[Discussion on Acupuncture as adjunctive therapy for chronic stable angina: a randomized clinical trial published in JAMA Internal Medicine].

Professor LIANG Fan-rong's team from Chengdu University of TCM published Acupuncture as adjunctive therapy for chronic stable angina: a randomized clinical trial in JAMA Internal Medicine on July 29, 2019, which demonstrates that acupuncture as an adjunctive therapy is safety and effective for mild and moderate chronic stable angina. Sixteen experts at home and abroad conducted serious discussions on the research design, evaluation methods, principles and mechanisms, clinical significance and enlightenment of future acupuncture research, and provided practical suggestions for acupuncture and moxibustion to go global and gain international recognition.

[Evidence-based medicine research on low-back pain with sham-acupuncture controlled design and the analysis on dynamic acupoint positioning].

To explore the positioning of acupoints, a research was done with PubMed for system reviews and clinical trials on treatment of low-back pain with sham-acupuncture controlled design from January 1, 2010 to October 27, 2017. Six system reviews and 12 sham-controlled acupuncture random trials were found. The statistical difference was not found in all the 6 trials with standard acupoint compared with the sham-acupuncture among the 8 penetrating skin sham-control trials. The statistical difference was found in the two trials with penetrating skin sham control, who was used individualized treatment, twirling for qi arrival or palpation for ashipoint. It is considered that sham-acupuncture penetrating skin is not a placebo, and needling with standard or dynamic acupoint may reduce low-back pain, and dynamic acupoint positioning may be better than standard acupoint positioning.

Glycogen synthase kinase 3 beta mediates high glucose-induced ubiquitination and proteasome degradation of insulin receptor substrate 1.

High glucose (HG) has been shown to induce insulin resistance in both type 1 and type 2 diabetes. However, the molecular mechanism behind this phenomenon is unknown. Insulin receptor substrate (IRS) proteins are the key signaling molecules that mediate insulin's intracellular actions. Genetic and biological studies have shown that reductions in IRS1 and/or IRS2 protein levels are associated with insulin resistance. In this study we have shown that proteasome degradation of IRS1, but not of IRS2, is involved in HG-induced insulin resistance in Chinese hamster ovary (CHO) cells as well as in primary hepatocytes. To further investigate the molecular mechanism by which HG induces insulin resistance, we examined various molecular candidates with respect to their involvement in the reduction in IRS1 protein levels. In contrast to the insulin-induced degradation of IRS1, HG-induced degradation of IRS1 did not require IR signaling or phosphatidylinositol 3-kinase/Akt activity. We have identified glycogen synthase kinase 3beta (GSK3 beta or GSK3B as listed in the MGI Database) as a kinase required for HG-induced serine(332) phosphorylation, ubiquitination, and degradation of IRS1. Overexpression of IRS1 with mutation of serine(332) to alanine partially prevents HG-induced IRS1 degradation. Furthermore, overexpression of constitutively active GSK3 beta was sufficient to induce IRS1 degradation. Our data reveal the molecular mechanism of HG-induced insulin resistance, and support the notion that activation of GSK3 beta contributes to the induction of insulin resistance via phosphorylation of IRS1, triggering the ubiquitination and degradation of IRS1.

Improved insulin sensitivity by calorie restriction is associated with reduction of ERK and p70S6K activities in the liver of obese Zucker rats.

Calorie restriction (CR) improves obesity-related insulin resistance through undefined molecular mechanisms. Insulin receptor substrate (IRS)-1 serine/threonine kinases have been proposed to modulate insulin sensitivity through phosphorylation of IRS proteins. The aim of this study is to test the hypothesis that changes in the activity of IRS1 serine/threonine kinases may underlie the molecular mechanism of CR in improving insulin sensitivity. Obese and lean Zucker rats were subjected to 40% CR or allowed to feed ad libitum (AL) for 20 weeks; body weight and insulin sensitivity were monitored throughout this period. The activity of IRS1 serine/threonine kinases - including JNK, ERK, MTOR/p70(S6K) (RPS6KB1 as listed in the MGI Database), glycogen synthase kinase 3beta (GSK3B), AMPK (PRKAA1 as listed in the MGI Database), and protein kinase C (PRKCQ) in liver tissue extracts was measured by an in vitro kinase assay using various glutathione-S-transferase (GST)-IRS1 fragments as substrates, while phosphorylation of IRS1 and serine kinases was determined by western blotting using phosphospecific antibodies. CR in obese rats significantly reduced body weight and increased insulin sensitivity compared to AL controls. Serine kinase activity toward IRS1(S612) (corresponding to S616 in human IRS1) and IRS1(S632/635) (corresponding to S636/639 in human IRS1) was increased in obese rats compared to lean littermates, and was markedly decreased following CR. Concomitantly, obesity increased and CR decreased the activity of hepatic ERK and p70(S6K) against IRS1. The close association between the activity of hepatic ERK and p70(S6K) with insulin resistance suggests an important role for ERK and p70(S6K) in the development of insulin resistance, presumably via phosphorylation of IRS proteins.

Facilitating effects of berberine on rat pancreatic islets through modulating hepatic nuclear factor 4 alpha expression and glucokinase activity.

AIM: To observe the effect of berberine on insulin secretion in rat pancreatic islets and to explore its possible molecular mechanism. METHODS: Primary rat islets were isolated from male Sprague-Dawley rats by collagenase digestion and treated with different concentrations (1, 3, 10 and 30 micromol/L) of berberine or 1 micromol/L Glibenclamide (GB) for 24 h. Glucose-stimulated insulin secretion (GSIS) assay was conducted and insulin was determined by radioimmunoassay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate cytotoxicity. The mRNA level of hepatic nuclear factor 4 alpha (HNF4alpha) was determined by reverse transcription polymerase chain reaction (RT-PCR). Indirect immunofluorescence staining and Western blot analysis were employed to detect protein expression of HNF4alpha in the islets. Glucokinase (GK) activity was measured by spectrophotometric method. RESULTS: Berberine enhanced GSIS rather than basal insulin secretion dose-dependently in rat islets and showed no significant cytotoxicity on islet cells at the concentration of 10 mumol/L. Both mRNA and protein expressions of HNF4alpha were up-regulated by berberine in a dose-dependent manner, and GK activity was also increased accordingly. However, GB demonstrated no regulatory effects on HNF4alpha expression or GK activity. CONCLUSION: Berberine can enhance GSIS in rat islets, and probably exerts the insulinotropic effect via a pathway involving HNF4alpha and GK, which is distinct from sulphonylureas (SUs).

Effect of berberine on expression of hepatocyte nuclear factor-4alpha in rats with fructose-induced insulin resistance.

The effects of berberine on the expression of hepatocyte nuclear factor-4alpha (HNF-4alpha) in liver of rats with fructose-induced insulin resistance and the molecular mechanism of berberine preventing insulin resistance were investigated. The experimental animals were divided into two groups of 16 animals each. The control group received a control routine diet containing 60% carbohydrate, and the study group a high-fructose diet containing 60% fructose as the sole source of carbohydrate. At the end of 6 weeks these were each subdivided into two groups. One was administered with berberine [187.5 mg/(kg x d) in 5 g/L carboxymethyl cellulose] by intragastric intubation and the other group was treated with a vehicle (5 g/L carboxymethyl cellulose). The rats were fed on the same dietary regimen for the next 4 weeks. After the experimental period of 10 weeks, plasma glucose, insulin and triglyceride levels were measured. HOMA insulin resistance index (HOMA-IR) was assayed. Immunohistochemistry, semiquantitative RT-PCR and western blot were used to detect the expression of HNF-4alpha in liver. Compared with control diet, fructose feeding induced hyperinsulinemia, HOMA-IR and increased triglyceride (all P<0.01). Berberine prevented the rise in plasma insulin (P<0.01), HOMA-IR (P<0.01) and triglyceride (P<0.05) in the fructose-fed rats. No change in plasma glucose was seen among these groups. The mRNA and protein expression of HNF-4alpha was decreased in the fructose-fed rats, but berberine could promote its expression. It was concluded that berberine could prevent fructose-induced insulin resistance in rats possibly by promoting the expression HNF-4alpha in liver.

[Effects of berberine on expression of hepatocyte nuclear factor 4alpha and glucokinase activity in mouse primary hepatocytes].

OBJECTIVE: To observe the expression of hepatocyte nuclear factor 4alpha (HNF4alpha) and the activity of key enzyme glucokinase (GK) in glucose metabolism, and further to investigate the possible mechanism of berberine in treating type 2 diabetes. METHOD: Mouse primary hepatocytes were isolated by an improved single two-step perfusion method. The murine hepatocytes were cultured and incubated with berberine (0, 1, 3, 10, 30, 100 micromol x L(-1)) and 1 mmol x L(-1) metformin for 24 h respectively. The mRNA expression of HNF4alpha were quantified by RT-PCR and the protein expression of HNF4alpha were quantified by Western-blot. And the activity of GK were detected with enzyme kinetics method. RESULT: As compared with the negative control group, at a certain concentration range, the expression of HNF4alpha mRNA and protein and the activity of GK were promoted by berberine. Both of them reached the top at the concentration of 30 micromol x L(-1) (P<0.01). But the metformin made no difference with the negative control group on the expression of HNF4alpha and the activity of GK. CONCLUSION: It is suggested that the effects of berberine on improving glucose metabolism can be mechanically associated with its up-regulating the HNF4a expression and inducing the activity of hepatic glucokinase.

Effect of Berberine on Expression of Hepatocyte Nuclear Factor-4α in Rats with Fructose-induced Insulin Resistance / 华中科技大学学报（医学）（英德文版）

The effects of berberine on the expression of hepatocyte nuclear factor-4α (HNF-4α) in liver of rats with fructose-induced insulin resistance and the molecular mechanism of berberine preventing insulin resistance were investigated. The experimental animals were divided into two groups of 16 animals each. The control group received a control routine diet containing 60% carbohydrate, and the study group a high-fructose diet containing 60% fructose as the sole source of carbohydrate. At the end of 6 weeks these were each subdivided into two groups. One was administered with berberine [187.5mg/(kg·d) in 5g/L carboxymethyl cellulosel] by intragastric intubation and the other group was treated with a vehicle (5g/L carboxymethyl cellulose). The rats were fed on the same dietary regimen for the next 4 weeks. After the experimental period of 10 weeks, plasma glucose, insulin and triglyceride levels were measured. HOMA insulin resistance index (HOMA-IR) was assayed. Immunohistochemistry, semiquantitative RT-PCR and western blot were used to detect the expression of HNF-4α in liver. Compared with control diet, fructose feeding induced hyperinsulinemia, HOMA-IR and increased triglyceride (all P<0.01). Berberine prevented the rise in plasma insulin (P<0.01), HOMA-IR (P<0.01) and triglyceride (P<0.05) in the fructose-fed rats. No change in plasma glucose was seen among these groups. The mRNA and protein expression of HNF-4α was decreased in the fructose-fed rats, but berberine could promote its expression. It was concluded that berberine could prevent fructose-induced insulin resistance in rats possibly by promoting the expression HNF-4α in liver.

[Protective effects of Huanglian Jiedu decoction on vascular endothelial function in type 2 diabetic rats].

OBJECTIVE: To investigate the effects of Huanglian Jiedu (HLJD) decoction on vascular endothelial function in type 2 diabetic rats and explore the prophylactic and therapeutic significance and pharmacological mechanism of HLJD decoction in type 2 diabetic angiopathic complication. METHOD: The murine type 2 diabetes models were induced by the intravenous injection of a small dose of streptozotocin plus high fat and high caloric laboratory chow. Then modeled diabetic animals were divided into model group, HLJD group, and aspirin group. Normal ratsfed with routine chow were designated as normal group. The oral glucose tolerance test (OGTT) were performed in all animals, 9 weeks after treatment, the changes of murine body weights and levels of fasting blood glucose (FBG), serum total cholesterol (TC), triglycerides (TG), high density lipoprotein-cholesterol(HDL-C), fasting serum insulin (FINS), serum nitric oxide, plasma endothelin, angiotensin II and von Willebrand Factor (vWF) were determined 10 weeks after treatment. RESULT: Compared with model group, the result of OGTT of HLJD group was improved. The levels of the body weights, TC, TG and ET in HLJD group weredecreased (P < 0.05). The levels of FBG,INS, AngII and vWFwere significantly decreased (P < 0.01), and the levels of HDL-C and NO were obviously increased (P < 0.05), as compared with those in model group. Furthermore. The levels of FBG was lower in HLJD group than in aspirin group (P < 0.05), and the improvement of TG, HDL-C,NO, AngII, vWF levels in HLJD group was more greatly than that in aspirin group, but there was not significant difference between two groups (P > 0.05). CONCLUSION: It is suggested by the present results that HLJD decoction could protect vascular endothelium from early damage in type 2 diabetes. The protective effects of HLJD on endothelium might be related to its ability of reducing the blood glucose, adjusting plasma lipids profiles, improving insulin resistance, antagonizing inflammatory mediators and inducing endothelium-dependent vascular relaxation.

Induction of pancreatic duct cells of neonatal rats into insulin-producing cells with fetal bovine serum: a natural protocol and its use for patch clamp experiments.

AIM: To induce the pancreatic duct cells into endocrine cells with a new natural protocol for electrophysiological study. METHODS: The pancreatic duct cells of neonatal rats were isolated, cultured and induced into endocrine cells with 15% fetal bovine serum for a period of 20 d. During this period, insulin secretion, MTT value, and morphological change of neonatal and adult pancreatic islet cells were comparatively investigated. Pancreatic beta-cells were identified by morphological and electrophysiological characteristics, while ATP sensitive potassium channels (K(ATP)), voltage-dependent potassium channels (K(V)), and voltage-dependent calcium channels (K(CA)) in beta-cells were identified by patch clamp technique. RESULTS: After incubation with fetal bovine serum, the neonatal duct cells budded out, changed from duct-like cells into islet clusters. In the first 4 d, MTT value and insulin secretion increased slowly (MTT value from 0.024+/-0.003 to 0.028+/-0.003, insulin secretion from 2.6+/-0.6 to 3.1+/-0.8 mIU/L). Then MTT value and insulin secretion increased quickly from d 5 to d 10 (MTT value from 0.028+/-0.003 to 0.052+/-0.008, insulin secretion from 3.1+/-0.8 to 18.3+/-2.6 mIU/L), then reached high plateau (MTT value >0.052+/-0.008, insulin secretion >18.3+/-2.6 mIU/L). In contrast, for the isolated adult pancreatic islet cells, both insulin release and MTT value were stable in the first 4 d (MTT value from 0.029+/-0.01 to 0.031+/-0.011, insulin secretion from 13.9+/-3.1 to 14.3+/-3.3 mIU/L), but afterwards they reduced gradually (MTT value <0.031+/-0.011, insulin secretion <8.2+/-1.5 mIU/L), and the pancreatic islet cells became dispersed, broken or atrophied correspondingly. The differentiated neonatal cells were identified as pancreatic islet cells by dithizone staining method, and pancreatic beta-cells were further identified by both morphological features and electrophysiological characteristics, i.e. the existence of recording currents from K(ATP), K(V), and K(CA). CONCLUSION: Islet cells differentiated from neonatal pancreatic duct cells with the new natural protocol are more advantageous in performing patch clamp study over the isolated adult pancreatic islet cells.

Therapeutic effects of berberine in impaired glucose tolerance rats and its influence on insulin secretion.

AIM: To explore the anti-diabetic effects of berberine and its influence on insulin secretion. METHODS: Impaired glucose tolerance rats induced by iv injection of streptozotocin 30 mg/kg were treated with berberine 187.5 and 562.5 mg/kg while fed with high fat laboratory chow. After rats were treated for 4 weeks, oral glucose tolerance was determined, and for 8 weeks, the fasting blood glucose, insulin, lipid series were determined. In insulin secretion experiments, berberine 93.75, 187.5, and 562.5 mg/kg was administered orally to BALB/c mice at a bolus. The murine serum was collected 2 h after the berberine administration for insulin determination. Insulin released from HIT-T15 cells and pancreatic islets incubated with berberine 1-100 micromol/L for 12 h was determined. RESULTS: The levels of fasting blood glucose (7.4+/-1.5 or 7.3+/-1.3 vs 9.3+/-1.3 mmol/L), triglycerides (0.61+/-0.22 or 0.63+/-0.17 vs 1.8+/-0.7 mmol/L), total cholesterol (1.8+/-0.3 or 1.9+/-0.3 vs 2.2+/-0.2 mmol/L), free fatty acid (456+/-93 or 460+/-72 vs 550+/-113 micromol/L) and apolipoprotein B (0.37+/-0.02 or 0.42+/-0.05 vs 0.46+/-0.04 g/L) were reduced greatly in berberine-treated groups at doses of 187.5 and 562.5 mg/g/d, respectively as compared with those in control group (P<0.05 or P<0.01), whereas high density lipoprotein-cholesterol (1.5+/-0.3 or 1.4+/-0.3 vs 1.1+/-0.1 g/L), apolipoprotein AI (0.80+/-0.08 or 0.87+/-0.08 vs 0.71+/-0.06 g/L) were significantly increased (P<0.05 or P<0.01), and oral glucose tolerance was improved. In vitro experiment showed that berberine 1-10 micromol/L facilitated insulin secretion of HIT-T15 cells and murine pancreatic islets in a dose-dependent manner. Meanwhile murine serum insulin level (27.5+/-2.7 or 29+/-4 or 29+/-4 vs 24.3+/-2.8 pIU/L) was undoubtedly promoted and blood glucose (4.52+/-0.31 or 4.45+/-0.29 or 4.30+/-0.19 vs 4.87+/-0.21 mmol/L) was reduced after berberine administration at doses of 93.75, 187.5, and 562.5 mg/kg, respectively in the BALB/c mice. CONCLUSION: Berberine possesses anti-diabetic effects, which is related to the property of stimulating insulin secretion and modulating lipids.