Early deficits in insulin secretion, beta cell mass and islet blood perfusion precede onset of autoimmune type 1 diabetes in BioBreeding rats

Abstract

Aims/hypothesis: Genetic studies show coupling of genes affecting beta cell function to type 1 diabetes, but hitherto no studies on whether beta cell dysfunction could precede insulitis and clinical onset of type 1 diabetes are available.

Methods: We used 40-day-old BioBreeding (BB) DRLyp/Lyp rats (a model of spontaneous autoimmune type 1 diabetes) and diabetes-resistant DRLyp/+ and DR+/+ littermates (controls) to investigate beta cell function in vivo, and insulin and glucagon secretion in vitro. Beta cell mass was assessed by optical projection tomography (OPT) and morphometry. Additionally, measurements of intra-islet blood flow were performed using microsphere injections. We also assessed immune cell infiltration, cytokine expression in islets (by immunohistochemistry and qPCR), as well as islet Glut2 expression and ATP/ADP ratio to determine effects on glucose uptake and metabolism in beta cells.

Results: DRLyp/Lyp rats were normoglycaemic and without traces of immune cell infiltrates. However, IVGTTs revealed a significant decrease in the acute insulin response to glucose compared with control rats (1685.3 ± 121.3 vs 633.3 ± 148.7; p < 0.0001). In agreement, insulin secretion was severely perturbed in isolated islets, and both first- and second-phase insulin release were lowered compared with control rats, while glucagon secretion was similar in both groups. Interestingly, after 5-7 days of culture of islets from DRLyp/Lyp rats in normal media, glucose-stimulated insulin secretion (GSIS) was improved; although, a significant decrease in GSIS was still evident compared with islets from control rats at this time (7393.9 ± 1593.7 vs 4416.8 ± 1230.5 pg islet^-1 h^-1; p < 0.0001). Compared with controls, OPT of whole pancreas from DRLyp/Lyp rats revealed significant reductions in medium (4.1 × 10⁹ ± 9.5 × 10⁷ vs 3.8 × 10⁹ ± 5.8 × 10⁷ μm³; p = 0.044) and small sized islets (1.6 × 10⁹ ± 5.1 × 10⁷ vs 1.4 × 10⁹ ± 4.5 × 10⁷ μm³; p = 0.035). Finally, we found lower intra-islet blood perfusion in vivo (113.1 ± 16.8 vs 76.9 ± 11.8 μl min^-1 [g pancreas]^-1; p = 0.023) and alterations in the beta cell ATP/ADP ratio in DRLyp/Lyp rats vs control rats.

Conclusions/interpretation: The present study identifies a deterioration of beta cell function and mass, and intra-islet blood flow that precedes insulitis and diabetes development in animals prone to autoimmune type 1 diabetes. These underlying changes in islet function may be previously unrecognised factors of importance in type 1 diabetes development.

Keywords: Beta cell dysfunction; Beta cell mass; Insulin secretion; Islet blood flow; Type 1 diabetes.

Fig. 1

(a) Daily glucose levels in 40-day-old female and male DRLyp/Lyp (circles), control (DRLyp/+, triangles and DR+/+, squares) rats presented as days before onset of type 1 diabetes. (b) Cumulative increase in diabetes incidence in male (solid line, squares) and female (dotted line, circles) DRLyp/Lyp rats. (c) Diabetes-free survival in male (solid line) and female (dotted line) DRLyp/Lyp rats. (d) Age at onset in female (F) and male (M) DRLyp/Lyp rats. Data shown as means ± SEM. **p < 0.01. DRLyp/Lyp: n = 225, 129M/96F; DRLyp/+ and DR+/+: n = 100, 50M/50F

Fig. 2

(a) Serum insulin over time in DRLyp/Lyp (black circles) and control rats (white squares). At 37–41 days of age: DRLyp/Lyp, n = 7 (4M/3F), control, n = 10 (5M/5F); at 50 days of age: DRLyp/Lyp, n = 6 (3M/3F), control, n = 10 (5M/5F); at 60 days of age: DRLyp/Lyp, n = 6 (3M/3F), control, n = 11 (6M/5F; at type 1 diabetes onset: DRLyp/Lyp, n = 7 (4M/3F), control n = 9 (5M/4F). (b–f) IVGTTs in 40-day-old DRLyp/Lyp (black circles/bars; n = 10, 6M/4F) and control rats (white squares/bars; n = 10, 6M/4F). (b) Plasma glucose, (c) plasma insulin, (d) AUC for glucose and (e) AUC for insulin. (f) AIR_Glucose. Data shown as means ± SEM. *p < 0.05, ***p < 0.001. T1D, type 1 diabetes

Fig. 3

(a) Isolated islets from 40-day-old DRLyp/Lyp (black circles; n = 14, 9M/5F) and control rats (white squares; n = 8, 4M/4Fe) were perifused with 2.8 mmol/l and 16.7 mmol/l glucose (G) with and without 35 mmol/l KCl (K⁺). (b–c) AUC for secreted insulin (b) 16–56 min at 16.7 mmol/l glucose and (c) 70–80 min at 16.7 mmol/l glucose + KCl. (d) Total insulin content in islets from DRLyp/Lyp (n = 6, 3M/3F) and control rats (n = 6, 3M/3F). (e–f) One-hour batch incubation of isolated islets cultured (e) over night or (f) for 5–7 days. Islets were stimulated with either 2.8 or 16.7 mmol/l glucose. Overnight incubation: DRLyp/Lyp, n = 6, (3M/3F), control, n = 6 (3M/3F); 5–7 day incubation: DRLyp/Lyp, n = 6 (3M/3F), control, n = 7 (3M/4F). White bars, control rats; black bars, DRLyp/Lyp rats. Data shown as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

(a) Whole pancreatic blood flow and (b) islet blood flow in 40-day-old DRLyp/Lyp (n = 11, 4M/7F) and control rats (n = 15, 6M/9F). White bars, control rats; black bars, DRLyp/Lyp rats. Data shown as means ± SEM. *p < 0.05

Fig. 5

(a) Overall beta cell volume in 40-day-old DRLyp/Lyp (n = 6, 4M/2F) and control rats (n = 4, 2M/2F). (b) Islet volumes of arbitrarily chosen islet size categories in DRLyp/Lyp and control rats. White bars, control rats; black bars, DRLyp/Lyp rats. Data shown as means ± SEM. *p < 0.05

Fig. 6

Representative OPT images from a splenic, duodenal and gastric pancreatic lobe from a 40-day-old heterozygote DRLyp/+ rat (control) and a DRLyp/Lyp rat. Scale bar, 2 mm

Fig. 7

(a) ATP/ADP ratio in beta cells from DRLyp/Lyp (black circles; n = 91 islets) and control rats (white squares; n = 70 islets). (b) Basal ATP/ADP ratio, (c) Δ_max ATP/ADP ratio, (d) slope increase of ATP/ADP ratio and (e) AUC for ATP/ADP measurements in beta cells from DRLyp/Lyp (black) and control rats (white). Data shown as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. AU, arbitrary units; G, glucose (mmol/l); FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; G, glucose (mmol/l)

Fig. 8

Pancreatic sections from (a,b) 40-day-old DRLyp/Lyp and (c,d) control rats (DRLyp/+ and DR+/+). Sections were stained for (a,c) insulin (green) and glucagon (red), and (b,d) CD3⁺ (red) with nuclear DAPI (blue). Scale bar, 50 μm