School of Medicine and Health Sciences Poster Presentations
Elucidating the Mechanism of Human Amylin Trafficking in the Pancreatic β Cells
Poster Number
13
Document Type
Poster
Publication Date
3-2016
Abstract
Type 2 Diabetes Mellitus (T2DM) is a complex metabolic disorder characterized by progressive loss of pancreatic β-cells secretory functions, β-cells death, peripheral insulin resistance and resulting hyperglycemia. Studies showed that, β-cell derived toxic aggregates of pancreatic hormone amylin contribute significantly towards the development of T2DM. The human amylin (hA) is a 37 amino acid hormone, which shares similar biosynthesis mechanism like insulin and is co-secreted with insulin by the pancreatic β-cells upon glucose stimulation. However, the cellular processes that regulate hA mediated cytotoxicity is far from clear. It is known that during T2DM, impaired folding, dysregulated ER to Golgi traffic or increased ER exit of proinsulin contribute to the development of insulin deficiency. It has also been suggested that similar to proinsulin, dysregulated processing and trafficking of pro-amylin contributes significantly towards the β cell failure during T2DM. However, detailed cellular trafficking mechanism of hA is unknown and hence investigated in the current study. In order to achieve the goal, I used lenti virus mediated gene delivery approach and validated hA and rat amylin (rA) overexpression in two rat pancreatic β-cells lines (RIN-m5f and INS 832/13) as well as in human islet cells. In order to identify the cellular compartments involved in hA trafficking and turnover cytosolic, membrane/organelle and nuclear/cytoskeletal fractions were prepared from the pancreatic β-cells following overexpression of hA and rA. WB analyses revealed the accumulation of hA in cytosol and organelle-enriched (Golgi/ER) fraction of RIN, INS and human islets cells. Interestingly, hA was also frequently found in the nuclear/cytoskeletal fraction of the RIN and human islet cells but not in the INS cells. Co-localization studies using immunofluorescence confocal microscopy confirmed significant accumulation of hA in Golgi, as well as nuclear accumulation of hA in RIN and human islet cells. Accumulation of hA in Golgi region rather than ER resembles trafficking pattern of proinsulin and possibly indicates the rate limiting role of Golgi during hA trafficking. Nuclear accumulation of hA mirrors trafficking pattern of other amyloid proteins and implies possible role/s of nucleus in turnover of hA in pancreatic β cells. This study, as well as future studies focused on determining the hA trafficking under normal and hyperglycemic condition, will help us to understand the strategies by which β-cells distribute and/sequester hA to prevent its toxicity, and if and how this protective mechanism may be altered and/or impaired under stress conditions like diabetes. This knowledge is important for development of new treatments against hA toxicity and T2DM.
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Open Access
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Elucidating the Mechanism of Human Amylin Trafficking in the Pancreatic β Cells
Type 2 Diabetes Mellitus (T2DM) is a complex metabolic disorder characterized by progressive loss of pancreatic β-cells secretory functions, β-cells death, peripheral insulin resistance and resulting hyperglycemia. Studies showed that, β-cell derived toxic aggregates of pancreatic hormone amylin contribute significantly towards the development of T2DM. The human amylin (hA) is a 37 amino acid hormone, which shares similar biosynthesis mechanism like insulin and is co-secreted with insulin by the pancreatic β-cells upon glucose stimulation. However, the cellular processes that regulate hA mediated cytotoxicity is far from clear. It is known that during T2DM, impaired folding, dysregulated ER to Golgi traffic or increased ER exit of proinsulin contribute to the development of insulin deficiency. It has also been suggested that similar to proinsulin, dysregulated processing and trafficking of pro-amylin contributes significantly towards the β cell failure during T2DM. However, detailed cellular trafficking mechanism of hA is unknown and hence investigated in the current study. In order to achieve the goal, I used lenti virus mediated gene delivery approach and validated hA and rat amylin (rA) overexpression in two rat pancreatic β-cells lines (RIN-m5f and INS 832/13) as well as in human islet cells. In order to identify the cellular compartments involved in hA trafficking and turnover cytosolic, membrane/organelle and nuclear/cytoskeletal fractions were prepared from the pancreatic β-cells following overexpression of hA and rA. WB analyses revealed the accumulation of hA in cytosol and organelle-enriched (Golgi/ER) fraction of RIN, INS and human islets cells. Interestingly, hA was also frequently found in the nuclear/cytoskeletal fraction of the RIN and human islet cells but not in the INS cells. Co-localization studies using immunofluorescence confocal microscopy confirmed significant accumulation of hA in Golgi, as well as nuclear accumulation of hA in RIN and human islet cells. Accumulation of hA in Golgi region rather than ER resembles trafficking pattern of proinsulin and possibly indicates the rate limiting role of Golgi during hA trafficking. Nuclear accumulation of hA mirrors trafficking pattern of other amyloid proteins and implies possible role/s of nucleus in turnover of hA in pancreatic β cells. This study, as well as future studies focused on determining the hA trafficking under normal and hyperglycemic condition, will help us to understand the strategies by which β-cells distribute and/sequester hA to prevent its toxicity, and if and how this protective mechanism may be altered and/or impaired under stress conditions like diabetes. This knowledge is important for development of new treatments against hA toxicity and T2DM.
Comments
Presented at: GW Research Days 2016