Autocrine regulation of glucose metabolism in pancreatic islets

María I. Borelli, Flavio Francini, and Juan José Gagliardino

Center of Experimental and Applied Endocrinology (Universidad Nacional de la Plata-Consejo Nacional de Investigaciones Cientificas y Tecnicas, Pan American Health Organization/World Health Organization Collaborating Center), National University of La Plata School of Medicine, 1900 La Plata, Argentina

Submitted 11 April 2003 ; accepted in final form 16 September 2003


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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We evaluated the possible autocrine modulatory effect of insulin on glucose metabolism and glucose-induced insulin secretion in islets isolated from normal hamsters. We measured 14CO2 and 3H2O production from D-[U-14C]glucose and D-[5-3H]glucose, respectively, in islets incubated with 0.6, 3.3, 8.3, and 16.7 mM glucose alone or with 5 or 15 mU/ml insulin, anti-insulin guinea pig serum (1:500), 25 µM nifedipine, or 150 nM wortmannin. Insulin release was measured (radioimmunoassay) in islets incubated with 3.3 or 16.7 mM glucose with or without 75, 150, and 300 nM wortmannin. Insulin significantly enhanced 14CO2 and 3H2O production with 3.3 mM glucose but not with 0.6, 8.3, or 16.7 mM glucose. Addition of anti-insulin serum to the medium with 8.3 and 16.7 mM glucose decreased 14CO2 and 3H2O production significantly. A similar decrease was obtained in islets incubated with 8.3 and 16.7 mM glucose and wortmannin or nifedipine. This latter effect was reversed by adding 15 mU/ml insulin to the medium. Glucose metabolism was almost abolished when islets were incubated in a Ca2+-deprived medium, but this effect was not reversed by insulin. No changes were found in 14CO2 and 3H2O production by islets incubated with 3.3 mM glucose and anti-insulin serum, wortmannin, or nifedipine in the media. Addition of wortmannin significantly decreased insulin release induced by 16.7 mM glucose in a dose-dependent manner. Our results suggest that insulin exerts a physiological autocrine stimulatory effect on glucose metabolism in intact islets as well as on glucose-induced insulin release. Such an effect, however, depends on the glucose concentration in the incubation medium.

insulin secretion; autocrine regulation; isolated islets


THE PRESENCE OF INSULIN RECEPTORS in normal islets and {beta}-cell lines has been shown by use of a variety of approaches such as radioautography combined with electron microscopy (28), binding techniques (10, 38), and, more recently, the knockout (KO) technology (17).

It has also been shown that insulin receptor substrates IRS-1 and -2, and phosphatidylinositol 3-kinase (PI3K) are present in islets from normal rats and cell lines (6, 11, 21-23, 37) and that glucose (11, 37) and insulin (1) stimulate their phosphorylation.

Despite evidence in the literature supporting the concept that these receptors are active and affect several {beta}-cell functions such as gene transcription, insulin content, insulin secretion, and cytosolic calcium content and proliferation, their positive or negative modulatory effect remains controversial (15, 20, 32). However, most data suggest that insulin may not only modulate the function of the classical insulin target tissues, i.e., muscle, fat, and liver, but also several {beta}-cell functions.

The release of insulin in response to glucose requires the hexose metabolism in the islets (2, 5, 25) that is geared by two phosphorylating enzymes, hexokinase and glucokinase (2, 24), with the latter acting as a glucose sensor (24, 26). The ATP generated in the course of glucose metabolism depolarizes the {beta}-cell membrane and increases the cytosolic concentration of Ca2+, finally triggering insulin secretion by exocytosis (7). Insulin stimulates many of these processes, namely, glucokinase activity (30, 31), transcription of glucokinase (21, 22, 23) and insulin (6, 22, 23) genes, PHAS-I phosphorylation (43), Ca2+ flux from the endoplasmic reticulum (3, 4, 30, 31, 41, 42), and insulin exocytosis (3, 4, 17). Considering that glucose is one of the main regulators of insulin secretion and other islet functions, it can be assumed that insulin also affects glucose metabolism in the islets.

To test such hypotheses, we evaluated the effect of insulin on glucose metabolism in islets isolated from normal hamster pancreases by use of a traditional metabolic two-pathway approach (glucose oxidation and utilization) based on the measurement of CO2 and H2O production from [14C]- and [3H]glucose (25). Our results show that insulin exerts an autocrine stimulatory effect on glucose metabolism (oxidation and utilization) in intact islets as well as on glucose-induced insulin release. Such an effect, however, is conditioned by the glucose concentration in the incubation medium.


    MATERIALS AND METHODS
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 MATERIALS AND METHODS
 RESULTS
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Chemicals and drugs. Collagenase was obtained from Serva Feinbiochemica (Heidelberg, Germany); D-[U-14C]glucose and D-[5-3H]glucose [10 µCi/ml (300 µCi/mmol)] were from New England Nuclear (Boston, MA); bovine serum albumin (BSA) fraction V and other reagents of the purest available grade were from Sigma Chemical (St. Louis, MO).

Animals. Adult male Syrian hamsters (30 ± 2 g body wt) were maintained in a temperature-controlled room (23°C) with a fixed 12:12-h light-dark cycle (0600-1800). The animals had free access to a standard commercial diet and water. At the time of euthanasia, the whole pancreas from each animal was removed to isolate islets by collagenase digestion (19).

Glucose oxidation and utilization. Groups of 20 islets were incubated in a plastic vial containing 40 µl of Krebs-Ringer bicarbonate (KRB) buffer supplemented with 10 mM HEPES (pH 7.4) containing D-[U-14C]glucose and D-[5-3H]glucose [10 µCi/ml (300 µCi/mmol)] in the presence of 0.6, 3.3, 8.3, and 16.7 mM glucose alone or with insulin [5 mU/ml (33.3 nM) or 15 mU/ml (100 nM) when indicated], anti-insulin (or normal) guinea pig serum (1:500), wortmannin (75 and 150 nM), or nifedipine (25 µM). The insulin concentrations tested were selected on consideration that the hormone's concentration in the {beta}-cell sorroundings may exceed 15 mU/ml during glucose stimulation (45). The anti-insulin serum dilution used (1:500) was able to neutralize 97% of the insulin released into the incubation medium when 20 islets were incubated for 2 h with 16.7 mM glucose (data not shown). Nifedipine (4, 30) and wortmannin (22) were used at concentrations previously tested by other authors using either intact isolated islets or cellular lines.

When glucose metabolism was measured at a 0.6 mM glucose concentration in the incubation medium, the buffer was supplemented with a mixture of sodium salts of pyruvic, glutamic, and fumaric acids (5 mM each) (5). In experiments performed to test the effect of an extracellular Ca2+-deprived medium upon glucose metabolism, the ion was replaced by equal concentrations of NaCl.

The vial with the islets was placed inside an airtight-sealed 20-ml glass scintillation vial (500 µl of distilled water at the bottom) containing another empty plastic vial; after 2 h at 37°C, the reaction was stopped by adding 20 µl of metabolic poison (400 mM citric acid, 10 mM rotenone, 10 mM antimycin, and 4.6 mM KCN, pH 4.9, injected through the rubber seal) to the incubation vial. At the same time, 250 µl of hyamine were added to the empty tube. After incubation for 60 min at 37°C, the 14CO2 fixed to hyamine was measured in vials containing 5 ml of scintillation liquid. The islets were then incubated overnight at room temperature, and glucose utilization was measured as 3H2O production captured by water in 5 ml of scintillation liquid (25). Every experiment was performed three times, including five replicates within each experiment.

Insulin secretion in vitro. Groups of five isolated islets were incubated for 60 min at 37°C in 0.6 ml of KRB, pH 7.4, previously gassed with a mixture of CO2-O2 (5:95) containing 1% (wt/vol) BSA and 3.3 mM glucose, 16.7 mM glucose alone, or different amounts of wortmannin (75, 150, and 300 nM) in the incubation media (9). Wortmannin was introduced in the media dissolved in DMSO; accordingly, the same final concentration of that vehicle was also added to the medium without wortmannin (control). At the end of the incubation period, aliquots were used to measure insulin by radioimmunoassay (12).

Statistical analysis. The experimental data were analyzed using Student's t-test. Data were expressed as means ± SE. Differences were considered significant when P < 0.05.


    RESULTS
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 MATERIALS AND METHODS
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Effect of exogenous insulin on glucose metabolism. The production of 14CO2 from D-[U-14C]glucose and of 3H2O from D-[5-3H]glucose by isolated islets increased significantly in a dose-dependent manner when the glucose concentration in the incubation medium was raised from 0.6 to 16.7 mM.

In the presence of 3.3 mM glucose, addition of 5 mU/ml insulin to the incubation medium enhanced significantly both 14CO2 and 3H2O production (P < 0.005). This stimulatory effect of insulin was not observed in the presence of 0.6, 8.3, or 16.7 mM glucose (Fig. 1). A higher insulin concentration (15 mU/ml) in the medium containing 16.7 mM glucose also failed to enhance glucose metabolism (data not shown).



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Fig. 1. Effect of exogenous insulin on glucose metabolism. Glucose oxidation (A) and utilization (B) were measured as 14CO2 and 3H2O production from D-[U-14C]glucose and D-[5-3H]glucose, respectively. Each bar represents the mean value ± SE of the percent changes above basal values from 3 different experiments (n = 15). Absolute basal values at 3.3 mM glucose: A, 5.42 ± 0.67 pmol glucose·islet-1·120 min-1; B, 30.4 ± 2.18 pmol glucose·islet-1·120 min-1. A, c vs. d, P < 0.005; c vs. a, e, and g, P < 0.001; B, c vs. d, P < 0.02; c vs. a, e, and g, P < 0.001.

 

Figure 2 shows the effect of increasing concentrations of exogenous insulin (0.005 to 5 mU/ml) on glucose metabolism measured in the presence of 3.3 mM glucose in the incubation medium. At 0.5 mU/ml, insulin started to enhance significantly the production of 14CO2 (P < 0.02) and 3H2O (P < 0.005), with no further enhancement after its concentration was increased to 5 mU/ml.



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Fig. 2. Effect of different insulin concentrations on glucose metabolism. Glucose oxidation (A) and utilization (B) were measured as 14CO2 and 3H2O production from D-[U-14C]glucose and D-[5-3H]glucose, respectively. Each bar represents the mean value ± SE of the percent changes above basal values (a) from 3 different experiments (n = 15). Absolute basal values at 3.3 mM glucose: A, 6.12 ± 0.67 pmol glucose·islet-1·120 min-1; B, 35.3 ± 3.5 pmol glucose·islet-1·120 min-1. A, a vs. d, P < 0.02, a vs. e, P < 0.001; B, a vs. d and e, P < 0.005.

 

Effect of anti-insulin serum, wortmannin, and nifedipine on glucose metabolism. We tested the possible existence of an autocrine effect of insulin on glucose metabolism by use of different experimental conditions. Anti-insulin guinea pig serum (1:500) significantly decreased both 14CO2 and 3H2O production in incubation media containing 8.3 mM (P < 0.05 and 0.02, respectively) and 16.7 mM glucose (P < 0.025 and 0.02, respectively) compared with islets incubated under the same conditions, but replacing the antiserum with normal guinea pig serum (1:500) (Table 1).


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Table 1. Effect of insulin antibody, wortmannin, and nifedipine on glucose metabolism at 3.3, 8.3, and 16.7 mM glucose

 

Wortmannin (150 nM), a competitive inhibitor of PI3K, induced a similar significant decrease in 14CO2 and 3H2O production when the islets were incubated with 8.3 mM (P < 0.02 and 0.005, respectively) and 16.7 mM glucose (P < 0.01 and 0.001, respectively; Table 1). Similar results were obtained with 75 nM wortmannin, thus suggesting that the effect was specific (data not shown).

Nifedipine (25 µM) significantly decreased 14CO2 and 3H2O production in the presence of 8.3 mM (P < 0.025 and 0.02, respectively) or 16.7 mM glucose (P < 0.02 and 0.01, respectively; Table 1). This effect was reversed with the addition of 15 mU/ml insulin to the incubation medium at 16.7 mM glucose (Fig. 3). Removal of extracellular Ca2+ from the medium also induced a significant decrease in glucose metabolism. In this case, however, the inhibition was not reversed by the addition of the same amount of insulin (15 mU/ml; Fig. 3).



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Fig. 3. Effect of nifedipine and a Ca2+-deprived medium on glucose metabolism. Glucose oxidation (A) and utilization (B) were measured as 14CO2 and 3H2O production from D-[U-14C]glucose and D-[5-3H]glucose, respectively. Each bar represents the mean value ± SE of the percent changes above basal values (a) from 3 different experiments (n = 15). N, nifedipine; I, insulin. Absolute basal values at 16.7 mM glucose: A, 25.6 ± 1.06 pmol glucose·islet-1·120 min-1; B, 115.9 ± 2.93 pmol glucose·islet-1·120 min-1. A, a vs. b, P < 0.005; b vs. c, P < 0.01; a vs. d, and e, P < 0.001; B, a vs. b, P < 0.01; b vs. c, P < 0.01; a vs. d and e, P < 0.001.

 

Addition of anti-insulin serum, wortmannin, or nifedipine to the incubation media did not produce any significant effect on either 14CO2 or 3H2O production in the presence of 3.3 mM glucose (Table 1).

Insulin secretion. Addition of wortmannin (75, 150, and 300 nM) to the incubation media significantly decreased the amount of insulin released in response to 16.7 mM glucose in a dose-dependent manner (P < 0.001; Fig. 4).



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Fig. 4. Effect of wortmannin on insulin secretion. Each bar represents the mean ± SE from 3 different experiments in absolute values expressed as ng·islet-1·h-1 (n = 15). a vs. b and b vs. c, d, and, e, P < 0.001; c vs. e, P < 0.02.

 


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our results show that insulin significantly affects glucose metabolism in the islets. Such an effect was demonstrated using a variety of experimental approaches: addition of insulin to the incubation medium and blockage of either insulin release or intracellular insulin messenger activity in the presence of different glucose concentrations.

In the presence of 3.3 mM glucose, addition of insulin to the incubation medium significantly increased glucose metabolism. This effect started at 0.5 mU/ml, without any significant changes being observed after increasing the concentration up to 5 mU/ml. However, for the sake of comparison with other reports in the literature, we performed most of our experiments using insulin at 5 mU/ml. Such a stimulatory effect of insulin was not observed at 0.6, 8.3, or 16.7 mM glucose.

On the other hand, blockage of insulin release (presence of nifedipine or absence of extracellular Ca2+), immune neutralization of the released insulin (anti-insulin serum), or blockage of the activity of the intracellular insulin messenger (PI3K by wortmannin) significantly decreased glucose metabolism in the islets (~50%) only in the presence of 8.3 and 16.7 mM glucose in the incubation medium. A similar decrease was obtained by other authors measuring insulin's effect on other parameters, such as glucokinase transcription (50%) (22) and {beta}-cell emiocytosis (50%) (3).

These data suggest that 1) insulin is an effective modulator, but not the main regulator, of glucose metabolism in normal islets; and 2) the autocrine stimulatory effect of insulin on glucose metabolism depends, at least in our model, on the glucose concentration in the medium.

In our experimental conditions, wortmannin significantly decreased the release of insulin in a dose-dependent manner. Wortmannin can inhibit PI3K as well as myosin light-chain kinase (MLCK) (44). Because it has been reported that MLCK is not involved in the regulation of insulin secretion (39), our data suggest that insulin modulates its own secretion, affecting PI3K activity.

Further support for this concept arises from experiments performed with a completely different approach, namely specific KO of the insulin receptors and IRS-1 and -2 in {beta}-cells (17, 40). However, it has been argued that, in some cases ({beta}IRKO mice), results would be secondary to changes induced by the KO procedure at the nervous system rather than specifically at the {beta}-cell level (34).

On the other hand, it has been shown that glucose still stimulated {beta}-cell membrane emiocytosis in mice with KO of the insulin receptors (17) and IRS-1 and -2 (18) even when insulin was unable to produce such an effect. All together, these results reinforce our proposal that, even though important, insulin autocrine modulation does not represent the unique regulatory mechanism of glucose metabolism in the islets. However, the fact that a simultaneous decrease in both glucose-stimulated insulin release and the hexose metabolism is induced by blocking the autocrine effect of insulin favors a physiological modulatory rather than a simple pharmacologically or experimentally induced effect of insulin on these two functions.

It is accepted that glucokinase activity is the limiting step of glucose metabolism in normal islets (24, 26); thus modulation of its activity could be a possible explanatory mechanism for the effect of insulin on glucose metabolism. The stimulatory effect of insulin on glucokinase activity (30, 31) favors this hypothesis. Nevertheless, such an assumption should be taken with caution, since other enzymes acting downstream of glucokinase, such as glyceraldehyde-3-phosphate dehydrogenase, also affect glucose metabolism and insulin secretion in the islets (33).

Glucose metabolism decreased to 48% when the islets were challenged with 16.7 mM glucose and nifedipine in the incubation medium. Such a decrease was corrected by adding insulin to the medium. A similar decrease in glucose metabolism was obtained when the islets were incubated with 16.7 mM glucose in a Ca2+-deprived medium, but in this case insulin did not restore the values to those observed in the control. The reported enhancing effect of insulin on the cytosolic Ca2+ concentration in {beta}-cells could account for our results (4, 30, 31, 41, 42), suggesting that glucose metabolism in the islets is closely linked to cytosolic Ca2+ concentrations, which may affect the activity of Ca2+-dependent enzymes (25). Because it has been reported that insulin can modify intracellular Ca2+ flow in the islets (4, 41, 42), this could be an alternative pathway to explain the effect of the hormone upon glucose metabolism. However, this mechanism would not be effective in conditions of extremely low Ca2+ concentrations (2-h incubation in a Ca2+-deprived medium) due to the depletion of intracellular Ca2+ pools.

Our results on the effect of insulin on its own secretion are in apparent conflict with those reporting no effect (36) or inhibition (8, 14, 29, 45). Some discrepancies, however, are only apparent, as is the case of Hügl et al. (13), who reported no insulin effect only in the presence of 15 mM glucose, thus obtaining the same results that we did. In other cases, differences could be ascribed to the use of a dynamic rather than a static model to measure insulin release (35, 45); the use of an insulin-mimetic compound instead of insulin (29); the use of a different glucose-to-insulin ratio in the experiments, since it has been reported that measurable insulin effects on islet function or cellularity are apparent only in the presence of a given ratio (27); the utilization of different animals, because the genetic background can significantly affect insulin action and {beta}-cell secretion (16); the existence of a different threshold for stimulation and inhibition, i.e., although lower insulin concentrations stimulate the hormone's secretion, higher concentrations inhibit it; and finally, according to the in vivo or in vitro model used, the effect of insulin would be masked due to the paracrine effect of the simultaneous somatostatin release (1). Cooccurence of some of these factors could probably explain the above-mentioned discrepancies concerning insulin's effect on its own secretion.

In summary, our results demonstrate for the first time that glucose metabolism in the islets is modulated, at least in hamsters and in our experimental model, by insulin in an autocrine fashion. This effect could be physiologically relevant under conditions of high extracellular glucose. Whether the impairment of insulin's effect on peripheral tissues in cases of insulin resistance also operates at islet level remains to be demonstrated.


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This work was supported by grants from Fondo Nacional para la Investigación Cientifica y Tecnológica, Consejo Nacional de Investigaciones Cientificas y Tecnicas, and Comisión de Investigaciones Cientificas de la Provincia de Buenos Aires.


    ACKNOWLEDGMENTS
 
Thanks are due to A. Díaz for insulin assays, E. Perez for graphic design, and A. Di Maggio for careful secretarial support.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. J. Gagliardino, CENEXA (UNLP-CONICET), Facultad de Ciencias Médicas, Calles 60 y 120, 1900 La Plata, Argentina (E-mail: gagliardino{at}infovia.com.ar).

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.


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