Loss of PDGF-B activity increases hepatic vascular permeability and enhances insulin sensitivity

Abstract

Hepatic vasculature is not thought to pose a permeability barrier for diffusion of macromolecules from the bloodstream to hepatocytes. In contrast, in extrahepatic tissues, the microvasculature is critically important for insulin action, because transport of insulin across the endothelial cell layer is rate limiting for insulin-stimulated glucose disposal. However, very little is known concerning the role in this process of pericytes, the mural cells lining the basolateral membrane of endothelial cells. PDGF-B is a growth factor involved in the recruitment and function of pericytes. We studied insulin action in mice expressing PDGF-B lacking the proteoglycan binding domain, producing a protein with a partial loss of function (PDGF-B(ret/ret)). Insulin action was assessed through measurements of insulin signaling and insulin and glucose tolerance tests. PDGF-B deficiency enhanced hepatic vascular transendothelial transport. One outcome of this change was an increase in hepatic insulin signaling. This correlated with enhanced whole body glucose homeostasis and increased insulin clearance from the circulation during an insulin tolerance test. In obese mice, PDGF-B deficiency was associated with an 80% reduction in fasting insulin and drastically reduced insulin secretion. These mice did not have significantly higher glucose levels, reflecting a dramatic increase in insulin action. Our findings show that, despite already having a high permeability, hepatic transendothelial transport can be further enhanced. To the best of our knowledge, this is the first study to connect PDGF-B-induced changes in hepatic sinusoidal transport to changes in insulin action, demonstrating a link between PDGF-B signaling and insulin sensitivity.

Fig. 1.

Enhanced vascular leakage resulting from reduced sinusoidal disruption in platelet-derived growth factor B (PDGF-B)^ret/ret liver. Eight-week-old male Leptin^ob (Lep^ob/+) PDGF-B^ret/+ control (A) and PDGF-B^ret/ret mice (B) were infused with FITC-dextran (green; molecular weight = 10 kDa) for 30 min. After circulation, livers were stained for platelet endothelial cell adhesion molecule-1 (PECAM-1; red) and imaged via confocal microscopy. Scale bars, 50 μm. C: the intensity of extravascular FITC-dextran was quantified. Liver sections from 8-wk-old male Lep^ob/+ wild-type (D) and PDGF-B^ret/ret mice (E) were stained for PECAM-1 (red) and desmin (green) and imaged via confocal microscopy. Scale bars, 75 μm. F: hepatic stellate cell (HSC) coverage of liver vessels was quantitated. G and H: transmission electron microscopy (TEM) of 8-wk-old male Lep^ob/+ wild-type (G) and PDGF-B^ret/ret liver sinusoids (H). Scale bars, 2 μm; RBC, red blood cell; KC, Kupffer cell; SD, space of Disse. Arrows represent fenestrae and endothelial cell gaps. I: sinsoidal disruption was quantified as loss of fenestrae and a disordered, electron-dense SD. All results are presented as means ± SE. *P < 0.05 by unpaired Student t-test.

Fig. 2.

Increased insulin signaling and glucose clearance in PDGF-B^ret/ret liver. A: representative Western blots of insulin receptor-β (IRβ) and Akt phosphorylation. Phosphotyrosine and total IRβ were measured in 8-wk-old Lep^ob/+ male wild-type and PDGF-B^ret/ret liver 5 min after insulin treatment. Phosphoserine 473 (Ser⁴⁷³) on Akt and total Akt were measured in 9- to 10-wk-old lean female wild-type and PDGF-B^ret/ret liver mice 15 min after insulin treatment. B: quantification of phosphorylation signal relative to total protein. C: quantification of glucokinase and glucose-6-phosphatase mRNA expression from 8-wk-old Lep^ob/+ wild-type and PDGF-B^ret/ret livers. D: hepatic glucose uptake in 8-wk-old lean female wild-type and PDGF-B^ret/ret liver. Insulin, glucose, and 2-[¹⁴C]deoxyglucose tracer were injected into the jugular vein, and accumulation of hepatic 2-[¹⁴C]deoxyglucose phosphate was measured after 15 min of circulation. E: plasma glucose during insulin tolerance tests on 8-wk-old female wild-type (n = 6) and PDGF-B^ret/ret (n = 6) mice. All results are presented as means ± SE. *P ≤ 0.05, ***P < 0.001 by unpaired Student t-test; #P ≤ 0.05 by area under the curve analysis through 30 min.

Fig. 3.

Enhanced whole body glucose and insulin clearance in PDGF-B^ret/ret Lep^ob mice. Plasma glucose (%time 0; A) and human insulin concentration in plasma (B) during insulin tolerance tests on 8-wk-old male control Lep^ob (n = 8) and PDGF-B^ret/ret Lep^ob mice (n = 4). Results are presented as means ± SE. *P ≤ 0.05 and **P ≤ 0.01 by unpaired Student t-test; vertical lines indicate area under the curve analysis.

Fig. 4.

Severely reduced plasma insulin without a change in glucose tolerance in PDGF-B^ret/ret Lep^ob mice. A and B: plasma insulin (A) and plasma glucose (B) from male control Lep^ob and PDGF-B^ret/ret Lep^ob mice measured after a 4-h fast. C and D: in vivo insulin secretion (C) and plasma glucose clearance (D) during glucose tolerance tests on 8-wk-old male control Lep^ob (n = 7) and PDGF-B^ret/ret Lep^ob mice (n = 5). All results are presented as means ± SE. *P ≤ 0.05, **P ≤ 0.01, and ***P < 0.001 by Mann Whitney (A) or unpaired Student t-test (B–D); vertical line indicates area under the curve analysis.

Fig. 5.

Intact β-cell secretory capacity in PDGF-B^ret/ret Lep^ob mice. A: C-peptide secretion during glucose tolerance tests on 8-wk-old male control Lep^ob (n = 6) and PDGF-B^ret/ret Lep^ob mice (n = 4). B: plasma C-peptide/insulin ratios during a glucose challenge were calculated from the C-peptide values in A and the insulin values in C. C: secreted insulin and total insulin content from 14-wk-old male control Lep^ob and PDGF-B^ret/ret Lep^ob isolated islets. Each data point represents the average of 3 biological replicates/animal. D: fractional release was calculated from the data in Fig. 4C. E: β-cell mass for 14-wk-old male control Lep^ob and PDGF-B^ret/ret Lep^ob mice. All results are presented as means ± SE. *P ≤ 0.05, **P ≤ 0.01, and ***P < 0.001 by unpaired Student t-test; vertical lines indicate area under the curve analysis.

Fig. 6.

Reduced PDGF-B signaling improves hepatic vascular delivery of insulin and enhances whole body insulin and glucose homeostasis. In C57BL/6J mice, normal PDGF-B retention and signaling leads to ultrastructural changes to the hepatic sinusoids, including loss of fenestration and extracellular matrix-like deposition in the SD. These alterations impede the normally rapid delivery of insulin to hepatocytes. Loss of PDGF-B retention leads to a reduction in these alterations, resulting in enhanced hepatic insulin delivery and signaling. The resulting upregulation of glucokinase and suppression of glucose-6-phosphatase should trigger elevated hepatic glucose flux while reducing glucose output into circulation, resulting in better maintenance of glucose homeostasis. In the obese state, this enhanced glycemic control likely triggers feedback mechanisms that signal to the β-cells to reduce insulin secretion, thereby alleviating the hyperinsulinemia typically associated with obesity. ECM, extracellular matrix.