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theses

Recently, it was reported in our laboratory that binge-like alcohol drinking for three weeks prior to conception by female rats produces poor birth outcome and defects in the offspring’s body stress response. These effects occur even after alcohol abstinence prior to conception, during pregnancy and postnatal care. Offspring born after preconception alcohol exposures (PCAE) show abnormalities in expression of the stress regulatory gene Pro-opiomelanocortin (Pomc) in the hypothalamus, resulting in higher stress hormone responses to a stress challenge and increased anxiety-like behavior. Since stress is often connected with increased body risk for metabolic diseases and since POMC-expressing neurons are known to control glucose homeostasis, I hypothesized that PCAE disrupts POMC neuronal function to increase susceptibility to developing hyperglycemia in animals. To test this hypothesis, I determined if PCAE had the ability to alter glucose homeostasis and increase the susceptibility to develop high fat diet (HFD)-streptozotocin (STZ)-induced diabetes in the offspring. The aims of my study were to determine the effects of PCAE on pancreatic, hepatic, and hypothalamic POMC/BEP neuronal abnormalities, and how the BEP replacement ameliorates these effects. In this study, 21 female adult Sprague Dawley rats were divided body weight-matched and randomly into three groups; the control group receiving rat chow and water ad libitum (AD; N=7), the alcohol-fed group (AF; N=7) receiving a liquid diet containing ethanol (6.7%), or the calorie-matched pair-fed (PF; N=7) group receiving an isocaloric liquid control diet. After 30 days of feeding, all rats were given normal chow ad libitum for three weeks; the estrus cycle was monitored and bred them with normal adult males to produce offspring. The pups were weaned at the age of postnatal day (PND) 25 and were randomly selected one animal from each litter to have three body weight-matched different groups (AD, PF, and AF, N=7). At the age of two months, offspring of AF exposed animals showed significantly higher fasting blood glucose and leptin and lower insulin and glucagon levels as compared with AD and PF offspring. Oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IPITT) data showed higher blood glucose and lower insulin production in AF in comparison with AD and PF offspring. Further, glucose-stimulated insulin secretion (GSIS) results demonstrated that AF group showed lowest insulin secretion as a response to an oral glucose solution in comparison with AD and PF groups. To evaluate the mechanistic link between PCAE effects on pancreatic homeostasis, I measured inflammatory markers such as; COX-2, IFN-γ, IL-6, and CD3 in whole (endocrine & exocrine) pancreatic tissue, insulin and glucagon in pancreatic islets by immunohistochemistry (IHC). Compared to AD and PF control groups, offspring from AF animals displayed higher levels of inflammatory markers accompanied with low pancreatic insulin and high glucagon expression, indicating the metabolic alterations at pancreas. However, there was no significant change in the expression of Ki-67 and caspase-3 as markers of cell proliferation and cell death, respectively in AF group compared with AD and PF. Further, gene expression analysis at liver showed that PCAE markedly increased several genes related to glucose homeostasis such as forkhead box O1 (Foxo1) and glucose-6-phosphatase (G6pc) and decreased insulin receptor (Insr) and protein kinase b (Akt) levels, which reflected an increase in glycogenolysis. However, Western blot results showed no significant changes in insulin receptors levels at muscular and adipose tissues among all of AD, PF, and AF groups. To evaluate the effect of PCAE on diabetes susceptibility, I induced type 2 diabetes in AD, PF, and AF groups by feeding them on 40% HFD for two weeks followed by single injection of STZ 40mg/kg. My results demonstrated that AF-HFD-STZ group showed the highest blood glucose and glucagon levels and lowest insulin in comparison with AD-HFD-STZ and PF-HFD-STZ groups. TNF-α, IL-6, and IL-1β as markers of inflammation were increased significantly in AF-HFD-ATZ in comparison with AD-HFD-STZ and PF-HFD-STZ animals which can reflect the high susceptibility to diabetes in AF group. In a previous study, our lab found that PCAE has the ability to disturb hypothalamic POMC/BEP neurons in the offspring and that might be the central regulation of initiating inflammatory processes in peripheral tissue, such as the pancreas. In this study, I measured the number of beta-endorphin expressing neurons in arcuate nucleus of the hypothalamus and found that AF group showed the lowest BEP secreting neurons in comparison with AD and PF. I therefore proposed that upregulating hypothalamic POMC/BEP would suppress inflammatory processes and reverse PCAE effect on glucose homeostasis by promoting pancreatic tissue functions and having a better glucose homeostasis. At age of two months, I did the intracerebroventricular (ICV) injection of dibutiryl cyclic adenosine monophosphate (db-cAMP)-delivering nanospheres and considered as a BEP subgroup or plain nanosphere (Sham subgroup) and left one subgroup of animals without surgery for all of AD, PF, and AF groups. I showed that AF-BEP animals demonstrated lower blood glucose and higher blood insulin in comparison with AF-no surgery and AF-Sham subgroups after overnight fasting. There were no changes in basal glucagon and leptin levels. OGTT and GSIS were improved in BEP subgroups of all of AD, PF, and AF. For type 2 diabetes model, I induced a non-insulin dependent diabetic-like state in all of control (no surgery), Sham, and BEP subgroups of AD, PF, and AF subgroups by feeding them on 40% HFD for two weeks followed by single injection of STZ 40mg/kg. BEP sub groups of AD, PF and AF showed lower blood glucose and highest blood insulin in comparison with control-no surgery and Sham surgery sub groups and did not find any change in blood glucagon and leptin level as well. Taken together, PCAE could significantly induce abnormal glucose homeostasis and increased the susceptibility to non-insulin dependent or type 2 diabetes state in the offspring by disturbing hypothalamic POMC/BEP neurons. One mechanism for this dysregulation of glucose homeostasis is a reduction of hypothalamic beta-endorphin (BEP) neuronal numbers and production in the PCAE offspring. The data from this study indicate that, improving BEP neuronal numbers/ expression could attenuate the abnormal glucose homeostasis and lower the risk to diabetes in AF offspring.

ETD_8311

Ph.D.

Includes bibliographical references

by Ali Mosa Rashid Al-Yasari

doi:10.7282/T3MW2M8F

ETD doctoral

The author owns the copyright to this work.