Angiopoietin-like protein 4 decreases blood glucose and improves glucose tolerance but induces hyperlipidemia and hepatic steatosis in mice

Abstract

Angiopoietin-like protein 4 (ANGPTL4) is a circulating protein predominantly expressed in adipose tissue and liver. Several recent studies demonstrated that ANGPTL4 is the target gene of peroxisome proliferation activators, the agonists of which are widely used as the antidiabetic and lipid-lowering drugs. Here we provide evidence that ANGPTL4 is a blood-borne hormone directly involved in regulating glucose homeostasis, lipid metabolism, and insulin sensitivity. Adenovirus-mediated expression of ANGPTL4 potently decreased blood glucose and improved glucose tolerance, whereas it induced hyperlipidemia, fatty liver, and hepatomegaly in C57 mice. In db/db diabetic mice, ANGPTL4 treatment reduced hyperglycemia to a normal level, and markedly alleviated glucose intolerance and hyperinsulinemia. Ex vivo studies on primary rat hepatocytes revealed that ANGPTL4 significantly decreased hepatic glucose production and enhanced insulin-mediated inhibition of gluconeogenesis. Serum levels of ANGPTL4 in human subjects inversely correlated with plasma glucose concentrations and HOMA IR, the homeostasis model assessment of insulin resistance. In patients with type 2 diabetes, serum levels of ANGPTL4 were significantly lower than those in healthy subjects, suggesting that the decreased ANGPTL4 could be a causative factor of this disease. These results collectively indicate that ANGPTL4 exerts distinct effects on glucose and lipid metabolism, and that its beneficial effect on glucose homeostasis might be useful for the treatment of diabetes.

Fig. 1.

Expression of ANGPTL4 protein in C57 mice following tail-vein injection with Adv-ANGPTL4. (A) The schematic diagram of mouse ANGPTL4 protein structure. Note that cysteines 76 and 80 are responsible for oligomerization (27). Asp-181, -232, and -320 are the three predicted N-glycosylation sites. SS, signal sequence. (B) Western blot analysis of FLAG-tagged ANGPTL4 in serum. One microliter of serum from mouse at 1 day before (-1) or different days after tail-vein injection with 5 × 10⁹ pfu of Adv-ANGPTL4 was separated by SDS/12% PAGE and probed with horseradish peroxidase (HRP)-conjugated anti-FLAG antibody.

Fig. 2.

Overexpression of ANGPTL4 induces hepatomegaly and fatty liver in mice. C57 mice were treated with 5 × 10⁹ pfu of Adv-ANGPTL4 or Adv-Luc for a period of 2 weeks. (A) Liver images of mice treated with Adv-ANGPTL4 or Adv-Luc. (B and C) The liver weights and TG levels in liver tissues from mice injected with adv-Luc (B) or Adv-ANGPTL4 (C). ^*, P < 0.05; ^**, P < 0.01, compared with Adv-Luc-treated mice (n = 5-8). (D) The representative image of Oil Red O-stained liver sections.

Fig. 3.

Overexpression of ANGPTL4 causes a sustained decrease of blood glucose levels and improves glucose tolerance in C57 mice. (A) Blood glucose levels of mice at various days after injection without (control) or with 5 × 10⁹ pfu of Adv-ANGPTL4 or Adv-Luc. (B) Glucose tolerance test at 2 weeks after treatment. ^*, P < 0.05; ^**, P < 0.01, compared with the Adv-Luc-treated group or control mice (n = 6-8).

Fig. 4.

The potent therapeutic effects of ANGPTL4 on hyperglycemia, hyperinsulinemia, and glucose intolerance associated with db/db diabetic mice. (A) Fasted and fed blood glucose levels of db/db diabetic mice at 1 and 2 weeks after injected without (control) or with 5 × 10⁹ pfu of Adv-ANGPTL4 or Adv-Luc. (B and C) Plasma insulin levels (B) and glucose tolerance test (C) at 2 weeks after treatment are also shown. ^*, P < 0.05; ^**, P < 0.01, compared with Adv-Luc-treated mice or control mice (n = 7-9).

Fig. 5.

ANGPTL4 exerts direct inhibitory effects on glucose production in primary rat hepatocytes. (A) Western analysis of ANGPTL4 secreted from primary rat hepatocytes. Cells grown in a 12-well plate were infected with Adv-ANGPTL4 or Adv-Luc (50 pfu per cell) for a period of 48 h. The conditioned culture media from these cells were subjected to immunoblotting analysis using horseradish peroxidase (HRP)-conjugated anti-FLAG monoclonal antibody. (B) The rates of hepatic glucose output in the absence or presence of different concentrations of insulin. (C) Percentage inhibition of hepatic glucose output (HGO) by various concentrations of insulin in cells infected with Adv-ANGPTL4 or Adv-Luc. ^*, P < 0.05; ^**, P < 0.01 compared with Adv-Luc-treated group (n = 6-8).

Fig. 6.

Serum levels of ANGPTL4 are decreased in patients with T2DM but not in obese individuals without T2DM. (A) Comparison of serum ANGPTL4 concentrations between T2DM patients and age-, BMI-, and sex-matched healthy individuals. (B) Serum ANGPTL4 levels in healthy lean subjects (BMI < 25) and obese individuals (BMI > 30) without or with T2DM. ^**, P < 0.01, compared with lean healthy subjects.