Berberine improves mesenteric artery insulin sensitivity through up-regulating insulin receptor-mediated signalling in diabetic rats

Abstract

Background and purpose: Berberine, a small molecule derived from Coptidis rhizome, has been found to be potent at lowering blood glucose and regulating lipid metabolism. Recent clinical studies have shown that berberine reduces blood pressure and increases systemic insulin sensitivity in patients with metabolic syndrome; however, the underlying mechanism is still unclear. Here, we investigated the mechanism by which berberine improves vascular insulin sensitivity in diabetic rats.

Experimental approach: Diabetes was induced in male Sprague–Dawley rats by feeding a high-fat diet and administration of a low dose of streptozotocin. These diabetic rats were treated with berberine (200 mg·kg(−1)·day(−1), gavage) for 4 weeks. Vascular dilation was determined in isolated mesenteric artery rings. Effects of berberine on insulin signalling were also studied in human artery endothelial cells cultured in high glucose (25 mmol·L(−1)) and palmitate (500 μmol·L(−1)).

Key results: Berberine treatment for 4 weeks significantly restored the impaired ACh- and insulin-induced vasodilatation of mesenteric arteries from diabetic rats. In isolated mesenteric artery rings, berberine (2.5–10 μmol·L(−1)) elicited dose-dependent vasodilatation and significantly enhanced insulin-induced vasodilatation. Mechanistically, berberine up-regulated phosphorylation of the insulin receptor and its downstream signalling molecules AMPK, Akt and eNOS, and increased cell viability and autophagy in cultured endothelial cells. Moreover, down-regulating insulin receptors with specific siRNA significantly attenuated berberine-induced phosphorylation of AMPK.

Conclusions and implications: Berberine improves diabetic vascular insulin sensitivity and mesenteric vasodilatation by up-regulating insulin receptor-mediated signalling in diabetic rats. These findings suggest berberine has potential as a preventive or adjunctive treatment of diabetic vascular complications.

Linked articles: This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23.

Figures

Figure 1

Berberine improved ACh‐induced vasodilatation of mesenteric arteries in diabetic rats. Mesenteric artery rings were isolated from control and diabetic rats treated with berberine for 4 weeks. (A) Berberine improved ACh‐induced vasodilatation of mesenteric arteries in diabetic rats. ACh‐induced relaxation in age‐matched control, berberine‐treated control, diabetic rats and berberine‐treated diabetic rat mesenteric arteries. (B) Sodium nitroprusside (SNP)‐induced relaxation in mesenteric artery. Relaxation is expressed as a percentage relative to the contraction induced by PE. Data are expressed as mean ± SEM (n = 12). Control, control rats; Control + BBR, control rats treated with berberine (200 mg·kg−1·day−1, gavage), Diabetes, diabetic rats; Diabetes + BBR, diabetic rats treated with berberine (200 mg·kg−1·day−1, gavage). **P < 0.01 versus Control; ##P < 0.01 versus Diabetes.

Figure 2

Berberine increased insulin‐induced vasodilatation in mesenteric arteries in diabetic rats via the PI3K/Akt pathway. Insulin‐induced relaxation was improved in berberine‐treated diabetic rat's mesenteric arteries. Relaxation is expressed as a percentage relative to the contraction induced by PE. Data are expressed as mean ± SEM (n = 12). Control, control rats; Diabetes, diabetic rats; Diabetes + BBR, diabetic rats treated with berberine (200 mg·kg−1·day−1, gavage); Diabetes + BBR + Wm, diabetic rats treated with berberine (200 mg·kg−1·day−1, gavage) and Wm (15 mg·kg−1·day−1, i.p.) for 4 weeks. **P < 0.01 versus Control; ##P < 0.01 versus Diabetes; ††P < 0.01 versus Diabetes + BBR.

Figure 3

Berberine combined with insulin alleviated the vascular dysfunction in isolated mesenteric arteries of diabetic rats. Mesenteric artery rings were isolated and the vasodilator responses were assessed. (A) Berberine improved vasodilatation in mesentery arteries in control and diabetic rats in a dose‐dependent (2.5 μmol·L−1, 5 μmol·L−1 and 10 μmol·L−1) manner. Data are expressed as mean ± SEM (n = 5). **P < 0.01 versus Control. (B) When combined with insulin (1 nmol·L−1), berberine (5 μmol·L−1) increased the vasodilatation of PE‐precontracted mesenteric artery rings isolated from control and diabetic rats. Data are expressed as mean ± SEM (n = 5). *P < 0.05 versus Control; ##P < 0.01 versus diabetic mesenteric artery rings treated with berberine (5 μmol·L−1).

Figure 4

Berberine increased cell viability and autophagy in HG/HF‐treated endothelial cells. EA.hy 926 cells were incubated for 24 h in medium supplemented with HG/HF (25 mmol·L−1 glucose + 500 μmol·L−1 palmitate) and 0.5% FBS in the presence or absence of insulin (1 nmol·L−1 or 100 nmol·L−1), or berberine (50 μmol·L−1). (A) Berberine increased the viability of HG/HF‐treated endothelial cells. Cell viability was assessed with MTT assay. Data are expressed as mean ± SEM (n = 6). *P < 0.05 versus Control; #P < 0.05 versus HG/HF; †P < 0.05 versus HG/HF + insulin (1 nmol·L−1). (B) Berberine increased autophagy in HG/HF‐treated HUVECs. HUVECs were fixed and examined using transmission electron microscopy for autophagosomes. Control, control culture medium; Control + BBR, control culture medium + berberine (50 μmol·L−1); HG/HF, HG/HF (25 mmol·L−1 glucose + 500 μmol·L−1 palmitate) culture medium; HG/HF + BBR, HG/HF culture medium treated with berberine (50 μmol·L−1). (C) Berberine increased autophagy‐related protein expression as analysed by western blots. Quantification of band intensity was normalized to GAPDH. The relative quantification of the Western blot band intensities are presented as mean ± SEM (n = 5–7). *P < 0.05 versus HG/HF.

Figure 5

Co‐treatment with berberine and insulin increased the HG/HF‐induced phosphorylation of InsR, Akt and eNOS in HG/HF‐treated endothelial cells. Berberine increased the protein expression of insulin receptor‐mediated signalling as analysed by western blots. (A) The effects of berberine on the phosphorylation of InsR, AMPK, Akt and eNOS. Quantification of band intensity was normalized to the corresponding total protein levels. The relative quantification of the western blot band intensities are presented as mean ± SEM (n = 5–9). *P < 0.05, **P < 0.01 versus HG/HF. (B) Co‐treatment with berberine and insulin increased HG/HF‐induced phosphorylation of InsR, Akt and eNOS in HG/HF‐treated endothelial cells. Representative bands are shown. Quantification of band intensity of p‐InsR, p‐Akt and p‐eNOS was normalized to InsR, Akt and eNOS protein levels respectively. The relative quantification of the western blot band intensities are presented as mean ± SEM (n = 5). **P < 0.01 versus Control; #P < 0.05, ##P < 0.01 versus HG/HF; †P < 0.05, ††P < 0.01 versus HG/HF + Ins (1 nmol·L−1).

Figure 6

Berberine‐induced phosphorylations of AMPK, Akt and eNOS were blunted following insulin receptor knockdown using siRNA in HG/HF‐treated endothelial cells. Semi‐quantification of p‐AMPK was normalized to the total AMPK protein levels and presented as mean ± SEM (n = 5). *P < 0.05 versus siRNA for InsR + BBR.

Figure 7

Schematic figure illustrating the enhancement by berberine of vascular insulin‐mediated signalling and vasodilatation. In addition to increasing AMPK‐induced autophagy, berberine up‐regulates the phosphorylation of the insulin receptor, resulting in enhanced activation of insulin‐mediated signalling and subsequent endothelial NO production with resultant vasodilatation in diabetic rats.