Biology subjects

Kebede, M. A.

Publications and source records attributed to Kebede, M. A..

6 recordsLinked to original sources

bioRxiv2024

Optimised proteomic analysis of insulin granules from MIN6 β-cells identifies Scamp3, a novel regulator of insulin secretion and content

Pancreatic {beta}-cells in the islets of Langerhans are key to maintaining glucose homeostasis, by secreting the peptide hormone insulin. Insulin is packaged within vesicles named insulin secretory granules (ISGs), that have recently been considered to have intrinsic structures and proteins that regulate insulin granule maturation, trafficking, and secretion. Previously, studies have identified a handful of novel ISG-associated proteins using different separation techniques. Here, this study combines an optimized ISG isolation technique and mass spectrometry-based proteomics, with an unbiased protein correlation profiling and targeted machine learning approach to uncover 211 ISG-associated proteins. Five of these proteins: Syntaxin-7, Synaptophysin, Synaptotagmin-13, Zinc transporter ZIP8 and SCAMP3 have not been previously ISG-associated. Through colocalization analysis of confocal imaging we validate the association of these proteins to the ISG in MIN6 and human {beta}-cells. We further validate the role for one (SCAMP3) in regulating insulin storage and secretion from {beta}-cells for the first time. SCAMP3 knock-down INS-1 cells show a reduction in insulin content and dysfunctional insulin secretion. These data provide the basis for future investigation into {beta}-cell biology and the regulation of insulin secretion. Article HighlightsO_LIWhy did we undertake this study? We undertook this study to optimize insulin granule isolation techniques alongside enhanced proteomics analyses to establish the first published murine insulin granule proteome. C_LIO_LIWhat is the specific question(s) we wanted to answer? We aimed to specifically answer and investigate what proteins are present on insulin granules from MIN6 cells to further our understanding of insulin granule biogenesis, trafficking, and secretion. C_LIO_LIWhat did we find? We find and validate the presence of 5 novel insulin granule-associated proteins. C_LIO_LIWhat are the implications of our findings? An extensive proteomics analysis of MIN6 insulin granules and implicate Scamp3 as a novel protein that regulates insulin content and secretion in beta-cells. C_LI

cell biology↗

bioRxiv2024

Loss of VPS41 triggers rapid insulin degradation and dysregulated autophagy in pancreatic beta-cells

Vacuolar protein sorting-associated protein 41 (VPS41) has been established as a requirement for normal insulin secretory function in pancreatic beta-cells. Genetic deletion of VPS41 in mouse pancreatic beta-cells results in diabetes, though the mechanisms are not understood. Presently, we show that VPS41 deletion results in rapid mature insulin degradation and downregulation of beta-cell identity. This phenotype is observed in vivo, with VPS41KO mice displaying progressive loss of insulin content and beta-cell function with age. In acute VPS41 depletion in vitro, the loss of insulin is associated with increased degradative pathway activity, increased Adapter Protein 3 complex colocalisation with lysosomes, increased nuclear localisation of transcription factor E3, and downregulation of PDX1 and INS mRNA expression. Inhibition of lysosomal degradation rescues the rapidly depleted insulin content. These data evidence a VPS41-dependent mechanism for both insulin content degradation and loss of beta-cell identity in beta-cells.

cell biology↗

bioRxiv2024

Phytochemical Screening, Investigation of the toxic and hepatoprotective effect of leaves of Prunus africana-using mice model

PurposeLiver disease is a growing public health problem both in developing and developed countries. Allopathic medicines available for liver disease are not accessible and affordable for 3rd world countries, like Ethiopia. There is a need to search for alternative therapy, in which herbals are widely used in traditional medicine and promising for the development of effective and less costly treatment. As a result, this study aimed to screen phytochemicals and investigate the toxic and hepatoprotective effects of Prunus africana which is traditionally used in Benchi sheko and Sheka Zones, Southwest Ethiopia. MethodsMethanolic extracts of Prunus africana, were used to evaluate the toxicity and hepatoprotective activity in both male and female swiss Albino Mice. Liver injury was induced by carbon tetrachloride (CCl4). The toxicity and hepatoprotective activity were examined biochemically, histologically, and through the analysis of the general features of the study animals. The phytochemical composition was screened qualitatively using standard chemical tests for secondary metabolites. The results were expressed as Mean {+/-} SD, and differences at P < 0.05 were considered significant. Differences between the experimental and control groups were analyzed using one-way analysis of variance (ANOVA) followed by Dunnetts T-test to determine their level of significance. ResultsThe extract of Prunus africana revealed positive for the presence of flavonoids and Saponins but negative for Anthraquinone glycoside. The current study showed that the median oral lethal dose of the plant was greater than 5000mg/kg. The general behavior of the animal and organ weight, gross morphology, and the biochemical and histological parameters confirmed that the Methanolic leaves extract of this plant is safe during the sub-acute toxicity tests with a dose of 600 and 1800mg/kg. Methanolic extract before and after the CCl4 administration caused a significant reduction in the values of Alanine transaminase, Aspartate transaminase, Alkaline phosphatase, and bilirubin (P<0.05) almost comparable to the standard drug Silymarine. The hepatoprotective activity was supported by histopathological examination of the liver tissue of control and treated animals. ConclusionThe methanolic extract of Prunus africana at the test doses did not show significant toxicity. The results of this study also demonstrate that the extract was effective for the prevention of CCl4-induced hepatic damage in Mice. This study thus justifies that, the use of Prunus africana in the treatment of liver diseases and points out that this plant warrants further detailed investigation as a promising hepatoprotective agent.

pharmacology and toxicology↗

bioRxiv2022

Insulin sensitivity is preserved in mice made obese by feeding a high starch diet

Obesity is generally associated with insulin resistance in liver and muscle and increased risk of developing type 2 diabetes, however there is a population of obese people that remain insulin sensitive. Similarly, recent work suggests that mice fed high carbohydrate diets can become obese without apparent glucose intolerance. To investigate this phenomenon further, we fed mice either a high fat (Hi-F) or high starch (Hi-ST) diet and measured adiposity, glucose tolerance, insulin sensitivity and tissue lipids compared to control mice fed a standard laboratory chow. Both Hi-ST and Hi-F mice accumulated a similar amount of fat and tissue triglyceride compared to chow-fed mice. However while Hi-F diet mice developed glucose intolerance as well as liver and muscle insulin resistance (assessed via euglycemic/hyperinsulinemic clamp), obese Hi-ST mice maintained glucose tolerance and insulin action similar to lean, chow-fed controls. This preservation of insulin action despite obesity in Hi-ST mice was associated with differences in de novo lipogenesis and levels of C22:0 ceramide in liver and C18:0 ceramide in muscle. This indicates that dietary manipulation can influence insulin action independently of the level of adiposity and that the presence of specific ceramide species correlate with these differences.

physiology↗

bioRxiv2020

Pancreatic beta-cell specific deletion of VPS41 causes diabetes due to defects in insulin secretion

Insulin secretory granules (SGs) mediate the regulated secretion of insulin, which is essential for glucose homeostasis. The basic machinery responsible for this regulated exocytosis consists of specific proteins present both at the plasma membrane and on insulin SGs. The protein composition of insulin SGs thus dictates their release properties, yet the mechanisms controlling insulin SG formation, which determines this molecular composition, remain poorly understood. VPS41, a component of the endo-lysosomal tethering HOPS complex, was recently identified as a cytosolic factor involved in the formation of neuroendocrine and neuronal granules. We now find that VPS41 is required for insulin SG biogenesis and regulated insulin secretion. Loss of VPS41 in pancreatic {beta}-cells leads to a reduction in insulin SG number, changes in their transmembrane protein composition, and defects in granule regulated exocytosis. Exploring a human point mutation, identified in patients with neurological but no endocrine defects, we show that the effect on SG formation is independent of HOPS complex formation. Finally, we report that mice with a deletion of VPS41 specifically in {beta}-cells develop diabetes due to severe depletion of insulin SG content and a defect in insulin secretion. In sum, our data demonstrate that VPS41 contributes to glucose homeostasis and metabolism.

physiology↗

bioRxiv2018

Fibroblast activation protein enzyme deficiency prevents liver steatosis, insulin resistance and glucose intolerance and increases fibroblast growth factor-21 in diet induced obese mice

Background & AimsFibroblast activation protein-a (FAP) is a post-proline peptidase closely related to dipeptidyl peptidase-4. FAP degrades bioactive peptides including fibroblast growth factor-21 (FGF-21) and neuropeptide Y. We examined metabolic outcomes of specific genetic ablation of FAP and its enzyme activity in a mouse model of diet-induced obesity (DIO) causing fatty liver.\n\nMethodsWildtype (WT) and genetically modified FAP deficient mice that specifically lacked either the FAP protein or FAP enzyme activity received chow, or an atherogenic diet for 8 to 20 weeks of DIO.\n\nResultsFAP deficient male and female mice in the DIO model were more metabolically healthy than controls. The FAP deficient mice had less glucose intolerance, liver lipid, adiposity, insulin resistance, pancreatic and plasma insulin, pancreatic {beta}-cell hyperplasia, serum alanine transaminase and circulating cholesterol compared to wild type controls. Furthermore, FAP deficiency lowered respiratory exchange ratio and greatly increased intrahepatic non-esterified free fatty acids, indicative of increased lipolysis and {beta}-oxidation. Concordantly, lipogenic genes (Pparg, Gck, Acc, Fasn) and hepatic triglyceride and fatty acid uptake genes (Cd36, Apoc3, Ldlr) and plasma low-density lipoprotein cholesterol were downregulated. Glucagon like peptide-1 levels were unaltered. FAP was localized to human pancreatic {beta}-cells and pancreas from diabetes mellitus patients contained elevated FAP activity. Comparable data from a FAP gene knockout mouse and a novel mouse lacking FAP enzyme activity indicated that these metabolic changes depended upon the enzymatic activity of FAP. These changes may be driven by FGF-21, which was upregulated in livers of FAP deficient DIO mice.\n\nConclusionThis is the first study to show that specific genetic ablation of FAP activity or protein protects against DIO-driven glucose intolerance, hyperinsulinaemia, insulin resistance, hypercholesterolaemia and liver steatosis in mice and provide mechanistic insights.

pathology↗