Investigating the effects of diet-induced pre-diabetes on calcium homeostasis in male Sprague Dawley rats.

Abstract

Diabetes mellitus (DM) affects over 400 million people worldwide with 90-95% being type 2 diabetes mellitus (T2DM) in South Africa. T2DM is positively correlated with the chronic consumption of a high caloric diet, often preceded by pre-diabetes. Pre-diabetes is a long-term intermediate stage of hyperglycaemia which is usually asymptomatic. One of the key aetiologies for the complications of physiological systems seen in T2DM has been found to be the chronic intake of high caloric diets. However, dysregulation of these physiological systems seen in T2DM have been reported to begin in pre-diabetes. Calcium homeostasis has been demonstrated to be one of the body's mechanisms that is disrupted in T2DM, leading to changes in calciotropic hormone levels and the functioning of calcium regulating organs. Altered levels of calciotropic hormones in diabetes have been shown to increase the risk of developing insulin resistance and hyperglycaemia. Furthermore, disrupted functioning of calcium-regulating organs in diabetes impairs their responsiveness to calciotropic hormones. A prediabetic rat model was utilized in our laboratory to explore numerous systems and mechanisms in the body, including glucose homeostasis, the cardiovascular system, and immunity, using a high-fat highcarbohydrate diet to induce pre-diabetes. However, there is a paucity in literature elucidating the changes to calcium homeostasis in pre-diabetes. Hence, the present study aimed to investigate the effects of diet-induced pre-diabetes on calcium homeostasis by looking at calciotropic hormones and the functioning of calcium-regulating organs. Materials and Methods Twelve male Sprague-Dawley rats were randomly divided into 2 groups (n=6, each group) whereby the first group: non-pre-diabetic (NPD) group was subjected to standard rat chow and the second group: pre-diabetic (PD) group was subjected to a high-fat high-carbohydrate (HFHC) for 20 weeks. At week 20, the American diabetes association criteria (ADA) were employed for pre-diabetes diagnosis. Plasma was collected for biochemical analysis to measure glucose, insulin, glycated haemoglobin (HbA1c) and the homeostatic model assessment for insulin resistance (HOMA-IR) in addition to urine and plasma calcium concentrations. This was accompanied by measurement of plasma parathyroid hormone (PTH), calcitonin, vitamin D, 1,25-dihydroxyvitamin D3 (calcitriol), osteocalcin and deoxypyridinoline via enzyme linked immunosorbent assay (ELISA). Correlation analysis of calciotropic hormones with HbA1c and HOMA-IR were performed. Furthermore, small intestine and kidney tissue were harvested after the experimental period for analysis of gene expression. Renal expressions of transient receptor potential vanilloid 5 (TRPV5), 1-alpha hydroxylase along with intestinal expressions of vitamin D receptor (VDR) and calbindin-D9k were measured via reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results and discussion

The HFHC diet resulted in moderate hyperglycaemia, elevated plasma insulin, elevated HbA1c and insulin resistance in the PD group by comparison to the NPD group. In the first study, there were increased calciotropic hormone concentrations; plasma PTH, calcitonin, calcitriol and vitamin D in addition to elevated urine calcium and unchanged plasma calcium in the PD group by comparison to NPD. This suggested that elevated calciotropic hormone concentrations in pre-diabetes may compensate for changes to plasma calcium. Furthermore, plasma PTH and calcitonin levels were positively correlated with HbA1c but not insulin resistance in the PD group. Plasma calcitriol concentrations were negatively correlated with HbA1c in the PD group. Altered levels of calciotropic hormones in pre-diabetes may exacerbate the moderate hyperglycaemia in pre-diabetes. In the second study, plasma fasting glucose, insulin, OGT response and HOMA-IR were higher in PD group compared to the NPD. It was observed that normal plasma calcium levels in the pre-diabetic group were accompanied by an upregulation in renal TRPV5, 1-alpha hydroxylase, intestinal VDR and calbindin-D9K expression in addition to increased plasma osteocalcin and decreased urine deoxypyridinoline. Calcium-regulating organs may have responded to disturbed calcium homeostasis by promoting increased intestinal calcium absorption, renal calcium reabsorption in addition to decreasing bone resorption and increasing bone formation. Conclusion The findings suggest that normocalcaemia is maintained in the pre-diabetic state due to compensation from calciotropic hormones and calcium-regulating organs. However, altered levels of calciotropic hormones in pre-diabetes may play a role in the onset of hyperglycemia in T2DM. Due to the cumulative evidence produced in study 1 and study 2, we accept the hypothesis which states that during the pre-diabetic state there will be changes to calciotropic hormones and calcium-regulating organs indicative of disturbed calcium homeostasis.