Effects of Ca2+, microRNAs, and rosuvastatin on insulin-secreting beta cell function

Type 2 diabetes (T2D) is a condition of high blood glucose levels due to insulin resistance and defective insulin secretion. Impaired insulin secretion plays a major role in the pathophysiology of T2D, it is mainly attributed to beta cell function i.e. failure to secrete insulin or reduced beta cell mass. The exocytotic process is crucial for beta cell function and its dysregulation leads to impaired insulin secretion. Therefore, understanding the central mechanisms involved in the regulation of exocytosis is essential to recognize possible targets for therapeutic intervention and treatment of T2D. In this thesis I have investigated the role of Ca2+, miRNAs and rosuvastatin in the regulation of ion channels, exocytosis and insulin secretion of beta cells. For this purpose, pancreatic rat INS-1 832/13 beta cells, rodent animal models, and islets from human cadaver donors has been used. Whole-cell patch clamp was used to study exocytosis measured as changes in cell membrane capacitance. In beta cells, biphasic exocytotic pattern was previously mainly attributed to insulin granule pool depletion. In paper I, we used the pulse length protocol and mixed-effect modelling; the latter takes care of cellular heterogeneity, to study exocytosis as a function of Ca2+ influx (measured as Q). The data suggests that pool depletion plays a minor role in observed biphasic exocytotic pattern in INS-1 832/13 cells; instead exocytosis is mostly determined by the kinetics of Ca2+ current inactivation. In paper II and III, we have investigated the effects of miRNA modulation on insulin secretion and exocytosis. First we investigated miRNA-regulation of voltage-gated Na+ channels since their role in beta cell function is not yet clear. Down-regulation of miR-375 differentially affected Na+ channel inactivation properties in INS-1 832/13 cells and miR-375 knock-out mice beta cells, suggesting species differences. As steady-state inactivation determines the number of channels available for generation of action potential, this study is a proof of principle that mir-375 could be important in regulating electrical activity in human beta cells. Next, miRNA-regulation of the exocytotic process was investigated. Overexpression of miR-335 reduced exocytosis and thereby insulin secretion through decreased expression of the exocytotic proteins STXBP1, SNAP25 and SYT11. In this paper I also made the novel observation that SYT11 increase basal insulin secretion and decrease rapid exocytosis, two phenomenons associated with T2D. The work on miR-335 emphasizes the importance of miRNAs in the regulation of the exocytotic process. In paper IV and V the effects of the cholesterol-lowering drug rosuvastatin was investigated. Rosuvastatin treatment dose dependently affected Ca2+ influx, exocytosis, basal and glucose-induced insulin secretion in INS-1 832/13 cells. Interestingly, most of this effect was though mevalonate pathway and not from the cholesterol lowering ability of rosuvastatin. In vivo rosuvastatin had an overall positive effect on glucose homeostasis in mice but negative effects on beta cell function such as disturbed Ca2+-signalling. In conclusion, the data in my thesis demonstrate the need for investigations of the mechanisms behind defective insulin secretion and exocytosis in order to understand and treat T2D

FORMULATION AND EVALUATION OF GASTRORETENTIVE TABLETS OF ANTIULCER DRUG

ABSTRACTGastroretentive floating drug delivery system is utilised to target drug release in the stomach or to the upper part of intestine. Lansoprazole is proton pump inhibitor intended for oral administration used as antiulcer agent. The present investigation involved formulation and evaluation of Gastroretentive floating tablets of Lansoprazole for prolongation of gastric residence time with a view to deliver the drug at sustained and controlled manner in gastrointestinal tract. The tablets of Lansoprazole were prepared by direct compression method using gas generating agent and different polymer combinations (HPMCK4M, HPMC K100M, Psyllium husk) . The prepared tablets of Lansoprazole were evaluated for hardness, thickness, friability, weight variation, drug content uniformity, buoyancy lag time, total floating time, swelling index, in-vitro dissolution study. The varying concentration of gas generating agent and polymers were found to affect on in-vitro drug release, floating lag time and swelling index. In vitro drug release of floating Gastroretentive tablet of Lansoprazole shown that the formulation F2 was found to be the best formulation as it releases 97.9% Lansoprazole in a controlled manner for extended period of time (upto 12 hrs.)Keywords: Lansoprazole, Gastroretentive, floating tablet, total floating time

FORMULATION AND EVALUATION OF MICROSPONGE GEL FOR TOPICAL DELIVERY OF ANTIFUNGAL DRUG

Objective: The purpose of present study aims to design novel drug delivery system containing oxiconazole nitrate microsponges and to prepare microsponge gel. Oxiconazole nitrate is an antifungal drug used in the treatment of fungal infection having a poor aqueous solubility, side effects and adverse reactions. The microsponge delivery system is unique technology for controlled release of active agents. Methods: The microsponges were prepared by quasi-emulsion solvent diffusion method by using polymer eudragit S-100 and eudragit L-100. All the formulated microsponges were subjected for various evaluation parameters such as production yield, encapsulation efficiency, particle size analysis and in vitro drug release study. The optimised microsponge formulation F3 and F9 were further formulated as gel formulation for topical delivery. Prepared gel was evaluated for physical parameters like pH, spreadability, viscosity, drug content and in vitro diffusion study and compared with the marketed formulation.Results: The Fourier transform infrared radiation measurement (FTIR) and Differential scanning colorimetry (DSC) of drug and excipient confirm compatibility. Results revealed that quasi-emulsion solvent diffusion method is a suitable technique for the preparation of microsponges as most of the formulations were discrete and spherical in shape with a good production yield of 61.44% to 80.45% and The highest drug release for F3 and F9 formulation was found to be 87.77 % and 83.24 % respectively for the 8 h. The microsponge gel formulation MGI (F3) showed the controlled release of oxiconazole nitrate for 12 h. The drug release data of optimised batch MGI (F3) were fitted into different kinetic models and showed that the drug release from gel formulation follows zero order release.Conclusion: As compared to conventional formulation, the prepared microsponge gel are expected to remain on the skin for a longer time, gradually releasing their contents over the time. Hence, oxiconazole nitrate microsponges and microsponge gel prepared in this study are promising as being more useful than conventional formulation therapy

Regulation of Pancreatic Beta Cell Stimulus-Secretion Coupling by microRNAs.

Increased blood glucose after a meal is countered by the subsequent increased release of the hypoglycemic hormone insulin from the pancreatic beta cells. The cascade of molecular events encompassing the initial sensing and transport of glucose into the beta cell, culminating with the exocytosis of the insulin large dense core granules (LDCVs) is termed "stimulus-secretion coupling." Impairment in any of the relevant processes leads to insufficient insulin release, which contributes to the development of type 2 diabetes (T2D). The fate of the beta cell, when exposed to environmental triggers of the disease, is determined by the possibility to adapt to the new situation by regulation of gene expression. As established factors of post-transcriptional regulation, microRNAs (miRNAs) are well-recognized mediators of beta cell plasticity and adaptation. Here, we put focus on the importance of comprehending the transcriptional regulation of miRNAs, and how miRNAs are implicated in stimulus-secretion coupling, specifically those influencing the late stages of insulin secretion. We suggest that efficient beta cell adaptation requires an optimal balance between transcriptional regulation of miRNAs themselves, and miRNA-dependent gene regulation. The increased knowledge of the beta cell transcriptional network inclusive of non-coding RNAs such as miRNAs is essential in identifying novel targets for the treatment of T2D

Doc2b Protects β-Cells Against Inflammatory Damage and Enhances Function

Loss of functional β-cell mass is an early feature of type 1 diabetes. To release insulin, β-cells require soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, as well as SNARE complex regulatory proteins like double C2 domain-containing protein β (Doc2b). We hypothesized that Doc2b deficiency or overabundance may confer susceptibility or protection, respectively, to the functional β-cell mass. Indeed, Doc2b+/- knockout mice show an unusually severe response to multiple-low-dose streptozotocin (MLD-STZ), resulting in more apoptotic β-cells and a smaller β-cell mass. In addition, inducible β-cell-specific Doc2b-overexpressing transgenic (βDoc2b-dTg) mice show improved glucose tolerance and resist MLD-STZ-induced disruption of glucose tolerance, fasting hyperglycemia, β-cell apoptosis, and loss of β-cell mass. Mechanistically, Doc2b enrichment enhances glucose-stimulated insulin secretion (GSIS) and SNARE activation and prevents the appearance of apoptotic markers in response to cytokine stress and thapsigargin. Furthermore, expression of a peptide containing the Doc2b tandem C2A and C2B domains is sufficient to confer the beneficial effects of Doc2b enrichment on GSIS, SNARE activation, and apoptosis. These studies demonstrate that Doc2b enrichment in the β-cell protects against diabetogenic and proapoptotic stress. Furthermore, they identify a Doc2b peptide that confers the beneficial effects of Doc2b and may be a therapeutic candidate for protecting functional β-cell mass

The actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAK1)-mediated glucose uptake into skeletal muscle cells

Defects in translocation of the glucose transporter GLUT4 are associated with peripheral insulin resistance, preclinical diabetes, and progression to type 2 diabetes. GLUT4 recruitment to the plasma membrane of skeletal muscle cells requires F-actin remodeling. Insulin signaling in muscle requires p21-activated kinase-1 (PAK1), whose downstream signaling triggers actin remodeling, which promotes GLUT4 vesicle translocation and glucose uptake into skeletal muscle cells. Actin remodeling is a cyclic process, and although PAK1 is known to initiate changes to the cortical actin-binding protein cofilin to stimulate the depolymerizing arm of the cycle, how PAK1 might trigger the polymerizing arm of the cycle remains unresolved. Toward this, we investigated whether PAK1 contributes to the mechanisms involving the actin-binding and -polymerizing proteins neural Wiskott-Aldrich syndrome protein (N-WASP), cortactin, and ARP2/3 subunits. We found that the actin-polymerizing ARP2/3 subunit p41ARC is a PAK1 substrate in skeletal muscle cells. Moreover, co-immunoprecipitation experiments revealed that insulin stimulates p41ARC phosphorylation and increases its association with N-WASP coordinately with the associations of N-WASP with cortactin and actin. Importantly, all of these associations were ablated by the PAK inhibitor IPA3, suggesting that PAK1 activation lies upstream of these actin-polymerizing complexes. Using the N-WASP inhibitor wiskostatin, we further demonstrated that N-WASP is required for localized F-actin polymerization, GLUT4 vesicle translocation, and glucose uptake. These results expand the model of insulin-stimulated glucose uptake in skeletal muscle cells by implicating p41ARC as a new component of the insulin-signaling cascade and connecting PAK1 signaling to N-WASP-cortactin-mediated actin polymerization and GLUT4 vesicle translocation

DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle

Aims/hypothesis: Skeletal muscle accounts for >80% of insulin-stimulated glucose uptake; dysfunction of this process underlies insulin resistance and type 2 diabetes. Insulin sensitivity is impaired in mice deficient in the double C2 domain β (DOC2B) protein, while whole-body overexpression of DOC2B enhances insulin sensitivity. Whether insulin sensitivity in the skeletal muscle is affected directly by DOC2B or is secondary to an effect on other tissues is unknown; the underlying molecular mechanisms also remain unclear. Methods: Human skeletal muscle samples from non-diabetic or type 2 diabetic donors were evaluated for loss of DOC2B during diabetes development. For in vivo analysis, new doxycycline-inducible skeletal-muscle-specific Doc2b-overexpressing mice fed standard or high-fat diets were evaluated for insulin and glucose tolerance, and insulin-stimulated GLUT4 accumulation at the plasma membrane (PM). For in vitro analyses, a DOC2B-overexpressing L6-GLUT4-myc myoblast/myotube culture system was coupled with an insulin resistance paradigm. Biochemical and molecular biology methods such as site-directed mutagenesis, co-immunoprecipitation and mass spectrometry were used to identify the molecular mechanisms linking insulin stimulation to DOC2B. Results: We identified loss of DOC2B (55% reduction in RNA and 40% reduction in protein) in the skeletal muscle of human donors with type 2 diabetes. Furthermore, inducible enrichment of DOC2B in skeletal muscle of transgenic mice enhanced whole-body glucose tolerance (AUC decreased by 25% for female mice) and peripheral insulin sensitivity (area over the curve increased by 20% and 26% for female and male mice, respectively) in vivo, underpinned by enhanced insulin-stimulated GLUT4 accumulation at the PM. Moreover, DOC2B enrichment in skeletal muscle protected mice from high-fat-diet-induced peripheral insulin resistance, despite the persistence of obesity. In L6-GLUT4-myc myoblasts, DOC2B enrichment was sufficient to preserve normal insulin-stimulated GLUT4 accumulation at the PM in cells exposed to diabetogenic stimuli. We further identified that DOC2B is phosphorylated on insulin stimulation, enhancing its interaction with a microtubule motor protein, kinesin light chain 1 (KLC1). Mutation of Y301 in DOC2B blocked the insulin-stimulated phosphorylation of DOC2B and interaction with KLC1, and it blunted the ability of DOC2B to enhance insulin-stimulated GLUT4 accumulation at the PM. Conclusions/interpretation: These results suggest that DOC2B collaborates with KLC1 to regulate insulin-stimulated GLUT4 accumulation at the PM and regulates insulin sensitivity. Our observation provides a basis for pursuing DOC2B as a novel drug target in the muscle to prevent/treat type 2 diabetes

STUDY THE ADVERTISEMENT AND PROMOTIONAL IMPACT ON FMCG SECTOR IN RURAL PUNE DISTRICT AND THE OVERALL POTENTIAL OF THE RURAL MARKET

In country like India, where the 70% of the people live in rural area, the rural market holds a lot of marketing potential. There is a wide spread difference in the standard of living between urban and rural India. In order to launch products and develop advertising for rural market there is a need to understand both the rural context and also the consumer very well. Promotion of brands in rural markets requires the special measures. Due to the social and backward condition the personal selling efforts have a challenging role to play in this regard. The word of mouth is an important message carrier in rural areas. In fact the opinion leaders are the most influencing part of promotion strategy of rural promotion efforts.The strong Indian brands have strong brand equity, consumer demand-pull and efficient and dedicated dealer network which have been created over a period of time. The rural market has a grip of strong country shops, which affect the sale of various products in rural market. The companies are trying to trigger growth in rural areas. They are identifying the fact that rural people are now in the better position with disposable income. The low rate finance availability has also increased the affordability of purchasing the costly products by the rural people. Marketer should understand the price sensitivity of a consumer in a rural area. This research paper will be therefore an attempt to study the Advertisement and Sales Promotion impact on FMCG Sector in rural India and the overall potential of the rural market

Technological Assessment on Steam Reforming Process of Crude Glycerol to Produce Hydrogen in an Integrated Waste Cooking-Oil-Based Biodiesel Production Scenario

The current scenario of society is to produce fuel from renewable energy resources. The purpose of this research work is to develop an integrated approach for glycerol valorization and biodiesel production. Employing a range of methodologies widely used in the industry, technical analysis and assessments of the process’s applicability in real-world situations are also made. The integrated process plant is simulated using Aspen Plus®. Several different sensitivity analyses are carried out to describe the process that improves efficiency and are designed to maximize hydrogen recovery from the reforming section. The integrated process results are compared with several existing standalone biodiesel production processes. Additionally, the results are verified with the theoretical studies on glycerol valorization. The outcomes of the process plant simulation reveal coherent results with the current industrial standards for the two processes. The results show that the amount of glycerol produced (stream 7) is 60.72 kmol/h in mass flow rate, this translates to 7272.74 kg/h. The hydrogen produced is 488.76 kmol/h and, in mass flow rate, this translates to 985.3 kg/h. The total yield of hydrogen produced is around 13%. The biodiesel yield is at 92.5%. It shows a realistic recovery that would be attained if the process is implemented, contrary to theoretical studies

Rosuvastatin treatment affects both basal and glucose-induced insulin secretion in INS-1 832/13 cells

Rosuvastatin is a member of the statin family. Like the other statins it is prescribed to lower cholesterol levels and thereby reduce the risk of cardiovascular events. Rosuvastatin lowers the cholesterol levels by inhibiting the key enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase) in the cholesterol producing mevalonate pathway. It has been recognized that apart from their beneficial lipid lowering effects, statins also exhibit diabetogenic properties. The molecular mechanisms behind these remain unresolved. To investigate the effects of rosuvastatin on insulin secretion, we treated INS-1 832/13 cells with varying doses (20 nM to 20 μM) of rosuvastatin for 48 h. At concentrations of 2 μM and above basal insulin secretion was significantly increased. Using diazoxide we could determine that rosuvastatin did not increase basal insulin secretion by corrupting the KATP channels. Glucose-induced insulin secretion on the other hand seemed to be affected differently at different rosuvastatin concentrations. Rosuvastatin treatment (20 μM) for 24-48 h inhibited voltage-gated Ca2+ channels, which lead to reduced depolarization-induced exocytosis of insulin-containing granules. At lower concentrations of rosuvastatin (≤ 2 μM) the stimulus-secretion coupling pathway was intact downstream of the KATP channels as assessed by the patch clamp technique. However, a reduction in glucose-induced insulin secretion could be observed with rosuvastatin concentrations as low as 200 nM. The inhibitory effects of rosuvastatin on glucose-induced insulin secretion could be reversed with mevalonate, but not squalene, indicating that rosuvastatin affects insulin secretion through its effects on the mevalonate pathway, but not through the reduction of cholesterol biosynthesis. Taken together, these data suggest that rosuvastatin has the potential to increase basal insulin secretion and reduce glucose-induced insulin secretion. The latter is possibly an unavoidable side effect of rosuvastatin treatment as it occurs through the same mechanisms as the lipid-lowering effects of the drug