Diabetes mellitus (DM) is a major chronic disease, affecting over 300 million people worldwide. Pancreatic islet β-cell dysfunction is known as the major cause of glucose metabolism disorder, which leads to hyperglycemia and eventually DM. Inside the cell, mitochondria perform a crucial role in coupling glucose metabolism to insulin secretion by the β-cell and on other cell functions requiring normal energy balance. Abnormal activity of mitochondria, such as increased apoptosis, contributes to β-cell dysfunction and diabetes related pathological changes in other cell types, for example, depressed growth hormone (GH) secretion was observed in the diabetic state. Cardiac complications of diabetes, also named diabetic cardiomyopathy, account for over 70% of the mortality among diabetic patients, which is characterized as cardiac systolic and diastolic dysfunctions. There are currently no effective targeted treatments. GH secretagogues (GHS) stimulate insulin secretion and GH release in the diabetic rodent model, and protect cardiomyocytes from ischemia/reperfusion injury. To further clarify the mechanism of GHS on diabetes and its cardiovascular complications, the present study employed mouse a β-cell line (MIN6) and streptozotocin (STZ)-induced diabetic rat model treated with the synthetic GHS, hexarelin (Hex), to investigate β-cell function, pulsatile GH secretion and cardiomyocyte contractility.
Diabetes was induced by a single dosage of STZ (65mg/kg) IP injection in male Wistar rats with vehicle control group. After 4 weeks of disease development, animals received Hex (100μg/kg) treatment for 2 weeks. In vitro cell study mimicked the in vivo treatment regime; MIN6 cells were exposed to STZ (1mM) for 4 hours followed by a 2 hour Hex (1μM) incubation. Cell viability and mitochondrial function were measured by MTS and Rhodamine assay. Immunohistochemistry of rat pancreas sections was performed to determine islet morphology and apoptosis signaling pathways. ELISA was conducted to examine pulsatile GH and circulating levels of insulin, insulin-like growth factor-1 (IGF-1) and free fatty acids (FFA). Rat cardiomyocyte contractility and intracellular Ca2+concentration were investigated by cell shortening and Ca2+ transient measurement after cardiomyocyte isolation. Whole-cell patch clamp was performed to study membrane potential and transient outward potassium current (Ito). The expression of the GHS receptor, GHS-R, was determined by Western blot. Apoptotic markers were also assessed by protein expression determination in both MIN6 cells and rat cardiomyocytes. STZ-treated MIN6 cells showed a decrease in cell proliferation and impaired mitochondrial function with increased expression of apoptotic markers, such as caspase-3, caspase-9 and the ratio of Bax/Bcl-2, while incubation of Hex recovered these parameters towards control levels.
STZ-induced diabetic rats showed evidence of symptoms of clinical diabetes such as hyperglycemia, polyphagia, polydipsia and polyuria. Body weight gain was decreased in STZ-treated rats with declines in the weight of heart and adipose tissue. Hex treatment ameliorated hyperglycemia and polyuria in the STZ group. Increased body weight gain was observed in both control and diabetic rats with elevated heart and adipose tissue weight. In addition, Hex elevated insulin release in both the control and diabetic groups, under fasting and normal conditions, which associated with a normalized structure of the rat pancreatic islets and reduced staining of apoptotic proteins, Bax and caspase-9. Correlated with normalized insulin release, increased circulating levels of FFAs in STZ rats were also reduced by Hex, but IGF-1 remained unchanged. Diminished pulsatile GH release was displayed in the STZ-treated group, whereas Hex attenuated this depressed release of GH and increase pulsatile GH levels in control animals. A diabetic cardiomyopathy study revealed a depression of measured parameters in cell shortening and intracellular Ca2+ transient with prolonged APD and suppressed Ito activity. Hex treatment normalized abnormal cell shortening, intracellular Ca2+ transients, APD and Ito activity with up-regulated GHS receptor (GHS-R) and protection against mitochondrial-mediated apoptosis.
In conclusion, Hex was able to ameliorated hyperglycemia via stimulation of insulin secretion and protection against β-cell apoptosis both in vivo and in vitro. The cardio-protective effect of Hex in diabetic cardiomyopathy was confirmed by improvement of contractility and regulation of intracellular Ca2+ homeostasis, associated with increased expression of GHS-R and decreased expression of apoptotic signal molecules. These results provided a potential therapeutic application of Hex in diabetic patients.