DHEA - PC Slows the Progression of Type 2 Diabetes ( NIDM ) in the ZDF /

The etiology of NIDDM is complex and development is manifested by initial insulin resistance coupled with elevated insulin levels in the early diabetic state with concomitant increases in circulating levels of glucose and triglycerides. This is followed by a decline in insulin levels due to pancreatic exhaustion. Our results show that administration of DHEA-PC, a conjugate of dehydroepiandrosterone (DHEA), delayed the development of NIDDM symptoms and the onset of type 2 diabetes in the ZDF/Gmi-fa/fa rat model. The treatment consisted of weekly implantation of subdermal osmotic infusion pumps in the rats starting at 6 weeks of age (n = 5 animals per group). Fr the first three weeks the pumps delivered 6 mg/day/rat followed by 12 mg/day/rat for 1 week (control group pumps delivered only carrier vehicle) after which the pumps were removed. Plasma was collected weekly from day 0 through day 58 and glucose, triglycerides, cholesterol, insulin, IGF-1 and IGF-BP3 levels were measured. Data was analyzed by two way ANOVA. Following 3 weeks of treatment with DHEA-PC, plasma glucose levels in the treated group remained low, 150 +/9 mg/dl while the level in the control animals steadily increased to 320 +/100 mg/dl (p < 0.05). After the DHEA-PC treatment ended plasma glucose plateaued for 10 days and then took 25 days to reach the level in the control animals (p < 0.05). After 2 weeks of DHEA-PC treatment, plasma triglyceride levels in the treated group remained low, 85 +/mg/dl while the level in the control rats increased increased to 180 +/35 mg/dl (p < 0.05). After the treatment was terminated triglyceride levels in the treated group increased to control levels within two days. Insulin, IGF-1,IGF-1-BP3, cholesterol, body weight and food consumption were not changed by DHEA-PC treatment (p < 0.05). Therefor the delay of increases in plasma glucose and triglycerides, caused by DHEA-PC, was not the result of differences in caloric intake, increased insulin, or increased IGF-1 levels. The data suggest that DHEA-PC delayed the onset of the two most important parameters of NIDDM, namely hperglycemia and hypertriglyceridemia. (ZDF/gmi-fa/fa rats and their care supplied by contract with Genetic Models Inc., Indianapolis, IN). INTRODUCTION The etiology of Non Insulin Dependent Diabetes Mellitus (NIDDM) is complex, even though it is the most common form of diabetes. Most people and rodents predisposed to developing NIDDM encounter initial insulin resistance in the early diabetic state. The consequences of advancing NIDDM is the appearance of hyperglycemia and hypertriglyceridemia in circulation. The obese male Zucker diabetic fatty strain of ZDF/Gmi-fa/fa rat are models of type 2 diabetes (1,2). They become hyperglycemic and hypertriglyceridemic as diabetes advances between 7-12 weeks of age and then plateau (3-5). Their diets consist of high fat (6 %) content food (1,5) and their level of physical activitiy is low. They therefor may adequately reciprocate their human diabetic counterpart. Thus, because of their sever diabetic state of hyperglycemia which is greater than other NIDDM models (2), the ZDF/Gmi-fa/fa rat may be appropriate for addressing the pathophysiology of human NIDDM. The ZDF rat like the human NIDDM condition becomes increas-ingly unresponsive to insulin secretion as the condition advances (2). In the hierarchy of circulalting hormones critical to the upor downglucoregulation there are redundancies, down-regulation by insulin is countered by up-regulation of glucagon, epinephrine, growth hormone, IGFs and glucocorticoids. Other factors including adrenal androgens are becoming recognized as regulators of circulating glucose. dehydroepiandrosterone (DHEA) and its3-sulfate ester (DHEAS) are both implicated in down-regulation of glucose. Furthermore, DHEA has a protective effect against development of insulin resistance in muscle of rats on high fat diets or accumulated visceral fat (6,7). In men free DHEAS or testosterone (T) were inversely correlated with glucose and insulin concentrations (8), but DHEA in men did not alter insulin sensitivity (9). In hyperandrogenic women insulin sensitivity seems to depend more on the ratio of DHEA to T (10). The inverse relationship of DHEA or DHEAS to insulin under physiological conditions occurs over a broad age range in men (30-80 yr.) (11). DHEA and DHEAS have been implicated in lipid metabolism. Pharmacological doses administered to animals or in cell culture have reduced the size of fat cells (12,13) and impaired wight gain or lipogenesis (13). Futhermore, DHEA and DHEAS increased protein content of the body mass (14). In adipocytes they inhibited differentiation from fibroblasts (15). DHEA has a protective effect against accumulation of visceral fat and development of muscle insulin resistance in rats fed a high fat diet (16); futhermore, DHEA does not alter food intake. Feeding DHEA to Zucker rats shows reduced hyperphagia and concomitant hypthalemic reduction (17). This dichotomy may indicate that obese animals eat less or that taste aversion may be a factor. DHEA-PC (androst-5-ene-3-phosphocholine-17-one) is a congener of DHEA whose identification and synthesis we have recently described (18). Also its biactivity has been demonstrated in its imune stimulation of Balb C mice sensitized cuntaneously with dinitrochlorobenzene (19). This effect was not suppressed by the potent glucocorticoid, dexamethasone. The aim of this study was to investigate the effects of this newly described hormone, DHEA-PC, on the most significant indicators of Type 2 diabetes in an animal model known for its hyperglycemia and hypertriglyceridemia, namely the ZDF/Gmi-fa/fa obese rat. MATERIAL and METHODS Synthesis: Dehydroepiandrosterone was reacted with chlorophosphoketal to yield the phosphoketal ester of DHEA as per patent # 5,703,063. Treatment with hypochlorite yields the pentavalent phophodioxolane ester. This was then reacted with trimethylamine in pyridine to yield the desired phosphocholine ester of DHEA. Animals: Spontaneously diabetic obese male ZDF/Gmi-fa/fa rats from Genetic Models Inc (Indianapolis, IN) were started out on this study at 6 weeks of age, 80 +/8 g and just weaned. ‘they were housed in individual ages and randomly assigned to treatment groups and maintained on Purina Mills lab diet 5008 and water ad-lib, with a 12 hr light-dark cycle at 25 C. Blood was collected by tail incision on days 0, 3 and at least weekly throughout the study. Food consumption and body weight were determined weekly through out the study. Animal care was given under IACUC oversight and guidelines at the Genetic Models Inc facilities (Indianapolis, IN). Treatment: The animals were individually surgically implanted with (2ML1) Alzet osmotic mini-pumps (Alza corp., Palo Alto, CA) delivering 10 μl/h. Animals were assigned to two groups (n = 5 each) with one receiving pumps filled with 25 mg/ml of DHEA-PC calibrated to deliver a dose of 6 mg per day or when two pumps were implanted 12 mg/day, while controls were implanted with pumps only containing water. the pumps were replaced weekly. anesthesia during the surgical procedure consisted of inhalation isoflurane; USP (Solvay Animal Health Inc.) delivered from the 2-5 minute procedure by an inhalation vaporization system, Isotec III (Matrix Medical Inc., Orchard Park, NY). Animals were allowed to rest for 2 two days post surgery before any blood was collected from the day. During this 54 day study , rats were first treated for 3 weeks at the lower dose of 6 mg.cay, then s the animals became more obese by week 4 they received 12 mg/day, On day 28, treatment was stopped, the pumps were removed, and the animals followed until the end of the study period. the controls were treated in exactly the same way except that the pumps contained no drug. Oral glucose tolerance test A 60% glucose solution at 3 g/kg was administered in the morning by gavage. Blood samples (100 μl) were taken at 0 30, 60, and 120 minutes. The samples were analyzed for glucose as described below. Assays: Plasma glucose, triglycerides, and cholesterol were measured enzymatically using the SychronCX systems, Pechman Instruments, Inc. (Brea, CA) (20-23). RIA of plasma insulin (24) used reagents from Linco Research Inc (St. Louis, MO). the RIAs for IGF-1 (24) and plasma IGF-BP3 (26) used reagents from diagnostic Systems lab (Webster TX). Each assay was done on unextracted plasma and used 125I tracer. The within and between assay percent coefficient of variation were respectively: insulin, 4% and 9%; IGF-1, 5% and 20%: IGF-BP3, 3.2% and 11.5%. Statistical Analysis: The data are preseted as mean +/SEM and statistical analysis were performed using two way ANOVA. post-hoc tests comparing individual differences beawteen means used Scheffe F statistic.