Mechanisms of gestational diabetes mellitus using rat model

PhD Thesis

Gestational diabetes mellitus (GDM) is a form of hyperglycemia due to carbohydrate intolerance that begins during pregnancy. This may be due to insulin resistance or impairment of insulin secretion during pregnancy. Knowledge on the pathophysiology of GDM is important for its management. Thus, the main objective of the current study was to explore the mechanisms of GDM development due to high fat diet (HFD) or heat stress (HST) in a rat model. Specifically, the study was done to evaluate the role of differential adipose tissue (AT) expansion, influence of oxidative stress (OS) and to determine the role of placental cytokine (tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6)) in the development of GDM. The study used Wistar rats as experimental animals. Rats of 8 - 10 weeks old were used and experiments were conducted at the Small Animal Research Unit (SARU), College of Veterinary Medicine and Biomedical Science (CVMBS), Sokoine University of Agriculture (SUA), Morogoro, Tanzania. Samples were analysed in research laboratories in the Departments of Physiology, Biochemistry and Pharmacology and Veterinary Anatomy and Pathology. This thesis is divided into three chapters. Chapter one is on introduction and literature review. It includes a general introduction, animal models for GDM, factors governing the development of GDM, establishing GDM models, histopathology of pancreas during GDM, placenta and GDM, objectives of the study, and organization of the thesis. Chapter two comprises three manuscripts describing the research findings. The first manuscript describes the contribution of HFD to the development of GDM. The second manuscript is on evaluation of how OS due to HST predisposes rats to GDM. Findings on assessment of theiv role of placental cytokines (TNF-α and IL-6) in the development of GDM are presented and discussed in the third manuscript. Lastly, general discussion, conclusion and recommendations of the study are provided in chapter three. Work described in the first manuscript evaluated contribution of HFD in differential AT expansion and subsequent development of GDM in Wistar rats. Pregnant and non- pregnant rats were given streptozotocin (STZ) by a single intraperitoneal injection or HFD throughout the experiment. The animals were sacrificed by a combination of ketamine 50 mg/kg and xylazine 5 mg/kg on days 1, 8, 15, and 21 of the experiment. Blood, AT (both visceral (VAT) and subcutaneous (SAT)) and pancreas were collected and analysed. In this study, STZ-treated animals had a significant increase (p<0.05) in serum glucose and a decrease in insulin, without changes in the size of adipocytes. The levels of both serum glucose and insulin were significantly high in HFD-fed animals (p<0.05); being higher in pregnant (p<0.05) than non-pregnant rats. The increase in glucose and insulin levels was associated with an increase in the size (hypertrophy) than the number (hyperplasia) of adipocytes. The increase in adipocytes was higher in VAT and corresponded to insulin resistance and GDM development than in SAT. Histologically, β-cells were decreased in number and deformed in STZ groups while maintained in HFD groups in both pregnant and non- pregnant animals. This study concluded that intake of HFD during pregnancy leads to AT expansion, which is one of the risk factors for the hyperglycemia and development of GDM. This study demonstrates in the second manuscript the association between HST and GDM. Pregnant and non- pregnant Wistar rats were maintained at 41 - 42°C for 21 days. On days 1, 8, 15 and 21 the animals were humanely sacrificed by a combination of ketamine 50 mg/kg andv xylazine 5 mg/kg. Blood samples were collected from the heart for glucose, insulin, malondialdehyde (MDA) and glutathione peroxidase (GPx) analyses. Pancreatic tissues were fixed in neutral buffered formalin, and processed for histopathology. The findings demonstrated that, in pregnant rats, HST induced a significant increase in glucose in conjunction with a drop in insulin levels than non-pregnant rats (p<0.05). In addition, heat treatment was accompanied by an increase in MDA and a drop in GPx levels. Histological examinations of the pancreas revealed damaged β-cells from day 15 and a reduction in the number of β-cells by day 21 of the experiment in pregnant rats. These results suggest that HST raises the levels of OS in pregnant rats more than in non-pregnant rats and increases the chances of GDM as it is associated with β-cell defects in the pancreas. Findings on serum concentration and placental production of TNF-α and IL-6 of HFD-given rats during pregnancy and their correlation with the development of GDM are presented and discussed in the third manuscript. Pregnant and non- pregnant rats were given STZ single IP injection or HFD throughout the experiment. On days 1, 8, 15 and 21, the animals were humanely sacrificed by a combination of ketamine 50 mg/kg and xylazine 5 mg/kg. Blood samples were collected from the heart for glucose, insulin, TNF-α and IL-6 analyses. Placenta samples were dissected, fixed in neutral buffered formalin, and processed for histopathological and immunohistochemical analyses for TNF-α and IL-6. The levels of serum glucose and insulin were significantly high in HFD-fed animals (p<0.05); being higher in pregnant (p<0.05) than non-pregnant rats. The increase in glucose and insulin levels was associated with an increase in serum levels of TNF-α and IL-6; which were higher in HFD pregnant than non-pregnant animals on day 21 of the experiment. Histologically, placenta tissues of STZ- treated animals were severely congested with blood vesselsvi on days 15 and 21 compared with those from HFD-fed rats which had low congestion on day 21. In both pregnant and non-pregnant rats, immunostaining intensity for TNF-α and IL-6 was high in HFD and STZ-treated animals on day 15 and 21. The findings of this study show that intake of HFD during pregnancy leads to an increase in the levels of IL-6 and TNF-α in the placenta towards the end of gestation resulting in insulin resistance and hyperglycemia that may predispose to GDM. Therefore, Intake of HFD during pregnancy causes AT expansion as well as increase in the levels of placental cytokines (TNF-α and IL-6) resulting in insulin resistance and hyperglycemia, which are risk factors for GDM development. In addition, exposing rats to HST during pregnancy raises the levels of OS which is associated with β-cell defects hence increasing the chances for GDM.

African Centre of Excellence for Rodent Pest Management and Biosensor Technology Development (ACEII-IRPM&BTD) project