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, 54 (3), 572-82

Glucose Stimulates Human Beta Cell Replication in Vivo in Islets Transplanted Into NOD-severe Combined Immunodeficiency (SCID) Mice

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Glucose Stimulates Human Beta Cell Replication in Vivo in Islets Transplanted Into NOD-severe Combined Immunodeficiency (SCID) Mice

H E Levitt et al. Diabetologia.

Abstract

Aims/hypothesis: We determined whether hyperglycaemia stimulates human beta cell replication in vivo in an islet transplant model

Methods: Human islets were transplanted into streptozotocin-induced diabetic NOD-severe combined immunodeficiency mice. Blood glucose was measured serially during a 2 week graft revascularisation period. Engrafted mice were then catheterised in the femoral artery and vein, and infused intravenously with BrdU for 4 days to label replicating beta cells. Mice with restored normoglycaemia were co-infused with either 0.9% (wt/vol.) saline or 50% (wt/vol.) glucose to generate glycaemic differences among grafts from the same donors. During infusions, blood glucose was measured daily. After infusion, human beta cell replication and apoptosis were measured in graft sections using immunofluorescence for insulin, and BrdU or TUNEL.

Results: Human islet grafts corrected diabetes in the majority of cases. Among grafts from the same donor, human beta cell proliferation doubled in those exposed to higher glucose relative to lower glucose. Across the entire cohort of grafts, higher blood glucose was strongly correlated with increased beta cell replication. Beta cell replication rates were unrelated to circulating human insulin levels or donor age, but tended to correlate with donor BMI. Beta cell TUNEL reactivity was not measurably increased in grafts exposed to elevated blood glucose.

Conclusions/interpretation: Glucose is a mitogenic stimulus for transplanted human beta cells in vivo. Investigating the underlying pathways may point to mechanisms capable of expanding human beta cell mass in vivo.

Figures

Fig. 1
Fig. 1
Experimental design. a NOD–SCID mice were rendered diabetic using streptozotocin (STZ) and then transplanted with a therapeutic dose of human islets under the kidney capsule. After a 2 week graft revascularisation period, mice were catheterised in the femoral artery and vein, and intravenously infused with BrdU to label replicating beta cells over a 4 day period. Serial blood samples were taken for measurement of blood glucose after transplant (tail snip, white circles) and during the infusion BrdU exposure (arterial catheter sample, black circles). After the 4 day infusion mice were killed, and the graft was removed and processed for histological analysis. b Mice in which the human islet graft normalised blood glucose (<11 mmol/l) received either 0.9% saline or 50% glucose co-infused with BrdU, to generate a range of blood glucose levels during the labelling period. Mice that remained diabetic after transplantation received infused saline during the BrdU exposure period
Fig. 2
Fig. 2
Human islet grafts supported the metabolism of recipient mice. a Serial blood glucose measurements in transplanted mice demonstrated resolution of diabetes in the majority of mice (blood glucose <11 mmol/l, n=14, circles). A subset of mice had persistent hyper-glycaemia (blood glucose >11 mmol/l, n=8, triangles). The dotted line denotes the blood glucose threshold of 11 mmol/l. b Mice with grafts from the same donor showed similar transplant outcome, as demonstrated by blood glucose 14 days after transplant. Mice engrafted with islets from donor 10 had no blood glucose measurement at this time point; all these grafts had blood glucose >11 mmol/l during the infusion period. c Histological examination of the endogenous mouse pancreas demonstrated ablation of mouse beta cells. Only rare, scattered insulin-positive cells (green) were found. d Circulating human insulin levels tended to be higher in mice with grafts that normalised blood glucose than in those in which the graft failed, although the difference did not achieve significance (p=0.18). Circulating rodent insulin levels were undetectable (n.d., not detectable)
Fig. 3
Fig. 3
Metabolic environment during the period of BrdU exposure. a Blood glucose remained elevated in the >11 mmol/l blood glucose group (triangles) and low in the normoglycaemic saline group (white circles), but increased slightly in mice receiving glucose infusions (black circles). *p=0.0009 vs saline by two-way ANOVA (pre-infusion sample at time zero excluded). b Plasma human insulin levels increased relative to baseline in the glucose-infused group (black circles); *p=0.04 vs saline (white circles) by two-way ANOVA (pre-infusion sample at time zero excluded). White triangles, >11 mmol/l blood glucose. c Average blood glucose and human insulin values for each mouse over the infusion period show that the majority of grafts maintained glucose and insulin within a defined region. Plasma insulin was not measured for one mouse in the >11 mmol/l blood glucose group (triangles) and in one mouse in the saline group (white circles). Black circles, glucose infusion. d During BrdU exposure, grafts in the >11 mmol/l blood glucose (BG) group showed relatively lower circulating human insulin than grafts in mice infused with glucose. *p<0.05 for blood glucose >11 mmol/l vs glucose infusion by one-way ANOVA
Fig. 4
Fig. 4
In grafts from the same donors, higher blood glucose stimulated human beta cell replication. a–d Graft sections stained for insulin (green) and BrdU (red) allowed quantification of human beta cell replication. Images are representative examples of BrdU-positive beta cells, acquired by confocal microscopy. Note that the 4 day BrdU exposure resulted in labelling of daughter cells. e–k To exclude the possibility that BrdU-positive and insulin-positive cells were cells of monocyte lineage that had incorporated BrdU, subsequently entering the graft and engulfing insulin-positive material, macrophages were immunolabelled with F4/80 (red). e, f Control liver sections labelled with F4/80 and Hoechst (blue) demonstrated appropriate staining without non-specific signal. g, h Graft sections stained for insulin (green) and F4/80 (red) also showed specific F4/80 label in and around grafts, which was not present in the absence of the F4/80 antibody. i–k Graft sections stained for insulin (green), BrdU (white), Hoechst (blue) and F4/80 (red) were imaged by confocal microscopy. Note (yellow arrowheads) the BrdU-positive nucleus that belongs to an insulin-positive cell that is not F4/80-positive. Scale bars, 10 μm. l Analysis restricted to donors for which saline-infused and glucose-infused mice were available showed a widely variable baseline replication rate among donors. However, human beta cell replication increased consistently across all donors with exposure to 4 days of higher blood glucose. m Despite the widely variable baseline replication rates, higher glucose significantly increased human beta cell replication (p=0.03 by paired t test). For these four donors, 9,041±1,907 beta cells were counted per donor
Fig. 5
Fig. 5
Across the cohort of human islet grafts, hyperglycaemia increased human beta cell replication. a Average blood glucose during 4 day BrdU exposure significantly correlated with human beta cell replication. The correlation was strongest for animals that maintained blood glucose <16.7 mmol/l (dashed line; p=0.0001, r2=0.60), but was highly significant even when all animals were included (continuous line; p=0.0019, r2=0.39). Grafts from a 7-year-old donor (+) were included as a positive control for replication, but were not included in analyses. b Bin analysis of adult human beta cell replication by blood glucose quartile found that replication increased for blood glucose values above 6.0 mmol/l, reaching significance at the highest quartile. *p<0.05 vs quartile 1 and p<0.05 vs quartile 2, calculated by ANOVA across all groups. c Graft beta cell replication showed no relationship with circulating plasma human insulin level; p=NS. Plasma insulin was not measured for one mouse in the >11 mmol/l blood glucose group and one mouse in the saline group; two grafts in the glucose infusion group had exactly the same plasma human insulin and replication rates (198 pmol/l insulin, 0.11% replication), and thus are superimposed. d Graft beta cell replication correlated with blood glucose during the week prior to BrdU exposure (p=0.03, r2=0.24). Three grafts (donor 10) did not have blood glucose measured during the week prior to BrdU exposure. Key (a, c): white circles, saline; black circles, glucose infusion; triangles, blood glucose >11 mmol/l
Fig. 6
Fig. 6
Human beta cell apoptosis was not increased in grafts exposed to hyperglycaemia. a Sections from a mouse pancreas 6 h after intraperitoneal streptozotocin injection and (b, c) from human islet grafts, stained for insulin (red) and TUNEL (green) allowed quantification of apoptosis. Confocal images are representative examples of TUNEL staining of positive control. Scale bars 10 μm. d Quantification of TUNEL staining did not reveal a relationship between beta cell apoptosis and circulating blood glucose or (e) plasma human insulin, p=NS for both. Two mice (e) did not have plasma insulin measured, and three points are not visible due to grafts with identical TUNEL and insulin values
Fig. 7
Fig. 7
Human beta cell replication was related to donor BMI but unrelated to donor age. a Replication showed a trend towards correlation with donor BMI in this cohort (p=0.056, r2=0.17). b No relationship between donor age and beta cell replication was observed across this cohort of human islet grafts. Two grafts from donor 8 (BMI 25.8 kg/m2, age 76) had identical replication rates (0.13%) and the points are therefore superimposed (a, b)

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