed before the initiation of the protocol at
2000. During the overnight period of the study (2000 to 0700), blood
was sampled for measurement of the plasma glucose concentration at
30-min intervals, and the plasma glucose concentration was clamped at 8 mmol/l on both occasions. During SAL, the plasma glucose concentration
was maintained via glucose infusion (50% dextrose, 0.9% normal saline
at 30 ml/h). During the overnight SMS, somatostatin was infused at 60 ng · kg1 · min
1 from 2000 to 0700 along with replacement infusions of growth hormone (3 ng · kg
1 · min
1), glucagon
(0.65 ng · kg
1 · min
1), and
insulin given at a variable rate (mean 0.19 mU · kg
1 · min
1; range 0.11 to 0.38 mU · kg
1 ·min
1) to
maintain the plasma glucose concentration at 8 mmol/l. At 0700, the
saline or somatostatin (and hormone replacement) infusions begun at 8 mmol/l by variable rate were discontinued, and the study period
(0700-0832) was begun. On both occasions,
blood was sampled from an arterialized dorsal hand vein at 5-min
intervals from 0700 to 0832 for measurement of the plasma glucose
concentration. The plasma glucose concentration was clamped at 8 mmol/l
by variable exogenous glucose infusion (50% dextrose). To assess the
pulsatility and orderliness of insulin secretion, blood was sampled at
1-min intervals from 0700 to 0800 from a forearm vein into chilled EDTA tubes that were immediately centrifuged, and the plasma was stored at
20°C for subsequent assay for insulin, C-peptide, and proinsulin. At 0820, a 1-mg bolus of glucagon was given intravenously to elicit first-phase insulin secretion. After the intravenous glucagon bolus,
blood was sampled at 1-min intervals for 12 min to allow measurement of
first-phase insulin secretion. The studies were concluded at 0832.
Control subjects.
Normal volunteers were also studied twice after overnight admission to
the GCRC. For control subjects, the overnight period (2000-0600) was identical on both occasions, with
admission to the GCRC by 1500, ingestion of the same standard meal as
the patients with type 2 diabetes at 1600, and two contralateral
forearm intravenous catheters placed by 2000. From 2000 to 0600, one
catheter was used to sample blood at 30-min intervals for measurement
of the plasma glucose concentration, which was allowed to remain at
euglycemia (~4 mmol/l); the other catheter was used to give a 0.9%
saline infusion (30 ml/h) identical to that given to patients with type 2 diabetes during the saline infusion night. At 0600, a dorsal hand
vein catheter was placed, and the hand was heated to obtain arterialized blood samples. On one occasion, the saline infusion was
continued until the end of the study, while blood samples were obtained
at 5-min intervals from the arterialized catheter to measure the plasma
glucose concentration (4-SAL). On the other occasion, at 0600, a
glucose infusion was started to increase the plasma glucose
concentration (again measured at 5-min intervals) to 8 mmol/l (8-SAL),
which level was clamped until the end of the study at 0820. To assess
the pulsatility and orderliness of insulin secretion, blood was sampled
at 1-min intervals from 0700 to 0820 into chilled EDTA tubes that were
immediately centrifuged, and the plasma was stored at 20°C for
subsequent assay for insulin, C-peptide, and proinsulin.
Assays
Plasma glucose concentrations were measured by the glucose oxidase method with a Beckman instrument. The plasma insulin concentration was measured in triplicate with a two-site immunospecific ELISA, as previously described (2). C-peptide was also measured by a two-site commercially available kit, as previously described (Dako Diagnostics, Cambridgeshire, UK) (27). Proinsulin was measured by a highly specific immunochemiluminometric assay, as previously described (11). Neither the proinsulin nor the insulin assay cross-reacted with the major split products of insulin.Calculations
Insulin secretion. Prehepatic insulin secretion rates were calculated by deconvolution of plasma C-peptide concentrations with standard parameters adjusted for age, body mass index, and sex, according to the method of Eaton et al. (4) and as modified by Van Cauter et al. (39), which validated these parameters for obese controls and subjects with type 2 diabetes like those included in this study. The mean insulin secretion rate for each individual during the last 20 min of the intensive sampling period (t = 60-80 min) was calculated.
Pulsatile insulin secretion. The dynamics of pulsatile insulin secretion were quantified by use of a deconvolution method specifically validated for this purpose (25, 26, 27). In the present study, we focused exclusively on the high-frequency pulses of insulin secretion (14, 21), rather than on the oscillations occurring with a periodicity of 60-90 min (23). In brief, the 1-min plasma (triplicate) insulin concentration time series were deconvolved by a multiparameter technique with the following assumptions. The venous plasma insulin concentration in each subject, as monitored at frequent intervals, results from five determinable and correlated parameters: 1) a finite number of discrete insulin secretory bursts occurring at specific times and having 2) individual amplitudes (maximal rate of insulin secretion within a burst), 3) a common half-duration (duration of an algebraically Gaussian secretory pulse at half-maximal amplitude) superimposed on 4) a basal time-invariant insulin secretory rate, and 5) a biexponential insulin disappearance model in the systemic circulation. The last-mentioned paramenter consisted of estimated insulin half-lives of 2.8 and 5.0 min and a fractional slow compartment of 0.28, as measured directly with the same assays as previously (27). Assuming the forgoing nominal insulin disappearance values, we estimated the numbers, locations, amplitudes, and half-duration of insulin secretory bursts, as well as a nonnegative basal insulin secretory rate for each data set by use of nonlinear least-squares fitting of the multiparameter convolution integral for each insulin time series. A modified Gauss-Newton quadratically convergent iterative technique was employed with an inverse (sample variance) weighting function (40). Parameters were estimated until their predicted values and the total fitted variance both varied by <1 part in 100,000. Asymmetric highly correlated joint variance spaces were calculated for each parameter by the Monte Carlo support plane procedure (40). Secretory rates are expressed as mass units of insulin (pmol) released per unit of distribution volume (liter) per unit of time (min). The mass of hormone secreted per burst (time integral of the calculated secretory burst) is thus computed as picomoles of insulin released per liter of volume of distribution. The percentage of insulin delivered into the circulation in discrete insulin bursts was calculated as previously described (27, 38).
Orderliness of insulin secretion. The orderliness of insulin secretion was calculated by a scale- and model-independent statistic termed approximate entropy (ApEn; Ref. 22). We have previously described in detail the use of this tool to determine the orderliness of insulin secretion (32). ApEn assigns a single nonnegative number to a time series in which larger values correspond to greater apparent process randomness, and smaller values, conversely, correspond to more instances of recognizable patterns or consistent features in the data. Briefly, ApEn measures the logarithmic likelihood that runs of patterns that are similar (within a certain threshold, which is here taken as 20% of each series SD, r) for m consecutive observations (plasma insulin concentrations) will remain similar on the next incremental comparisons.
First-phase insulin secretion. First-phase insulin secretion in response to glucagon injection was quantified by calculating the area under the curve of the plasma insulin concentration profile during the 12 min after glucagon injection.
Proinsulin-to-insulin ratio. The plasma proinsulin-to-insulin molar ratio was calculated by dividing the concentration of proinsulin (pmol/l) by insulin (pmol/l) obtained during the end of the 60-min intensive sampling period in each patient. To examine the trend of this with time, the corresponding values were also calculated at the beginning (t = 0) and middle (t = 30) of the intensive sampling period.
Statistical Analysis
Data are presented as means ± SE. Statistical comparison between groups was made with the Student's two-tailed t-test (nonpaired between diabetic or control groups and paired for comparisons within groups). Linear regression analysis was used to determine correlation between parameters within a group. A probability of <5% due to occurrence by chance alone (P < 0.05) was taken as evidence of statistical significance. ![]() |
RESULTS |
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Experimental Conditions
In patients with type 2 diabetes, the plasma glucose concentrations were successfully clamped at equivalent hyperglycemia overnight during the SMS and SAL nights (Fig. 1), and this clamp was maintained after discontinuation of these infusions during the intensive sampling period. Suppression of insulin secretion by SMS was confirmed by suppression of the plasma C-peptide concentration during SMS vs. SAL. In control subjects, the plasma glucose concentration remained at ~5 mmol/l during both nights and was then increased to ~8 mmol/l during the intensive sampling period on the SAL visit (Fig. 1).
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Insulin Secretion
The calculated insulin secretion rates by deconvolution of C-peptide kinetics are shown in Fig. 2. As previously reported, insulin secretion in patients with type 2 diabetes is defective compared with relevantly matched controls (3.03 ± 0.35 vs. 5.10 ± 0.78 pmol · kg
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Pulsatile Insulin Secretion
The calculated proportion of posthepatic insulin delivery derived from discrete insulin bursts was ~40% in both diabetic and nondiabetic subjects. This lower percentage than the ~90% observed with prehepatic sampling (32) presumably reflects hepatic extraction of the insulin pulses delivered into the portal circulation (31). Inspection of the 1-min plasma insulin concentration profile in control subjects and patients with type 2 diabetes revealed high-frequency oscillations (Figs. 3 and 4). These oscillations were markedly enhanced after discontinuation of overnight inhibition of insulin secretion with SMS. Formal pulse detection and quantification by deconvolution revealed that the frequency of pulses was comparable in patients with type 2 diabetes after either overnight SAL or SMS treatment (interburst interval 5.9 ± 0.4 vs. 4.7 ± 0.3 min, SAL vs. SMS, P = NS). This frequency was also comparable with that observed in control subjects studied at euglycemia (7.3 ± 1.5 min, 4-SAL) or hyperglycemia (6.6 ± 1.1 min, 8-SAL). In contrast, there were major differences in the pulse mass (and parallel changes in pulse amplitude) between groups. Thus the pulse mass (Fig. 5) was similar in controls studied during euglycemia to that in patients with type 2 diabetes studied at hyperglycemia (27.4 ± 5.8 vs. 18.8 ± 3.0 pmol/l, SAL vs. 4-SAL, P = NS). In contrast, when controls were studied at comparable plasma glucose concentrations (8 mmol/l) to those observed in patients with type 2 diabetes, the defect in insulin pulse mass was evident (27.4 ± 5.8 vs. 67.6 ± 15.5 pmol/l, SAL vs. 8-SAL, P < 0.02). However, after overnight SMS treatment, this defect in pulse mass was overcome (77.8 ± 19.3 vs. 67.6 ± 15.5 pmol/l, SMS vs. 8-SAL). Both the increase in pulse mass (increment in pulse mass in each patient) (r = 0.8, P < 0.01) and the increase in total insulin secretion rate (r = 0.9, P < 0.01) after overnight SMS infusion (Fig. 6) was strongly related to the basal C-peptide concentration (Fig. 6). The increment in pulse mass and total insulin secretion was also positively correlated with the basal insulin secretion rate (P < 0.01). This observation suggests that the extent of the increment in insulin pulse mass after
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Orderliness of Insulin Secretion
Under the conditions studied, the orderliness of insulin secretion measured by ApEn was not significantly higher in patients with type 2 diabetes compared with control subjects (Fig. 8). However, the orderliness of insulin secretion increased markedly in patients with type 2 diabetes after SMS treatment (1.34 ± 0.05 vs. 1.09 ± 0.05, SAL vs. SMS, P < 0.05). Acute hyperglycemia in control subjects did not cause an increase in ApEn.
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Proinsulin-to-Insulin Ratio
The proinsulin-to-insulin molar ratio was increased in patients with type 2 diabetes, as previously reported (0.61 ± 0.2 vs. 0.29 ± 0.04, SAL vs. 4-SAL, P < 0.05). However, after overnight inhibition of insulin secretion, the proinsulin-to-insulin ratio fell markedly to normal in patients with type 2 diabetes (0.61 ± 0.2 vs. 0.14 ± 0.04, SAL vs. SMS, P < 0.01). Acute hyperglycemia did not affect the molar proinsulin-to-insulin ratio in nondiabetic controls (0.29 ± 0.04 vs. 0.24 ± 0.01, 4-SAL vs. 8-SAL, P = NS). Inspection of Fig. 9 reveals that the proinsulin-to-insulin ratio was stable for 60 min of observation after discontinuation of somatostatin.
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DISCUSSION |
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The present study affirms the hypothesis that the pattern of
multiply dysregulated insulin secretion typical in patients with type 2 diabetes is, at least temporarily, reversible by short-term -cell
rest achieved via an overnight somatostatin infusion. The results of
this clinical experiment thus imply that
-cell rest is a logical
therapeutic strategy to restore the pattern of insulin secretion in
patients with type 2 diabetes.
In the present studies, we applied validated deconvolution techniques
(4, 25, 26, 39) to
identify and quantify total and pulsatile insulin secretion and
approximate entropy to estimate the orderliness of the insulin release
process (32, 22) in patients with type 2 diabetes and in control subjects, who were matched for body mass index
with patients with diabetes and were therefore obese. The insulin
secretion dynamics in the controls are therefore those of obese
insulin-resistant subjects. In contrast to earlier comparisons of
pulsatile insulin secretion in patients with type 2 diabetes and
controls (14, 21), we studied patients with
type 2 diabetes and control subjects at comparable plasma glucose
concentrations. Also, we used a novel, highly sensitive insulin assay
that has minimal cross-reactivity with insulin split products or
proinsulin (2). Under these experimental conditions, in
contrast to previous reports (14), we observe that the
frequency of insulin secretory pulses in humans with type 2 diabetes is no different from that in control subjects (interpulse interval ~6
min). The frequency reported here is comparable with that observed by
direct sampling of the portal vein in dogs (25,
26) and humans (35) by previously available
insulin radioimmunoassays but greater than that observed in the
systemic circulation with those assays (14,
21, 25). This pulse frequency is also comparable with that reported in the isolated perfused pancreas (7) and periperfused islet (3,
20). The normal insulin pulse frequency in patients with
type 2 diabetes reported here implies that the pacemaker intensity
driving pulsatile insulin secretion and the system for coordination of
insulin secretory bursts among islets are both intact in these diabetic
patients. In contrast, we were able to record a markedly decreased
amount of insulin secreted with each burst (burst mass) in patients
with type 2 diabetes studied at blood glucose concentrations comparable with those in controls. These data allow the speculation that the mechanism underlying defective insulin secretion in patients with
type 2 diabetes is insufficiently released insulin by -cells in
response to each ~6-min (synchronous) wave of depolarization through
the endocrine pancreas. The observation that this defect can be
resolved by a period of overnight somatostatin-induced inhibition of
insulin secretion supports the concept that defective insulin secretion
in type 2 diabetes may be partly caused by depletion of the immediately
secretable insulin pool in
-cells of these patients.
We also report that orderliness of insulin secretion is enhanced in response to previous overnight inhibition of insulin secretion by somatostatin. However, in contrast to conditions where the plasma glucose concentration in patients with type 2 diabetes is allowed to fluctuate liberally during the night before study (31), under the conditions of the present study, where the glucose concentration was fixed overnight (and during intensive sampling) at 8 mmol/l, the calculated approximate entropy was no higher in patients with diabetes than it was in control subjects. Therefore, one explanation for the increased disorderliness of insulin concentration behavior in patients with type 2 diabetes could be the greater disorderliness of the plasma glucose concentration.
Although it is well established that the molar ratio of proinsulin to
insulin concentrations in the systemic circulation is increased in
patients with type 2 diabetes (30, 36,
43), the cause of this anomaly is controversial. On the
one hand, this may represent a primary defect in the
proinsulin-to-insulin conversion present in patients with type 2 diabetes (28); alternatively, it may reflect release of
immature insulin granules as a consequence of chronically increased
-cell demand (1, 16). The current study
supports the latter hypothesis, because the proinsulin-to-insulin molar
ratio measured by two highly specific sandwich assays decreased to
normal in patients with type 2 diabetes after overnight
-cell inhibition. Such data are also consistent with the observation that a
decreased
-cell mass in humans with a partial pancreatectomy (33) results in increased circulating levels of proinsulin
relative to insulin. Given the longer half-life of proinsulin compared with insulin, one possible theoretical explanation for this apparent restoration of the proinsulin-to-insulin ratio is a non-steady-state artifact. However, the proinsulin-to-insulin ratio remained quite stable during the 60 min of study.
Many of the characteristics of abnormal insulin secretion present in
type 2 diabetes are observed in a variety of models of decreased
-cell mass. These features include decreased first-phase insulin
secretion (8, 18, 19,
33), reduced amplitude of pulsatile insulin secretion
(8), and an elevated molar ratio of the proinsulin/insulin
concentration in the systemic circulation (33). In the
present study, we report that overnight inhibition of insulin secretion
in humans with type 2 diabetes results in restoration of these dynamic
abnormalities of insulin secretion. Such experimental observations
support the hypothesis that many characteristics of abnormal insulin
secretion typical of type 2 diabetes are, at least in part, a
consequence of chronically increased demand (hyperglycemia and insulin
resistance) on a decreased
-cell mass.
In summary, we report that, in humans with type 2 diabetes, impaired
pulsatile insulin secretion is marked by an alteration of insulin pulse
mass, which defect can be overcome by -cell rest. Furthermore, we
report that
-cell rest enhances first-phase insulin secretion,
decreases the abnormally elevated proinsulin-to-insulin ratio in
plasma, and restores a significantly more orderly time/mode of insulin
release. Taken together, these data suggest that, in humans with type 2 diabetes, the immediately secretable insulin pool, consisting of docked
vesicles, is depleted and replaced by relatively less mature vesicles
with incompletely processed proinsulin. Somatostatin and/or
-cell
rest reconstitutes effective islet cell secretory dynamics, at least in
the short term, pointing to key role(s) of relatively deficient
somatostatin restraint of
-cell secretory activity and/or attrition
of total
-cell mass in this disease.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the excellent technical skills of Jane Kahl and Karen Laasko, the superb care given by the nursing staff of the Mayo Clinic GCRC, and especially the volunteers who participated in the research studies.
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FOOTNOTES |
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The current study was funded by a grant from the US Public Health Service, National Institutes of Health Grants DK-4434 (P. C. Butler) and MO1 R00858 (GCRC Mayo Clinic), the Mayo Foundation (P. C. Butler), the National Science Foundation Science and Technology Center in Biological Timing (University of Virginia), National Institutes of Health Research Career Development Award IK04 HD-00634 (J. Veldhuis), The Wellcome Trust Grant 049853 (P. C. Butler), and the University of Edinburgh.
Address for reprint requests and other correspondence: P. C. Butler, Div. of Endocrinology and Diabetes, Keck School of Medicine, Univ. of Southern California, 1333 San Pablo St., BMT-B11, Los Angeles, CA 90089-9113 (E-mail: pbutler{at}hsc.usc.edu).
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. §1734 solely to indicate this fact.
Received 20 December 1999; accepted in final form 24 March 2000.
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