BJP
British Journal of Pharmacology
British Journal of Pharmacology (2016) 173 1569–1579
1569
Themed Section: Chinese Innovation in Cardiovascular Drug Discovery
RESEARCH PAPER Berberine improves mesenteric artery insulin sensitivity through up-regulating insulin receptor-mediated signalling in diabetic rats Correspondence Ling Dong, School of Aerospace Medicine, The Fourth Military Medical University, Xi’an 710032, China. E-mail: [email protected] or Feng Gao, School of Aerospace Medicine & Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, Xi’an, China. E-mail: [email protected]
Received 11 June 2014; Revised 25 January 2016; Accepted 11 February 2016
Feng-Hao Geng1*, Guo-Hua Li1*, Xing Zhang1, Peng Zhang2, Ming-Qing Dong3, Zhi-Jing Zhao4, Yuan Zhang1, Ling Dong1 and Feng Gao1,4 1
School of Aerospace Medicine, The Fourth Military Medical University, Xi‘an, China, 2Department of Orthopedic Surgery, Urumqi General Hospital,
Urumqi, China, 3Department of Pathophysiology, The Fourth Military Medical University, Xi‘an, China, and 4Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, Xi‘an, China *Feng-Hao Geng and Guo-Hua Li contributed equally to this work.
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.
© 2016 The British Pharmacological Society
DOI:10.1111/bph.13466
BJP
F-H Geng et al.
Abbreviations AMPK, AMP-activated protein kinase; eNOS, endothelial NOS; IRS, insulin receptor substrate; InsR, insulin receptor; MTT, 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide thiazolyl blue; PE, phenylephrine; PI3K, phosphatidylinositol 3-kinase; SNP, sodium nitroprusside
Tables of Links LIGANDS
TARGETS Catalytic receptors InsR
a
Enzymes
b
ACh
Palmitic acid (palmitate)
Akt (PKB)
Insulin glargine
Wortmannin
AMPK
Phenylephrine (PE)
eNOS PI3K
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology. org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise a,b Guide to PHARMACOLOGY 2015/16 ( Alexander et al., 2015a,b).
Introduction Type 2 diabetes characterized by insulin resistance is associated with impaired endothelium-dependent vasodilatation (Steinberg et al., 1996). Berberine is a natural isoquinoline alkaloid, which is present in medicinal herbs Coptidis rhizome (Huanglian in Chinese). Recent clinical studies have shown that berberine reduces blood pressure and increases systemic insulin sensitivity in patients with metabolic syndrome (Perez-Rubio et al., 2013). Berberine has been shown to lower blood glucose and modulate lipid metabolism by activating AMP-activated protein kinase (AMPK) (Ko et al., 2005; Chang et al., 2013). Berberine has also vasodilator effects (Ko et al., 2000; Lau et al., 2001), preventing hyperglycaemiainduced endothelial injury via AMPK (Wang et al., 2009). In a previous study, we showed that berberine protects the diabetic heart from ischaemic/reperfusion injury through activating the Akt-eNOS signalling pathway and inhibiting the apoptosis of myocardium cells in rats (Chen et al., 2014). Therefore, we hypothesized that berberine may improve insulin sensitivity in diabetes by up-regulating insulin receptor-mediated signalling. The present study aimed to investigate the effects of berberine on insulin-induced vasodilatation and its underlying mechanism in a streptozotocin (STZ)-treated, high-fat diet-fed type 2 diabetes animal model. We found that berberine significantly ameliorated impaired endothelial function and improved insulin-induced vasodilatation in diabetic mesentery arteries mainly through up-regulating insulin receptormediated signalling.
Methods Male Sprague–Dawley rats, 120–150 g, were housed in a temperature controlled room with a 12 h light dark cycle. Rats 1570
British Journal of Pharmacology (2016) 173 1569–1579
were fed a casein-cornstarch-sucrose-based diet containing a high-fat content consisting of 20% fat, 62% carbohydrate and 17% protein, ad libitum, with free access to dwater. After 1 week of adaptation, rats were fasted overnight and then injected i.p. with 30 mg·kg 1 body weight of STZ (Sigma-Aldrich, Shanghai, China) dissolved in citrate buffer (pH 4.5) to induce diabetes. Normal control animals (Control) were deprieved of food (fasted) overnight and injected with the citrate buffer vehicle. The fasting blood glucose of STZ-treated rats was measured 3days later, and a second injection was applied if rats were not diabetic. Fasting blood glucose was measured again 3 days after the first STZ injection. Rats with fasting blood glucose levels over 7.0 mmol·L 1 were randomly divided into two groups and continued to be fed the high-fat diet for 8 weeks. These two groups were matched for body weight and blood glucose levels. One group was used as the diabetic control (Diabetes) and the other was treated with berberine chloride (gavage) dissolved in 0.9% saline solution at a dose of 200 mg·kg 1·day 1(Chen et al., 2014). The diabetic rats and the normal control rats were treated with the vehicle of 0.9% saline solution (gavage) as in our previous study (Chen et al., 2014). At the end of the study, animals were fasted overnight and killed by administration of an overdose of anaesthetic, sodium pentobarbital (30 mg·kg 1, i.p.). All experimental procedures were performed in accordance with International Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Care and Research Ethics Committee of the Fourth Military Medical University Research Council. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath & Lilley, 2015).
Isometric force measurement After the rats had been killed, the mesenteric artery (inner diameter 100 to 150 μm) was dissected out, any adhering
Berberine improves diabetic vascular insulin sensitivity
connective tissues were removed, and then it was cut into ring segments (1 mm in length). Rings were suspended on a micromyograph in organ baths containing oxygenated (95% O2; 5% CO2) physiological saline solution (composition in mmol·L 1: NaCl 119, KCl 4.7, KH2PO4 1.18, MgSO4 · 7H2O 1.17, CaCl2 2H2O 2.5, NaHCO325, EDTA 0.03 and D-glucose 5.5, pH 7.4, 37°C). Following equilibration for 90 min under a resting tension of 0.25 g, rings were twice contracted with KCl (60 mmol·L 1) (Xing et al., 2013a,b). All rings were initially contracted by addition of 10 nmol·L 1 phenylephrine (PE) (to evaluate the contractility) and then relaxed to 1 mmol·L 1 ACh (to assess the endothelial integrity). Then a sustained contraction was induced by PE (10 5 mol·L 1), and ACh was added cumulatively to evoke relaxations. Contraction to PE (10 5 mol·L 1) and subsequent vasodilatation to insulin (10 10 to 10 6 mol·L 1), ACh (10 9 to 10 4 mol·L 1) and sodium nitroprusside (SNP, 10 10 to 10 5 mol·L 1) were tested in all vessels. Subsequently, we evaluated the vasodilator responses of mesentery artery rings preconstricted with PE; vasorelaxation was evoked by increasing doses of insulin (10 10 to 10 6 mol·L 1). The maximal contraction induced by PE was considered as the baseline and vasorelaxant responses are expressed as % reduction in contraction. We obtained concentration-response curves to ACh, insulin and SNP on different segments from the same artery.
Cell culture Human artery endothelial cell line EA.hy 926 (Summers et al., 2014) and HUVECs (obtained from American Type Culture Collection) were used. Endothelial cells at passages 4–8 were cultured on gelatin-coated flasks in Dulbecco’s minimum essential medium supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) under endotoxin-free conditions (Armani et al., 2014). Cells were cultured in an atmosphere of 5% CO2 at 37°C. One day before treatment, cells were trypsinized and allowed to grow to about 80% confluence. Afterwards, fresh media supplemented with vehicle (DMSO) or berberine were added to the cells as indicated for 1 h. We used HG/HF (25 mmol·L 1 glucose + 500 μmol·L 1 palmitate)-treated endothelial cells to simulate the in vivo diabetic cell condition. For the insulin stimulation experiments, 1 or 100 nmol·L 1 of bovine insulin was added to the culture medium (Tanigaki et al., 2009). Cells were treated for an additional 60 min before harvest. Berberine was dissolved in DMSO and was added to the medium at the indicated concentrations.
Cell viability The 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide thiazolyl blue (MTT) assay was conducted as described previously (Hao et al., 2011). Briefly, the MTT was dissolved in PBS at a concentration of 5 mg·mL 1 and sterilized by passage through a 0.22 μm filter. EA.hy 926 cells were seeded in wells of a 96-well plate containing 200 μL of the culture medium, and 20 μL MTT stock solution was then added to each well. After incubation for 4 h at 37°C, 150 μL of a DMSO solution were added to all of the wells and mixed thoroughly to lyse the cells and dissolve the dark blue crystals. After 10 min, the absorbance was read on a
BJP
microplate reader (Beckman DU640, Brea, CA, USA) at a wavelength of 570 nm.
Transmission electron microscopy HUVECs were quickly fixed with 2.5% glutaraldehyde in 0.1 mol·L 1 phosphate buffer (pH 7.4) overnight at 4°C. After fixation, cells were then immersed in 1% osmium tetroxide for 2 h, dehydrated in graded ethanol and then embedded in epoxy resin. After that, cells were post-stained with uranyl acetate and lead citrate, and then examined under a JEM1230 transmission electron microscope (JEOL, Tokyo, Japan).
Small interfering (si) RNA design and transfection The cognate siRNA against insulin receptors (InsR-homo3428) was designed as described previously and purchased from Genepharm (Shanghai, China) along with a scrambled control siRNA (siCONTROL non-targeting siRNA, scrambled) (Xing et al., 2013a,b). HUVECs were seeded and transfected with siRNA to a final concentration of 20 nmol·L 1 using RNA mate (Genepharma, Shanghai, China) when cells reached 30–50% confluence. Protein knockdown efficiencies were assessed at 72 h after transfection.
Western blot HUVECs were washed twice with ice-cold 1× PBS solution and lysed in ice-cold lysis buffer and proteins were extracted as described previously (Dong et al., 2008). Protein samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes by a semi-dry transfer cell (Bio-Rad, Hercules, CA, USA). For detection of InsR-β-1150, Ser473phosphorylated Akt and total Akt, LC3B and p62 were detected by specific rabbit polyclonal antibodies (Cell Signaling Technology, Danvers, MA, USA). Anti-phosphoeNOS (Ser1177), anti-eNOS antibodies, anti-phospho-AMPK and anti-AMPK were obtained from Abcam (San Francisco, CA, USA) with GADPH as internal control. Berberine, PE, acetylcholine, insulin, sodium palmitic acid, MTT and PI3K inhibitor wortmannin were obtained from Sigma-Aldrich.
Statistical analysis All values are presented as mean ± SEM. Differences were compared by ANOVA followed by Bonferroni correction for post hoc t-test, where appropriate. Probabilities of
British Journal of Pharmacology
British Journal of Pharmacology (2016) 173 1569–1579
1569
Themed Section: Chinese Innovation in Cardiovascular Drug Discovery
RESEARCH PAPER Berberine improves mesenteric artery insulin sensitivity through up-regulating insulin receptor-mediated signalling in diabetic rats Correspondence Ling Dong, School of Aerospace Medicine, The Fourth Military Medical University, Xi’an 710032, China. E-mail: [email protected] or Feng Gao, School of Aerospace Medicine & Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, Xi’an, China. E-mail: [email protected]
Received 11 June 2014; Revised 25 January 2016; Accepted 11 February 2016
Feng-Hao Geng1*, Guo-Hua Li1*, Xing Zhang1, Peng Zhang2, Ming-Qing Dong3, Zhi-Jing Zhao4, Yuan Zhang1, Ling Dong1 and Feng Gao1,4 1
School of Aerospace Medicine, The Fourth Military Medical University, Xi‘an, China, 2Department of Orthopedic Surgery, Urumqi General Hospital,
Urumqi, China, 3Department of Pathophysiology, The Fourth Military Medical University, Xi‘an, China, and 4Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, Xi‘an, China *Feng-Hao Geng and Guo-Hua Li contributed equally to this work.
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.
© 2016 The British Pharmacological Society
DOI:10.1111/bph.13466
BJP
F-H Geng et al.
Abbreviations AMPK, AMP-activated protein kinase; eNOS, endothelial NOS; IRS, insulin receptor substrate; InsR, insulin receptor; MTT, 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide thiazolyl blue; PE, phenylephrine; PI3K, phosphatidylinositol 3-kinase; SNP, sodium nitroprusside
Tables of Links LIGANDS
TARGETS Catalytic receptors InsR
a
Enzymes
b
ACh
Palmitic acid (palmitate)
Akt (PKB)
Insulin glargine
Wortmannin
AMPK
Phenylephrine (PE)
eNOS PI3K
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology. org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise a,b Guide to PHARMACOLOGY 2015/16 ( Alexander et al., 2015a,b).
Introduction Type 2 diabetes characterized by insulin resistance is associated with impaired endothelium-dependent vasodilatation (Steinberg et al., 1996). Berberine is a natural isoquinoline alkaloid, which is present in medicinal herbs Coptidis rhizome (Huanglian in Chinese). Recent clinical studies have shown that berberine reduces blood pressure and increases systemic insulin sensitivity in patients with metabolic syndrome (Perez-Rubio et al., 2013). Berberine has been shown to lower blood glucose and modulate lipid metabolism by activating AMP-activated protein kinase (AMPK) (Ko et al., 2005; Chang et al., 2013). Berberine has also vasodilator effects (Ko et al., 2000; Lau et al., 2001), preventing hyperglycaemiainduced endothelial injury via AMPK (Wang et al., 2009). In a previous study, we showed that berberine protects the diabetic heart from ischaemic/reperfusion injury through activating the Akt-eNOS signalling pathway and inhibiting the apoptosis of myocardium cells in rats (Chen et al., 2014). Therefore, we hypothesized that berberine may improve insulin sensitivity in diabetes by up-regulating insulin receptor-mediated signalling. The present study aimed to investigate the effects of berberine on insulin-induced vasodilatation and its underlying mechanism in a streptozotocin (STZ)-treated, high-fat diet-fed type 2 diabetes animal model. We found that berberine significantly ameliorated impaired endothelial function and improved insulin-induced vasodilatation in diabetic mesentery arteries mainly through up-regulating insulin receptormediated signalling.
Methods Male Sprague–Dawley rats, 120–150 g, were housed in a temperature controlled room with a 12 h light dark cycle. Rats 1570
British Journal of Pharmacology (2016) 173 1569–1579
were fed a casein-cornstarch-sucrose-based diet containing a high-fat content consisting of 20% fat, 62% carbohydrate and 17% protein, ad libitum, with free access to dwater. After 1 week of adaptation, rats were fasted overnight and then injected i.p. with 30 mg·kg 1 body weight of STZ (Sigma-Aldrich, Shanghai, China) dissolved in citrate buffer (pH 4.5) to induce diabetes. Normal control animals (Control) were deprieved of food (fasted) overnight and injected with the citrate buffer vehicle. The fasting blood glucose of STZ-treated rats was measured 3days later, and a second injection was applied if rats were not diabetic. Fasting blood glucose was measured again 3 days after the first STZ injection. Rats with fasting blood glucose levels over 7.0 mmol·L 1 were randomly divided into two groups and continued to be fed the high-fat diet for 8 weeks. These two groups were matched for body weight and blood glucose levels. One group was used as the diabetic control (Diabetes) and the other was treated with berberine chloride (gavage) dissolved in 0.9% saline solution at a dose of 200 mg·kg 1·day 1(Chen et al., 2014). The diabetic rats and the normal control rats were treated with the vehicle of 0.9% saline solution (gavage) as in our previous study (Chen et al., 2014). At the end of the study, animals were fasted overnight and killed by administration of an overdose of anaesthetic, sodium pentobarbital (30 mg·kg 1, i.p.). All experimental procedures were performed in accordance with International Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Care and Research Ethics Committee of the Fourth Military Medical University Research Council. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath & Lilley, 2015).
Isometric force measurement After the rats had been killed, the mesenteric artery (inner diameter 100 to 150 μm) was dissected out, any adhering
Berberine improves diabetic vascular insulin sensitivity
connective tissues were removed, and then it was cut into ring segments (1 mm in length). Rings were suspended on a micromyograph in organ baths containing oxygenated (95% O2; 5% CO2) physiological saline solution (composition in mmol·L 1: NaCl 119, KCl 4.7, KH2PO4 1.18, MgSO4 · 7H2O 1.17, CaCl2 2H2O 2.5, NaHCO325, EDTA 0.03 and D-glucose 5.5, pH 7.4, 37°C). Following equilibration for 90 min under a resting tension of 0.25 g, rings were twice contracted with KCl (60 mmol·L 1) (Xing et al., 2013a,b). All rings were initially contracted by addition of 10 nmol·L 1 phenylephrine (PE) (to evaluate the contractility) and then relaxed to 1 mmol·L 1 ACh (to assess the endothelial integrity). Then a sustained contraction was induced by PE (10 5 mol·L 1), and ACh was added cumulatively to evoke relaxations. Contraction to PE (10 5 mol·L 1) and subsequent vasodilatation to insulin (10 10 to 10 6 mol·L 1), ACh (10 9 to 10 4 mol·L 1) and sodium nitroprusside (SNP, 10 10 to 10 5 mol·L 1) were tested in all vessels. Subsequently, we evaluated the vasodilator responses of mesentery artery rings preconstricted with PE; vasorelaxation was evoked by increasing doses of insulin (10 10 to 10 6 mol·L 1). The maximal contraction induced by PE was considered as the baseline and vasorelaxant responses are expressed as % reduction in contraction. We obtained concentration-response curves to ACh, insulin and SNP on different segments from the same artery.
Cell culture Human artery endothelial cell line EA.hy 926 (Summers et al., 2014) and HUVECs (obtained from American Type Culture Collection) were used. Endothelial cells at passages 4–8 were cultured on gelatin-coated flasks in Dulbecco’s minimum essential medium supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) under endotoxin-free conditions (Armani et al., 2014). Cells were cultured in an atmosphere of 5% CO2 at 37°C. One day before treatment, cells were trypsinized and allowed to grow to about 80% confluence. Afterwards, fresh media supplemented with vehicle (DMSO) or berberine were added to the cells as indicated for 1 h. We used HG/HF (25 mmol·L 1 glucose + 500 μmol·L 1 palmitate)-treated endothelial cells to simulate the in vivo diabetic cell condition. For the insulin stimulation experiments, 1 or 100 nmol·L 1 of bovine insulin was added to the culture medium (Tanigaki et al., 2009). Cells were treated for an additional 60 min before harvest. Berberine was dissolved in DMSO and was added to the medium at the indicated concentrations.
Cell viability The 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide thiazolyl blue (MTT) assay was conducted as described previously (Hao et al., 2011). Briefly, the MTT was dissolved in PBS at a concentration of 5 mg·mL 1 and sterilized by passage through a 0.22 μm filter. EA.hy 926 cells were seeded in wells of a 96-well plate containing 200 μL of the culture medium, and 20 μL MTT stock solution was then added to each well. After incubation for 4 h at 37°C, 150 μL of a DMSO solution were added to all of the wells and mixed thoroughly to lyse the cells and dissolve the dark blue crystals. After 10 min, the absorbance was read on a
BJP
microplate reader (Beckman DU640, Brea, CA, USA) at a wavelength of 570 nm.
Transmission electron microscopy HUVECs were quickly fixed with 2.5% glutaraldehyde in 0.1 mol·L 1 phosphate buffer (pH 7.4) overnight at 4°C. After fixation, cells were then immersed in 1% osmium tetroxide for 2 h, dehydrated in graded ethanol and then embedded in epoxy resin. After that, cells were post-stained with uranyl acetate and lead citrate, and then examined under a JEM1230 transmission electron microscope (JEOL, Tokyo, Japan).
Small interfering (si) RNA design and transfection The cognate siRNA against insulin receptors (InsR-homo3428) was designed as described previously and purchased from Genepharm (Shanghai, China) along with a scrambled control siRNA (siCONTROL non-targeting siRNA, scrambled) (Xing et al., 2013a,b). HUVECs were seeded and transfected with siRNA to a final concentration of 20 nmol·L 1 using RNA mate (Genepharma, Shanghai, China) when cells reached 30–50% confluence. Protein knockdown efficiencies were assessed at 72 h after transfection.
Western blot HUVECs were washed twice with ice-cold 1× PBS solution and lysed in ice-cold lysis buffer and proteins were extracted as described previously (Dong et al., 2008). Protein samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes by a semi-dry transfer cell (Bio-Rad, Hercules, CA, USA). For detection of InsR-β-1150, Ser473phosphorylated Akt and total Akt, LC3B and p62 were detected by specific rabbit polyclonal antibodies (Cell Signaling Technology, Danvers, MA, USA). Anti-phosphoeNOS (Ser1177), anti-eNOS antibodies, anti-phospho-AMPK and anti-AMPK were obtained from Abcam (San Francisco, CA, USA) with GADPH as internal control. Berberine, PE, acetylcholine, insulin, sodium palmitic acid, MTT and PI3K inhibitor wortmannin were obtained from Sigma-Aldrich.
Statistical analysis All values are presented as mean ± SEM. Differences were compared by ANOVA followed by Bonferroni correction for post hoc t-test, where appropriate. Probabilities of
