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j.jcjd.2017.08.200

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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
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
S72
Abstracts / Can J Diabetes 41 (2017) S22–S83
on dispersed human islets from healthy donors and donors with
T2D.
Results: mRNA analysis of glycine receptors α1 subunit reveal splice
variation producing Glycine receptor α1 variant 1 and Glycine receptor α1 variant 3 (henceforth referred to as variant 1 and variant 3 respectively), where variant 1 is the full length protein while variant 3 contains
a truncation on the N-terminus. Plasmids for both variant 1 and variant
3 were created and transfected into HEK cells. Variant 3 is unable to
produce a current when stimulated with 300uM glycine. Molecular
dynamic modeling of the receptor was employed to further study the
structural differences between variant 1 and variant 3. Binding analysis demonstrated that glycine cannot bind to the binding site in variant
3. We suspect that variant 3 maybe upregulated in T2D and may explain
the reduced glycine current associated with T2D and increased plasma
glucose concentration.
Conclusion: glycine receptor mediated current is known to be
reduced in islets from human donors with T2D compared to human
donors without T2D. Although insulin resistance is suspected to play
a role in the reduced glycine current, our evidence suggests that
upregulation of a non-functional receptor variant 3 may also contribute to reduced glycine current.
193
Gestational Diabetes Mellitus (GDM) Exposure and Postnatal
Diet Influence Pancreatic Islet Function of the Rat Offspring
TAYLOR MORRISEAU, NAVDEEP BRAR, PRASOON AGARWAL,
MARIO FONSECA, STEPHANIE KERELIUK, LAURA COLEBO XIANG,
NIVEDITA SESHADRI, KRISTIN HUNT, GRANT HATCH,
CHRISTINE DOUCETTE, VERNON DOLINSKY
Winnipeg, MB
Introduction: As the obesity epidemic worsens, GDM rates are
rapidly increasing. GDM exposure and obesity are strong risk factors
for type 2 diabetes (T2D) development; however, the connections
between GDM exposure, postnatal diet and islet dysfunction in offspring metabolic health remain unclear.
Hypothesis: GDM exposure alters gene expression in islets and
impairs beta cell function, which is worsened by a postnatal highfat and sucrose (HFS) diet.
Methods: GDM was induced in female rats using a HFS diet, according to our established protocol. Litters of LEAN or GDM dams were
divided and pups were weaned onto a low-fat (LF) or HFS diet.
Pancreata and islets were isolated from 15-week-old offspring to
analyze pancreas morphology, gene expression (RNA-seq), glucosestimulated insulin secretion (GSIS), and insulin content.
Results: GDM exposure reduced GSIS by 15%, which was
exacerbated by postnatal HFS exposure (85% GSIS reduction). Beta
cell mass was reduced 2-fold from GDM exposed and HFS-fed offspring, compared to GDM-exposed LF-fed offspring. Islets from LF-fed
GDM-exposed offspring exhibited 93 upregulated genes (including Il-6, Ppp1r3a, and Angptl4) and 30 downregulated genes. In the
GDM-exposed HFS-diet group, a unique set of 510 genes (including Cel and Rbp4) were upregulated and 174 genes were
downregulated.
Conclusion: GDM exposure followed by postnatal over-nutrition
additively impair islet function and induce greater changes in gene
expression. Together, our findings suggest that GDM exposure and
post-natal diet cumulatively interact to worsen metabolic health
outcomes in offspring.
194
A Case of Hypertriglycideremia-induced Pancreatitis in
Diabetic Ketoacidosis
AURELIE PARE*, MICHAEL TSOUKAS
Montreal, QC
Figure 1. Evolution of Triglyceride Level in Response to Insulin Therapy.
Clinical Presentation: An 54 year-old female known for poorly controlled diabetes mellitus type 2 presented to the emergency room
for vomiting. She was found to have diabetic ketoacidosis (DKA) and
hypertriglyceridemia. Computed tomography of the abdomen
revealed grade D pancreatitis.
Clinical Outcome: The patient’s pancreatitis was believed to be secondary to hypertriglyceridemia in the context of DKA. The patient
was known for a similar episode having occurred one year prior.
She improved following treatment of hypertriglyceridemia with
intravenous insulin.
Discussion: Hypertriglyceridemia causing acute pancreatitis is a
known complication of DKA, having been previously reported in case
reports and further examined in retrospective cohort studies of
patients presenting with DKA. The pathophysiology, clinical presentation, and treatment of this condition are explored.
Conclusion: Hypertriglyceridemia-induced pancreatitis associated with DKA is an uncommon entity that can present a diagnostic challenge for clinicians.
195
Identification of Novel Adipogenic Factors in the 14–3-3ζ
Interactome During Adipocyte Differentiation
YVES MUGABO, JASON T. LEE, NANCY N. FANG,
AMPARO ACKER-PALMER, THIBAULT MAYOR, JAMES D. JOHNSON,
GARETH E. LIM
Repentigny, QC
Adipogenesis involves a signaling network that requires strict temporal and spatial organization of effector molecules. We previously identified critical roles for the 14–3-3 protein family, and in
particular 14–3-3ζ, as an essential upstream factor in adipocyte differentiation. 14–3-3ζ interact with diverse proteins to form its
interactome suggesting that it can regulate several cellular processes that may be involved in adipogenesis. Thus, determining this
interactome may help in further understanding the regulatory roles
of 14–3-3ζ on adipocyte differentiation.
Mouse embryonic fibroblasts from TAP-epitope-tagged 14–3-3ζ
transgenic mice were generated, followed by an unbiased proteomic
analysis to determine how adipocyte differentiation influences the
14–3-3ζ interactome. Interacting proteins were categorized using
gene ontology and their role in adipogenesis, as assessed by Oil
Red-O incorporation or quantitative PCR, were evaluated with a functional siRNA screen in 3T3-L1 cells.
Proteomic analysis of the 14–3-3ζ interactome identified over 120
proteins that were unique to adipocyte differentiation. An
enrichment of proteins related to RNA metabolism, processing,
and splicing were found in the differentiation-associated interactome. Our screening approach revealed essential roles for RNA
splicing-related proteins, Hnrnpf, Hnrnpk, Ddx6, and Sfpq in
adipogenesis.
This study demonstrates the ability of identifying novel adipogenic
factors within the 14–3-3ζ interactome and reveals alternative path-
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