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Detection of Islet Autoantibodies in Diabetes Mellitus Patients in Accra
Abstracts / Can J Diabetes 41 (2017) S22–S83
of Ucp2 at ZT4 was associated with reduced ATP production and impaired GSIS, indicative of reduced mitochondrial uncoupling. Conclusions: The circadian clock regulates daily cycles of Ucp2 expression in MIN6 cells, which is a part of an important metabolic switch that aligns GSIS capacity with the time of day. Future studies will explore if these findings are translatable in vivo by generating and characterizing a β cell-specific Bmal1 knockout mouse model. 189 Glucose Suppresses Calcium Currents to Reduce α-Cell Exocytosis in Mouse and Human XIAOQING DAI, ALIYA SPIGELMAN, PATRICK MACDONALD Edmonton, AB
S71
disruption of lysosomal homeostasis. Interestingly, this loss was accelerated in Atg5-deficient cells. Treatment with chloroquine (CHQ) or Bafilomycin A1 (Baf-A1) results in lysosomal alkalization and impairment of autophagic turnover. To determine if lysosomal failure affects β-cell metabolic integrity, we cultured MIN6 cells for 24 hrs in CHQ (0.5, 1, 5 and 10 μM) and Baf-A1 (1, 5 and 10 nM) and measured mitochondrial oxygen consumption rates (OCR). Basal OCR in 3 mM glucose was not affected by pre-culture in Baf-A1 but was reduced by CHQ at higher concentrations. Both Baf-A1 (10 nM) and CHQ (5 μM and 10 μM) lowered the acute OCR response to 25 mM glucose. Conclusion: Our data reveal a protective role of autophagy in β-cells under hypoxic stress and suggest hypoxia may impair lysosomal homeostasis with possible consequences for β-cell metabolic function.
Glucagon is synthesized and released by pancreatic α cells and, along with insulin, maintains blood glucose levels within the physiological range. Glucagon release is stimulated at low glucose concentrations (hypoglycemia). In type 2 diabetes (T2D), in addition to impaired insulin secretion, the release of glucagon becomes dysregulated and this exacerbates hyperglycemia. Glucagon secretion may be regulated by either intrinsic and/or paracrine mechanisms, but their relative significance and the conditions under which they operate are highly debated. Here, we used whole-cell patch clamp to study the exocytosis and Ca2+-channel function of single mouse and human α cells. Opposite to what is observed in β cells, depolarization-induced exocytosis in α cells is stimulated by low glucose (LG, 1 mM G), and inhibited by high glucose (HG, 5–20 mM G). However, this is reversed in α cells (identified by glucagon immunostaining) from T2D donors, which display a β-cell-like phenotype where HG amplifies exocytosis and LG inhibits it. Using the pharmacological antagonists of Ca2+ channels, we found that LG increases and HG suppresses, P/Q-type Ca2+ currents in human α cells, which are specifically linked to glucagon exocytosis although they make a minor contribution in total α cell Ca2+ current (~30%). While the majority of Ca2+ current flows through L-type Ca2+ channels (~60%), the pharmacological blockade of these Ca2+ channels has no effect on a-cell exocytosis. Finally, perifusion of mouse islets indicated that depolarization-induced (30 mM KCl) glucagon secretion was much increased at 1 versus 5 mM G, whereas insulin secretion was largely similar. Thus, we find that LG increases P/Q-type Ca2+ currents in α cells, which results in amplification of depolarization-induced glucagon exocytosis. In T2D this regulation is lost. Better understanding of the glucose-regulated glucagon secretion is critical towards understanding glucose homeostasis and the development of diabetes.
The factors that lead to the decline of β-cell function are still largely unknown, but most studies have identified islet autoantibodies in serum as predictors of future β-cell decline. The aim of this study was to determine serum levels of Glutamic acid decarboxylase- 65 autoantibody (GAD-65A), Insulinoma associated- 2 antibody (IA–2A), and Islet cell cytoplasmic autoantibody (ICA) in Ghanaian diabetics. One hundred and ten (110) participants were used for this study and were divided into high-risk subjects (obese persons, hypertensives, relatives of diabetic patients, women with previous history of gestational diabetes) (55); negative controls (apparently healthy persons) (28); and positive controls (diabetics on insulin therapy) (27). Lipid profile, fasting blood glucose (FBG), glycated haemoglobin (HbA1c), blood pressure, body mass index (BMI), C-peptide and islet autoantibodies (GAD-65A, IA–2A and ICA) were measured. ICA, IA-2A, and GAD-65A were significantly increased in the positive controls (18.5%, 14.8% and 40.7%). The negative controls were seronegative to the auto antibodies. FBG and HbA1c showed a strong positive correlation with ICA, GAD-65A, and IA-2A. However, C-peptide showed a negative correlation with the ICA (r=−0.301, p=0.023), GAD-65A (r=−0.496, p=0.010) and IA-2A (r=−0.116, p=0.038). ICA and GAD-65A could be useful markers for the prediction of β-cell dysfunction among Ghanaians.
191 Detection of Islet Autoantibodies in Diabetes Mellitus Patients in Accra HENRY ASARE-ANANE, RICHMOND O. ATEKO, LARYEA E. TORGBOR Accra, Ghana
190
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Autophagy and Lysosomal Function Protect β-Cells under Hypoxic Stress YUANJIE ZOU*, MEI TANG, DAN S. LUCIANI Vancouver, BC
Pancreatic Islet Glycine Receptor Variants RICHARD YAN-DO, KUNIMASA SUZUKI, TYLER GRIESON, MOURAD FERDAOUSSI, CATHY HAJMRLE, PATRICK MACDONALD Edmonton, AB
Background: Autophagy helps protect β-cells from lipotoxicity, but the importance of β-cell autophagy and lysosomal homeostasis under other forms of diabetes- and islet transplantation-related stress is unclear. We investigated if impairment of autophagosome formation or lysosomal physiology affect β-cell function and survival under normoxia and hypoxia. Results: We deleted the essential autophagy gene Atg5 using adenovirus-mediated Cre expression in Atg5flox/flox mouse islet cells. Time-lapse recordings showed that loss of Atg5 markedly amplified hypoxia (1% O2)-induced islet-cell death. Hypoxia-like conditions, induced by CoCl2, reduced β-cell LysoTracker Red staining, suggesting a reduction in the number of lysosomes or severe
Background: Insulin, the only blood-glucose lowering hormone in the body, is secreted from pancreatic β-cells and is regulated by neuronal, nutritional, and hormonal signals. Glycine is an inhibitory neurotransmitter but recent metabolic studies identify glycine as a potential biomarker of type 2 diabetes (T2D) risk. A strong correlation exists between plasma glycine concentrations and insulin sensitivity, glucose disposal, and obesity. Circulating plasma glycine concentrations are inversely related to T2D risk. The mechanism for glycine’s action in diabetes is unknown. Methods: Human islets were isolated in the Alberta Diabetes Institute Islet Core and the Clinical Islet Laboratory at the University of Alberta from donor organs. Electrical recordings were performed
of Ucp2 at ZT4 was associated with reduced ATP production and impaired GSIS, indicative of reduced mitochondrial uncoupling. Conclusions: The circadian clock regulates daily cycles of Ucp2 expression in MIN6 cells, which is a part of an important metabolic switch that aligns GSIS capacity with the time of day. Future studies will explore if these findings are translatable in vivo by generating and characterizing a β cell-specific Bmal1 knockout mouse model. 189 Glucose Suppresses Calcium Currents to Reduce α-Cell Exocytosis in Mouse and Human XIAOQING DAI, ALIYA SPIGELMAN, PATRICK MACDONALD Edmonton, AB
S71
disruption of lysosomal homeostasis. Interestingly, this loss was accelerated in Atg5-deficient cells. Treatment with chloroquine (CHQ) or Bafilomycin A1 (Baf-A1) results in lysosomal alkalization and impairment of autophagic turnover. To determine if lysosomal failure affects β-cell metabolic integrity, we cultured MIN6 cells for 24 hrs in CHQ (0.5, 1, 5 and 10 μM) and Baf-A1 (1, 5 and 10 nM) and measured mitochondrial oxygen consumption rates (OCR). Basal OCR in 3 mM glucose was not affected by pre-culture in Baf-A1 but was reduced by CHQ at higher concentrations. Both Baf-A1 (10 nM) and CHQ (5 μM and 10 μM) lowered the acute OCR response to 25 mM glucose. Conclusion: Our data reveal a protective role of autophagy in β-cells under hypoxic stress and suggest hypoxia may impair lysosomal homeostasis with possible consequences for β-cell metabolic function.
Glucagon is synthesized and released by pancreatic α cells and, along with insulin, maintains blood glucose levels within the physiological range. Glucagon release is stimulated at low glucose concentrations (hypoglycemia). In type 2 diabetes (T2D), in addition to impaired insulin secretion, the release of glucagon becomes dysregulated and this exacerbates hyperglycemia. Glucagon secretion may be regulated by either intrinsic and/or paracrine mechanisms, but their relative significance and the conditions under which they operate are highly debated. Here, we used whole-cell patch clamp to study the exocytosis and Ca2+-channel function of single mouse and human α cells. Opposite to what is observed in β cells, depolarization-induced exocytosis in α cells is stimulated by low glucose (LG, 1 mM G), and inhibited by high glucose (HG, 5–20 mM G). However, this is reversed in α cells (identified by glucagon immunostaining) from T2D donors, which display a β-cell-like phenotype where HG amplifies exocytosis and LG inhibits it. Using the pharmacological antagonists of Ca2+ channels, we found that LG increases and HG suppresses, P/Q-type Ca2+ currents in human α cells, which are specifically linked to glucagon exocytosis although they make a minor contribution in total α cell Ca2+ current (~30%). While the majority of Ca2+ current flows through L-type Ca2+ channels (~60%), the pharmacological blockade of these Ca2+ channels has no effect on a-cell exocytosis. Finally, perifusion of mouse islets indicated that depolarization-induced (30 mM KCl) glucagon secretion was much increased at 1 versus 5 mM G, whereas insulin secretion was largely similar. Thus, we find that LG increases P/Q-type Ca2+ currents in α cells, which results in amplification of depolarization-induced glucagon exocytosis. In T2D this regulation is lost. Better understanding of the glucose-regulated glucagon secretion is critical towards understanding glucose homeostasis and the development of diabetes.
The factors that lead to the decline of β-cell function are still largely unknown, but most studies have identified islet autoantibodies in serum as predictors of future β-cell decline. The aim of this study was to determine serum levels of Glutamic acid decarboxylase- 65 autoantibody (GAD-65A), Insulinoma associated- 2 antibody (IA–2A), and Islet cell cytoplasmic autoantibody (ICA) in Ghanaian diabetics. One hundred and ten (110) participants were used for this study and were divided into high-risk subjects (obese persons, hypertensives, relatives of diabetic patients, women with previous history of gestational diabetes) (55); negative controls (apparently healthy persons) (28); and positive controls (diabetics on insulin therapy) (27). Lipid profile, fasting blood glucose (FBG), glycated haemoglobin (HbA1c), blood pressure, body mass index (BMI), C-peptide and islet autoantibodies (GAD-65A, IA–2A and ICA) were measured. ICA, IA-2A, and GAD-65A were significantly increased in the positive controls (18.5%, 14.8% and 40.7%). The negative controls were seronegative to the auto antibodies. FBG and HbA1c showed a strong positive correlation with ICA, GAD-65A, and IA-2A. However, C-peptide showed a negative correlation with the ICA (r=−0.301, p=0.023), GAD-65A (r=−0.496, p=0.010) and IA-2A (r=−0.116, p=0.038). ICA and GAD-65A could be useful markers for the prediction of β-cell dysfunction among Ghanaians.
191 Detection of Islet Autoantibodies in Diabetes Mellitus Patients in Accra HENRY ASARE-ANANE, RICHMOND O. ATEKO, LARYEA E. TORGBOR Accra, Ghana
190
192
Autophagy and Lysosomal Function Protect β-Cells under Hypoxic Stress YUANJIE ZOU*, MEI TANG, DAN S. LUCIANI Vancouver, BC
Pancreatic Islet Glycine Receptor Variants RICHARD YAN-DO, KUNIMASA SUZUKI, TYLER GRIESON, MOURAD FERDAOUSSI, CATHY HAJMRLE, PATRICK MACDONALD Edmonton, AB
Background: Autophagy helps protect β-cells from lipotoxicity, but the importance of β-cell autophagy and lysosomal homeostasis under other forms of diabetes- and islet transplantation-related stress is unclear. We investigated if impairment of autophagosome formation or lysosomal physiology affect β-cell function and survival under normoxia and hypoxia. Results: We deleted the essential autophagy gene Atg5 using adenovirus-mediated Cre expression in Atg5flox/flox mouse islet cells. Time-lapse recordings showed that loss of Atg5 markedly amplified hypoxia (1% O2)-induced islet-cell death. Hypoxia-like conditions, induced by CoCl2, reduced β-cell LysoTracker Red staining, suggesting a reduction in the number of lysosomes or severe
Background: Insulin, the only blood-glucose lowering hormone in the body, is secreted from pancreatic β-cells and is regulated by neuronal, nutritional, and hormonal signals. Glycine is an inhibitory neurotransmitter but recent metabolic studies identify glycine as a potential biomarker of type 2 diabetes (T2D) risk. A strong correlation exists between plasma glycine concentrations and insulin sensitivity, glucose disposal, and obesity. Circulating plasma glycine concentrations are inversely related to T2D risk. The mechanism for glycine’s action in diabetes is unknown. Methods: Human islets were isolated in the Alberta Diabetes Institute Islet Core and the Clinical Islet Laboratory at the University of Alberta from donor organs. Electrical recordings were performed