Loss of coupling between calcium influx, energy consumption and insulin secretion associated with development of hyperglycaemia in the UCD-T2DM rat model of type 2 diabetes

Abstract

Aims/hypothesis: Previous studies on isolated islets have demonstrated tight coupling between calcium (Ca(2+)) influx and oxygen consumption rate (OCR) that is correlated with insulin secretion rate (ISR). To explain these observations, we have proposed a mechanism whereby the activation of a highly energetic process (Ca(2+)/metabolic coupling process [CMCP]) by Ca(2+) mediates the stimulation of ISR. The aim of the study was to test whether impairment of the CMCP could play a role in the development of type 2 diabetes.

Methods: Glucose- and Ca(2+)-mediated changes in OCR and ISR in isolated islets were compared with the time course of changes of plasma insulin concentrations observed during the progression to hyperglycaemia in a rat model of type-2 diabetes (the University of California at Davis type 2 diabetes mellitus [UCD-T2DM] rat). Islets were isolated from UCD-T2DM rats before, 1 week, and 3 weeks after the onset of hyperglycaemia.

Results: Glucose stimulation of cytosolic Ca(2+) and OCR was similar for islets harvested before and 1 week after the onset of hyperglycaemia. In contrast, a loss of decrement in islet OCR and ISR in response to Ca(2+) channel blockade coincided with decreased fasting plasma insulin concentrations observed in rats 3 weeks after the onset of hyperglycaemia.

Conclusions/interpretation: These results suggest that phenotypic impairment of diabetic islets in the UCD-T2DM rat is downstream of Ca(2+) influx and involves unregulated stimulation of the CMCP. The continuously elevated levels of CMCP induced by chronic hyperglycaemia in these islets may mediate the loss of islet function.

Fig. 1

Circulating glucose and insulin concentrations at 2 weeks before, and 1 and 3 weeks after, the onset of diabetes in the UCD-T2DM rat. Non-fasting blood glucose (black circle), fasting plasma glucose (white circle) and fasting plasma insulin concentrations (triangle) were plotted as a function of time before and after onset of hyperglycaemia. Values are mean±SEM. *p<0.05 by Student’s t test compared with value at 2 weeks before onset

Fig. 2

Effect of blocking Ca²⁺-influx through L-type Ca²⁺ channels on OCR, Ca²⁺ and ISR by islets isolated from normoglycaemic Sprague–Dawley rats that were stimulated with 20 mmol/l glucose. Islets were perifused in the presence of 3 mmol/l glucose for 90 min then 20 mmol/l for 60 min, followed by the addition of 5 μmol/l nimodipine. OCR and ISR were measured concomitantly using the flow culture system (n=5). Detection of cytosolic Ca²⁺ by fluorescence imaging was measured in separate experiments (n=3). Data are means±SEM and are displayed as the change in signal relative to the steady-state value obtained at 3 mmol/l glucose. Steady-state values of OCR and ISR at 3 mmol/l glucose are listed in Table 2. Statistical analysis was carried out by comparing steady-state values before and after each change in media composition using a paired t test

Fig. 3

Effect of blocking Ca²⁺-influx through L-type Ca²⁺ channels on glucose-stimulated islets from UCD-T2DM rats isolated before (a), and 1 week (b) and 3 weeks (c) after, the onset of hyperglycaemia. Islets were perifused using identical protocols to those described in Fig. 2 except Ca²⁺ was not measured for (b). Data are displayed and were analysed as described in Fig. 2 legend, except that n=8 for OCR and ISR measurements, and n=4 for Ca²⁺ measurements

Fig. 4

Assessment of uncoupling in islets from UCD-T2DM rats. (a) Islets were perifused in the presence of 3 mmol/l glucose for 90 min. The glucose in the perifusate was subsequently raised to 20 mmol/l for 45 min, followed by the addition of a blocker of the F₀F₁ATPase (5 μmol/l oligomycin) and subsequent addition of a mitochondrial uncoupler (1 μmol/l FCCP). OCR and cytochrome c reduction were measured concomitantly using the flow culture system. OCR data is displayed as the change in signal relative to the steady-state value obtained at 3 mmol/l glucose (determined by averaging data obtained in the final 10 min before the increase in glucose). Steady-state OCR at 3 mmol/l glucose was 0.20±0.06 nmol min⁻¹ (100 islets)⁻¹ (n=6). Cytochrome c data were normalised to values obtained in the presence of a blocker of electron flow into cytochrome c (antimycin A) and of electron transfer from cytochrome c (KCN) as 0% and 100% respectively, as done previously [18]. The cytochrome c data were inherently more inaccurate than OCR, and data smoothing was carried out by calculating moving averages over 6-min intervals. Therefore, the kinetics of the cytochrome c data are delayed relative to the OCR data, although the algorithm did not affect the steady-state values that were reached. Data were plotted as mean±SEM. ***p<0.001, comparing steady-state values before and after each change in buffer composition (paired t test). (b) Using the same protocol as in (a), islets harvested from pre-onset (lower curve) and 3 weeks post-onset rats were assessed (n=4). To facilitate comparison, data was normalised to the average value attained during the final 5 min of the high-glucose period. To maintain clarity, error bars (representing the SEM) are only shown in a single direction for each curve. No statistical difference between the two curves was detected

Fig. 5

Insulin content of isolated islets harvested from control rats and UCD-T2DM rats before, and 1 and 3 weeks after, becoming hyperglycaemic. Data are means±SEM, n=3 or 4. ***p<0.001 (Student’s t test)

Fig. 6

Quantification of islet area from pancreas harvested from rats before, and 2 weeks and 10 weeks after, onset of hyperglycaemia. (a–d) Representative images from pancreas sections stained for insulin (brown staining) showing islets from Sprague–Dawley controls (a) and UCD-T2DM rats before the onset of diabetes (b), with recent-onset diabetes (2 weeks after hyperglycaemia) (c) or with long-term diabetes (greater than 10 weeks after onset of hyperglycaemia) (d). The images show the increased damage to islet morphology as diabetes progressed. (e, f). Representative example of output from Visiopharm software where (e) is a stained pancreatic section and (f) is the computer-generated identification of insulin-positive (green) or insulin-negative (pancreas; blue) areas. (g). Beta cell area expressed as total insulin-positive area / total pancreas area assessed. Number of replicates were 4, 5, 7 and 8, for control, pre-onset, 2 weeks post-onset and 10 weeks post-onset of hyperglycaemia, respectively. **p<0.01 (ANOVA), where insulin-positive area for islets harvested 10 weeks post-onset was significantly different from insulin-positive area for both 2 weeks post-onset and pre-onset islets

Fig. 7

Schematic depiction of the normal coupling between Ca²⁺, OCR and ISR, and the observed effects seen in the UCD-T2DM rat model. In normal islets, ISR is controlled by Ca²⁺ influx through L-type Ca²⁺ channels, and a factor generated when metabolic rate is increased, via a highly energetic process we have termed the CMCP. Sustained ISR only occurs when the CMCP is activated by both Ca²⁺ and increased metabolic rate. During the transition from hyper- to hypo-insulinaemia occurring between 1 and 3 weeks after onset of hyperglycaemia in the UCD-T2DM rat, islet function is characterised by continually activated CMCP (i.e. not inhibited by Ca²⁺ channel blockade) and a time-dependent decrease in coupling between CMCP and insulin secretion. We speculate that the continuous operation of the CMCP is energetically unfavourable to islet function and survival