Browsing by Author "Khathi, Andile."

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The acute effects of dioxidovanadium on blood glucose concentration and oxidative stress in the hippocampus of non-diabetic male Sprague Dawley rats and the chronic effects of dioxidovanadium on selected markers associated with hippocampal dysfunction in male streptozotocin-induced diabetic rats.
(2022) Dayanand, Yalka.; Ngubane, Phikelelani Siphosethu.; Khathi, Andile.; Sibiya, Ntethelelo Hopewell.
Diabetes mellitus is a disease associated with derangements in glucose metabolism and chronic hyperglycaemia. Chronic hyperglycaemia induces oxidative stress and inflammation that affect glucose sensitive hippocampal neurons resulting in generation of amyloid plaques and tau tangles. These are the primary markers used in the detection of neurodegenerative diseases such as Alzheimer’s and dementia. Hence, there is a strong correlation between diabetes and memory impairment. Current therapeutic options such as bolus insulin have been successful in the management of the disease. Despite the efficacy of these therapies, they however have been shown to possess undesirable effects that exacerbate the secondary pathological effects of diabetes on the hippocampus thereby contributing to the detriment of cognitive tasks such as learning and memory. Therefore, there is a need to explore alternative treatments. Transition metals have been shown to possess therapeutic effects with vanadium possessing the greatest potency in lowering blood glucose concentrations. However, studies have demonstrated toxic accumulation of vanadium in the hippocampus which result in the generation of oxidative stress and neurodegeneration. In our laboratory, we have synthesised dioxidovanadium (V) complex by attaching organic ligands to reduce the toxicity and improve potency of the metal. This complex has been shown to efficiently reduce blood glucose and elicit cardio and reno-protective properties. Despite these advancements the effects of this complex on the hippocampus and learning and memory are yet to be established. Therefore, in this study the aim was to evaluate the effect of dioxidovanadium complex on selected learning and memory parameters. Methodology The effect of vanadium on the brain was studied acutely and chronically. In the acute study, animals were separated into 2 groups, non-diabetic control group and a non-diabetic animal group which was were treated with vanadium complex (40 mg.kg-1 p.o). The treatment was administered at time 0. Subsequently an n=3 from each group was sacrificed at regular time intervals (1 hour, 2 hours, 6 hours, 24 hours, 5 days, 10 days) in each group. Blood glucose concentration was monitored before sacrificing and hippocampal tissue was harvested for malonaldehyde (MDA) analysis and glutathione peroxidase (GPx1) and tumour necrosis alpha (TNF-α). The second study was conducted over 5 weeks and consisted of an untreated non-diabetic control, a diabetic control, a positive insulin treated group (0.175 mg.kg-1 s.c) and two dioxidovanadium (V) treated groups (40 mg.kg-1 p.o), a non-diabetic and a diabetic group. Blood glucose was monitored weekly and the Morris water maze was conducted on the last week of the study. After 5 weeks the animals were sacrificed and hippocampal tissue was harvested for malonaldehyde (MDA) analysis, glutathione peroxidase (GPx1) tumour necrosis alpha (TNF-α), amyloid beta (Aβ) and hyperphosphorylated tau (pTau) ELISA’s. Results Acutely, dioxidovandium (V) did not lower blood glucose significantly in comparison to the control group. Interestingly, MDA, GPx1 and (TNF-α) were also not significantly different from the control group over all time periods in the study. Chronically, the glucose concentration of the dioxidovandium (V) treated diabetic group was significantly lowered when compared to the untreated group which displayed significantly increased glucose concentration in comparison to the non-diabetic control. The non-diabetic dioxidovanadium (V) treated group did not show a significant difference in glycaemic level. Increased MDA concentration in the diabetic group was significantly lowered by dioxidovanadium(V) treatment. GPx1 concentration in the dioxidovanadium (V) treated group significantly improved in comparison to the diabetic untreated control. The non-diabetic dioxidovandium (V) treated group showed no significant change in MDA and Gpx1 after the 5-week period. There was no significant difference in TNF-α in dioxidovanadium (V) treated groups, diabetic and non-diabetic. The concentration of Amyloid β was significantly lower in the diabetic control when compared to the non-diabetic control. The dioxidovanadium (V) treated groups, both diabetic and non-diabetic did not have a significant difference in comparison to the diabetic control. pTau concentrations in all groups did not significantly differ. Latency times for the last day of training the Morris water maze followed the same trend. The probe test results, which measured spatial memory, for the diabetic untreated and dioxidovanadium (V) treated groups were significantly reduced in comparison to the non-diabetic control group. The non-diabetic untreated and non-diabetic dioxodivanadium (V) treated were not significantly different. Conclusion Dioxidovanadium (V) treatment in non-diabetic animals did not induce hypoglycaemia acutely however reduced blood glucose concentration in diabetic animals when administered chronically. Dioxidovanadium (V) did not induce oxidative stress and may protect against neurodegeneration by enhancing antioxidant status and therefore was considered as a pro-oxidant in the hippocampus.
An investigation on the effects of a rhenium (V) compound with uracil-derived ligands on markers associated with hepatic, cardiovascular and renal complications in diet-induced prediabetic rats.
(2023) Siboto, Angezwa.; Khathi, Andile.; Ngubane, Phikelelani Siphosethu.; Sibiya, Ntethelelo Hopewell.
Prediabetes is a metabolic disorder that often precedes the onset of type 2 diabetes mellitus. This development of this asymptomatic condition is associated with chronic consumption of high calorie diets and sedentary lifestyles. Prediabetes results in decreased insulin sensitivity in the peripheral tissues resulting in elevated blood glucose levels that are not high enough for a diagnosis of type 2 diabetes. This moderate hyperglycaemia has been shown to lead to trigger complications such as non-alcoholic fatty liver disease, renal dysfunction and cardiovascular disease which are generally only diagnosed during type 2 diabetes. The current management strategy for prediabetes consists of a combination of pharmacological and lifestyle intervention. The pharmacological agents such as metformin while lifestyle intervention involves dietary modification to lower calorie diets. Studies show that prediabetic patients tend to be more dependent pharmacological intervention and struggle with changing diets thus lowering the efficacy of drugs such as metformin. This often leads to the eventual development of prediabetes. Therefore, there is a need for new pharmacological agents that can remain effective in both the presence and absence of dietary intervention. In our laboratory we have synthesised a novel rhenium (v) compound with uracil-derived ligands that has shown promising biological activities that include anti-hyperglycaemic effects in diet-induced prediabetic rats. This compound was shown to improve insulin sensitivity in peripheral tissues in prediabetic rats. To advance from this knowledge, this study sought to investigate the effects of the rhenium (V) compound with uracil-derived ligands on markers associated with hepatic, cardiovascular and renal complications in diet induced prediabetic rats model.
The association between incretin hormones concentrations and the development of diet-induced Prediabetes.
(2023) Mzimela, Nhlakanipho Mphatheni.; Khathi, Andile.; Sosibo, Aubrey Mbulelo.
The increase in the prevalence of type two diabetes mellitus(T2DM) is attributed to unhealthy lifestyles and high-calorie diets. T2DM is a chronic metabolic condition characterised by impaired insulin function and high blood glucose concentration. Prediabetes is an intermediate hyperglycemic condition that frequently occurs before the onset of T2DM. This condition is characterised by a gradual reduction of insulin sensitivity by insulin receptors in insulin-dependent cells, frequently followed by significantly high plasma glucose levels. In this condition, the blood glucose concentration is insufficient to diagnose T2DM. Studies have looked at how incretin peptides affect the pathology of T2DM. However, the link between incretin peptide levels and the onset of prediabetes remains unknown. Additionally, the effect of a low carbohydrate, high unsaturated fat diet on incretin levels during the reversal of prediabetes has not been established. Thus, this study aimed to assess the role of incretin levels in the emergence of prediabetes and the effect of a low carbohydrate, high unsaturated fat diet on incretin levels during the reversal of prediabetes. Methods The first study was conducted using 24 male Sprague-Dawley rats, divided into two groups given a standard rat diet (NPD) (=12), while the other group was given a high-fat high carbohydrate (HFHC) (n=12) diet. Six animals from each group were sacrificed at week 10 and week 20, and blood was collected for biochemical analysis at each time interval. After 20 weeks, the HFHC fed group was found to be prediabetic and were therefore named the prediabetic group (PD). At week 10, the NPD group had the following mean measurements for the NPD and HFHC groups respectively: Glucose (4.3mmol/L and 5.9mmol/L), Insulin (40.26 and 118.32pmol/L), HbAc1 (4.9 and 5.15%), GIP (9.308 and 12.91pmol/L),GLP-1 (18.53 and 15.73pmol/L), Leptin(1.92 and 1.08mmol/L), and Ghrelin (122.1 and 186.5pmol/L ). At week 20, the PD group had the following mean measurements for the NPD and PD groups: Glucose (4.4mmol/L and 7.35mmol/L), Insulin (41.18 and 159.42pmol/L), HbAc1 (4.7 and 6.65%), GIP (10.03 and15.1pmol/L),GLP-1 (21.52 and 6.73pmol/L), Leptin (2.16 and 0.78mmol/L ), Ghrelin (124.2and 210.63pmol/L). After 20 weeks of pre-diabetes induction, the second study began with 18 male Sprague-Dawley rats. Group A continued with the standard diet and was used as a non-prediabetic control (NPDC) (n=6). The pre-diabetic group B (n=12) was split into two experimental groups. One of the groups continued a HFHC diet and served as the pre-diabetic control group (PD)(n=6). In contrast, the other group had a diet intervention where the diet was changed to a low carbohydrate-high unsaturated fats diet (PD+DI) (n=6). All groups were then maintained on their respective diets for a further 12 weeks. At week 32, the PD+DI group had the following mean measurements: Glucose (5.367mmol/L), Insulin (188.5ng/ml), HbAc1 (4.62%), GIP (24.08pg/ml) , GLP-1, Leptin (1.267ng/ml) , and Ghrelin (17.09pg/ml). XVI Results In the first study, after 20 weeks, the HFHC diet resulted in moderate hyperglycaemia, elevated plasma insulin, elevated HbA1c and insulin resistance in the PD group compared to the NPD group. There were also significantly increased GIP and ghrelin concentrations with significantly low GLP-1 and leptin concentrations in the PD group compared to the NPD group. Interestingly, at week 10, there was moderate hyperglycaemia, elevated plasma insulin, elevated HbA1c and insulin resistance in the HFHC group. There were also significant GIP and ghrelin levels with significantly low GLP-1 and leptin concentrations, but there was no prediabetes. In the second study, there were significantly reduced blood glucose levels, plasma insulin levels, HOMA-IR index, and HbA1c in the PD+DI group compared to the PD group. In the PD+DI group, there are significantly reduced GIP and ghrelin levels with significantly increased GLP-1 and leptin concentrations compared to the PD group. However, when the PD-DI group is compared to the NPDC group, there is no significant difference in all measured parameters. Conclusion The first study's findings show that chronic ingestion of a HFHC diet causes dysregulation of incretin hormones from week 10, while prediabetes was only diagnosed at week 20. This dysregulation of incretin hormones precedes the onset of prediabetes and may trigger chronic insulin stimulation, leading to prediabetes development. In the second study, we observed the effect of diet on incretins as they play a significant role in developing and reversing pre-diabetes. Chronic consumption of a HFHC diet led to elevated blood glucose and insulin concentration, resulting in abnormal concentrations of incretins. The abnormal incretins then maintained this state of hyperglycemia and hyperinsulinemia, resulting in prediabetes. Chronic consumption of a LCHF by the pre-diabetic rats led to reduced concentrations of incretins, which could have led to a reduced HbA1c and eventually to the reversal of pre-diabetes. The results of this study suggest that incretin concentrations preceded the development of prediabetes and may even have a role in its development as well as its reversal.
The changes in immune cells concentration during the progression of pre-diabetes to type 2 diabetes in a diet-induced pre-diabetic rat model.
(2019) Mzimela, Nomusa Christina.; Khathi, Andile.; Ngubane, Phikelelani Siphosethu.
T2D has been discovered to be preceded by a long-lasting condition known as prediabetes. The primary cause of prediabetes and T2D has also been shown to be continuous consumption of unhealthy diets and living a sedentary lifestyle. Type 2 diabetic patients have been discovered to have a supressed immune system, but it is still debatable whether immune activation begins at the pre-diabetes stage or during overt T2D. According to literature, T2D is a result of elevated levels of glucose known as hyperglycaemia caused by a condition called insulin resistance. Additionally, T2D has also been shown to be characterised by increased levels of triglycerides, low density lipoproteins (LDL) and decreased levels of high-density lipoproteins (HDL). Insulin resistance then causes metabolic and signalling pathways such as oxidative stress, activation of PKC pathway, formation of advanced glycation end products (AGEs) and shunting of polyol pathway which trigger metabolic inflammation resulting in a dysregulated innate immunity. Dysregulated innate immunity in T2D patients has also been discovered to be due immune response caused by hyperglycaemia. However, it has not been discovered if immune activation occurs at the pre-diabetes stage. It has not been discovered if upregulation of inflammatory markers occurs at prediabetes stage. This study envisaged to characterise the changes that occur in immune cell concentration during the progression of pre-diabetic stage and if there is upregulation of inflammatory markers such as fibrinogen, CRP, CD40L, p-selectin, IL-6 and TNF-α during pre-diabetic stage. To accomplish this, male Sprague Dawley rats were divided into two groups. The first group was fed a high-fat high-carbohydrate diet for 20 weeks to induce pre-diabetes and the second group was fed a normal rat diet for 20 weeks. To confirm if the animals were pre-diabetic, criteria according to American Diabetes Association were used. The animals were then divided into 2 groups which is the pre-diabetic group with 6 animals and a non-diabetic control with another 6 animals. The animals were then further monitored for another 12 weeks (experimental period) while fed the same diet. Blood was collected for haemocytometer analysis on week 0,4,8 and 12 of the experimental periods after which the animals were sacrificed. Plasma was collected from centrifuged blood for ELISA (TNF- α, CRP, P-selectin, CD40 L, fibrinogen & IL-6). Adipose tissue was collected for histology. The results showed a significant decrease in blood percentage count of neutrophils and eosinophils at week 12 experimental period and these immune cells were further observed embedded in-between the adipocytes of adipose tissue. This indicated that neutrophils and eosinophils are produced due to hyperglycaemia and then recruited to the inflamed area such as adipose tissue. The blood percentage count of lymphocytes, basophils and monocytes showed a significant increase at week 12, indicating their increase in production in the bone marrow during immune response. Additionally, the results showed a significant increase in inflammatory cytokines such as TNF-α, IL-6, CRP and P-selectin. The results also showed a slight increase in inflammatory markers such as CD40L and fibrinogen. These finding indicate that there is immune activation during pre-diabetes stage due to changes in immune cells concentration and upregulation of inflammatory markers.
Effects of Momordica balsamina methanolic extract on cardiovascular and haematological function in streptozotocin-induced diabetic rats: effects on selected markers.
(2018) Ludidi, Asiphaphola.; Ngubane, Phikelelani Siphosethu.; Khathi, Andile.
Background The hyperglycaemia-induced haemanetic changes reduces the oxygen-carrying capacity of erythrocytes, thus aggravating cardiovascular disorders in diabetic patients. The conventional therapies have been shown to be associated with the progression of haematological and cardiovascular dysfunction, which may not be favorable for patients with congestive heart failure. We have previously shown the anti-hyperglycaemic and antioxidant properties of Momordica balsamina (MB) methanolic extract which may be of benefit in alleviating cardiovascular disorders, thus providing an effective alternative therapy. The current study therefore, investigated the short-term effects of MB methanolic extract on cardiovascular and haematological function in streptozotocin-induced diabetic rats. Methods Briefly, air-dried MB leaves were extracted with methanol to yield a methanolic extract. STZ-induced diabetic rats were divided into untreated and treated groups with insulin (170 μg kg-1 s.c.) and metformin (500 mg kg-1 p.o.) as standard drugs. MB (250 mg kg-1 p.o.) was administered twice daily for 5 weeks. Blood glucose concentration, body weight and blood pressure were monitored weekly for 5 weeks. Terminally, animals were sacrificed after which blood, heart and kidneys were collected for haematological and biochemical analysis. Histological analysis was also performed on the hearts. Results MB significantly decreased blood glucose concentration from week 3-5 by comparison with diabetic untreated animals. Treatment with MB reduced oxidative stress in the plasma, kidney and heart while improving their antioxidant status compared with untreated diabetic animals. This was associated with increased EPO secretion by the kidneys thus improving RBC production and haemoglobin concentrations. MB moderately increased erythrocyte indices: mean cell volume (MCV), mean cell haemoglobin concentration (MCHC) and mean corpuscular haemoglobin (MCH) by comparison with untreated diabetic animals. MB ameliorated heart hypertrophy and decreased CRP, CT-I and Ang-II concentrations by comparison with untreated diabetic animals. MB also decreased MAP by comparison with untreated diabetic animals.Conclusion MB administration protects against hyperglycaemia-induced cardiovascular and haematological changes by attenuating hyperglycaemia, oxidative stress in both the kidney and heart tissues of STZ-induced diabetic rats, which may reduce the risks of cardiac myopathology complications in diabetes mellitus.
Effects of momordica balsamina on glucose handling in high fat high carbohydrate induced prediabetic rat model and glucose handling in C2C12 induced insulin resistant cell lines: effects on selected metabolic markers.
(2020) Khumalo, Bongiwe.; Ngubane, Phikelelani Siphosethu.; Khathi, Andile.; Sibiya, Ntethelelo Hopewell.
Increased consumption of fat and high carbohydrate coincided with increased prevalence in type 2 diabetes, a condition whose onset is always preceded by prediabetes. Prediabetes is best characterised by hyperglycaemia, insulin resistance and glucose intolerance. Prolong chronic hyperglycaemia exacerbates complications of increased oxidative stress, advanced glycation end products (AGE), dyslipidaemia, hyperinsulinemia and increased inflammatory markers. Once diagnosed, life style and dietary interventions strategies are one of the cornerstones in management of prediabetes. However, patients do not adhere to these life style changes. Hence the present study investigated the effects of Momordica balsamina (MB) on glucose handling in insulin resistance in C2C12 palmitic acid induced insulin resistant cell lines and in high fat high carbohydrate induced prediabetic rat model. Methods Briefly, air-dried MB leaves were extracted with methanol to yield methanolic extracts. The study was divided into 2 experimental series invitro, first series investigated the effects of MB compounds on cell viability in skeletal muscle cell lines. The second series investigated the effects of MB on glucose uptake in palmitic acid induced insulin resistant skeletal muscle cell lines. Invivo studies encompassed HFHC-induced diabetic rats which were divided into untreated and treated groups. The rats were treated with metformin (500 mg kg-1 p.o.) as standard drug and MB (250 mg kg-1 p.o.) a test drug. MB (250 mg kg-1 p.o.) was administered once every third day. Blood glucose concentration, body weight and calorie intake were monitored every fourth week for a period of 12 weeks. Terminally, animals were sacrificed after which blood, liver and skeletal muscle were collected for biochemical analysis. Results MB significantly decreased media glucose concentration whilst glycogen concentration was improved by comparison with insulin resistant cells. Treatment with MB reduced tissue damage which was shown MDA in the plasma while also improving their antioxidant status compared with insulin resistant cells. In vivo study, we measured caloric intake, body weights, ghrelin concentration, OGT response, glycogen concentration, GLUT 4, glycogen synthase, HOMA2- IR value and glycated haemoglobin (HbA1c) concentration. Interestingly, Momordica balsamina coupled with dietary intervention resulted in decreased fasting glucose concentration, suggesting improvement in insulin sensitivity. Reduced caloric intake and restored a steady constant weight growth, thus preventing obesity. This was associated with decreased plasma ghrelin levels. Additionally, there was a significant decrease in HOMA2-IR value. This was further evidenced by decreased levels of glycated haemoglobin in the MB-treated rats. Conclusion The results obtained suggests that Momordica balsamina (MB) possesses anti-hyperglycaemic and protective properties in vivo and in vitro, therefore could be potent in the management of prediabetes, impaired glucose homeostasis induced hyperglycaemia. In addition, these findings provide new scope to comprehensively delineate the medicinal plant, Momordica balsamina mechanism of activity.
Evaluation of the therapeutic properties of a Ruthenium(ll) uracil-derived diimine complex on selected complications associated with diet-induced pre-diabetes.
(2019) Mabuza, Lindokuhle Patience.; Khathi, Andile.; Ngubane, Phikelelani Siphosethu.
Pre-diabetes is a chronic metabolic condition where blood glucose levels are above the upper threshold considered normal but below the threshold for a diagnosis of diabetes. Pre-diabetes predisposes individuals to a high probability of future progression to overt T2DM. Pre-diabetic patients are at increased risk of developing other pathologies such as NAFLD, diabetic nephropathy (DN) and immune dysregulation complications. As a chronic disease, the long-term implications of diabetes contribute to poor quality of life and significantly increase costs associated with healthcare. Pre-diabetes may however be reversible, through the implementation of lifestyle modification programmes based around dietary modification and increased physical activity. Where lifestyle modifications are ineffective, both pharmacotherapy and lifestyle modification are recommended. However, there is reported poor patient compliance in terms of dietary intervention as patients tend to heavily rely on the pharmacological treatment, thus reducing the efficacy of the drug and increasing the possibility to develop T2DM. Hence, there is a need for novel drugs that will remain therapeutic even in the absence of dietary modification. In our laboratory, we have synthesized a novel ruthenium(II) uracil-derived diimine complex that has been shown to improve insulin sensitivity and restore glucose homeostasis in diet-induced prediabetes. In this study, we further sought to evaluate the effects of this compound on selected complications associated with diet-induced pre-diabetes. Estimating whether ruthenium(II) uracil-derived diimine complex in both the presence and/or absence of dietary intervention will show to be effective in the management of prediabetic-related complications. A high fat high carbohydrate (HFHC) diet was used to induce pre-diabetes for 20 weeks. After the induction, pre-diabetic rats were randomly allocated to the following treatment groups: non-prediabetes (NPD); pre-diabetic (PD); metformin plus HFHC; metformin plus normal diet (ND); Ruthenium plus HFHC and ruthenium plus ND. The animals were treated with subcutaneous injection of ruthenium complex (15 mg/ kg) and oral dose of metformin (500 mg/ kg). The rats were treated once a day every third day at 09:00 am for 12 weeks. Every 4 weeks, parameters such as body weight, food intake, fasting blood glucose, fluid intake and urinary output were monitored for 12 weeks treatment period. In study 1, the administration of ruthenium(II) complex resulted in the restoration of liver and body weights in the pre-diabetic treated rats when compared to the PD group. This treatment also reduced liver damage enzyme biomarkers and plasma total bilirubin levels in the pre-diabetic treated rats when compared to the PD group whilst administration of ruthenium(II) with dietary intervention reduced plasma sterol regulatory element binding protein 1c (SREBP-1c) concentration in the pre-diabetic treated rats when compared to the PD group. These findings were further supported by the histological analysis of the liver, showing reduced hepatic lipid droplet accumulation, hepatocyte ballooning and locular disarray in the ruthenium(II)-treated rats when compared to the PD group as seen in chapter 2 of the study. In study 2, the administration of ruthenium(II) complex resulted in reduced blood glucose, aldosterone, fluid intake and urinary output in the pre-diabetic treated rats when compared to the PD group which positively correlated with a restoration in plasma and urinary electrolytes along with plasma antioxidants concentrations in the ruthenium(II)-treated rats. Furthermore, there was a decrease in kidney injury molecular-1 (KIM-1) concentration, albumin excretion rate (AER) albumin creatinine ratio (ACR) and mRNA expression of podocin in urine in the ruthenium(II)-treaded rats. These observetions were further demonstrated by the histological analysis of the kidney, displaying improved histology of renal glomerulus in ruthenium-treated rats when compared to the PD group as seen in chapter 3 of the study. In study 3, treatment with ruthenium(II) complex resulted in reduction of platelet activation markers mean platelet volume (MPV) and CD40 Ligand (CD40 L) concentrations, which positively correlated with decreased plasma triglycerides (TG) and very low-density lipoproteins (VLDL) levels in the pre-diabetic treated rats when compared to the PD group. Whilst administration of ruthenium(II) with dietary intervention reduced plasma fibrinogen concentration in the pre-diabetic treated rats when compared to the PD group. These was further evidenced by normalization of immune cell counts in the ruthenium(II)-treated rats. Furthermore, there was a decrease in pro-inflammatory cytokines, tumor necrosis factor α (TNF-α) concentration in the ruthenium(II)-treated rats and decreased interleukin-1β (IL-1β) concentration in the ruthenium(II) with dietary interventiontreated rats when compared to the PD group as seen in chapter 4 of the study. Taken together, the results observed suggest that ruthenium(II) complex exhibited hepato and renoprotective effects while ameliorating immune dysregulation underlying pre-diabetes in diet-induced pre-diabetic rats. However, further studies are still required to find out the exact mechanism behind potential effect of this metal-based compound.
Investigating changes to the insulin signalling pathway in a diet-induced pre-diabetic rat model: effects on selected markers.
(2019) Sosibo, Aubrey Mbulelo.; Khathi, Andile.; Ngubane, Phikelelani Siphosethu.
No abstract available.
Investigating the association between diet-induced “leaky gut” and the development of prediabetes.
(2023) Dimba, Nosipho Rosebud.; Khathi, Andile.; Ngubane, Philani Smangaliso.
Introduction Type 2 diabetes (T2D) is the most common type of diabetes mellitus, which is reported to be associated with life-threatening co-morbidities. This condition is characterized by hyperglycaemia due to a defect of the insulin receptor, and its often preceded by prediabetes. Chronic consumption of a calorie diets is the primary cause of T2D, and this diet has been associated with altered intestinal permeability in diabetic patient. However, it remains unknown whether increased intestinal permeability complications begin in the prediabetic state. Previous studies done in our laboratory developed a high-fat high carbohydrate (HFHC) diet-induced prediabetic animal model, using male Sprague Dawley rats. This model was found to mimic the human condition of prediabetes. In this model, the animals develop prediabetes after 20 weeks of ingesting a HFHCdiet. Using this HFHC diet-induced animal model of prediabetes, this study sought to investigate the changes on gut microbiota and the association between prediabetes and markers associated with intestinal permeability. Furthermore, this study sought to investigate changes in concentration level of markers associated with a leaky gut and glucose homeostasis, following change to a low carbohydrate, high unsaturated fat (LCHUF) diet. Method and Materials 12 male Sprague Dawley rats (3 weeks old) were randomly assigned into the non-prediabetic group and diet-induced prediabetic group (n=6). Group A animals were exposed to a standard diet with normal drinking water for 20 weeks, and group B animals were exposed to a HFHC diet supplemented with 15% fructose for a period of 20 weeks. After 20 weeks, the American Diabetes Association criteria for diagnosis of prediabetes was used to diagnose prediabetes in all animals. The fecal samples were analyzed to measure the gut microbiota composition of Firmicutes, Bacteroidetes, and Proteobacteria in both animal groups. Furthermore, blood glucose, plasma insulin, serum zonulin, plasma lipopolysaccharide (LPS), soluble CD14 (sCD14), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP), and intestinal fatty-acid binding protein (IFABP) concentrations were measured. The first manuscript measured all these parameters at 20 weeks. In the second manuscript, 12 male Sprague Dawley rats were used and fed a HFHC diet for 20 weeks. The prediabetic animals were subdivided into two groups. Group A animals remained on the HFHC diet (the prediabetic control group), while the other 6 animals in group B were switched to a low carbohydrate, high unsaturated fat (LCHUF) diet. Group B was then categorized as the prediabetic group that had dietary intervention (PD+DI). All animals were then maintained on their respective diets and monitored for further 12 weeks. The fecal samples were analyzed to measure the gut microbiota composition of Firmicutes, Bacteroidetes, and Proteobacteria in both animal groups. Furthermore, blood glucose, glycated haemoglobin (HbA1c), serum zonulin, plasma LPS, sCD14, TNFxvii α, IL-6, CRP, and IFABP concentrations were measured. The second manuscript measured all these parameters at 32 weeks. Results and Discussion Prolonged consumption of a HFHC diet results in the development of prediabetes. This was evidenced by a significant increase in fasting blood glucose and plasma insulin in the prediabetic animals compared to the non-prediabetic animals. The HFHC diet also showed to dysregulate the gut microbiota causing gut dysbiosis which enhances translocation of endotoxins from the gut lumen into the bloodstream that elicits an inflammatory response. In the prediabetic group (PD), there was a reduction in the Firmicutes levels and an increase in Bacteroidetes and Proteobacteria compared to the nonprediabetic group (NPD). Serum zonulin, plasma sCD14, TNF-α, IL-6, CRP, and IFABP concentrations in the PD group were increased compared to the NPD group, while plasma LPS concentrations were similar. The low-grade inflammation that is observed in the prediabetic state is suggested to further progresses onset of prediabetes. However, to reverse prediabetes and to combat a leaky gut problem, the second manuscript illustrated that switching to a LCHUF diet can effectively improve glucose homeostasis thus reverse prediabetes. This was evidenced by a significant decrease in fasting blood glucose and HbA1c concentration. These results were accompanied by a decrease of Firmicutes and an increase of Bacteroidetes and Proteobacteria suggesting that a LCHUF diet effectively improved gut microbiome composition. This caused the release of serum zonulin and its effect on disassembling the tight junctions to decrease. This was evidenced by a decrease in plasma LPS and sCD14 concentration. In addition, we also observed a decrease in plasma TNF-α, IL-6, CRP, and IFABP indicating another beneficial effect of this diet on reducing intestinal inflammation, and risks of insulin resistance. Conclusion Taken together, these results suggest that chronic consumption of the HFHC diet may be associated with the dysregulation of gut microbiota, leading to increased intestinal permeability. This could be associated with chronic subclinical inflammation that have been reported to result in the development of insulin resistance in the pre-diabetic stage. In addition, a LCHSF diet markedly improved intestinal permeability as well as the glucose regulation
Investigating the effects of bredemolic acid on selected markers of some prediabetes-associated dysfunctions in diet-induced prediabetic rats.
(2019) Akinnuga, Akinjide Moses.; Khathi, Andile.; Sibiya, Ntethelelo Hopewell.; Ngubane, Phikelelani Siphosethu.
Prediabetes is an abnormal glycaemic state between normoglycaemia and chronic hyperglycaemia which is currently prevalent in developing and developed countries due to increased consumption of high caloric diet coupled with sedentary lifestyle. Prediabetes is associated with abnormal glucose metabolism. Additionally, the risk of developing prediabetes-associated complications such as non-alcoholic fatty liver disease (NAFLD), cardiovascular and renal diseases is not only present in overt diabetes mellitus but also in prediabetes. Management of prediabetes involves the combination of dietary and pharmacological interventions, however there is reported low compliance among patients as they tend to become overly dependent on the pharmacological interventions. Consequently, the pharmacological intervention efficacy is reduced as patients still progress to having overt diabetes. Therefore, managing prediabetes with anti-diabetic agents that will remain effective even in the absence of dietary intervention is considered necessary. Triterpenes have been found to have potential as anti-diabetic agents. Bredemolic acid (BA), a pentacyclic triterpene, has been reported to have increased biological activity relative to some other triterpenes. In this study, we sought to investigate the effects of BA on selected markers of some prediabetes-associated dysfunctions such as abnormal glucose homeostasis, hepatic, cardiovascular and renal dysfunctions in a prediabetic rat model in both the presence and absence of dietary intervention. Materials and Methods Thirty six (36) Sprague Dawley male rats that weighed 150 – 180g were divided into two groups: the non-prediabetic (n=6) and the prediabetic groups (n=30) which were fed a normal diet (ND) and high fat high carbohydrate (HFHC) diet respectively for 20 weeks to induce prediabetes. At 20th week, prediabetes was confirmed by assessment of fasting blood glucose (FBG) and oral glucose tolerance test (OGTT). The prediabetic rats were further sub-divided into five groups (n=6) and treated with either BA (80 mg/kg) or metformin (MET, 500 mg/kg) in the presence and absence of diet intervention for 12 weeks. Every 4 weeks of treatment, all the animals were placed in metabolic cages to determine caloric and fluid intake as well as urine output. Also, the body mass index (BMI), waist circumference (WC), blood pressure and heart rate were measured at every 4 weeks of treatment. After the 12 weeks of treatment, the animals were sacrificed, blood samples were collected into EDTA sample bottles and centrifuged to obtain plasma. Also, the skeletal muscle, liver, heart and kidney were collected, weighed, snapped frozen with liquid nitrogen and stored at -80°C before the biochemical analysis of selected markers of glucose homeostasis, hepatic, cardiovascular and renal functions. Results In the first study, the untreated diet-induced prediabetic rats had a significantly increased body weight, increased caloric intake, elevated glycated haemoglobin, increased ghrelin plasma concentration, decreased muscle glycogen concentration, insulin resistance and hyperinsulinaemia compared to the non-prediabetic rats. However, BA treatment with or without diet intervention ameliorated the body weight, caloric intake, glycated haemoglobin, muscle glycogen, glucose tolerance, plasma insulin and increased the expression of glucose transporter 4 (GLUT 4) in the skeletal muscle by comparison to the untreated prediabetic rats. Prediabetic induction in the second study resulted into elevated plasma concentration of liver enzymes, increased liver glycogen and triglyceride concentrations, increased oxidative stress in the liver and decreased sterol regulatory element binding protein (SREBP1c) by comparison to the non-prediabetic animals. Conversely, administration of BA with or without dietary intervention ameliorated liver functions by decreased oxidative stress, decreased liver enzymes, decreased liver glycogen and triglyceride as well as increased hepatic SREBP1c concentration in comparison to the untreated prediabetic animals. The results in the third study showed that the untreated prediabetic rats had a significantly increased body mass index (BMI), waist circumference (WC), blood pressure, heart rate, lipid profile, oxidative stress and inflammatory markers with significantly decreased endothelial nitric oxide synthase (eNOS) by comparison to the non-prediabetic control rats. On the other hand, the administration of BA with or without diet intervention improved cardiovascular functions by a decrease in BMI, WC, total cholesterol concentration, triglyceride concentration, blood pressure, heart rate, oxidative stress and inflammation with significant increase in eNOS plasma concentration in comparison to the untreated prediabetic rats. In the fourth study, the untreated prediabetic rats had a significantly increased fluid intake, urine output, sodium retention, potassium loss, aldosterone concentration, albuminuria, proteinuria, kidney injury molecule (KIM-1) and urinary podocin mRNA expression in comparison to non-prediabetic control and BA treated rats with or without diet intervention. Also, the untreated prediabetic rats presented increased albumin, total protein, urea, uric acid, creatinine and oxidative stress markers concentrations with a significant decrease in glomerular filtration rate (GFR). However, administration of BA with or without diet intervention attenuated oxidative stress, decreased urinary podocin mRNA expression and the aforementioned renal dysfunctions parameters. Conclusion This study showed that long term consumption of high caloric diet-induced prediabetes and resulted in abnormal glucose homeostasis, hepatic, cardiovascular and renal dysfunctions. Also, the results of this study showed that these dysfunctions are not only present during overt type 2 diabetes mellitus but already present at the prediabetic stage due to insulin resistance or hyperinsulinaemia that triggered oxidative stress in the physiological systems that we examined in this study. However, due to amelioration of insulin resistance via improved insulin sensitivity and earlier reported antioxidant activities that are common to all pentacyclic triterpenes, administration of BA significantly ameliorated the prediabetes-associated dysfunctions (abnormal glucose homeostasis, hepatic, cardiovascular and renal dysfunctions) with or without diet intervention in the prediabetic stage.
Investigating the effects of diet-induced pre-diabetes on calcium homeostasis in male Sprague Dawley rats.
(2022) Naidoo, Karishma.; Khathi, Andile.; Ngubane, Phikelelani.
Diabetes mellitus (DM) affects over 400 million people worldwide with 90-95% being type 2 diabetes mellitus (T2DM) in South Africa. T2DM is positively correlated with the chronic consumption of a high caloric diet, often preceded by pre-diabetes. Pre-diabetes is a long-term intermediate stage of hyperglycaemia which is usually asymptomatic. One of the key aetiologies for the complications of physiological systems seen in T2DM has been found to be the chronic intake of high caloric diets. However, dysregulation of these physiological systems seen in T2DM have been reported to begin in pre-diabetes. Calcium homeostasis has been demonstrated to be one of the body's mechanisms that is disrupted in T2DM, leading to changes in calciotropic hormone levels and the functioning of calcium regulating organs. Altered levels of calciotropic hormones in diabetes have been shown to increase the risk of developing insulin resistance and hyperglycaemia. Furthermore, disrupted functioning of calcium-regulating organs in diabetes impairs their responsiveness to calciotropic hormones. A prediabetic rat model was utilized in our laboratory to explore numerous systems and mechanisms in the body, including glucose homeostasis, the cardiovascular system, and immunity, using a high-fat highcarbohydrate diet to induce pre-diabetes. However, there is a paucity in literature elucidating the changes to calcium homeostasis in pre-diabetes. Hence, the present study aimed to investigate the effects of diet-induced pre-diabetes on calcium homeostasis by looking at calciotropic hormones and the functioning of calcium-regulating organs. Materials and Methods Twelve male Sprague-Dawley rats were randomly divided into 2 groups (n=6, each group) whereby the first group: non-pre-diabetic (NPD) group was subjected to standard rat chow and the second group: pre-diabetic (PD) group was subjected to a high-fat high-carbohydrate (HFHC) for 20 weeks. At week 20, the American diabetes association criteria (ADA) were employed for pre-diabetes diagnosis. Plasma was collected for biochemical analysis to measure glucose, insulin, glycated haemoglobin (HbA1c) and the homeostatic model assessment for insulin resistance (HOMA-IR) in addition to urine and plasma calcium concentrations. This was accompanied by measurement of plasma parathyroid hormone (PTH), calcitonin, vitamin D, 1,25-dihydroxyvitamin D3 (calcitriol), osteocalcin and deoxypyridinoline via enzyme linked immunosorbent assay (ELISA). Correlation analysis of calciotropic hormones with HbA1c and HOMA-IR were performed. Furthermore, small intestine and kidney tissue were harvested after the experimental period for analysis of gene expression. Renal expressions of transient receptor potential vanilloid 5 (TRPV5), 1-alpha hydroxylase along with intestinal expressions of vitamin D receptor (VDR) and calbindin-D9k were measured via reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results and discussion The HFHC diet resulted in moderate hyperglycaemia, elevated plasma insulin, elevated HbA1c and insulin resistance in the PD group by comparison to the NPD group. In the first study, there were increased calciotropic hormone concentrations; plasma PTH, calcitonin, calcitriol and vitamin D in addition to elevated urine calcium and unchanged plasma calcium in the PD group by comparison to NPD. This suggested that elevated calciotropic hormone concentrations in pre-diabetes may compensate for changes to plasma calcium. Furthermore, plasma PTH and calcitonin levels were positively correlated with HbA1c but not insulin resistance in the PD group. Plasma calcitriol concentrations were negatively correlated with HbA1c in the PD group. Altered levels of calciotropic hormones in pre-diabetes may exacerbate the moderate hyperglycaemia in pre-diabetes. In the second study, plasma fasting glucose, insulin, OGT response and HOMA-IR were higher in PD group compared to the NPD. It was observed that normal plasma calcium levels in the pre-diabetic group were accompanied by an upregulation in renal TRPV5, 1-alpha hydroxylase, intestinal VDR and calbindin-D9K expression in addition to increased plasma osteocalcin and decreased urine deoxypyridinoline. Calcium-regulating organs may have responded to disturbed calcium homeostasis by promoting increased intestinal calcium absorption, renal calcium reabsorption in addition to decreasing bone resorption and increasing bone formation. Conclusion The findings suggest that normocalcaemia is maintained in the pre-diabetic state due to compensation from calciotropic hormones and calcium-regulating organs. However, altered levels of calciotropic hormones in pre-diabetes may play a role in the onset of hyperglycemia in T2DM. Due to the cumulative evidence produced in study 1 and study 2, we accept the hypothesis which states that during the pre-diabetic state there will be changes to calciotropic hormones and calcium-regulating organs indicative of disturbed calcium homeostasis.
Syzygium aromaticum-derived triterpenes modulate intestinal glucose handling in streptozotocin-induced diabetic rats.
(2014) Khathi, Andile.; Musabayane, Cephas Tagumirwa.
Polyphagia in diabetes mellitus ascribed to elevated plasma ghrelin concentrations is associated with prolonged postprandial hyperglycaemia due to increased activities of intestinal carbohydrate hydrolyzing enzymes and glucose transporters. Postprandial hyperglycaemia is a major risk factor in the development of diabetic complications, and as such, should be managed to prevent chronic vascular complications. Previous studies in our laboratory have shown that Syzygium aromaticum-derived oleanolic acid (OA) and maslinic acid (MA) use various mechanisms to lower blood glucose concentrations in experimental diabetes. The effects of these triterpenes, however, on intestinal glucose handling remain unknown. Accordingly, this study was designed to investigate the effects of these triterpenes on intestinal glucose handling in STZ-induced diabetic rats. Materials and methods OA and MA were extracted from Syzygium aromaticum cloves using a previously validated protocol. Briefly, S. aromaticum-derived OA and MA were sequentially extracted with dichloromethane and ethyl acetate to obtain ethyl acetate solubles which contained mixtures of OA/ursolic acid (UA) and methyl maslinate/methyl corosolate. These solubles were purified by silica gel 60 column chromatography with hexane: ethyl acetate solvent systems to produce OA and MA. The structures of these triterpenes were confirmed by using 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy and were comparable with those previously reported in literature. The in vitro studies investigated the inhibitory effects of OA and MA against enzymes such as α-amylase, α-glucosidase and sucrase. Additionally, the effects of various concentrations of OA and MA (0.82 - 6.56 mmol/L) on intestinal glucose transport were investigated using the everted intestinal sacs protocol. The in vivo studies investigated the effects of OA and MA on intestinal carbohydrate handling in separate groups of non-diabetic and STZdiabetic male Sprague Dawley rats. These studies were subdivided into oral glucose tolerance (OGT) responses which were carried out over two hours following loading with various carbohydrates as well as sub-chronic studies that were carried out over 5-weeks where the rats were kept on standard rat chow. OGT responses were monitored in separate groups of nondiabetic and STZ-induced diabetic animals the rats treated with OA and MA (80 mg/kg, p.o.). The rats were loaded with monosaccharides, disaccharides and polysaccharides after an 18-hour fast. The sub-chronic studies investigated the effects of the triterpenes on blood glucose concentrations over 5-weeks in groups of non-diabetic and STZ-induced diabetic male Sprague- Dawley rats. In those animals in which the effects of OA/MA were investigated, the rats were administered with OA/MA (80 mg/kg, p.o.) twice daily. Blood glucose, body weights as well as food and water intake were assessed every third day for the duration of the experimental period. At the end of the experimental period, the rats were killed and blood was collected for plasma insulin and ghrelin measurements. Furthermore, mid portions of the small intestine were snap frozen in liquid nitrogen and stored in a BioUltra freezer at -70 °C for Western blot analysis of glucose transporters, carbohydrate hydrolyzing enzymes and ghrelin expression. Additionally, the effects of OA and MA on intestinal oxidative stress were evaluated through malondealhyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) measurements.Results The in vitro studies revealed that OA and MA possess inhibitory effects on the activity of α- amylase, α-glucosidase and sucrase comparable with those of the standard drug acarbose. In addition, OA and MA significantly (p<0.05) inhibited intestinal glucose transport in the everted intestinal sacs in a dose-independent manner. The OGT studies showed that OA and MA had no significant effects on blood glucose concentrations in non-diabetic rats loaded with the various carbohydrates by comparison with the non-diabetic control. However, the triterpene-treated STZdiabetic rats loaded with the various carbohydrate showed significant (p<0.05) reductions in blood glucose concentrations by comparison with untreated STZ-diabetic rats. OA and MA progressively reduced blood glucose concentration as well as food and water intake over the 5- week study in STZ-induced diabetic rats by comparison with untreated STZ-diabetic rats. Treatment with OA and MA had no effects on plasma insulin concentration in STZ-induced diabetic rats. However, these triterpenes significantly (p<0.05) reduced plasma ghrelin concentrations by comparison with untreated STZ-induced diabetic rats. Furthermore, rats treated with OA and MA showed significant (p<0.05) decreases in ghrelin, SGLT1, GLUT2, α- amylase and α-glucosidase expression in the gastrointestinal tract by comparison with untreated STZ-diabetic rats. This was accompanied by improvements in their intestinal antioxidant status as there were significant (p<0.05) reductions in MDA concentrations with significant (p<0.05) increases in SOD and GPx by comparison with the STZ-diabetic control. Additionally, OA and MA-treated rats showed significant (p<0.05) increases in intestinal glycogen concentrations with concomitant significant (p<0.05) increases in the intestinal expression of glycogen synthase by comparison with untreated STZ-diabetic animals. Discussion The results of the present study indicate that the blood glucose lowering effects of OA and MA in STZ -induced diabetic rats are mediated, in part, via modulating postprandial hyperglycaemia. These findings suggest that this is achieved through the ghrelin-mediated reduction in food intake leading to decreased expression of intestinal carbohydrate hydrolyzing enzymes as well as intestinal glucose transporters. This was followed by significant improvements in the antioxidant status in the rats suggesting that these triterpenes could, by preventing chronic postprandial hyperglycaemia, prevent the onset of the development of diabetic complications. The results of this study are of considerable importance as they suggest another mechanism for the anti-diabetic properties of the triterpenes and further explain the role of the gastrointestinal tract in the management of diabetes mellitus. Conclusion The results of the present study suggest that the S. aromaticum-derived triterpenes possess antidiabetic properties that arise, in part, through the modulation of intestinal glucose handling.