Uncoupling protein-2 mediates the protective action of berberine against oxidative stress in rat insulinoma INS-1E cells and in diabetic mouse islets

BACKGROUND AND PURPOSE

Uncoupling protein-2 (UCP2) may regulate glucose-stimulated insulin secretion. The current study investigated the effects of berberine, an alkaloid found in many medicinal plants, on oxidative stress and insulin secretion through restoration of UCP2 expression in high glucose (HG)-treated INS-1E cells and rat islets or in db/db mouse islets.

EXPERIMENTAL APPROACH

Mouse and rat pancreatic islets were isolated. Nitrotyrosine, superoxide dismutase (SOD)-1 and UCP2 expression and AMPK phosphorylation were examined by Western blotting. Insulin secretion was measured by elisa. Mitochondrial reactive oxygen species (ROS) production was detected by confocal microscopy.

KEY RESULTS

Incubation of INS-1E cells and rat islets with HG (30 mmol·L⁻¹; 8 h) elevated nitrotyrosine level, reduced SOD-1 and UCP2 expression and AMPK phosphorylation, and inhibited glucose-stimulated insulin secretion. HG also increased mitochondrial ROS in INS-1E cells. Co-treatment with berberine inhibited such effects. The AMPK inhibitor compound C, the UCP2 inhibitor genipin and adenovirus ucp2 shRNA inhibited these protective effects of berberine. Furthermore, compound C normalized berberine-stimulated UCP2 expression but genipin did not affect AMPK phosphorylation. Islets from db/db mice exhibited elevated nitrotyrosine levels, reduced expression of SOD-1 and UCP2 and AMPK phosphorylation, and decreased insulin secretion compared with those from db/m⁺ mice. Berberine also improved these defects in diabetic islets and genipin blocked the effects of berberine.

CONCLUSIONS AND IMPLICATIONS

Berberine inhibited oxidative stress and restored insulin secretion in HG-treated INS-IE cells and diabetic mouse islets by activating AMPK and UCP2. UCP2 is an important signalling molecule in mediating anti-diabetic effects of berberine.     

黄连素的降血糖药效学试验与临床观察

目的探讨中药小檗碱(黄连素)的降血糖作用。方法用四氧嘧啶建立糖尿病小鼠模型,以小檗碱不同剂量和西药降糖灵对照治疗观察糖尿病小鼠,采用葡萄糖氧化酶法测定血糖和糖耐量,用放射免疫法测定血清胰岛素;并采用黄连素与对照组达美康随机分配73例糖尿病患者进行临床对比观察。结果小檗碱对糖尿病小鼠有降血糖作用,有提高其糖耐量和提高血清胰岛素作用,且有量效关系。黄连素治疗糖尿病43例,临床有效率为81.39%,对照组达美康治疗同症30例,临床有效率为80.00%,两组疗效明显,但无明显差异(P<0.05)。结论小檗碱对糖尿病小鼠和糖尿病患者有降血糖作用。     

Berberine in type 2 diabetes therapy: a new perspective for an old antidiarrheal drug?

Type 2 diabetes mellitus (T2DM) and dysglycemia (impaired glucose tolerance and/or impaired fasting glucose) are increasingly contributing to the global burden of disease. Despite the continued introduction of hypoglycemic drugs, intervention in diabetes and its related complications remains a major global medical problem. Traditional Chinese medicine offers a number of potential candidates for developing hypoglycemic drugs. Berberine (BER), an isoquinoline alkaloid extract, has been commonly used as an oral drug to treat gastroenteritis and diarrhea for more than 1400 years. Although the antidiabetic effect of berberine has been noted in diabetic patients and demonstrated diabetic animal models in the last decade, its use is not yet accepted in the general medical community, for two reasons: its mechanism of action remains to be determined, and its bioavailability is low. Therefore, characterization of its mechanism of action and enhancement of its bioavailability are most important and the subject of current investigations. Recent studies have also revealed beneficial effects of berberine on diabetic complications. In this review the antidiabetic mechanism of action of berberine, its effect on diabetic complications, and efforts to improve its bioavailability are summarized. These studies may lead to its wider use for the treatment of type 2 diabetes mellitus and its complications.     

Traditional chinese medicine in treatment of metabolic syndrome

In management of metabolic syndrome, the traditional Chinese medicine (TCM) is an excellent representative in alternative and complementary medicines with a complete theory system and substantial herb remedies. In this article, basic principle of TCM is introduced and 25 traditional Chinese herbs are reviewed for their potential activities in the treatment of metabolic syndrome. Three herbs, ginseng, rhizoma coptidis (berberine, the major active compound) and bitter melon, were discussed in detail on their therapeutic potentials. Ginseng extracts made from root, rootlet, berry and leaf of Panax quinquefolium (American ginseng) and Panax ginseng (Asian ginseng), are proved for anti-hyperglycemia, insulin sensitization, islet protection, anti-obesity and anti-oxidation in many model systems. Energy expenditure is enhanced by ginseng through thermogenesis. Ginseng-specific saponins (ginsenosides) are considered as the major bioactive compounds for the metabolic activities of ginseng. Berberine from rhizoma coptidis is an oral hypoglycemic agent. It also has anti-obesity and anti-dyslipidemia activities. The action mechanism is related to inhibition of mitochondrial function, stimulation of glycolysis, activation of AMPK pathway, suppression of adipogenesis and induction of low-density lipoprotein (LDL) receptor expression. Bitter melon or bitter gourd (Momordica charantia) is able to reduce blood glucose and lipids in both normal and diabetic animals. It may also protect beta cells, enhance insulin sensitivity and reduce oxidative stress. Although evidence from animals and humans supports the therapeutic activities of ginseng, berberine and bitter melon, multi-center large-scale clinical trials have not been conducted to evaluate the efficacy and safety of these herbal medicines.     

Insulin sensitizing and insulinotropic action of berberine from Cortidis rhizoma

Our preliminary study demonstrated that 70% ethanol Cortidis Rhizoma extracts (CR) had a hypoglycemic action in diabetic animal models. We determined whether CR fractions acted as anti-diabetic agent, and a subsequent investigation of the action mechanism of the major compound, berberine ([C(20)H(18)NO(4)](+)), was carried out in vitro. The 20, 40 and 60% methanol fractions from the XAD-4 column contained the most insulin sensitizing activities in 3T3-L1 adipocytes. The common major peak in these fractions was berberine. Treatment with 50 microM berberine plus differentiation inducers significantly reduced triglyceride accumulation by decreased differentiation of 3T3-L1 fibroblasts to adipocytes and triglyceride synthesis. Significant insulin sensitizing activity was observed in 3T3-L1 adipocytes which were given 50 microM berberine plus 0.2 nM insulin to reach a glucose uptake level increased by 10 nM of insulin alone. This was associated with increased glucose transporter-4 translocation into the plasma membrane via enhancing insulin signaling pathways and the insulin receptor substrate-1-phosphoinositide 3 Kinase-Akt. Berberine also increased glucose-stimulated insulin secretion and proliferation in Min6 cells via an enhanced insulin/insulin-like growth factor-1 signaling cascade. Data suggested that berberine can act as an effective insulin sensitizing and insulinotropic agent. Therefore, berberine can be used as anti-diabetic agent for obese diabetic patients.     

Berberine suppresses intestinal disaccharidases with beneficial metabolic effects in diabetic states, evidences from in vivo and in vitro study

Clinical reports have demonstrated that berberine is a potential antidiabetic agent, but the underlying mechanism is unclear. The purpose of this study was to investigate if berberine exerts its hypoglycemic action via inhibiting intestinal disaccharidases using in vivo and in vitro experiments. Streptozotocin-induced diabetic rats received berberine (100 or 200 mg/kg) orally once daily or acarbose (40 mg/kg) orally twice daily for 5 weeks. Disaccharidase activities and sucrase–isomaltase (SI) complex messenger RNA (mRNA) expression in intestinal regions were assessed. The same treatment was operated in normal rats. Sucrose and maltose loading tests were also documented. In addition, Caco-2 cells were cultured in medium containing berberine or berberine plus chelerythrine. Compound C or H-89 for 5 days, disaccharidase activities, and SI complex mRNA levels were measured. The animal experiments showed that berberine significantly decreased the disaccharidase activities and SI complex mRNA expression both in diabetic rats and normal rats. Berberine can also significantly lower postprandial blood glucose levels induced by sucrose or maltose loading in normal rats. The cellular results showed that berberine may suppress disaccharidase activities and downregulate SI complex mRNA expression in a concentration-dependent manner. Only H-89, an inhibitor of protein kinase A (PKA), may reverse the decrease in disaccharidase activities and SI complex mRNA expression induced by berberine. In conclusion, berberine suppresses disaccharidase activities and SI complex mRNA expression with beneficial metabolic effects in diabetic states. The inhibitory effect, at least partly, involves the PKA-dependent pathway.     

小檗碱对正常小鼠血糖调节的影响

本文探讨了小檗碱降低正常小鼠血糖的作用机制。实验表明,小檗碱未影响胰岛素的分泌与释放,也未影响肝细胞膜胰岛素受体的数目与亲和力,小檗碱的作用可能为受体后效应。实验观察到小檗碱可以对抗注射葡萄糖引起的血糖升高,可以抑制以丙氨酸为底物的糖原异生,以及小檗碱的降血糖作用与血乳酸的升高密切相关,因而小檗碱可能通过抑制糖原异生和/或促进糖酵解产生降血糖作用。     

小檗碱调脂作用机制的研究进展

小檗碱是从黄连等植物中提取的一种异喹啉类生物碱,作为传统的抗炎、抑菌药物在临床应用已久。近年来研究发现其具有调脂作用,可通过调节肠道菌群结构、影响胆汁酸的合成与代谢、上调低密度脂蛋白受体(LDLR)的表达、激活内脏脂肪组织相关基因的表达、升高血清脂联素的表达、激活腺苷酸活化蛋白激酶(AMPK)途径、改善胰岛素抵抗等多个作用靶点发挥调脂作用,主要针对小檗碱的调脂作用及其机制进行综述。     

Ameliorative effect of berberine on endothelial dysfunction in diabetic rats induced by high-fat diet and streptozotocin

Berberine can improve insulin resistance, lower blood glucose, and regulate lipid metabolism disorders which cause endothelial dysfunction, leading to vascular complications of type 2 diabetes mellitus. The aim of the present study was to investigate the effects of berberine on endothelial dysfunction of aortas in type 2 diabetes mellitus rats and its mechanism. Wistar rats were randomly divided into four groups: diabetic rats, control rats, diabetic rats treated with berberine (100 mg/kg), and control rats treated with berberine. The serum fasting blood glucose, insulin, total cholesterol, triglyceride and nitric oxide (NO) levels were tested. Acetylcholine-induced endothelium-dependent relaxation and sodium nitroprusside induced endothelium-independent relaxation were measured in aortas for estimating endothelial function. The expression of endothelial nitric oxide synthase (eNOS) mRNA was measured by RT-PCR, and the protein expressions of eNOS and NADPH oxidase (NOX4) were analyzed by western blot. The results showed that berberine significantly decreased fasting blood glucose, and triglyceride levels in diabetic rats. Berberine also improved endothelium-dependent vasorelaxation impaired in aorta. The expressions of eNOS mRNA and protein were significantly increased, while NOX4 protein expression was decreased in aortas from diabetic rats with berberine treatment. Moreover, serum NO levels were elevated after berberine treatment. In conclusion, berberine restores diabetic endothelial dysfunction through enhanced NO bioavailability by up-regulating eNOS expression and down-regulating expression of NADPH oxidase.     

瓜蒌子油对糖尿病小鼠降血糖作用的研究

目的 研究瓜蒌子油对糖尿病小鼠模型的降血糖作用及其可能的作用机制。 方法 ICR小鼠尾静脉注射四氧嘧啶(100 mg/kg)造成糖尿病模型,给予不同剂量的瓜蒌子油灌胃,另设正常对照组、模型对照组和阳性药对照组,连续给药4周,每周监测小鼠体重及血糖,末次给药18 h后,摘眼球采血,分离血清,用化学比色法测血清总胆固醇(TC)、三酰甘油(TG)、一氧化氮(NO)和一氧化氮合酶(NOS)含量,用放射免疫法测血清胰岛素含量;另采用四氧嘧啶糖尿病小鼠对瓜蒌子油进行单次给药的糖耐量测定。 结果 给予瓜蒌子油不同剂量(5、10、20 ml/kg)的各组小鼠体重增长略有加快,并使血糖值呈剂量依赖性下降,能显著升高模型小鼠的血清胰岛素含量,降低血清TC、TG、NO和NOS含量;从糖耐量试验看,瓜蒌子油不同剂量组小鼠喂以淀粉后各时段血糖值呈剂量依赖性降低。 结论 瓜蒌子油具有降血糖以及改善糖耐量的作用,其降血糖的作用可能与升高血清胰岛素含量、降低血清的NO和NOS水平有关。     

小檗碱调控AMPK-TSC1/LC3-Ⅱ信号通路促进高糖高脂应激下NIT-1胰岛β细胞的自噬

目的：观察小檗碱（berberine,BBR）对高糖高脂应激条件下的NIT-1胰岛β细胞自噬的影响并探讨其潜在的分子机制。方法：分别采用高浓度葡萄糖（25 mmol·L^-1）和软脂酸（0.25 mmol·L^-1）诱导NIT-1胰岛β细胞建立高糖、高脂模型,予以小檗碱处理,用放射免疫法检测胰岛素水平,通过电镜观察自噬体的形成;然后予以腺苷酸活化蛋白激酶（AMPK）激动剂AICAR,腺苷酸活化蛋白激酶（AMPK）抑制剂Compound C,自噬诱导剂雷帕霉素（Rapa）进行干预,Western blot方法检测各组细胞AMPK的活化、自噬相关蛋白结节性硬化复合物1（TSC1）、微管相关蛋白1轻链蛋白3（LC3-Ⅱ）的表达。结果：在高糖高脂诱导的NIT-1胰岛β细胞中,小檗碱可以促进胰岛素的分泌和自噬体的形成,促进自噬相关蛋白TSC1,LC3-Ⅱ的表达,并活化其上游的AMPK。结论：小檗碱在高糖高脂的应激条件下可能通过促进自噬来发挥保护胰岛β细胞功能的作用,其分子机制可能与小檗碱调控AMPK-TSC1/LC3-II信号通路相关蛋白有关。     

Mitochondrial inhibitor as a new class of insulin sensitizer

Insulin resistance is a major risk factor for type 2 diabetes. AMP-activated protein kinase (AMPK) is a drug target in the improvement of insulin sensitivity. Several insulin-sensitizing medicines are able to activate AMPK through inhibition of mitochondrial functions. These drugs, such as metformin and STZ, inhibit ATP synthesis in mitochondria to raise AMP/ATP ratio in the process of AMPK activation. However, chemicals that activate AMPK directly or by activating its upstream kinases have not been approved for treatment of type 2 diabetes in humans. In an early study, we reported that berberine inhibited oxygen consumption in mitochondria, and increased AMP/ATP ratio in cells. The observation suggests an indirect mechanism for AMPK activation by berberine. Berberine stimulates glycolysis for ATP production that offsets the cell toxicity after mitochondria inhibition. The study suggests that mitochondrial inhibition is an approach for AMPK activation. In this review article, literature is critically reviewed to interpret the role of mitochondria function in the mechanism of insulin resistance, which supports that mitochondria inhibitors represent a new class of AMPK activator. The inhibitors are promising candidates for insulin sensitizers. This review provides a guideline in search for small molecule AMPK activators in the drug discovery for type 2 diabetes.     

黄连及小蘖碱降血糖作用的研究

实验证明,黄连水煎剂可以降低正常小鼠血糖,黄连的主要成分小蘖碱可以降低正常小鼠、四氧嘧啶糖尿病小鼠和自发性糖尿病KK小鼠的血糖。给正常小鼠腹腔注射葡萄糖或肾上腺素可以引起血糖升高,小蘖碱可以对抗这两种作用。给小蘖碱后,可以改善KK小鼠的葡萄糖耐量。此外,小蘖碱可以降低喂高胆固醇乳剂小鼠的血清胆固醇水平,在体外可以抑制家兔血小板聚集。     

The metabolism of berberine and its contribution to the pharmacological effects

Berberine, a bioactive alkaloid isolated from several herbal substances, possesses multiple pharmacological effects, including antimicrobial, antidiabetic, anticancer activities. Meanwhile, berberine undergoes extensive metabolism after oral administration which results in its extremely low plasma exposure. Therefore, it is believed that the metabolites of berberine also contribute a lot to its pharmacological effects. Along these lines, this review covers the metabolism studies of berberine in terms of its metabolic pathways and metabolic organs based on the identified metabolites, and it also covers the pharmacological activities of its active metabolites. In brief, the predominant metabolic pathways of berberine are demethylation, demethylenation, reduction, hydroxylation and subsequent conjugation in vivo. Active metabolites such as columbamine, berberrubine and demethyleneberberine also exhibit similar pharmacological effects by comparison with berberine, such as antioxidant, anti-inflammatory, antitumor, antimicrobial, hepatoprotective, neuroprotective, hypolipidemic and hypoglycemic effects. Overall, berberine together with its metabolites formed the material basis of berberine in vivo.     

Berberine inhibits the growth of human colorectal adenocarcinoma in vitro and in vivo

Berberine is an alkaloid isolated from the Chinese herbal medicine Huanglian, and has long been used as an antibiotic. Its antineoplastic properties were subsequently discovered in vitro. The purpose of this study was to investigate the effects of berberine on the growth of human colorectal carcinoma cells in vitro and in vivo. The results showed that berberine inhibited human colorectal adenocarcinoma (LoVo) cell growth in a time- and dose-dependent manner. A WST-1 assay showed that the IC₅₀ value after 72 h was 40.79 ± 4.11 μM. Cell cycle analysis of 40 μM berberine-treated LoVo cells by flow cytometry showed accumulation of cells in the G₂/M phase. The inhibition of LoVo cell growth by berberine was associated with the suppression of cyclin B1, cdc2, and cdc25c proteins. Berberine at a dose of 50 mg kg^?1 day^?1 showed inhibitory rates of 45.3 % in a human colorectal adenocarcinoma xenograft in nude mice. The combination of berberine and 5-fluorouracil (5-FU) had a higher inhibitory rate (59.8 %) than the berberine group (36.4 %, P = 0.01), but no significant difference was observed between the 5-FU group (43.0 %, P = 0.06) and the combination group. These results support the possibility that berberine may be useful as an alternative therapy for colorectal carcinoma.     

Hypoglycemic effect of <Emphasis Type="Italic">Gynostemma pentaphyllum</Emphasis> saponins by enhancing the Nrf2 signaling pathway in STZ-inducing diabetic rats

Gynostemma pentaphyllum (GP) is a natural plant resources for diabetes therapy, however, there is little research on the mechanisms of GP. The present study was undertaken to characterize if G. pentaphyllum saponins (GPs) is the principal active compound of GP responsible for anti-diabetes, and to examine the relativity between blood glucose modulate and antioxidation. The GPs-treated streptozotocin diabetic rats had a more effective hypoglycemic status than those of diabetic control rats, which also ameliorate dyslipidemia. GPs has increased SOD and GSH-px activities, and the spleen and thymus indexes in diabetic rats. The insulin levels in the GPs-treated groups were significantly higher than diabetic control group. Our finding provides a new insight into the application of GPs for the treatment of oxidative stress related diseases.     

黄连素作为抗糖尿病药物的临床应用及优势

目的对黄连素的降糖作用进行综述,为进一步研究和推广黄连素的作用提供参考。方法首先对黄连素降糖作用的研究报告和众多患者亲身体验的有关资料进行广泛收集,然后进行归纳整理和综合分析。结果黄连素作为抗糖尿病药物具有六大优势。结论黄连素具有被开发成新型降糖药品的价值。