Diabetes UK Young Investigator Award

Generation and characterisation of a novel beta cell-specific GPR56 knockout mouse model

EO Olaniru¹, AJ King¹, PM Jones¹, X Piao², SJ Persaud¹

¹Department of Diabetes, King's College London, London, UK, ²Department of Medicine, Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA

Aims We have previously shown that GPR56 is the most abundant islet-expressed GPCR and demonstrated that it is activated by an exogenous extracellular matrix protein collagen III. We have now generated beta cell-specific GPR56 knockout (KO) mice and determined the effect of deletion of this receptor only in beta cells on glucose homeostasis.

Methods Transgenic mice with GPR56 deletion specifically in islet beta cells were achieved by crossing Ins1^Cre mice with LoxP-GPR56 mice flanking exons 4–6. Genotyping and GPR56 expression were carried out by PCR and western blotting respectively. Glucose tolerance tests were carried out in GPR56-β-cell KO mice and their wild-type (WT) littermates at 8 weeks.

Results Genotyping showed a 465kb LoxP band and the presence of Cre in GPR56-βKO mice, as expected, and GPR56 deletion was confirmed by western blotting in isolated islets. There was no significant difference in the weights of GPR56-βKO mice and WT littermates at 8 weeks (WT: 24.5 ± 1.9g, bKO: 25.5 ± 1.4g, n = 6, p > 0.2), nor in fasting glucose levels (WT: 8.4 ± 0.7mM, bKO: 7.9 ± 0.4mM, n = 6, p > 0.2). In addition, GPR56 deletion in beta cells did not significantly affect glucose tolerance of lean 8-week-old mice (AUC; WT: 1,292 ± 58.2, bKO: 1,114 ± 81.2, n = 4–7, p > 0.2).

Conclusions These data demonstrate that we have successfully generated beta cell-specific GPR56 KO mice, which are phenotypically normal and show normal glucose tolerance. These mice will allow us to evaluate the requirement of beta cell GPR56 in beta cell development and compensatory responses of beta cells to metabolic dysregulation.

Early development of insulinopenia in Black African men with dysglycaemia

M Ladwa¹, O Bello¹, C Mohandas¹, F Shojee-Moradie², JL Peacock³, AM Umpleby², SA Amiel¹, KGMM Alberti¹, LM Goff¹

¹Diabetes Research Group, Kings College London, London, UK, ²Health and Medical Sciences, University of Surrey, Guildford, UK, ³Population Health and Environmental Sciences, Kings College London, London, UK

Background and Aim The heterogeneity of type 2 diabetes is increasingly recognised, particularly among different ethnic groups. We sought to compare glucose-stimulated insulin responses between Black West African (BWA) and White European (WE) men during the early development of diabetes.

Methods In total, 27 BWA and 25 WE men with either impaired glucose tolerance (n = 11 vs 10) or type 2 diabetes diagnosed within the last five years, treated with metformin and/or lifestyle only (n = 16 vs 15), underwent a two hour hyperglycaemic clamp (fasting glucose + 6.9 mmol/l).

Results Ethnic groups were comparable in age (51.1 ± 8.7 vs 54.4 ± 8.6 years, p = 0.18), BMI (29.6 ± 2.5 vs 30.6 ± 3.3kg/m², p = 0.24), fasting plasma glucose (6.3 ± 1.0 vs 6.5 ± 1.3mmol/l, p = 0.52), HbA1c (47.6 ± 7.6 vs 45.8 ± 7.8mmol/mol, p = 0.41) and insulin sensitivity (M/I ratio during last 30min of clamp; 0.038 vs 0.037mg/kg/min pmol/L, p = 0.94). Incremental area under the curve insulin over 120min was lower in BWA compared with WE (23.7 ± 17.4 × 10³ vs 42.2 ± 42.5 × 10³pmol/L/min, p = 0.046).

Conclusions Our data indicate that Black African men with IGT and type 2 diabetes have greater insulin deficiency than their white European peers, even at mild levels of dysglycaemia. Further work is needed to understand the impact this may have on clinical presentations and therapeutic strategies in this population.

SUPPORTING INFORMATION The conference poster for this abstract is available online in the Supporting Information section at the end of this page.

Recovery of counterregulatory response to subsequent hypoglycaemia after dishabituation with cold exposure in recurrently hypoglycaemic rodents

K Vickneson, J Blackburn, J Gallagher, A McNeilly, RJ McCrimmon

Division of Systems Medicine, University of Dundee, Dundee, UK

Aims Impaired awareness of hypoglycaemia (IAH) is associated with reduced counterregulatory responses (CRR) to hypoglycaemia. Recently, our group hypothesised that IAH develops through habituation of CRR to recurrent hypoglycaemia (RH). Consistent with this hypothesis, we demonstrated a single exposure to high-intensity exercise, at least temporarily restored CRR in non-diabetic rodents exposed to RH; called dishabituation. In this study, we sought to determine if exposure to an alternate novel stimulus, cold, would mediate a similar dishabituating effect and restore CRR.

Methods Three groups of male rats were studied (n = 10/group). Two groups experienced RH (1U/kg insulin i.p.; x3 per week for 4 weeks) while group 3 received saline injections (acute hypoglycaemia; AH). RH rats were then randomised to a single episode of intense cold exposure (3^oC for 4.5h) or to room temperature. After a further 24h, all animals underwent a hyperinsulinaemic-hypoglycaemic clamp at 2.8mmol/l with serial measurement of CRR. Data are expressed as mean ± SEM.

Results Epinephrine (0.94 ± 0.26 vs 0.09 ± 0.04 vs 0.74 ± 0.13ng/ml; AH vs RH vs RH + cold, respectively; p < 0.05) and corticosterone (166 ± 30 vs 58.6 ± 24 vs 167 ± 24ng/ml; p < 0.05) responses to hypoglycaemia were restored to near normal levels following acute cold dishabituation. Glucagon responses did not differ significantly.

Conclusion Dishabituation with acute cold exposure restores adapted hormonal CRRs to hypoglycaemia. These findings lend support to the habituation hypothesis and suggest that dishabituation may offer an alternative therapeutic approach for the management of IAH.

Favourable adiposity genes pinpoint to the role of adipose tissue function in mechanisms protecting obese individuals from type 2 diabetes

S Martin, A Pitt, L Weymouth, A Barnes, M Hudson, K Kos, TM Frayling, H Yaghootkar

College of Medicine and Health, University of Exeter, Exeter, UK

Background Genetic studies have identified 14 variants associated with ‘favourable adiposity’ – higher adiposity but lower risk of type 2 diabetes. Abdominal MRI scans suggest that ‘favourable adiposity’ alleles likely protect against developing type 2 diabetes by storing the extra fat in subcutaneous depots. One explanation is that these variants are associated with healthier adipose tissue function in the form of smaller adipocytes since smaller cells are associated with better insulin sensitivity.

Aims To test the hypothesis that ‘favourable adiposity’ alleles are associated with smaller adipocytes.

Methods We recruited 162 participants from the Exeter 10,000 study, measured body size, skinfold thickness, body composition (using BODPOD^TM system) and collected subcutaneous abdominal fat biopsies. Average adipocyte area was calculated from adipocyte histology images. We tested for associations between each genetic variant and these outcomes, adjusting for age, sex and BMI.

Results The mean adipocyte area was 1,664μm² (95% confidence interval (CI): 1,603–1,725) in 99 men and 1,666μm² (1,575–1,757) in 63 women, and was positively correlated with BMI (r = 0.50; p < 0.01), skinfold (r = 0.40; p < 0.01) and body fat percentage (r = 0.44; p < 0.01). Adiposity-increasing alleles at PPARγ, KLF14 and FAM13A were associated with smaller adipocytes (p = 0.03, 0.03 and 0.046, respectively) but in larger numbers consistent with hyperplasia (p = 0.2, 0.05 and 0.007 respectively). The association with FAM13A was female specific.

Conclusions Our results provide evidence that three ‘favourable adiposity’ variants (PPARγ, KLF14 and FAM13A) are linked with adipose tissue function. Adiposity-increasing alleles at these variants possibly protect from type 2 diabetes by increasing the pool of small adipocytes in the subcutaneous fat depots.

Gender-specific difference in fat handling during weight loss in type 2 diabetes

A Jesuthasan¹, S Zhyzhneuskaya², C Peters², A Barnes³, KG Hollingsworth², N Sattar⁴, MEJ Lean⁵, R Taylor², A Al-Mrabeh²

¹Medical School, Newcastle University, Newcastle upon Tyne, UK, ²Magnetic Resonance Centre, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK, ³Institute of Health and Society, Newcastle University, Newcastle upon Tyne, UK, ⁴Institute of Cardiovascular & Medical Sciences, Glasgow University, Glasgow, UK, ⁵School of Medicine, Dentistry and Nursing, Glasgow University, Glasgow, UK

Objectives Type 2 diabetes and NAFLD are more prevalent in men than women at any given BMI. We have evaluated the difference in lipid parameters at baseline and post-weight loss between men and women in a subgroup of DiRECT.

Methods Fifty-six participants were studied (30M/26F; 53.1 ± 1.1 years; weight 100.9 ± 2.3, diabetes duration 3.1 ± 0.2 years). Magnetic resonance was used for fat quantification, and hepatic VLDL1-TG production was measured via Intralipid infusion method.

Results At baseline men and women had the same level of liver fat (15.1 ± 1 vs 16.9 ± 1.9%, p = 0.49), VLDL1-TG production (553.5 ± 38.7 vs 559.3 ± 32.9mg/kg/day, p = 0.91), pancreas fat (8.5 ± 0.4 vs 8.4 ± 0.5%, p = 0.61) and total plasma TG (2.0 ± 0.2 vs 1.7 ± 0.11mmol/l, p = 0.14). However, there were differences in plasma VLDL1-TG concentration (0.83 ± 0.1 vs 0.58 ± 0.1mmol/l, p = 0.014) and TG pool size (3176 ± 339 vs 1894 ± 295mg, p = 0.003). Women had higher SAT (397.4 ± 21.8 vs 239.3 ± 15.0cm², p < 0.0001) and lower VAT areas (226.6 ± 12.3 vs 320.9 ± 12.4cm², p < 0.0001). This difference in adipose tissue distribution may explain the high plasma NEFA in women (0.67 ± 0.04 vs 0.53 ± 0.03mmol/l, p = 0.004). Following weight loss, there was a major decrease in all lipid parameters irrespective of gender. VLDL-TG and pool size became similar in men and women (0.41 ± 0.06 vs 0.54mmol/l, p = 0.29, and 1937 ± 354 vs 1495 ± 274mg, p = 0.93, respectively). SAT remained significantly higher in women after weight loss. However, both VAT and NEFA became similar to men (158.2 ± 12.2 vs 158.1 ± 12.7cm², p = 0.92 and 0.6 ± 0.04 vs 0.52, p = 0.41mmol/l, respectively).

Conclusions The clear differences between men and women in fat handling point to a potential mechanism involving the higher SAT volumes in women.

Acknowledgement DiRECT study