Pancreatic islet vasculature adapts to insulin resistance through dilation and not angiogenesis - PubMed (original) (raw)

. 2013 Dec;62(12):4144-53.

doi: 10.2337/db12-1657. Epub 2013 Apr 29.

Affiliations

Pancreatic islet vasculature adapts to insulin resistance through dilation and not angiogenesis

Chunhua Dai et al. Diabetes. 2013 Dec.

Abstract

Pancreatic islets adapt to insulin resistance through a complex set of changes, including β-cell hyperplasia and hypertrophy. To determine if islet vascularization changes in response to insulin resistance, we investigated three independent models of insulin resistance: ob/ob, GLUT4(+/-), and mice with high-fat diet-induced obesity. Intravital blood vessel labeling and immunocytochemistry revealed a vascular plasticity in which islet vessel area was significantly increased, but intraislet vessel density was decreased as the result of insulin resistance. These vascular changes were independent of islet size and were only observed within the β-cell core but not in the islet periphery. Intraislet endothelial cell fenestration, proliferation, and islet angiogenic factor/receptor expression were unchanged in insulin-resistant compared with control mice, indicating that islet capillary expansion is mediated by dilation of preexisting vessels and not by angiogenesis. We propose that the islet capillary dilation is modulated by endothelial nitric oxide synthase via complementary signals derived from β-cells, parasympathetic nerves, and increased islet blood flow. These compensatory changes in islet vascularization may influence whether β-cells can adequately respond to insulin resistance and prevent the development of diabetes.

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Figures

FIG. 1.

FIG. 1.

Reduced islet vessel density but increased vessel size in insulin-resistant ob/ob mice. A–F: Islet vasculature in wt/wt and ob/ob mouse pancreas was visualized by intravital labeling with FITC-conjugated endothelium-binding tomato lectin. Pancreatic sections (60 µm) were optically sectioned, and islet vasculature was three-dimensionally reconstructed. The scale bar in A represents 50 µm and applies to B_–_F. G and H: Three-dimensional projections of islet capillaries visualized after a bolus of rhodamine-conjugated dextran by live imaging in vivo. The arrowheads in D_–_H point to capillaries in the islet periphery. The scale bar in G represents 50 µm and applies to H. Images in A_–_H are representative three-dimensional projections of islet capillaries obtained from 3 ob/ob mice and 3 wt/wt controls at 16 weeks of age with 3–5 islets/mouse. I_–_K: Vascular morphometry was performed on 10-µm cryosections, with blood vessels visualized by intravital labeling with lectin-FITC (6,19). MetaMorph 6.1 software (Universal Imaging) was used to apply integrated morphometry analysis to at least 40 islets per tissue block or comparable areas of acinar tissue (n = 4–5 mice/genotype) to determine ratio of islet capillary area to islet area, capillary density, and area per capillary. Capillaries were counted using a technique described by Weidner (49), where any fluorescently labeled EC or EC cluster clearly separate from adjacent microvessels was considered a single, countable microvessel. L: Islet capillary diameter was measured on 20 capillaries visualized by a bolus of sulforhodamine-conjugated dextran in wt/wt and ob/ob mice at 16 weeks of age (n = 3 islets/group). Capillary density (M) and area per capillary (N) in acinar tissue were not statistically different in wt/wt and ob/ob mice at 8 weeks of age (at least 40 acinar tissue areas were analyzed per mouse; n = 4 mice/genotype). ***P < 0.001 ob/ob compared with wt/wt.

FIG. 2.

FIG. 2.

Reduced islet vessel density and increased vessel area in mice with insulin resistance due to HFD or GLUT4 heterozygosity. Islet vasculature in C56BL/6 mice fed chow (A) or HFD (B) visualized by EC labeling with caveolin-1. C_–_E: Morphometric analysis of islet vasculature in mice fed chow or HFD. Islet vasculature in GLUT4+/+ (F) and GLUT4+/− (G) was visualized by EC labeling with caveolin-1. H_–_J: Morphometric analysis of islet vasculature in GLUT4+/+ and GLUT4+/− mice. The scale bar in A represents 50 µm and applies to B, F, and G. ***P < 0.001 HFD or GLUT4+/− mice compared with respective controls (_n_ = 4–5 mice and >100 islets/genotype).

FIG. 3.

FIG. 3.

Islet angiogenic factor expression and EC fenestrations are not altered in insulin resistance. Gene expression profile of angiogenic factors (A) and their receptors (B) in wt/wt and ob/ob islets at 8 weeks of age was measured by quantitative RT-PCR. Gene expression analysis was performed on islets isolated from five separate mice per genotype (P > 0.05). Quantitative RT-PCR data were normalized to endogenous B2m control and then expressed relative to wt/wt control. Ins1 mRNA level was not different in ob/ob and wt/wt islets, and Ins2 mRNA level was modestly increased in ob/ob islets (40 ± 10% increase in ob/ob compared with wt/wt). C: VEGF-A secretion in isolated size-matched wt/wt (n = 5 samples) and ob/ob islets (n = 4 samples) was assessed as described previously (6) and normalized per 100 islet equivalents (IEQs) (50). Pancreatic sections from wt/wt (D and E) and ob/ob (F and G) mice at 4 weeks of age were stained for insulin (Ins, green), Ki67 (red), and CD31 (green) and counterstained with DAPI (blue). No Ki67+/CD31+ ECs were detected in wt/wt or ob/ob pancreatic sections. The scale bar in G represents 50 µm and applies to D_–_F. Pancreatic sections from wt/wt (H and I) and ob/ob (J and K) mice at 8 weeks of age were stained for insulin (Ins, green), BrdU (red), and caveolin-1 (Cav-1, green) and counterstained with DAPI (blue). No BrdU+/caveolin-1+ ECs were detected in wt/wt or ob/ob pancreatic sections. BrdU (Sigma-Aldrich) was administered in drinking water at 0.8 mg/mL for 7 days before tissue collection. The scale bar in K represents 50 µm and applies to H_–_J. EC ultrastructure of islet vasculature in wt/wt (L) and ob/ob (M) was assessed by transmission electron microscopy. The arrowheads point to EC fenestrations; L, capillary lumen; RBC, red blood cell. The scale bar in L and M represents 500 nm. N: Basement membrane thickness in wt/wt and ob/ob was analyzed by transmission electron microscopy and morphometry (n = 10 capillaries/genotype, n = 2 mice/genotype). **P < 0.01.

FIG. 4.

FIG. 4.

Pericytes associated with intraislet capillaries become hypertrophied during insulin resistance. Pancreatic sections from wt/wt (A_–_C) and ob/ob mice (D_–_F) at 16 weeks of age were colabeled with the pericyte markers NG2 (green) and PDGFRβ (red). The dashed line denotes the islet perimeter. The boxes in C and F denote enlargements in C′ and F′. The scale bar in F represents 50 µm and also applies to A_–_E. Transmission electron microscopy micrographs of wt/wt (G) and ob/ob islet (H). The arrows point to normal pericytes in G and to hypertrophied pericytes in H; L, capillary lumen. The scale bar in G and H represents 500 nm. I: Pdgfb and Pdgfrb mRNA expression was determined by quantitative RT-PCR. Gene expression analysis was performed on islets isolated from four separate mice per genotype. Quantitative RT-PCR data were normalized to endogenous B2m control and then expressed relative to wt/wt. *P < 0.05.

FIG. 5.

FIG. 5.

Enhanced parasympathetic innervation and blood flow in ob/ob islets. Islets from ob/ob mice (B and D) and their wt/wt controls (A and C) were immunolabeled for insulin (green) and neuronal class III β-tubulin (TUJ1; red/grayscale in A′ and B′) or VAChT (red/grayscale in C′ and D′). The scale bars are 100 μm. E_–_G: Global islet innervation density was quantified by calculating the number of TUJ1+ fibers within the insulin+ islet area. Islet parasympathetic innervation was quantified by calculating the number of VAChT+ varicosities per insulin+ area and by calculating the VAChT+ area as a percentage of the insulin+ islet area. Quantification is shown for islet TUJ1+ nerve fiber density (E), islet VAChT+ varicosity density (F), and islet VAChT+ area (G) (n = 100 islets/genotype). H: Islet blood flow was measured by tracking sulforhodamine-labeled RBCs in wt/wt and ob/ob mice at 16 weeks of age (n = 90/genotype). Ins+, insulin+. *P < 0.05 and ***P < 0.001 ob/ob compared with wt/wt.

FIG. 6.

FIG. 6.

Expression of NOS isoforms and NO production in wt/wt and ob/ob islets. A: The expression level of three NOS isoforms in wt/wt islets was measured by quantitative RT-PCR (n = 5 islet samples) and expressed relative to Actb. B: Expression profile of Nos1, Nos2, and Nos3 in wt/wt and ob/ob islets at 8 weeks of age (n = 5 samples/genotype). B2m was used as an endogenous control in A and B. C_–_G: NO production in wt/wt and ob/ob islets was measured by DAF-2T fluorescence in vivo. Phase contrast images of pancreatic cryosections from wt/wt (C) and ob/ob mice (E), with islet boundaries denoted by the dashed line. D and F: DAF-2T fluorescence within islet boundaries marked in C and E. G: Intensity of DAF-2T fluorescence. AU, arbitrary unit. *P < 0.05; ***P < 0.001 ob/ob compared with wt/wt.

FIG. 7.

FIG. 7.

Proposed model of mechanisms leading to islet vascular changes in insulin-resistant mice. Details are outlined in the

DISCUSSION

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References

    1. Lifson N, Lassa CV, Dixit PK. Relation between blood flow and morphology in islet organ of rat pancreas. Am J Physiol 1985;249:E43–E48 - PubMed
    1. Murakami T, Miyake T, Tsubouchi M, Tsubouchi Y, Ohtsuka A, Fujita T. Blood flow patterns in the rat pancreas: a simulative demonstration by injection replication and scanning electron microscopy. Microsc Res Tech 1997;37:497–508 - PubMed
    1. Adams RH, Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol 2007;8:464–478 - PubMed
    1. Augustin HG, Koh GY, Thurston G, Alitalo K. Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system. Nat Rev Mol Cell Biol 2009;10:165–177 - PubMed
    1. Lammert E, Gu G, McLaughlin M, et al. Role of VEGF-A in vascularization of pancreatic islets. Curr Biol 2003;13:1070–1074 - PubMed

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