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Glucose Stimulates Pulsatile Insulin Secretion from Human Pancreatic Islets by Increasing Secretory Burst Mass: Dose-Response Relationships

Division of Endocrinology and Diabetes (R.A.R., P.C.B.), Keck School of Medicine, University of Southern California, Los Angeles, California 90033; and Endocrine Division (J.D.V.), Mayo Medical and Graduate Schools of Medicine, Mayo Clinic, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Peter C. Butler, M.D., Division of Endocrinology and Diabetes, Keck School of Medicine, University of Southern California, 1333 San Pablo Street, BMT-B11. E-mail: pbutler{at}usc.edu.

	Abstract

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Insulin is secreted almost exclusively in discrete bursts, and physiological regulation is accomplished by modulation of the pulse mass. How the integrity of contiguous anatomic structures in the human pancreas (islets, splanchnic innervation, exocrine tissue, local hormones) directs the coordinated insulin secretion is not known. We posed the hypothesis that glucose stimulates insulin secretion from isolated human islets by an amplification of insulin pulse mass with no change in pulse frequency and that the glucose dose-response curve for the regulation of insulin pulse mass mirrors that recognized in vivo. Islets from five nondiabetic cadaveric donors were perifused in a recently validated perifusion system at 4 mM and subsequently at 8, 12, 16, or 24 mM glucose. The effluent was collected in 1-min intervals and used for the measurement of insulin (ELISA). Pulsatile insulin secretion was analyzed by deconvolution analysis. Total insulin secretion increased progressively (P < 0.0001). This augmentation was due to amplified pulse mass (3-fold, 24 mM vs. 4 mM glucose; P < 0.0001) with no change in pulse interval (4 min). Pulsatile insulin secretion was stimulated most effectively in a physiologic concentration range of 4–8 mM. The islet insulin content was significantly correlated to the magnitude of first and second phase insulin secretion (P < 0.0001). The quantifiable orderliness of pulsatile insulin secretion rose with escalating glucose concentration (P = 0.02). In conclusion, glucose stimulates pulsatile insulin secretion from isolated human islets by amplification of insulin pulse mass without altering pulse interval. The in vitro concentration-response relationship is comparable with that observed in vivo. These data imply that transplanted human islets should be able to reproduce glucose-regulated insulin secretion as observed in the intact human pancreas.

	Introduction

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INSULIN IS SECRETED in a glucose-regulated manner (1, 2). Insulin release is pulsatile with an interpulse interval of approximately 6 min (3, 4). In vivo insulin secretion in response to glucose ingestion and glucose infusion is driven by amplification of insulin pulse mass with no or little change in event frequency (5). It is of interest that pulsatile insulin secretion is demonstrable in isolated perifused islets thus pointing to an intrinsic pacemaker system (6, 7, 8, 9, 10). This pacemaker has been ascribed to oscillations in second-messenger signals and glycolysis (11). In vivo, interislet coordination of pulsatile insulin may be mediated by an intrapancreatic neural network (12, 13).

With increasing interest in the potential to use human islets in therapeutic transplantation (14), understanding the mechanism subserving glucose-regulated insulin secretion becomes important. The present study employs a recently validated method (9) for quantifying pulsatile insulin secretion from perifused islets studied at physiological glucose concentrations. We use this to test the hypothesis that increased glucose selectively amplifies insulin pulse mass in perifused human islets via an agonist dose-response curve function mirroring that inferred for the intact human pancreas in vivo. This notion has relevance to the conjecture that islet transplantation may reproduce some of the key mechanisms of normal physiological control.

	Materials and Methods

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Design

Islets were studied in an islet perifusion system, which has previously been described in detail and validated for quantification of pulsatile insulin release from human islets (9). The islets were exposed to a basal glucose concentration of 4 mM (n = 51 runs) for 40 min (0–40 min) followed by a step increase to 8 (n = 11 runs), 12 (n = 16 runs), 16 (n = 14 runs), or 24 mM (n = 11 runs) for 60 min (40–100 min). An equilibration period of 40 min without sample collection was allowed before the experiments (-40 to 0 min). Islets were recovered from the perifusion chamber and islet insulin content measured. The mean islet insulin content was not significantly different in islets studied at any of the stimulatory glucose concentrations 8, 12, 16, 24 mM (1344 ± 102; 1838 ± 236; 1398 ± 250; 2132 ± 151; P = not significant). Insulin concentrations in the effluent and insulin secretion from the islets are reported in mass units per islet to account for different islet sizes. Islet sizes were also measured at the beginning and the end of each perifusion experiment. For glucose dose-response relationships pulse mass is reported as a percentage-fraction of islet insulin content to isolate the effect of glucose from the impact of insulin stores on pulsatile insulin secretion.

Islet culture

Islets from the pancreas of five heart-beating organ donors (four male and one female; age, 47 ± 6 yr; body mass index, 31.5 ± 7.8 kg/m²) were isolated in the Northwest Tissue Center Seattle (R. Paul Robertson) and University of Minnesota Diabetes Institute for Immunology and Transplantation (Bernhard J. Hering). The islets were maintained in Roswell Park Memorial Institute culture medium with 5 mM glucose and 10% FBS at 37 C in humidified air containing 5% CO_2. Experiments were performed in random order starting after a minimum recovery period of 4 d following the islet isolation process.

Islet perifusion

Human islets were preincubated for 2 h at 4 mM glucose, then suspended in Bio-Gel P-2 beads (Bio-Rad Laboratories, Inc., Hercules, CA) and placed in perifusion chambers in aliquots of five to six islets. The perifusion system (ACUSYST-S, Cellex Biosciences, Inc., Minneapolis, MN) consisted of a multi-run peristaltic pump that delivered perifusate through six parallel tubing sets via a heat exchanger and six perfusion chambers at a constant rate of 0.3 ml/min. The perifusion buffers (Krebs Ringer bicarbonate buffer: NaCl 115 mM; KCl 4.7 mM; CaCl₂ 2.5 mM; MgCl₂ 1.2 mM; NaHCO₃ 5 mM, pH 7.4) were supplemented with 0.2% human serum albumin, preheated to 37 C, and oxygenized with 95% O₂ and 5% CO₂. The perifusate was delivered to the perifusion chambers containing the human islets, and the effluent was collected in 1-min intervals for determination of insulin concentrations.

Laboratory determinations

Glucose concentrations in the perifusion buffer were measured using the glucose oxidase method with a Glucose Analyzer 2 (Beckman Instruments, Brea, CA). Insulin was measured in duplicate with a two-site immunospecific ELISA as previously described (15). There is no cross-reactivity with proinsulin and split 32,33 and des-31,32 proinsulins. The less frequently occurring proinsulin intermediates split 65,66 and des-64,65 proinsulin react with a frequency of 30% and 63%, respectively. The lower detection limit is 4 pmol/liter, and the assay range is 5–2000 pmol/liter. The intraassay coefficient of variation ranged from 1.5–3.0%. The interassay coefficient of variation ranged from 3.5–4.5%.

Calculations and statistical analysis

The insulin concentration time series were analyzed by deconvolution analysis and approximate entropy (ApEn).

Deconvolution analysis is a multiparameter technique (16) to detect and quantify insulin secretory bursts, as described previously (17) and expressly validated for this perifusion system (9). Briefly, deconvolution analysis calculates underlying insulin secretion by way of two families of measures; the position, duration, mass, and amplitude of insulin secretory bursts; and a time-invariant nonpulsatile insulin secretion rate. Computations are based on the known half-life of exit of insulin from the perifusion system (0.63 min; Ref. 9).

ApEn is a model-independent and scale-invariant statistic designed to quantify the regularity or orderliness of (hormone) time series (18). Technically, ApEn measures the logarithmic likelihood that runs of patterns that are close (within r) for m contiguous observations remain close (within the tolerance width r) on subsequent incremental comparisons. This regularity metric is validated for parameter choices of r = 0.2 x SD in the individual time series and m = 1, as used here (19, 20, 21, 22). Larger ApEn indicates a higher degree of process randomness. A precise mathematical definition is given by Pincus (18).

Statistical analysis. Time evolution of measures was examined by ANOVA. ANOVA and the two-tailed Student’s unpaired t test were used to test for glucose effects on parameters of insulin pulsatility. A P value of less than 0.05 denoted significant contrasts. The EC₅₀ of glucose for insulin pulse mass and total insulin secretion was calculated by nonlinear regression analysis. The best-fit curve to the glucose concentration-response relationship for the relative proportion of the pulsatile component of insulin secretion was derived by nonlinear regression analysis. The curves of best-fit values for all other relationships were derived by linear regression analysis.

	Results

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Overall pattern of insulin secretion

Islets exposed to basal glucose (4 mM) secreted insulin at a relatively steady state (Fig. 1). Following the step increase in perfusate glucose concentration there was a rapid early phase of insulin secretion, which lasted approximately 10 min, and a more prolonged second phase of secretion, which lasted until the end of the experiment (Fig. 1). Insulin secretion increased in response to increased glucose concentrations between 4 and 15 mM, reaching a maximal rate at glucose concentrations between 15 and 20 mM (Fig. 2). Both first and second phase insulin secretion were augmented with increasing stimulatory glucose concentrations (Fig. 3).

View larger version (28K): [in this window] [in a new window]   Figure 1. Insulin concentration profiles (top panels) and insulin secretion rates (derived by deconvolution analysis, bottom panels) for two representative human-islet perifusion experiments (6 islets each). Islets were maintained at 4 mM glucose during the interval 0–40 min and 12 and 24 mM glucose from 40–100 min.

View larger version (17K): [in this window] [in a new window]   Figure 2. Glucose concentration-response relationships depicted separately for insulin pulse interval (top panel), secretory burst mass (middle panel), and total insulin secretion (bottom panel) by isolated human islets in perifusion (n = 103 runs). The solid lines indicate the curve of best-fit values derived by nonlinear regression analysis.

View larger version (29K): [in this window] [in a new window]   Figure 3. Glucose concentration-response relationships for first and second-phase insulin secretion (left panels). Relationship between the mass of insulin pulses during first and second-phase secretion and absolute magnitude of first and second-phase insulin secretion (right panels) by perifused human islets (n = 52 runs). First phase insulin secretion was calculated from insulin concentration data 0–10 min after raising glucose and second phase insulin secretion from the data 10–55 min after raising glucose concentration in the perifusion buffer. The solid lines indicate the curve of best-fit values derived by linear regression analysis. The dashed lines indicate the 95% confidence intervals of the regression line.

As previously reported (8, 9, 10), insulin release from perifused human islets showed a pulsatile pattern indicated by oscillations in insulin concentration in the perifusate. Pulse height rose at higher glucose concentrations (Fig. 1). Deconvolution analysis of insulin concentration profiles confirmed that insulin is secreted from human islets in a pulsatile pattern with a mean pulse interval of 3.9 ± 0.1 min. Stepwise increases in glucose concentration induced a volley of discrete high-amplitude insulin pulses (Fig. 1, bottom panels), which we defined as early phase of insulin secretion (Fig. 1, top panels). Volleys were followed by lower amplitude insulin pulses that give rise to second phase insulin secretion.

The mass of insulin secretory bursts increased in a concentration-dependent manner with increasing glucose exposure, reaching a plateau at approximately 15 mM (Fig. 2). The EC₅₀ of perifusate glucose concentration for insulin pulse mass and total insulin secretion was in a physiologic range of glucose concentrations (5–8 mM). In contrast, the pulse interval was unchanged with increasing glucose perifusate concentration. When the proportion of insulin secretion derived from pulsatile release vs. nonpulsatile release was compared, the relative proportion attributable to pulsatile insulin secretion increased from 26.2 ± 1.5% at 4 mM to 37.6 ± 2.2% at 24 mM glucose (Fig. 4), whereas the relative proportion of nonpulsatile insulin secretion decreased (P < 0.01).

View larger version (15K): [in this window] [in a new window]   Figure 4. Glucose concentration-response relationship for the relative proportion of the pulsatile component of total insulin secretion by perifused human islets (n = 103 runs). The solid line indicates the curve of best-fit values derived by nonlinear regression analysis.

Moreover, the insulin content of the perifused islets correlated with the pulse mass of insulin secretion (Fig. 5) and first and second-phase insulin secretory-response over the glucose range studied (Fig. 6).

View larger version (23K): [in this window] [in a new window]   Figure 5. Relationship between the islet insulin content and the mean pulse mass of insulin secretion by perifused human islets (n = 52 runs). The solid line indicates the curve of best-fit values derived by linear regression analysis. The dashed lines indicate the 95% confidence intervals of the regression line.

View larger version (33K): [in this window] [in a new window]   Figure 6. Relationship between the islet insulin content and the magnitude of first (top panel) and second-phase (bottom panel) insulin secretion by perifused human islets (n = 52 runs). First phase insulin secretion was calculated from insulin concentration data 0–10 min after raising glucose and second phase insulin secretion from the data 10–55 min after raising glucose concentration in the perifusion buffer. The solid lines indicate the curve of best-fit values derived by linear regression analysis. The dashed lines indicate the 95% confidence intervals of the regression line.

The regularity of patterns of perifusate insulin concentrations was assessed with ApEn. The process randomness of insulin release decreased (lower ApEn) in a glucose concentration-dependent fashion (Fig. 7).

View larger version (14K): [in this window] [in a new window]   Figure 7. Glucose enhances the quantifiable orderliness of insulin concentration time-series (n = 103 runs) in perifused human islets. Lower ApEn denotes greater secretory regularity or reduced relative randomness of the release process. The solid line indicates the curve of best-fit values derived by linear regression analysis. The dashed lines indicate the 95% confidence intervals of the regression line.

	Discussion

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In the present study, we document that increasing perifusate glucose concentration results in an approximately 2- to 3-fold augmentation of insulin pulse mass (Fig. 2). These data are consistent with prior studies in rodent and human islets showing an increment in pulse mass with increased glucose, although a concentration-response relationship has not previously been established (6, 7, 8, 9, 10, 24, 25). The data are also consistent with previous studies in vivo, which have reported increased insulin pulse mass in response to glucose ingestion and glucose infusion (5, 26). These and other studies (27, 28) employed similar methods as used in the present study to analyze insulin secretion in nondiabetic humans and showed a 3- to 5-fold stimulation. This difference might arise from the fact that human islets that were kept in culture for several days have lost their intraislet capillary network and insulin release from the islet by diffusion into the buffer rather than by direct release into the bloodstream might be slowed or limited particularly from the cells in the core of the islet. Also, in the case of oral glucose ingestion in vivo, the incretin effect, which is absent in isolated islets, has been found to contribute to the insulin secretory response with 30–60% (29).

One question arising from the present and other studies showing pulsatile insulin secretion from groups of perifused islets is: how is the pulsatile insulin secretion between these islets coordinated? Whereas individual islets can secrete insulin in a pulsatile manner (10), the mechanisms that coordinate pulsatile insulin secretion from groups of islets are not yet clear, including in perifused islets (as in the present experiments). Insulin secretion arises largely (80%) from discrete insulin bursts in vivo (4, 30, 31) and in vitro in single rodent islets (25). The present estimate that approximately 35% of insulin secretion by groups of perifused islets is pulsatile presumably denotes partial coordination of in vitro insulin secretion. As discussed previously (9), the mechanisms that lead to partial coordination of pulsatile insulin secretion by perifused isolated islets may include local paracrine effects and/or electrical coupling between adjacent islets. However, it is unlikely that these are the same factors that lead to such a high (100%) degree of coordination of islets scattered in the exocrine pancreas in vivo. In vivo, available data suggest that coordination of pulsatile insulin secretion involves intra-organ neural networks. For example, following transplantation of rat islets into the liver via the portal vein, we observed a delay of approximately 30–50 d before coordinate pulsatile insulin secretion was reestablished. This interval coincided with reinnervation of the transplanted islets (32).

Estimates of the physiological frequency of pulsatile insulin secretion have evolved historically. Initial predictions yielded interpulse intervals of approximately 15 min based on RIA, sampling intervals of 1–2 min and Fourier analysis to detect stable oscillations (33, 34). Subsequent studies employing more sensitive insulin assays, neuroendocrine methods of pulse detection, direct sampling from the portal vein in dogs and humans (3, 30, 31) and in the systemic circulation in humans and pigs without or with type 2 diabetes or alloxan-induced insulinopenia report interpulse times of 4–6 min (4, 26, 35, 36). This study as well as other recent human islet perifusion studies (9, 10) describe a similar pulse frequency (5 min). The present data corroborate the stability of insulin pulse-renewal time across a wide range of glucose availability.

We report here that pulse mass is highly correlated with islet insulin content, an observation recognized recently (37). In accordance with the reduction of insulin stores in patients with type 2 diabetes (38, 39), there is a concomitant decline in posthepatically estimated insulin pulse mass in this disorder (26). Overnight inhibition by somatostatin of insulin secretion (but not synthesis) in type 2 diabetes restores pulsatile and first-phase insulin secretion (26). Accordingly, islet insulin stores are primary determinants of normal insulin secretion both in vivo and in vitro (present data).

The orderliness of pulsatile insulin secretion as determined by ApEn increases in a glucose concentration-dependent manner. This suggests that, besides an augmentation of insulin release, synchronization of oscillatory insulin release by islets (5–6 per chamber) may be enhanced by glucose. On theoretical grounds, greater (interislet) feedback signal number and/or strength would account for enhanced orderliness of the insulin secretory process (40, 41). If, as previously proposed (11), oscillations in glycolysis are the pulse generator for pulsatile insulin release by islets, an increased glycolytic flux under conditions of elevated glucose concentrations might selectively enhance the pulsatile component of insulin secretion, as observed in the present experiments.

In summary, glucose stimulates insulin secretion from isolated human islets by amplifying the mass of peptide released per burst without changing pulse frequency. The EC₅₀ of this in vitro effect is comparable to that inferred in vivo. Both first- and second-phase insulin secretion are regulated by this amplitude-specific mechanism. For any given glucose stimulus, the islet insulin content predicts the magnitude of the secretory response. Moreover, glucose acts as an interislet coordinating trigger, which enhances the ensemble orderliness of the insulin release process.

In conclusion, perifused human islets, which are independent of vascularization and innervation, reproduce key features of glucose-regulated insulin secretion observed in the intact pancreas.

	Acknowledgments

 
We acknowledge the technical expertise of Lucretia Howard (insulin assays).

	Footnotes

 
This work was supported by NIH Grant DK-61539 and the Juvenile Diabetes Research Foundation. R.A.R. is a recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (Ri 1055).

Abbreviation: ApEn, Approximate entropy.

Received August 12, 2002.

Accepted October 29, 2002.

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