First and foremost, I would like to thank my Lord and Savior, Jesus Christ of Nazareth, for this opportunity to continue my studies. To Dr. To Khathi, my mentor and main supervisor for this project, I would like to express my sincere gratitude for your academic/research input, trust in me, patience and understanding. I would also like to thank Dr. Ngubane, my co-supervisor, for your academic/research input and regular check-ups.
To the endocrinology laboratory and the department of Human Physiology, I would like to thank everyone for their academic and social support. And Cleo from the neuroscience group, Prenan and Simeon for their help during my experiments. I would like to thank Mr Dennis Makhubela, Ms Kogi Moodley and the BRU staff for their assistance with laboratory work and animal work whenever I needed it.
Finally, I would like to thank the College of Health Science and the National Research Foundation for the financial support provided to me during my postdoctoral studies. OGT Oral glucose tolerance PKC Protein kinase C KOH Potassium hydroxide PI3K Phosphoinositol 3-kinase p70S6K Protein S6 kinase.
The diagram depicts the process of glycogen synthesis which depends on a coordinated action of several enzymes. Findings show no significant difference between the ND and HFHCID groups during the 120-minute interval. However, the AUC for the HFHC-D group is approximately four times greater than the ND group.
Bar graph representing the expression (2-ΔCt) of three genes of interest expressed in skeletal muscle of diet-induced prediabetic Sprague Dawley rats.
List of Tables
Manuscript 1
List of appendices
Conference proceedings
Literature review
- Background
- Insulin
- Insulin signalling pathway
- Cbl-associated protein (CAP) pathway
- Phosphatidylinositol-3 kinase (PI3K) pathway
- Type 2 Diabetes mellitus
- GLUT-4
- Pre-diabetes
- Justification
- Aim & Objectives
- List of references
This causes harmful effects in the body and can result in type 2 diabetes mellitus (T2DM), which often leads to death. Thus, this study sought to investigate whether there are any changes in the expression of genes encoded for proteins involved in the insulin signaling pathway during pre-diabetes using a diet-induced pre-diabetic rat model characterized by moderate impairment of insulin sensitivity. In the absence of insulin, glucose uptake decreases and thus increases the mobilization of lipids in adipocytes [29, 32].
The PI3K pathway depicted in Figure 2 begins with the binding of insulin to the α subunit of the insulin receptor, which then causes tyrosine residues in the intracellular β domain of the insulin receptor to undergo autophosphorylation [ 40 ]. Glycogen is a branched polymer of glucose stored primarily in the liver and skeletal muscle [3], which supplies glucose to the bloodstream during fasting periods and to muscle cells during muscle contraction [48]. Therefore, any dysfunction in the insulin pathway can lead to metabolic complications such as T2DM.
In T2DM states, the insulin signaling pathway is generally disrupted, and proteins involved in the insulin pathway, such as protein kinase beta/Akt, mTOR, and GLUT-4, are often affected [ 4 , 52 ]. A human study showed that a mutation in the gene encoding Akt2/PKBβ causes severe IR [ 54 , 59 ]. The literature suggests that changes in the activation of mTOR complexes may be one mechanism to explain altered insulin signal transduction in response to lipid availability [63].
The current study is particularly interested in the mTORC1 pathway because of its association with insulin resistance when it is hyperactivated [63]. The muscle-specific GLUT4 knockout mice characterized by IR are an example that confirms the importance of GLUT-4 in the insulin pathway [72]. Furthermore, macro/microvascular complications seen in overt T2DM have also been demonstrated in the pre-diabetic state [69].
Therefore, the findings will reveal whether the insulin pathway experiences any changes in the prediabetic model used and will expand the understanding of the mechanism leading to insulin resistance and its relationship to genetics in HFHC-D-induced prediabetes. Moreover, the findings will help research to create an effective strategy for pharmacological therapy targeting diabetes/pre-diabetes. High levels of palmitic acid lead to insulin resistance due to changes in the level of phosphorylation of the insulin receptor and insulin receptor substrate-1.
Selective modulation of the CAP/Cbl pathway in the adipose tissue of high-fat diet-treated rats. Akt/PKB activation and insulin signaling: a novel insulin signaling pathway in the treatment of type 2 diabetes.
Manuscript)
Prologue
Title
Abstract
- Introduction
- Materials and methods
- Chemicals and reagents
- Animals
- Experimental diet
- Experimental protocol
- Oral glucose tolerance (OGT) response
- Blood collection and tissue harvesting
- Glycogen Assay
- HOMA2-IR index
- Biochemical analysis 1 Plasma insulin
- Analysis of data
- Results
- Calorie intake and Body weights
The insulin signaling pathway is one of the processes involved in the regulation of glucose homeostasis by facilitating the entry of glucose into tissues, such as skeletal muscle, to either be used as fuel or stored as glycogen [12–14]. This is achieved by activating a downstream target of the insulin pathway known as protein kinase B (PKB, also known as Akt), which in turn phosphorylates and inactivates glycogen synthase kinase-3 (GSK3), an enzyme involved in glycogen synthase phosphorylation and inactivation results in an increase in glucose storage as glycogen [5, 15]. Akt is also responsible for the translocation of glucose transporter-4 (GLUT-4) to the plasma membrane, thereby promoting glucose uptake [5].
Finally, Akt is responsible for the activation of the mTORC1 protein that results in protein synthesis and fatty acid synthesis [16]. Therefore, we sought to investigate the changes that occur in the insulin signaling pathway and in the gene expression of proteins involved in the insulin pathway, using a pre-diabetes rat model. The study used male Sprague-Dawley rats (150-250 g), bred and housed at the University of KwaZulu-Natal Biomedical Research Unit (BRU).
All animal experiments were approved by the University of KwaZulu-Natal Animal Research Ethics Committee (Ethics number: AREC/026/017M; see Appendix 1). Procedures involving animal care were performed in accordance with the University of KwaZulu-Natal Institutional Animal Care Guidelines. The OGT response of all animal groups was monitored in animals according to our established laboratory protocol (21).
The qRT-PCR results were analyzed using the 2-ΔΔCq comparative method to compare the Cq values of the treated groups with the control group. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used to normalize the Cq values of the treated and control groups. The table shows the calorie intake and body weights measured in the last week for both the ND and HFHC-D groups.
Caloric intake along with body weight of the HFHC-D group was significantly higher compared to that of the ND group (p <0.05). OGTT was performed at the culmination of the diet-induced prediabetes period in ND and HFHC-D groups. However, there was a significant difference in the area under the curve (AUC) between the ND and HFHC-D groups.
OGTT
The results show that there was no significant difference between the ND and HFHC-D groups during the OGT response.
OGTT AUC
HFHC-D
The OGTT was performed at the peak of the diet-induced pre-diabetes period in ND and. The glycogen concentration levels were measured at the terminal of diet-induced pre-diabetes period. The results showed that HFHC-D group had significantly higher skeletal muscle glycogen compared to ND (p < 0.05).
Skeletal Muscle Glycogen
- Discussion
- Conclusion
- Shortfalls and future studies
- Acknowledgements
- List of references
- Synthesis)
Compared to ND, there is a significant increase in triglyceride levels in the HFHC-D fed group (p < 0.05). Expression of three genes of interest (GLUT-4, Akt & P70S6) was measured using a quantitative real-time polymerase chain reaction (RT-qPCR). This study investigated potential changes in the insulin signaling pathway in a Sprague Dawley rat model induced by HCHF-D pre-diabetes.
This weight gain is expected during prediabetes because insulin resistance is still moderate in the prediabetic state. The high levels of triglycerides in the blood of the HFHC-D-fed group suggest that their accumulation may be responsible for the development of moderate IR. Interestingly, intermediates of the citric acid cycle shift towards insulin-controlled lipid synthesis in muscle [41], further explaining the high triglyceride levels observed in the HFHC-D group (Table 8).
The findings of this study show increased glycogen concentrations in the HFHC-D fed group compared to the ND group. This correlates with the high insulin levels observed in the HOMA2-IR index table, as insulin is thought to promote glycogen synthesis. We suggest that there is a partial disruption of the insulin pathway, probably due to the twofold increase in the expression of the P70S6 gene encoding the P70S6 protein.
We suggest that the elevated triglyceride concentration observed in HFHC-D-induced prediabetic mice is a contributing factor to changes in the insulin signaling pathway. We suggest that during pre-diabetes, moderate changes in the insulin signaling pathway are already present, due to the current findings of a twofold increase in gene expression of P70S6, which is responsible for the perturbation of the insulin pathway in the IRS . -1 resulting in IR. Also, in the future, western blot analysis of proteins involved in the insulin pathway can be performed to further validate the current findings.
Therefore, the aim of this study is to investigate diet-induced changes in the insulin signaling pathway in prediabetic Sprague Dawley rats. This was supported by higher AUC of the OGT test plot, higher plasma triglycerides and higher HOMA2-IR index in the HFHC-D group compared to ND/control. This is a sign of a poor response to the hormone insulin in skeletal muscle tissue of the HFHC-D group, because the ideal response to insulin would be to return glucose levels to baseline after oral glucose administration.
After this process of prediabetic induction, the next step was to assess the status of the insulin signaling pathway by analyzing the gene expression of genes coding for proteins involved in the insulin pathway and the expression of these proteins. Together, the gene expression findings of Akt, GLUT-4 and p70S6 suggest that there are changes in the insulin pathway, although still at a moderate level.
List of appendixes
Ethical clearance
