Calcium homeostasis is regulated by calciotropic hormones which act on the bone, kidney and intestine . Several studies have shown that calcium homeostasis is disturbed in type 2 diabetes mellitus (T2DM), a condition that is often preceded by pre-diabetes [12, 22]. Furthermore, disrupted calcium homeostasis has shown to promote the onset of hyperglycaemia and insulin resistance . Pre-diabetes was induced in rats in our laboratory using a well-established protocol that included the chronic intake of a high-fat high-carbohydrate (HFHC) diet [14, 15]. These studies have shown that complications in T2DM such as cardiovascular disorders, immune dysregulation and renal failure begin in the pre-diabetic state [15, 16]. The changes to calcium homeostasis that are associated with T2DM have been well-characterised; however, there is paucity in information for the pre-diabetic state [24, 25]. Therefore, in this study the effects of diet-induced pre-diabetes on calciotropic hormones were investigated. Furthermore, this study sought to also investigate the association of calciotropic hormones with glycated haemoglobin and insulin resistance in the pre-diabetic state. Pre-diabetes is a metabolic disorder characterised by early insulin resistance and blood glucose levels that are over the homeostatic range . Physiologically, increased glucose concentrations promote insulin release and increase insulin in circulation, allowing peripheral tissue to absorb glucose . This accounts for plasma glucose concentrations been conserved within the homeostatic range in normal glucose tolerant (NGT) individuals . After plasma glucose levels become conserved, plasma insulin levels return towards the homeostatic range . Furthermore, glucose levels are not constantly elevated in NGT individuals to promote increased glycation of haemoglobin and insulin resistance . However, in the pre-diabetic state there is increased glucose concentration, insulin secretion, HbA1c and early insulin resistance by comparison to normal glucose tolerant individuals [2, 15]. During pre-diabetes, plasma insulin fails to stimulate a response in insulin-targeted peripheral tissue such as skeletal muscle . The accumulation of plasma glucose results in enhanced glycation with haemoglobin . As a result, pancreatic β-cells respond by secreting a greater quantity of insulin to overcome the high glucose concentrations resulting in compensatory hyperinsulinemia . In this study, the fasting glucose concentration, plasma insulin concentration, HbA1c and HOMA-IR index were significantly higher in the PD group than the NPD group. These results corroborated with previous findings that have shown elevated plasma glucose, insulin, HOMA-IR and HbA1c in pre- diabetic patients by comparison to normal glucose tolerant individuals [29, 30]. This study further validates that the consumption of the HFHC diet promotes the development of pre-diabetes, as seen by the impaired fasting glucose, insulin, HbA1c and HOMA-IR value in the range of insulin resistance. This suggests that the body's ability to use glucose in insulin-dependent tissues has been disturbed . The excessive intake of dietary fats, which has shown to increase triacylglycerides levels, may have contributed to insulin resistance . The increased exposure of triacylglycerides to insulin-dependent peripheral tissue induces insulin resistance . Consequently, pancreatic beta (β)- cells may have produced more insulin to compensate for the elevated plasma glucose that is found in insulin-resistant tissue . Calcium homeostasis has been found to be disrupted by elevated plasma glucose concentrations and insulin resistance in T2DM [22, 32].
Calcium is needed for a variety of bodily functions, including signal transmission, secretion of hormones and mineralization of bone . Plasma calcium levels are regulated by PTH, calcitonin and 1,25-dihydroxyvitamin D3 also known as calcitriol which act on the bone, kidney and small intestine . When plasma calcium levels fall below normal, the chief cells of the parathyroid gland produce PTH, which stimulates calcitriol synthesis [6, 22]. The parafollicular cells of the thyroid gland on the other hand, are triggered to release calcitonin when plasma calcium levels rise above normal [6, 22]. Some studies have shown that plasma calcium concentrations are within the homeostatic range in type 2 diabetic patients [33, 34]. These studies have stated that plasma calcium levels do not change significantly due to calciotropic hormones maintaining a constant plasma calcium concentration, despite variations in calcium excretion [33, 34]. In this study, the PD group displayed no significant change to the plasma calcium concentrations by comparison to the NPD group. These results coincided with prior literature that showed no significant change in calcium levels in plasma among T2DM patients [33, 34]. However, these results contradicted other studies that have found either hypocalcaemia or hypercalcaemia among T2DM patients [7, 25]. The possible reason for no significant change to plasma calcium levels in the PD group of this study may have been due to calciotropic hormones compensating for the changes to plasma calcium concentration. The significant alterations to plasma calcium levels in studies on T2DM patients may have been due to the failure of the body to compensate for the changes to plasma calcium levels [32, 35]. In the current study, it was observed that calciotropic hormones had compensated for changes to plasma calcium levels which may have occurred as a result of pre-diabetes.
The concentrations of plasma calcium are kept within the homeostatic range by calciotropic hormones . Calcium levels in the plasma control the secretion of PTH, calcitriol and calcitonin . Parathyroid hormone with aid from calcitriol increase plasma calcium levels by promoting an increase in calcium absorption in intestine, renal calcium reabsorption and bone resorption whereas calcitonin promotes the opposite . Previous research has shown increased concentrations of PTH and calcitonin in plasma of type 2 diabetic individuals [37, 38]. According to studies the increased PTH concentration may have compensated for the increased calcium loss in urine and the increased plasma calcitonin concentration may have protected the body against the adverse effects of PTH oversecretion [13, 39]. The harmful effects associated with elevated PTH secretion include hypercalcaemia and bone loss . In this study, the plasma PTH and calcitonin concentrations in the PD group were significantly higher than the NPD group. These findings validated prior research that has shown elevated levels of PTH and calcitonin in plasma of T2DM patients [11, 40]. In the PD group of this study, the elevated plasma PTH and calcitonin concentrations may have been ascribed to the coordination between PTH and calcitonin, in order to stabilize plasma calcium levels. Additionally, PTH levels in plasma may have risen to compensate for the increased urine calcium loss. This may lead to secondary hyperparathyroidism which has shown to be associated with impaired glucose tolerance, decreased insulin sensitivity and increased risk of T2DM . Plasma calcitonin concentration may have increased to compensate for the elevated plasma PTH levels in the PD group.
The kidneys, bone and small intestine regulate urinary calcium homeostasis under the influence of PTH and calcitriol . Nearly 98% of filtered calcium by the kidneys is reabsorbed back into the bloodstream via the renal tubules, which helps to maintain plasma calcium levels . About 60–70% of filtered calcium is reabsorbed in the kidney's proximal tubule . The remaining calcium is reabsorbed along the ascending limb of Henle and distal convoluted under the influence of PTH . It is estimated that less than 2% of filtered calcium is lost in urine due to renal calcium reabsorption . Type 2 diabetes mellitus promotes renal calcium wastage by altering renal functioning, calcium transport mechanisms and damaging renal tubular cells [7, 32]. Furthermore, compensatory hyperinsulinemia as a consequence of insulin resistance has shown to inhibit renal calcium reabsorption in T2DM patients [42, 43]. In this study, the urinary calcium levels in the PD group were significantly higher by comparison to NPD. Furthermore, the urinary calcium levels in the PD group were above the homeostatic range of 15 mmol/L-20 mmol/L, indicative of hypercalciuria . These findings coincide with recent studies that have shown increased urine calcium levels in T2DM patients [45, 46]. Renal impairment is present in the pre-diabetic state, according to studies conducted in our laboratory [16, 19]. The elevated urinary calcium concentration in the PD group may have been due to impaired renal regulatory function induced by pre-diabetes. Furthermore, high glucose levels in the PD state may have directly damaged mechanisms of calcium transport in the renal tubule . In addition, studies in human and rodent models have shown that increased urinary calcium levels were associated with elevated plasma insulin levels [7, 10]. Compensatory hyperinsulinemia as a consequence of insulin resistance has shown to inhibit renal calcium reabsorption . Hyperinsulinemia-induced by pre-diabetes may have promoted increased urinary calcium loss by inhibiting renal calcium reabsorption. Additionally, increased renal calcium load may have also contributed to the higher concentrations of calcium in the urine of the PD group. Diets that have content high in protein have been demonstrated to cause metabolic acidosis, which is manifested by the elevated loss of calcium in urine . The increased renal acid load contributes to bone breakdown and consequently promote hypercalcaemia . Therefore, the kidneys may try to compensate for the increased plasma calcium by excreting it into urine.
Vitamin D is a fat-soluble vitamin that plays a significant role in regulating whole body calcium homeostasis within the endocrine system . Vitamin D is the precursor molecule needed for calcitriol synthesis . Calcitriol is vitamin D in its hormonally active form responsible for increasing plasma calcium concentrations . Previous studies have found reduced plasma vitamin D and calcitriol levels among T2DM individuals [49, 50]. These studies have shown that impaired renal reabsorption of vitamin D binding proteins, impaired intestinal vitamin D absorption and increased sequestration of vitamin D by adipose tissue may be responsible for decreased plasma vitamin D and calcitriol concentrations in T2DM [39, 43]. Furthermore, studies have shown that calcitriol production is impaired due to renal damage, low plasma PTH levels and PTH resistance in T2DM [47, 51]. In this study, the PD group had significantly higher plasma vitamin D and calcitriol levels by comparison to the NPD group. These results contrasted previous findings which have shown decreased plasma vitamin D and calcitriol in T2DM individuals [49, 50]. The PD group in this study was fed a diet high in saturated fats and carbohydrate content by comparison to the standard diet. Diets that contain high fat content have shown to stimulate bile secretion and consequently enhanced intestinal vitamin D absorption [52, 53]. It is plausible that the increased dietary fat content in the HFHC diet may have promoted an increase in vitamin D absorption, accounting for the higher plasma vitamin D levels in the PD group. In addition, the increased calcitriol levels in the PD group may have been a compensatory response to reduced plasma calcium levels and elevated plasma PTH levels. Elevated plasma PTH levels induce an increase in renal-1 alpha hydroxylase expression . Renal1- alpha hydroxylase catalyses’ the conversion of calcifediol to calcitriol, promoting an increase in plasma calcitriol levels as seen in this study .
Previous studies have shown that calciotropic hormones participate in the regulation of glucose homeostasis by modulating effects on gluconeogenesis, glycogenolysis and insulin-signaling [23, 39]. Studies have shown that plasma PTH and calcitonin levels were positively correlated with hyperglycaemia and insulin resistance in T2DM [40, 47]. Conversely, previous studies have shown that calcitriol levels in plasma were inversely correlated with hyperglycemia and insulin resistance [12, 40]. Elevated PTH and calcitonin levels were found to promote insulin resistance and hyperglycaemia by disrupting the insulin signaling pathway, increasing hepatic glycogenolysis and gluconeogenesis . In contrast, calcitriol has been shown to improve the sensitivity to insulin and tolerance of glucose . In this study, plasma PTH and calcitonin levels were positively correlated with HbA1c but not with insulin resistance in the PD state. Furthermore, plasma calcitriol levels were negatively correlated with HbA1c in the PD group. These results corroborated with previous findings that reported that plasma PTH, calcitonin and calcitriol were associated with HbA1c [39, 40]. However, the lack of association between plasma PTH, calcitonin and calcitriol with insulin resistance contrasted previous studies that have reported associations between these hormones with insulin resistance [23, 32]. These observations may suggest that the elevated plasma PTH and calcitonin levels due to disrupted calcium homeostasis may contribute to the elevated HbA1c concentrations in the PD state. Elevated plasma PTH and calcitonin levels from disrupted calcium homeostasis may stimulate gluconeogenesis and glycogenolysis . It may be speculated that early insulin resistance and beta dysfunction in the PD state may create an unfavourable environment, where increased glucose production stimulated by elevated PTH and calcitonin levels may not be compensated for in a pre-diabetic individual. This may lead to a vicious cycle where disrupted calcium homeostasis in the PD state may promote the development of hyperglycaemia in T2DM. The significant negative correlation between plasma calcitriol and HbA1c in the PD state may suggest a protective role of calcitriol against intermediate hyperglycaemia in the PD state.
The findings of this study may serve of clinical importance in the early detection of type 2 diabetes mellitus. These results may increase our understanding of calcium homeostasis and may provide a possible insight into potential target sites in the treatment of overt T2DM and its associated complications. Furthermore, these findings have elucidated the mechanism of calcium homeostasis in a pre-diabetic rat model. Due to the similarity in the genetic variability of Sprague Dawley rats and humans, these findings may provide an understanding of calcium homeostasis in pre-diabetic humans. Since the current study indicated that some of the complications associated in T2DM such as hyperparathyroidism and hypercalciuria begins in the pre-diabetic state, it is worth translating this research to human studies as a future prospective.
It was evident that hypercalciuria is present in the PD state, an indication of disturbed calcium homeostasis. In addition, plasma calcium levels may have been conserved due to elevated plasma PTH, calcitonin, vitamin D and calcitriol levels in the PD state. Furthermore, plasma PTH and calcitonin levels were positively correlated with HbA1c but not with insulin resistance in the PD state. In addition, plasma calcitriol levels were negatively correlated with HbA1c in the PD state. Although calciotropic hormones try to maintain calcium homeostasis in pre-diabetes, elevated levels of PTH and calcitonin may promote the development of hyperglycaemia in T2DM.