A role for pancreatic beta-cell secretory hyperresponsiveness in catch-up growth hyperinsulinemia: Relevance to thrifty catch-up fat phenotype and risks for type 2 diabetes
- Marina Casimir†2,
- Paula B de Andrade†1,
- Asllan Gjinovci2,
- Jean-Pierre Montani1,
- Pierre Maechler†2 and
- Abdul G Dulloo†1, 3Email author
© Casimir et al; licensee BioMed Central Ltd. 2011
Received: 12 November 2010
Accepted: 18 January 2011
Published: 18 January 2011
Current notions about mechanisms by which catch-up growth predisposes to later type 2 diabetes center upon those that link hyperinsulinemia with an accelerated rate of fat deposition (catch-up fat). Using a rat model of semistarvation-refeeding in which catch-up fat is driven solely by elevated metabolic efficiency associated with hyperinsulinemia, we previously reported that insulin-stimulated glucose utilization is diminished in skeletal muscle but increased in white adipose tissue. Here, we investigated the possibility that hyperinsulinemia during catch-up fat can be contributed by changes in the secretory response of pancreatic beta-cells to glucose. Using the rat model of semistarvation-refeeding showing catch-up fat and hyperinsulinemia, we compared isocalorically refed and control groups for potential differences in pancreatic morphology and in glucose-stimulated insulin secretion during in situ pancreas perfusions as well as ex vivo isolated islet perifusions. Between refed and control animals, no differences were found in islet morphology, insulin content, and the secretory responses of perifused isolated islets upon glucose stimulation. By contrast, the rates of insulin secretion from in situ perfused pancreas showed that raising glucose from 2.8 to 16.7 mmol/l produced a much more pronounced increase in insulin release in refed than in control groups (p < 0.01). These results indicate a role for islet secretory hyperresponsiveness to glucose in the thrifty mechanisms that drive catch-up fat through glucose redistribution between skeletal muscle and adipose tissue. Such beta-cell hyperresponsiveness to glucose may be a key event in the link between catch-up growth, hyperinsulinemia and risks for later type 2 diabetes.
A large body of evidence indicate that subjects who had low birth weight or who showed reduced growth rate during childhood, but who subsequently showed catch-up growth, have higher susceptibility for type 2 diabetes or cardiovascular diseases later in life [1–5]. While the nature of this association between catch-up growth and later disease risks remains obscure , it is intricately linked to the state of hyperinsulinemia and accelerated recovery of body fat (catch-up fat) that characterizes catch-up growth [5–7]. There is a well-described rat model of semistarvation-refeeding in which catch-up fat and hyperinsulinemia occur in absence of hyperphagia and could be linked to an elevated metabolic efficiency due to suppressed thermogenesis . Using this model, we previously showed that insulin-mediated glucose utilization is diminished in skeletal muscle but enhanced in white adipose tissue , thereby suggesting that catch-up fat is characterized by glucose redistribution from skeletal muscle to adipose tissue. The suppressed thermogenesis is thus associated with establishment of a thrifty metabolism which spares glucose for catch-up fat via coordinated induction of insulin resistance in skeletal muscle, insulin hyperresponsiveness in adipose tissue and a state of hyperinsulinemia. In this context, putative implication of insulin-secreting cells remains unknown. Here, we tested the hypothesis that the hyperinsulinemic state of catch-up fat might also be contributed by pancreatic beta-cell hyperresponsiveness to glucose. To this end, we investigated the semistarvation-refeeding rat model for pancreatic endocrine function and morphology. In particular, the secretory responses of perfused pancreases and isolated islets were analyzed.
Animals and Diet
Male Sprague Dawley rats (Elevage Janvier, France), caged singly in a temperature-controlled room (22 ± 1°C) with 12-h light/dark cycle, were maintained on chow diet (Kliba, Cossonay, Switzerland) consisting, by energy, of 24% protein, 66% carbohydrates, and 10% fat, and had free access to tap water. Animals were maintained in accordance with our institute's regulations and guide for the care and use of laboratory animals.
Design of study
Pancreas perfusions (in situ)
To evaluate insulin-secretory capacity of the endocrine pancreas, refed and control rats were anesthetized with sodium pentothal and prepared for pancreas perfusion as previously described . Briefly, the pancreas was perfused with a Krebs-Hank's buffer (KHB) at a constant rate of 5 ml/min via mesenteric and transileac arteries, and the perfusate was collected every minute from a catheter placed in the portal vein. After an initial equilibration period with no sample collected, the effluent was collected in 1-min fractions from the portal vein. The pancreas was perfused at 37°C with the KHB buffer supplemented with the following concentrations of glucose: period I (basal, last 4 min) 2.8 mmol/l glucose, periods II and III (15 min each) 16.7 mmol/l glucose, period IV (recovery, 15 min) 2.8 mmol/l glucose. Aliquots of perfusates were collected on ice and stored at -20°C until insulin assay by radioimmunoassay (RIA) using rat insulin as standard.
Isolated islet perifusions (ex vivo)
To evaluate the kinetics of insulin secretion in islet-perifusion experiments, pancreatic islets were isolated by collagenase digestion and handpicking from refed and control rats as described previously . Isolated islets were cultured free-floating in RPMI 1640 medium before experiments. Insulin levels were determined by RIA and insulin secretion collected every min was normalized per islet number. Islet perifusions were carried out using 15 to 20 hand-picked islets per chamber of 250 μl volume thermostated at 37°C (Brandel, Gaithersburg, MD, USA). The flux was set at 0.5 ml/min and fractions were collected every min following a 20-min washing period at basal glucose. Rat islets were perifused with Krebs-Ringer bicarbonate HEPES buffer at basal 2.8 mmol/l glucose for 20 min, then stimulated with 8.0 mmol/l glucose (20 min) and 16.7 mmol/l glucose (20 min), returning to 2.8 mmol/l glucose (last 10 min).
Pancreata were harvested in cold PBS and treated overnight at 4°C in 4% paraformaldehyde before embedding in paraffin and 5 μm-thick tissue sections were mounted on adhesive-coated slides. Pancreata sections were incubated with a diluted primary antibody for 2 hours at room temperature, and with an appropriate Cy3- (Jackson ImmunoResearch Laboratories, Inc, WestGrove, PA, USA) or ALEXA-conjugated (Molecular Probes, Inc., Eugene, OR, USA) anti IgG serum for 1 hour. The antibodies and their dilution used in the present analysis were as follows: guinea pig anti-insulin (Dako, Carpinteria, CA, USA; dilution 1/400), rabbit anti-glucagon (Dako, Carpinteria, CA, USA; dilution 1/100). Sections were analyzed on a Zeiss Axiophot microscope equipped with an Axiocam color CCD camera (Carl Zeiss, Feldbach, Switzerland).
Data are expressed as mean ± SE, and were analyzed by either unpaired t-test or analysis of variance, using computer software STATISTIK 8 (Analytical Software, St. Paul, Minnesota).
Pancreatic perfusions (in situ)
Isolated islet perifusions (ex vivo)
The kinetics of insulin secretion in islet-perifusion experiments, shown in Figure 2 (panel c and d) indicated that, once isolated, islets from refed and control animals responded similarly to 8.0 and 16.7 mmol/l glucose.
Islets and whole pancreas
Plasma concentrations of hormones on day 7 of refeeding
Glucagon-like peptide-1 (ng/ml)
0.28 ± 0.02
0.24 ± 0.02
Gastric inhibitory peptide (ng/ml)
0.49 ± 0.04
0.55 ± 0.07
1.91 ± 0.10
2.17 ± 0.18
7.34 ± 0.41
11.7 ± 1.60
p < 0.01
Beta-cell function was investigated in a rat model of semistarvation-refeeding in which a high metabolic efficiency for body fat recovery (i.e., thrifty metabolism driving catch-up fat) is intricately associated with hyperinsulinemia . Data show that the hyperinsulinemic state of catch-up growth is characterized at the beta-cell level by enhanced secretory response to glucose stimulation. No difference was observed between refed and controls in the weight of the pancreas, pancreatic islet morphology or insulin content. Accordingly, pancreatic insulin hypersecretion during catch-up growth cannot be attributed to an increase in beta-cell mass or pancreatic insulin content and hence in functional cells, but rather resides primarily in an in situ beta-cell hyperresponsiveness.
Interestingly, such insulin hypersecretion during catch-up growth was observed in the in situ pancreatic perfusion preparation, although not in isolated islets. Therefore, hyperresponsiveness cannot be explained by intra-cellular alterations in metabolism-secretion coupling per se nor in the insulin exocytosis mechanisms. The observed phenomenon is likely to reside in differential modulation of the secretory response, possibly through negative modulators of insulin secretion being repressed during catch-up growth, resulting in the observed hyperresponsiveness of the pancreatic response to glucose. Such in situ islet tuning could be contributed by neuro-hormonal effectors (e.g glucagon-like peptide 1 ), paracrine systems (e.g. dopamine [13, 14]), or even composition of surrounding fatty acids , all these factors being lost once islets are isolated.
The pancreatic insulin hypersecretion during catch-up growth is, however, unlikely be attributed to glucagon-like peptide 1 and gastric inhibitory peptide since these incretins did not differ in refed and control groups in the post-absorptive state (Table 1) nor after a glucose load (data not shown). It is also unlikely to be consequential to excess adiposity and the associated elevation in circulating leptin since our between-group comparison was conducted on day 7 of refeeding, i.e. at a time-point when body fat and plasma leptin in the refed animals had not yet exceeded those of controls (see Figure 1, panel b and Table 1), respectively. Whether our findings of an elevated plasma adiponectin in the refed group versus controls (Table 1) can be implicated in the increased pancreatic hyperresponsiveness to glucose is at present unknown. This is an avenue for further research, particularly in the light of emerging evidence that adiponectin may act directly on pancreatic beta-cells to enhance insulin secretion .
An enhanced beta-cell function, as evidenced by an increased insulin release in response to glucose stimulation, has been observed early in the pathogenesis of type 2 diabetes in animal models [19–21]. It has also been shown to be an early characteristic of ethnic groups and people with normal glucose tolerance at higher risks for diabetes [22–27], and is embodied in the concept that β-cell hyperfunction is an early stage in the progression to β-cell failure . The pancreatic β-cell hyperresponsiveness to glucose during catch-up fat may therefore be a key component in the link between catch-up growth and later risks for type 2 diabetes.
This work was supported by the Swiss National Science Foundation, (grants # 3200B0-113634 to AGD and #310030-120584 to PM).
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