stress; and 2) that prolonged/severe hypoxia impairs lysosomal homeostasis and autophagic flux, which may further exacerbate metabolic failure and death of the β-cells. 35 A Pipeline for 2D and 3D Confocal Analysis of Mitochondrial Morphology and Network Connectivity in β-cells ROCKY SHI, AHSEN CHAUDHRY, DAN S. LUCIANI Vancouver, BC Background: Increasing evidence suggests mitochondrial dynam- ics are important for glucose-stimulated insulin secretion and that quantification of mitochondrial network characteristics can provide insights into β-cell physiology and pathophysiology. To facilitate research in this area, we established a pipeline for accurate 2D and 3D quantification of β-cell mitochondria based on confocal micros- copy and the open source software ImageJ/FIJI. Methods and Results: We expressed mitochondria-targeted YFP in MIN6 cells and built test-sets consisting of 84 2D images and 29 3D image stacks. First, we compared different thresholding approaches for identification and separation of mitochondria within networks. Via manual assessment, and photo-labelling of indi- vidual mitochondria, we identified adaptive thresholding as supe- rior. We manually separated our 2D and 3D image sets into 3 morphological categories (filamentous, intermediate and frag- mented), and established that these could be distinguished statis- tically based on calculations of select shape/networking parameters. The validity of these results was verified by comparing them to an analysis of the same images after grouping them in an unsuper- vised manner using Spanning-tree Progression Analysis of Density- normalized Events (SPADE). We next applied this pipeline to MitoTracker-labelled mouse islet cells and could detect subtle mito- chondrial fission upon glucose stimulation. Finally, we extended the analysis to time-lapse data and demonstrated the feasibility of quan- titating the dynamics of the entire mitochondrial network. Conclusion: We have developed and validated an image analysis pipeline that can accurately quantify β-cell mitochondrial mor- phology and network connectivity in 2D and 3D. 36 Normoglycemia in Mice Lacking Beta-Cell Prohormone Convertase 1/3 AUSTIN TAYLOR, BRUCE VERCHERE Vancouver, BC Introduction: In islet beta cells, the endoprotease activity of prohormone convertases (PC) PC1/3 and PC2 are believed neces- sary for efficient processing of proinsulin to mature insulin. PC1/3 expression is a marker of beta-cell maturity and function, and poly- morphisms in PC1/3 are associated with obesity, impaired glucose tolerance and elevated circulating proinsulin:insulin. Given PC1/ 3’s key role in beta-cell function, we hypothesized that beta-cell PC1/3 deficiency would lead to beta-cell dysfunction and glucose intolerance. Methods: Male and female PC1/3flox/flox Ins1+/cre, PC1/3flox/ +Ins1+/cre, PC1/3+/+Ins1+/cre and PC1/3flox/flox Ins1+/+mice were tracked for changes in body weight and fasting blood glucose. Glucose and insulin tolerance tests were administered at 12 and 14 weeks, respectively, and fasting plasma proinsulin was assessed by ELISA at 10 weeks. Results: Beta-cell PC1/3 deficiency drastically increased fasting plasma proinsulin in both male (PC1/3flox/flox Ins1+/cre vs. PC1/ 3+/+Ins1+/cre:>820 vs. 17 pM) and female mice, but did not influ- ence fasted blood glucose (9.9±1.2 vs. 9.9±1.1 mM; p=NS) or body weight up to 21 weeks of age. There was a trend toward glucose intolerance in PC1/3flox/flox Ins1+/cre male mice (AUC: 521±185 vs. 360±146; p=NS), and no differences in insulin sensitivity. Conclusions: Beta-cell PC1/3 deficiency does not severely impair glucose homeostasis in mice fed a chow diet, despite highly elevated circulating proinsulin. Our data suggest that beta cells can com- pensate for marked impairment in proinsulin processing machin- ery caused by PC1/3 deficiency. 37 Effects of 6-Month Vitamin D Supplementation on Insulin Sensitivity and Secretion: A Randomized, Placebo-Controlled Trial PATRICIA LEMIEUX*, S. JOHN WEISNAGEL, ANNABELLE Z. CARON, ANNE-SOPHIE JULIEN, ANNE-SOPHIE MORISSET, JONATHAN POIRIER, ANNE-MARIE CARREAU † , ANDRÉ TCHERNOF † , JULIE ROBITAILLE, JEAN BERGERON, MARIE-CLAUDE VOHL, CLAUDIA GAGNON † Quebec, QC Primary Objective: To determine whether vitamin D3 supplemen- tation improves insulin sensitivity in vitamin D-deficient and insulin- resistant individuals, using the gold-standard euglycemic- hyperinsulinemic clamp. Methods: A single-centre, double-blind randomized placebo- controlled trial was conducted. Ninety-six participants without known diabetes and with serum 25-hydroxyvitamin D (25(OH)D)≤55 nM, increased waist circumference (men:≥102 cm; women:≥88 cm) and ≥1 metabolic abnormality associated with insulin resistance were randomized to vitamin D3 5,000 IU daily or placebo for 6 months. We assessed at baseline and 6 months: periph- eral insulin sensitivity (M-value) using a 2-h euglycemic- hyperinsulinemic clamp; other insulin sensitivity (HOMA2%S, Matsuda) and insulin secretion (insulinogenic index, C-peptide area under the curve, HOMA2-B) indices using a 2h-OGTT; ß-cell func- tion (disposition index: M-value x insulinogenic index); weight; fasting and 2-h glucose post-OGTT; HbA1c and blood pressure. Results: Baseline characteristics were similar between groups (% or mean±SD): women 38.5%; age 58.7±9.4 years; BMI 32.2±4.1 kg/ m 2 ; prediabetes 35.8%; diabetes 20.0%; 25(OH)D 51.1±14.2 nM. At 6 months, the mean 25(OH)D was 127.6 nM and 52.0 nM in the treat- ment and placebo groups, respectively (p<0.001). A significant ben- eficial effect of vitamin D3 compared with placebo was observed on peripheral insulin sensitivity (M-value: +17.5% vs. -11.2%; p=0.003) and on disposition index (-2.8% vs. -14.8%; p=0.011). No effect was seen on other insulin sensitivity and secretion indices, metabolic markers and anthropometric measures. Conclusion: In vitamin D-deficient and insulin-resistant individu- als, vitamin D supplementation significantly increased peripheral insulin sensitivity, as assessed by the euglycemic-hyperinsulinemic clamp. 38 ★ Clinical Prediction Score to Identify Hepatic Steatosis in Adolescents with Polycystic Ovarian Syndrome ANNE-MARIE CARREAU, LAURA PYLE, YESENIA GARCIA-REYES, HASEEB RAHAT, THOMAS JENSEN, KRISTEN J. NADEAU, MELANIE CREE-GREEN Denver, CO Background: Hepatic steatosis (HS) is common in obese girls with polycystic ovarian syndrome (PCOS) and may best predict future development of type 2 diabetes. To date, there are no non-invasive and inexpensive ways to identify HS in youth with PCOS. Objective: To develop a new simple clinical prediction score for HS in young women with PCOS. Design: Eighty-seven overweight/obese girls with PCOS (age 12–19 years) had a fasting blood sample drawn, anthropometrics Abstracts / Can J Diabetes 42 (2018) S2–S17 S13