Low-density lipoprotein receptor-related protein 5 (LRP5) is essential for normal cholesterol metabolism and glucose-induced insulin secretion

Abstract

A Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) plays an essential role in bone accrual and eye development. Here, we show that LRP5 is also required for normal cholesterol and glucose metabolism. The production of mice lacking LRP5 revealed that LRP5 deficiency led to increased plasma cholesterol levels in mice fed a high-fat diet, because of the decreased hepatic clearance of chylomicron remnants. In addition, when fed a normal diet, LRP5-deficient mice showed a markedly impaired glucose tolerance. The LRP5-deficient islets had a marked reduction in the levels of intracellular ATP and Ca(2+) in response to glucose, and thereby glucose-induced insulin secretion was decreased. The intracellular inositol 1,4,5-trisphosphate (IP3) production in response to glucose was also reduced in LRP5-- islets. Real-time PCR analysis revealed a marked reduction of various transcripts for genes involved in glucose sensing in LRP5-- islets. Furthermore, exposure of LRP5++ islets to Wnt-3a and Wnt-5a stimulates glucose-induced insulin secretion and this stimulation was blocked by the addition of a soluble form of Wnt receptor, secreted Frizzled-related protein-1. In contrast, LRP5-deficient islets lacked the Wnt-3a-stimulated insulin secretion. These data suggest that WntLRP5 signaling contributes to the glucose-induced insulin secretion in the islets.

Figure 1

Generation of LRP5-deficient mice. (A) Diagram of the targeting strategy. Only the relevant restriction sites are indicated. (B) Southern blot analysis of HindIII-digested DNA from LRP5+/+, LRP5−/−, and LRP5+/− mice. Southern blotting was performed with the probe indicated in A. HindIII digestion resulted in an 18-kb fragment in wild-type DNA and a 13-kb fragment in homologous recombinants. A typical autoradiogram is shown. (C) Northern blot analysis of LRP5 transcripts. Total RNA (15 μg) from the livers of LRP5+/+ and LRP5−/− mice was hybridized with a mouse LRP5 cDNA probe (extended from nucleotide 2401 to nucleotide 2991). A typical autoradiogram (48-h exposure) is shown. RNA loading was consistent among the lanes as judged by ethidium bromide staining and reprobing with glyceraldehyde-3-phosphate dehydrogenase. (D) Immunoblot analysis, using an anti-mouse LRP5 antibody, of LRP5+/+, LRP5+/−, and LRP5−/− mouse liver membrane fractions. Each lane was loaded with 500 μg of crude membrane fraction from the liver homogenates. Protein loading was consistent among the lanes as judged by Ponceau staining.

Figure 2

Diet-induced hypercholesterolemia in LRP5-deficient mice. (A) Total plasma cholesterol levels in mice that were fed a high-fat diet. Mice (7–8 weeks of age) heterozygous (LRP5+/−) and homozygous (LRP5−/−) for LRP5 deficiency and their wild-type littermates (LRP5+/+) were fed a high-fat diet for 16 weeks, during which plasma total cholesterol levels of each mouse were measured at the indicated times. The values are the mean ± SE for six mice. *, P < 0.05 compared with LRP5+/+. (B and C) Plasma clearance (B) and liver uptake (C) of injected chylomicron remnants (CMR). The values are the mean ± SE for six mice. *, P < 0.01; Student's t test.

Figure 3

Impaired glucose-induced insulin secretion in LRP-deficient mice and amelioration by AdLRP5. (A and B) Blood glucose (A) and serum insulin (B) levels in LRP5+/+, +/−, and −/− mice after glucose injection. (C) Impaired insulin secretion from the islets of LRP5−/− mice. Insulin secretion was induced by different concentrations of glucose and 0.2 mM tolbutamide (Tol), or 10 mM α-ketoisocaproate (KIC), in the presence of 5.6 mM glucose. (D) Restoration of insulin secretion by AdLRP5. Pancreatic islets were isolated from LRP5+/+ and −/− mice, infected with recombinant adenoviruses encoding LRP5 (AdLRP5) or LacZ (AdLacZ), and insulin secretion was measured at various glucose concentrations. The values in A and B are the mean ± SE for six mice; those in C and D are the mean ± SE for four mice. *, P < 0.01; Student's t test.

Figure 4

Impaired glucose-induced [Ca²⁺]_i increase and IP3 production in LRP5-deficient islets. (A and B) LRP5+/+ (A) and −/− (B) islets infected with AdLRP5 or control AdLacZ. Changes in [Ca²⁺]_i were measured under low glucose (2.8 mM; LG), high glucose (20 mM; HG), or 20 mM KCl (KCl). Representative data from 30 experiments are shown. (C) Time course of IP3 content in response to 20 mM glucose in LRP5+/+ and −/− islets. Values are the mean ± SE from quadruplicate determinations. (D) Restoration of IP3 production by AdLRP5. Pancreatic islet cells were isolated from LRP5+/+ and −/− mice and infected with AdLRP5 or AdLacZ, and the IP3 content was measured at 5 min after exposure to 20 mM glucose. The values are the mean ± SE from quadruplicate determinations. *, P < 0.01; Student's t test.

Figure 5

Effects of Wnt-3a and Wnt-5a on glucose-induced insulin secretion. (A) Insulin secretion from the islets. LRP5+/+ islets were pretreated with 5-fold diluted Wnt-3a-CM, Wnt-5a-CM, or control neo-CM in the presence of the indicated concentration of sFRP-1 for 16 h before measuring insulin secretion induced by glucose. (B) Lack of Wnt-3a stimulation of insulin secretion from LRP5-deficient islets and restoration by AdLRP5. LRP5−/− islets were infected with AdLRP5 or AdLacZ and exposed to Wnt-3a- or control neo-CM for 16 h before measuring insulin secretion in the presence of 16.5 mM glucose. The values are the mean ± SE for four mice from quadruplicate determinations. *, P < 0.01; Student's t test.