Biochemical, Histopathological and Therapeutic Studies in Alloxan- and Streptozotocin-induced Diabetes Mellitus in Rabbits.

The present experimental study was designed to establish diabetes mellitus in New Zealand white rabbits using diabetogenic drugs so as to investigate/elucidate biochemical, histopathological and behavioural changes/complications. In one group of rabbits diabetes mellitus was induced by intraperitoneal administration of alloxan (@ 80 mg/kg b.w.) and the other group of rabbits was made diabetic using intravenous administration of streptozotocin (@ 65 mg/kg b.w.).Another group of rabbits was kept as control (normal healthy) which received normal saline. The establishment of diabetes mellitus in rabbits was confirmed by periodical elevated levels of fasting blood glucose, blood urea and serum creatinine. The subsequent effect of hyperglycemia on tissue morphology of diabetic rabbits was studied by processing of different organs viz., pancreas, kidneys, liver, lungs, heart, brain and gut of both diabetic and normal rabbits for histological/hiostopathological study using Haematoxylin and Eosin stain and modified Gomori’s staining technique.Digital copy of Thesis.University of Kashmir

Evaluation of antihyperglycemic effect of aloe vera gel extract in normal rats and streptozotocin induced diabetic rats

AIM OF THE STUDY: The aim is to evaluate the antihyperglycemic effect of aloe vera gel extract in normal rats and streptozotocin induced diabetic rats by tail puncture method. METHODS: 24 adult male albino rats weighing 150-200g were selected from central animal house, Madurai Medical College, Madurai. Initially, 18 animals will be divided into 3 groups of 6 animals each. Group I received normal feed, Group II and Group III received aloe vera gel extract 200mg/kg and 400mg/kg orally for 14 days. After washout period of one month, 24 albino rats will be divided into 4 groups of 6 animals each. Group I received normal feed. Group II received Tab. Glibenclamide1mg/kg orally. Group III and Group IV received aloe vera gel extract 200mg/kg and 400mg/kg orally for 14days. The blood glucose level was monitored on day 1, 7 and 14 by tail vene puncture method RESULTS: Aloe vera gel extract of 200mg/kg and 400mg/kg did not produce hypoglycemic effect on day 1, day 7 and day 14 in normal rats. The antihyperglycemic effect of standard drug is highly significant (p < 0.001) & aloe vera gel 200mg/kg and 400mg/kg shown significant antihyperglycemic effect when compared with control group (p < 0.05). The percentage fall in blood glucose levels with standard was 64.1% and aloe vera gel 400mg/kg treated group was 24.6% when compared with control group. CONCLUSION: Aloe vera gel extract 200 mg/kg and 400 mg/kg produce significant reduction in blood glucose level in streptozotocin induced diabetic rats when compared with control group but not in normal rats

Exploring the Role of Insulin Receptor Signaling in Hippocampal Learning and Memory, Neuronal Calcium Dysregulation, and Glucose Metabolism

In the late 90’s, emerging evidence revealed that the brain is insulin-sensitive, highlighted by broad expression of brain-specific insulin receptors and reports of circulating brain insulin. Contemporary literature robustly supports the role of insulin signaling in normal brain function and suggests that insulin-related processes diminish with aging, evidenced by decreased signaling markers, reduced insulin receptor density, and lower levels of insulin transport across the blood-brain barrier. In the context of pathological cognitive decline, clinical trials using intranasal insulin delivery have reported positive outcomes on memory and learning in patients with mild cognitive decline or early-stage Alzheimer’s disease. However, while the importance of insulin and its related actions in the brain are robustly supported, the distinct mechanisms and pathways that mediate these effects remain unclear. To address this, I conducted a series of experiments exploring the impact of insulin on memory and learning in two models: primary hippocampal cell cultures and the Fisher 344 animal model of aging. These studies attempted to identify relationships between insulin receptor signaling, neuronal gene expression, glucose metabolism, and calcium homeostasis in the hippocampus using either expression of a constitutively active human insulin receptor or administration of intranasal insulin. The following dissertation summarizes this work and provides valuable insights into the potential pathways mediating these relationships. Of note, intranasal studies reported that insulin is able to significantly alter gene expression patterns in the hippocampus of both young and aged rats following chronic, repeated exposure to the ligand. In cell culture, constitutive insulin signaling correlated with significantly elevated neuronal glucose uptake and utilization, as well as with significant alterations in the overall expression and localization of the neuron-specific glucose transporter 3. Interestingly, continued activity of the insulin receptor did not appear to alter voltage-gated calcium channels in hippocampal neurons despite prior evidence of the ligand’s role in other calcium-related processes. The results reported in this manuscript suggest that in the brain, insulin may be involved in a myriad of complex and dynamic events dependent on numerous variables, such as age, length of the exposure, and/or the insulin formulation used. Nevertheless, this work highlights the validity of using insulin to ameliorate age-related cognitive decline and supports the need for further studies exploring alternative approaches to enhance insulin receptor signaling in the brain

Glucose Tolerance

The progression from normal glucose tolerance (NGT) to type 2 diabetes involves intermediate stages of impaired fasting glucose (IFG) and impaired glucose tolerance (IGT), also known as prediabetes. The pathophysiology underlying the development of these glucose metabolic alterations is multifactorial, leading to an alteration in the balance between insulin sensitivity and insulin secretion. Our knowledge of the molecular basis of the signaling pathways mediating the various physiologic effects of insulin is steadily advancing. New substrates and signaling molecules have been identified and potential mechanisms involved in the pathophysiology of type 2 diabetes have been revealed. This book summarises the current state of knowledge on the pathophysiology underlying the progression from normal glucose tolerance to type 2 diabetes and therapeutic advances in the improvement of glycaemic control in prediabetic and diabetic states

Effects of Kv1.3 inhibition in type 2 diabetes-induced cardiac electrical remodeling.

217 p.Background: Diabetic patients have prolonged cardiac repolarization and a higher risk ofarrhythmia. Besides, diabetes activatesthe innate immune system, resulting in higher levels of plasmatic proinflammatory cytokinessuch as TNFa and IL1b, which are described to prolong ventricular repolarization.Methods: Most of the current knowledge on diabetic cardiac electrical remodeling derivesfrom type 1 diabetic animals. Here, we characterize a metabolic model of type 2 diabetes withprolonged cardiac repolarization. Sprague-Dawley rats were fed on high fat diet (45% Kcalfrom fat) for 6 weeks, and a low dose of streptozotozin was intraperitoneally injected at week2. Body weight and fasting blood glucose were measured and electrocardiograms to consciousanimals were recorded weekly. Plasmatic lipid profile, insulin, cytokines and arrhythmiasusceptibility were determined at the end of the experimental period. The transient outwardK+ current and action potentials were recorded in isolated ventricular myocytes by patchclamp.Animals were also treated with PAP-1, an inhibitor of Kv1.3 channel that has animmunomodulatory role. Results: Type 2 diabetic animals showed insulin resistance,hyperglycemia and higher levels of plasma cholesterol, triglycerides, TNFa and IL1b thancontrols. They also developed bradycardia and prolonged QTc-interval duration that resultedin increased susceptibility to severe ventricular tachycardia under cardiac challenge. Actionpotential duration (APD) was prolonged in control cardiomyocytes incubated 24h with plasmaisolated from diabetic rats. However, adding TNFa and IL1b receptor blockers to the serum ofdiabetic animals prevented the increased APD. Inhibition of Kv1.3 reduced the circulaitingcytokines including TNFa and IL1B. It had an antidiabetic effect improving insulin resistanceand controlling the glucose while reduced the prolongation of QTc and the susceptibility toarrhythmia in the diabetic rats.Conclusions: The elevation of the circulating levels of TNFa and IL1b are responsible forimpaired ventricular repolarization and higher susceptibility to cardiac arrhythmia in ourmetabolic model of type 2 diabetes. Thus, immunomodulation by inhibition of Kv1.3 hasantidiabetic effects while improving the electrical alterations of the diabetic heart. Theseresults make the immune system a potential target for the treatment of diabetes and itsassociated alterations

Characterization of Murine Cardiac Cholinergic Innervation and Its Remodeling in Type 1 Diabetes.

Murine models have become increasingly popular to study various aspects of cardiovascular diseases due to their ease of genetic manipulation. Unfortunately, there has been little effort put into describing the distribution of autonomic nerves in the mouse heart, making it difficult to compare current findings from clinical and experimental models related to cardiovascular diseases. Furthermore, determination of the requirements for the development of this system and its maintenance in adult mice remains largely unexplored. This study represents the first detailed mapping of cholinergic neuroanatomy of the mouse heart based on immunohistochemical staining using true cholinergic markers. We found cholinergic innervation of the mouse heart to be largely focused in the atrium and conducting system. We investigated the involvement of the neurotrophic factor neurturin (NRTN) in the development of cholinergic innervation, because there was indirect evidence that implicated it as a crucial factor. Results from our work definitively demonstrate that NRTN plays a major role in the development of cardiac parasympathetic ganglia and cholinergic innervation of the mouse heart. Adult NRTN knockout mice exhibited a drastic reduction in the number of intracardiac neurons with decreased atrial acetylcholine, cholinergic nerve density at the sinoatrial node and negative chronotropic responses to vagal stimulation. The presence of NRTN and its receptors in hearts from adult wild-type mice suggests that this neurotrophic factor might also be required for maintenance of cardiac cholinergic innervation. Finally, we wanted to determine how intracardiac neurons and their processes change during diseased states, specifically type 1 diabetes. This work has shown that the cardiac cholinergic nervous system in the mouse undergoes structural and functional remodeling when challenged with streptozotocin-induced diabetes. Cholinergic nerves in diabetic hearts undergo extensive sprouting at the sinoatrial node with no change in the number of intracardiac neurons. Cholinergic function appears to be enhanced in diabetic mice, based on pharmacological testing, despite decreased response to direct vagal nerve stimulation. Evidence also suggests that diabetic mice have an imbalance in autonomic control of heart rate. The latter findings suggest that disruption of central input into intrinsic cardiac ganglia also contributes to the neuropathology of type 1 diabetes

Effects of Streptozotocin-Induced Diabetes on gene and protein expression in Sinoatrial node of rat heart

Diabetes mellitus is one of the most common endocrine disorders and its global prevalence is increasing at an alarming rate. Cardiovascular disease is the major cause of morbidity and mortality among diabetic patients. In addition to defects in contraction, the diabetic heart also frequently suffers from disturbances to the electrical conduction system. This study investigates the effects of diabetes on the sinoatrial node (SAN) of the rat heart. Previous in vivo and in vitro experiments have demonstrated reduced heart rate, longer SAN action potential duration, prolonged pacemaker cycle length and Sino-atrial conduction time in streptozotocin (STZ) - induced diabetic rat heart. Thus it is of paramount importance to identify the molecular basis of electrical disturbances in SAN of diabetic heart. The hypothesis is that alterations in mRNA encoding various proteins associated with the generation and transmission of electrical signals in the heart may be responsible for the functional disturbances in the SAN of diabetic heart. Experiments were performed in male Wistar rats 10-12 weeks of age after STZ treatment with a single intraperitoneal injection (60 mg/kg body weight). Heart rate was measured in isolated perfused heart with an extracellular suction electrode. Expression of mRNA encoding a variety of cardiac proteins, involved in electrical transmission, were measured in SAN and right atrial biopsies using real time reverse transcription polymerase reaction technique. Expression of proteins in SAN biopsies was measured using sodium dodecyl sulphate polyacramide gel electrophoresis and Western blotting technique. Ultrastructure of the STZ-SAN was studied using transmission electron microscopy. The heart rate was significantly lower in STZ compared to controls. Of the 85 genes studied, there were some changes of particular interest. These include an increase in the expression of Gja7 (connexin), Cacna1g, Cacna1h, Cacnb3 (calcium channel), Nppa, Nppb (natriuretic peptide) Kcnj5 and Kcnk3 (potassium channel), in STZSAN. There was also decrease in the expression of Scn7a (sodium channel), Kcna2 (potassium channel), a 6-fold decrease in Kcnd2 and a 7-fold decrease in Cacng4. Some of the proteins encoded by these genes showed similar changes in expression including Ryr3, Cav3.1 and Kv4.2 while others like Pro-ANP and BNP did not. The ultrastructure of STZ-SAN had reduced mitochondria. These changes in gene and protein expression may contribute to the electrophysiological disturbances seen in the diabetic SAN. Further studies will be required to investigate whether these changes in mRNA and protein translate into changes in electrophysiological function