Role of AMP-activated protein kinase in pancreatic islet beta-cell death during type 1 and type 2 diabetes
Lead Research Organisation:
University of Bristol
Department Name: Biochemistry
Abstract
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Technical Summary
Diabetes mellitus currently affects ~ 5 % of the population in westernised societies, an incidence expected to double by 2020. Destruction of beta-cells, achieved respectively through immune-mediated attack or other mechanisms, is involved in the aetiology of type 1 and type 2 diabetes respectively.
Our laboratories and others have demonstrated that AMP-activated protein kinase (AMPK) plays a key role in pancreatic islet beta-cells as a sensor of glucose. We propose here to explore the potential role of forced changes in AMPK activity, achieved using recombinant adenoviruses or transgenesis, in modulating beta-cell survival and function during a variety of pathophysiological pro-apoptotic assaults. Assessment of beta-cell function, necrosis and apoptosis will be achieved using a wide range of assays. Transplantation into mice of islets in which AMPK activity has been blocked or enhanced will also serve to assess the potential therapeutic value of these changes for human islet transplantation. Our principal aims are as follows:
Aim 1: The role of AMPK will be studied in three models relevant to beta cell death in type 1 diabetes:
(a) Non-immune attack in vitro: isolated mouse islets will be treated with the inflammatory cytokines, IL-1beta and TNFalpha
(b) Immune-mediated destruction of islet beta-cells in vitro: islets isolated from non-obese diabetic (NOD) mice and incubated with insulin-reactive CD8+ T cells.
(c) Recurrent immune-mediated destruction of islet beta-cells in vivo. Syngeneic islets that have been infected with viral constructs (or null virus control) will be transplanted under the kidney capsule of diabetic NOD mice, and changes in glycaemia monitored as a read-out of beta-cell survival and function.
Aim 2: We propose to examine the role of AMPK in ex vivo models of beta cell failure, as follows:
(a)?Glucolipotoxicity? in vitro: achieved either over-expression of the lipogenic transcription factor, sterol regulatory element binding protein 1c (SREBP1c) or by treatment of MIN6 beta cells with high glucose and saturated fatty acid concentrations
(b) Insulin or IGF-1 receptor depletion from cells. MIN6 cells will be deployed and receptor silencing achieved using specific, validated siRNAs
Aim 3: What mechanisms are involved in the execution of apoptosis during (a) pathophysiological stimulation (Aims 1 and 2) and (b) after activating AMPK? The role in both cases of downstream signalling pathways will be explored using classical biochemical approaches.
Aim 4: Generation and characterisation of transgenic mice expressing constitutively-active or dominant-negative AMPK selectively in pancreatic beta cells.
Our laboratories and others have demonstrated that AMP-activated protein kinase (AMPK) plays a key role in pancreatic islet beta-cells as a sensor of glucose. We propose here to explore the potential role of forced changes in AMPK activity, achieved using recombinant adenoviruses or transgenesis, in modulating beta-cell survival and function during a variety of pathophysiological pro-apoptotic assaults. Assessment of beta-cell function, necrosis and apoptosis will be achieved using a wide range of assays. Transplantation into mice of islets in which AMPK activity has been blocked or enhanced will also serve to assess the potential therapeutic value of these changes for human islet transplantation. Our principal aims are as follows:
Aim 1: The role of AMPK will be studied in three models relevant to beta cell death in type 1 diabetes:
(a) Non-immune attack in vitro: isolated mouse islets will be treated with the inflammatory cytokines, IL-1beta and TNFalpha
(b) Immune-mediated destruction of islet beta-cells in vitro: islets isolated from non-obese diabetic (NOD) mice and incubated with insulin-reactive CD8+ T cells.
(c) Recurrent immune-mediated destruction of islet beta-cells in vivo. Syngeneic islets that have been infected with viral constructs (or null virus control) will be transplanted under the kidney capsule of diabetic NOD mice, and changes in glycaemia monitored as a read-out of beta-cell survival and function.
Aim 2: We propose to examine the role of AMPK in ex vivo models of beta cell failure, as follows:
(a)?Glucolipotoxicity? in vitro: achieved either over-expression of the lipogenic transcription factor, sterol regulatory element binding protein 1c (SREBP1c) or by treatment of MIN6 beta cells with high glucose and saturated fatty acid concentrations
(b) Insulin or IGF-1 receptor depletion from cells. MIN6 cells will be deployed and receptor silencing achieved using specific, validated siRNAs
Aim 3: What mechanisms are involved in the execution of apoptosis during (a) pathophysiological stimulation (Aims 1 and 2) and (b) after activating AMPK? The role in both cases of downstream signalling pathways will be explored using classical biochemical approaches.
Aim 4: Generation and characterisation of transgenic mice expressing constitutively-active or dominant-negative AMPK selectively in pancreatic beta cells.