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Background: Zerumbone is one of the pungent constituents of Zingiber zerumbet (L) Smith (Zingiberaceae family). The aim of the present study was to examine the effects of zerumbone in rats with streptozotocin-induced diabetic nephropathy (DN)., Methods: Diabetic rats were treated orally with zerumbone (20 or 40 mg/kg/day) for 8 weeks. Changes in renal function-related parameters in plasma and urine were analyzed at the end of the study. Kidneys were isolated for pathology histology, immunohistochemistry, and Western blot analyses., Results: Diabetic rats exhibited renal dysfunction, as evidenced by reduced creatinine clearance, increased blood glucose, blood urea nitrogen and proteinuria, along with marked elevation in the ratio of kidney weight to body weight, that were reversed by zerumbone. Zerumbone treatment was found to markedly improve histological architecture in the diabetic kidney. Hyperglycemia induced p38 mitogen-activated protein kinase activation, leading to increased infiltration of macrophages and increased levels of interleukin (IL)-1, IL-6 and tumor necrosis factor-α. All of the above abnormalities were reversed by zerumbone treatment, which also decreased the expression of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, transforming growth factor-β1 and fibronectin in the diabetic kidneys., Conclusions: The beneficial effect of zerumbone in rats with DN is at least in part through antihyperglycemia which was accompanied by inhibition of macrophage infiltration via reducing p38 mediated inflammatory response.

With the increasing patients and limited therapeutic options, diabetic nephropathy (DN) is a long-term complication of diabetic mellitus. The precise mechanism of DN is not yet fully understood and the effective blockade of the progression of nephropathy remains a therapeutic challenge. Application of traditional Chinese medicine (TCM) for diabetes and its related complications has received increasing attention due to its wide availability, low side effects, and proven therapeutic mechanisms and benefits. In the current review, we mainly focus on the recent laboratory studies of the TCM formulas including Wu-Ling-San (Poria Five Powder; Wǔ Líng Sǎn), Danggui-Buxue-Tang (Tangkuei and Astragalus Decoction; Dāng Guī Bǔ Xuè Tang), and Danggui-Shaoyao-San (Tangkuei and Paeonia Formula; Dāng Guī Sháo Yào Sǎn), conducted by the Committee on Chinese Medicine and Pharmacy at the Department of Health of Taiwan Government, in the amelioration of DN. These selected TCM formulas have anti-diabetic properties, with antihyperglycemic activity accompanied by amelioration of advanced glycation end product-mediated renal damage in streptozotocin-induced diabetic rats. However, the renoprotective effects of the selected TCM formulas did not correlate with suppressing renal renin-angiotensin system hyperactivity in diabetic rats. These TCM formulas also have the capacity to ameliorate the defective antioxidative defense system, leading to modulation of the oxidative stress, thereby resulting in downregulation of nuclear factor-kB as well as transforming growth factor-β1 and, consequently, attenuation of extracellular matrix components such as fibronectin or type IV collagen expression in diabetic renal cortex tissue. More detailed mechanistic researches and long-term clinical evaluations, as well as evaluation of safety of the selected TCM formulas are needed for their future applications in DN therapy.

6-Gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone) is one of the pungent constituents of Zingiber zerumbet (L) Smith (Zingiberaceae family). In this study, we investigated the effects of 6-gingerol on the inhibition of adipogenesis in 3T3-L1 cells. After treatment with 6-gingerol in differentiation medium for 4 or 8 days, the 3T3-L1 cells were lysed for experimental analysis. Cells were stained with Oil-Red-O to detect oil droplets in adipocytes. The 3T3-L1 cells were lysed and measured for triglyceride contents. The protein expression of adipogenesis-related transcription factor was evaluated by Western blot analysis. 6-Gingerol suppressed oil droplet accumulation and reduced the droplet size in a concentration (5-15 μg/ml)- and time-dependent manner. Treatment of 3T3-L1 cells with 6-gingerol reduced the protein levels of peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer-binding protein (C/EBP)α. Additionally, the protein levels of fatty acid synthase (FAS) and adipocyte-specific fatty acid binding protein (aP2) decreased upon treatment with 6-gingerol. Meanwhile, 6-gingerol diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3β (Ser9). These results suggest that 6-gingerol effectively suppresses adipogenesis and that it exerts its role mainly through the significant down-regulation of PPARγ and C/EBPα and subsequently inhibits FAS and aP2 expression. 6-Gingerol also inhibited differentiation in 3T3-L1 cells by attenuating the Akt/GSK3β pathway. Our findings provide important insights into the mechanisms underlying the anti-adipogenic activity of 6-gingerol., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

We investigated the effects of zerumbone, a natural cyclic sesquiterpene, on hepatic lipid metabolism in Syrian golden hamsters fed on high-fat diet (HFD). After being fed HFD for 2 weeks, hamsters were dosed orally with zerumbone (75, 150, and 300 mg kg(-1)) once daily for 8 weeks. After treatment with zerumbone, the plasma levels of total cholesterol (TC) and triglycerides (TGs) and the contents of TC and TG in hepatic tissue as well as homeostasis model assessment of insulin resistance were lowered, especially in the zerumbone-treated group (300 mg kg(-1)). Moreover, the histological evaluation of liver specimens demonstrated that the steatosis and inflammation in liver of zerumbone-treated groups were improved. Zerumbone exhibited the ability to decrease hepatic mRNA levels of sterol regulatory element-binding protein-1c and its lipogenic target genes, such as fatty acid synthase, acetyl-CoA carboxylase 1, and stearoyl-CoA desaturase 1. The hepatic mRNA expression of peroxisome proliferator-activated receptor α , together with its target genes including carnitine palmitoyl transferase-1, acyl-CoA oxidase, and acyl-CoA oxidase 1, was also upregulated by zerumbone. In conclusion, zerumbone improves insulin sensitivity, decreases lipogenesis, and increases lipid oxidation in the liver of HFD-fed hamsters, implying a potential application in the treatment of nonalcoholic fatty liver disease.

The tuberous root of Liriope spicata var. prolifera (TRLS; Liliaceae family) is valued for the ability to promote glucose homeostasis, and it may therefore be utilized as an adjuvant therapy in the control of diabetic complications. The aim of the present study was to examine the effects of an aqueous ethanol extract from TRLS (TRLS-ext) (100 or 200 mg kg(-1) per day for eight weeks) on rats with streptozotocin-induced diabetic nephropathy (DN). Renal dysfunction in diabetic rats was ameliorated by TRLS-ext as evidenced by reduced creatinine clearance, as well as increased blood urea nitrogen and proteinuria. Treatment with TRLS-ext was found to markedly improve histological architecture in the diabetic kidney. Hyperglycemia induced degradation of inhibitory kappa B and reduced nuclear factor kappa B activation, leading to increased infiltration of macrophages and increased levels of proinflammatory cytokines, including interleukin-1 and tumor necrosis factor- α . All of the above abnormalities were reversed by TRLS-ext treatment, which also decreased the expression of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and fibronectin in the diabetic kidneys. These findings provide a perspective on the renoprotective effects of TRLS-ext in DN.

The ethanol extract from the rhizome of Zingiber zerumbet (L.) Smith (EEZZR) has been indicated to possess an insulin-like property by ameliorating hyperglycemia in diabetes. We aimed to investigate whether EEZZR exerts an ameliorative effect on renal damage in diabetes induced by streptozotocin (STZ). Diabetic rats were treated orally with EEZZR (200 and 300 mg kg(-1) per day) or metformin (100 mg kg(-1) per day) for 8 weeks. The plasma glucose, creatinine, and blood urea nitrogen as well as urine protein levels and the ratio of kidney weight to body weight were significantly elevated in diabetic rats. EEZZR displayed similar characteristics to those of metformin in reducing hyperglycemia and renal dysfunction in diabetic rats. The histological examinations revealed amelioration of diabetes-induced glomerular pathological changes following the treatment with EEZZR. In addition, the protein expressions of renal nephrin and podocin in diabetic rats were significantly increased following the treatment with EEZZR. The AMP-activated protein kinase (AMPK) protein phosphorylation and expression levels were remarkably reduced in diabetic renal tissues. EEZZR treatment significantly rescued the AMPK phosphorylation compared to nontreated diabetic group. This study suggested that the renoprotective effects of EEZZR may be similar, with the action of metformin, to the prevention of AMPK dephosphorylation and upregulate the expressions of renal nephrin and podocin.

Background: Lonicera japonica Thunb. (Caprifoliaceae), a widely used traditional Chinese medicinal plant, is used to treat some infectious diseases and it may have uses as a healthy food and applications in cosmetics and as an ornamental groundcover. The ethanol extract of the flowering aerial parts of L. japonica (LJEE) was investigated for its healing efficiency in a rat excision wound model., Methods: Excision wounds were inflicted upon three groups of eight rats each. Healing was assessed by the rate of wound contraction in skin wound sites in rats treated with simple ointment base, 10% (w/w) LJEE ointment, or the reference standard drug, 0.2% (w/w) nitrofurazone ointment. The effects of LJEE on the contents of hydroxyproline and hexosamine during healing were estimated. The antimicrobial activity of LJEE against microorganisms was also assessed. The in vivo anti-inflammatory activity of LJEE was investigated to understand the mechanism of wound healing., Results: LJEE exhibited significant antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Candida albicans, and Candida tropicalis. The ointment formulation prepared with 10% (w/w) LJEE exhibited potent wound healing capacity as evidenced by the wound contraction in the excision wound model. The contents of hydroxyproline and hexosamine also correlated with the observed healing pattern. These findings were supported by the histopathological characteristics of healed wound sections, as greater tissue regeneration, more fibroblasts, and angiogenesis were observed in the 10% (w/w) LJEE ointment-treated group. The results also indicated that LJEE possesses potent anti-inflammatory activity, as it enhanced the production of anti-inflammatory cytokines that suppress proinflammatory cytokine production., Conclusions: The results suggest that the antimicrobial and anti-inflammatory activities of LJEE act synergistically to accelerate wound repair.

The aim of this study was to investigate the antiobesity and antihyperlipidemic effects of Angelica acutiloba root (Japanese Dong Quai). High-fat diet (HFD)-induced obese rats were treated orally with the polyphenolic-rich extract of Angelica acutiloba root (AARE) once daily for 8 weeks. The AARE (300 mg/kg per day) supplementation significantly lowered body weight gain, visceral fat-pad weights and plasma lipid levels, as well as the coronary artery risk index and the atherogenic index of HFD-fed rats. The AARE caused dose related reductions in the hepatic triglyceride and cholesterol contents, as well as lowered hepatic lipid droplet accumulation and epididymal adipocyte size in the HFD-fed rats. The AARE reversed the HFD-induced down-regulation of the hepatic peroxisome proliferator activated receptor-α (PPARα). The HFD-induced decreases of the hepatic protein level of acyl-CoA oxidase (ACO), and the cytochrome P450 isoform 4A1 (CYP4A1) was up-regulated by AARE. The elevated expressions of hepatic sterol regulatory element binding proteins (SREBPs) of HFD-fed rats were lowered by AARE. These results suggest that AARE attenuated visceral fat accumulation and improved hyperlipidemia in HFD-induced obesity by increasing lipid metabolism through the down-regulation of SREBPs and enhanced the expression of ACO and CYP4A1 in the liver, which was likely mediated by up-regulation of the expression of hepatic PPARα., (Copyright © 2011 John Wiley & Sons, Ltd.)

The objective of this study was to evaluate the acute and subacute toxicity (28 days) of the ethanol extract of Z. zerumbet rhizomes (EEZZ) via the oral route in Wistar rats of both sexes. In the acute toxicity study, Wistar rats were administered a single dose of 15 g kg(-1) of body weight by gavage, and were monitored for 14 days. EEZZ did not produce any toxic signs or deaths; the 50% lethal dose must be higher than 15 g kg(-1). In the subchronic toxicity study, EEZZ was administered by gavage at doses of 1000, 2000 and 3000 mg/kg daily for 4 weeks to Wistar rats. The subacute treatment with EEZZ did not alter either the body weight gain or the food and water consumption. The hematological and biochemical analysis did not show significant differences in any of the parameters examined in female or male groups. Necropsy and histopathological examination, did not reveal any remarkable and treatment related changes. A no-observed adverse-effect level for EEZZ is 3000 mg kg(-1) for rats under the conditions of this study. Hence, consumption of EEZZ for various medicinal purposes is safe.

The present study evaluated the potential genotoxicity of the ethanol extracts from the rhizome of Zingiber zerumbet (L.) Smith (EEZZR) using a standard battery of tests. Chemical analysis with liquid chromatography-tandem mass spectrometry revealed that EEZZR contained Zerumbone (200.3 ± 0.37 μg/g) and 6-gingerol (102.5 ± 0.28 μg/g). There were no increases in the number of revertant colonies with EEZZR at concentrations of 150-5000 μg per plate, regardless of the metabolic activation system (S-9 mix) used in the histidine-dependent auxotrophic mutants of Salmonella typhimurium (strains TA97, TA98, TA100, TA102, and TA1535) compared to the vehicle control. Furthermore, EEZZR at doses of 150-5000 μg mL(-1) did not increase the number of structural aberrations in Chinese hamster lung cells in the presence or absence of S-9 mix. An oral administration of EEZZR to ICR mice, with doses of up to 2000 mg/kg, caused no significant increases in the number of micronucleated polychromatic erythrocytes (MNPCEs) and mean ratio of polychromatic erythrocytes to total erythrocytes. Lastly, RZZEE did not increase the incidence of MNPCEs in bone marrow. Based on these findings, it may be concluded that the use of EEZZR in traditional medicine poses no risk of genotoxicity.

The aim of this study was to investigate the antiobesity and antihyperlipidaemic effects of myricetin. Myricetin exhibited a significant concentration-dependent decrease in the intracellular accumulation of triglyceride in 3T3-L1 adipocytes. The high-fat diet (HFD)-fed rats were dosed orally with myricetin or fenofibrate, once daily for eight weeks. Myricetin (300 mg kg(-1) per day) displayed similar characteristics to fenofibrate (100 mg kg(-1) per day) in reducing lowered body weight (BW) gain, visceral fat-pad weights and plasma lipid levels of HFD-fed rats. Myricetin also reduced the hepatic triglyceride and cholesterol contents, as well as lowered hepatic lipid droplets accumulation and epididymal adipocyte size in HFD-fed rats. Myricetin and fenofibrate reversed the HFD-induced down-regulation of the hepatic peroxisome proliferator activated receptor (PPAR)α. HFD-induced decreases of the hepatic protein level of acyl-CoA oxidase and cytochrome P450 isoform 4A1 were up-regulated by myricetin and fenofibrate. The elevated expressions of hepatic sterol regulatory element binding proteins (SREBPs) of HFD-fed rats were lowered by myricetin and fenofibrate. These results suggest that myricetin suppressed BW gain and body fat accumulation by increasing the fatty acid oxidation, which was likely mediated via up-regulation of PPARα and down-regulation of SREBP expressions in the liver of HFD-fed rats.

The aim of this study was to investigate the antiobesity and antihyperlipidemic effects of vinegar-baked Radix Bupleuri (VBRB) on high-fat diet- (HFD-) induced obese rats. After being fed HFD for two weeks, rats were dosed orally with VBRB or fenofibrate, once daily for further twelve weeks. VBRB (1.0 g kg(-1) per day) produced effects similar to fenofibrate (100 mg kg(-1)) in reducing body weight (BW) gain, visceral fat-pad weights, plasma lipid levels, as well as hepatic TG and cholesterol content of HFD-fed rats. VBRB also lowered hepatic lipid droplet accumulation and the size of epididymal adipocytes in HFD-fed rats. VBRB and fenofibrate reversed the HFD-induced downregulation of hepatic peroxisome proliferator-activated receptor (PPAR)α. HFD-induced reductions in the hepatic levels of acyl-CoA oxidase (ACO) and cytochrome P450 isoform 4A1 (CYP4A1) proteins were reversed by VBRB and fenofibrate. The elevated expression of hepatic sterol regulatory element binding proteins (SREBPs) in HFD-fed rats was lowered by VBRB and fenofibrate. The results of this study show that VBRB suppresses BW gain and body fat accumulation by increasing fatty acid oxidation, an effect which is likely mediated via upregulation of PPARα and downregulation of SREBP expression in the liver of HFD-fed rats.

Angelica acutiloba root (Japanese Dong Quai), used for treatment of gynecological disorders, is currently cultivated in Taiwan. The present study evaluated the preventative effect of Angelica acutiloba root (Japanese Dong Quai) on the induction of insulin resistance. Insulin resistance was induced in rats by feeding a high fructose diet for 6 weeks. Thereafter, the rats were maintained on the same diet and treated with oral A. acutiloba root extract or pioglitazone once daily for 8 weeks. At the end of treatment, the degree of basal insulin resistance was measured by homeostasis model assessment (HOMA-IR). Insulin sensitivity was calculated using the composite whole body insulin sensitivity index (ISIcomp). Protein expression was evaluated by immunoblotting. A. acutiloba (300 mg/kg/day) displayed similar characteristics to pioglitazone (20 mg/kg/day) in reducing HOMA-IR and elevating ISIcomp. Elevated glycosylated hemoglobin levels and hyperinsulinemia were ameliorated by A. acutiloba treatment without hepatotoxic or nephrotoxic effects. A. acutiloba treatment improved dyslipidemia, induced lipoprotein lipase activity and enhanced hepatic glycogen accumulation. Further, A. acutiloba treatment enhanced the action of insulin on muscle glucose transporter subtype 4 translocation and attenuated hepatic phosphoenolpyruvate carboxykinase expression. The findings suggest that A. acutiloba may be an effective ethnomedicine for improving insulin sensitivity., (Copyright © 2011 John Wiley & Sons, Ltd.)

Tanshinone IIA is one of the active principles in danshen (Salvia miltiorrhiza Bge) widely used in treatment of cardiovascular disorders. We investigated the effect of danshen or tanshinone IIA on blood pressure and its possible mechanisms. An i.p. injection of danshen at 10 mg kg(-1) significantly lowered systolic blood pressure (SBP) of spontaneously hypertensive rats (SHRs) but failed to modify the SBP in normotensive Wistar-Kyoto rats (WKY). Oral administration of tanshinone IIA also decreased SBP in SHR but not in WKY. Tanshinone IIA produced a concentration-dependent relaxation in isolated SHR aortic rings precontracted with phenylephrine (10 nmol l(-1)) or potassium chloride (KCl) (40 mmol l(-1)). The relaxing effect of tanshinone IIA on tonic contraction of phenylephrine in isolated aortic rings without endothelium remained produced. Glibenclamide at concentration sufficient to block adenosine triphosphatase (ATP)-sensitive potassium (K(+)) channel attenuated this tanshinone IIA-induced relaxation that was not influenced by other inhibitors. We further investigated the effect of tanshinone IIA on the changes of intracellular calcium concentration ([Ca(2+)](i)) in cultured aortic smooth muscle (A7r5) cells using fura-2 as indicator. Tanshinone IIA decreased [Ca(2+)](i) elicited by phenylephrine (10 nmol l(-1)) or KCl (40 mmol l(-1)) in a concentration-dependent manner; glibenclamide, but not other inhibitors for K(+) channel, abated this effect. Our results suggest that tanshinone IIA acts as an active principle of danshen showing vasodilation through ATP-sensitive K(+) channel to lower [Ca(2+)](i).

Dang Gui Bu Xue Tang (DBT), a Chinese medicinal decoction contains Radix Angelicae sinensis (Danggui) and Radix Astragali (Huangqi) at a ratio of 1 : 5, is used commonly for treating women's ailments. This study was conducted to explore the effects of this preparation on insulin resistance in rats fed with 6-week diet containing 60% fructose. Similar to the action of rosiglitazone (4 mg kg(-1) per day by an oral administration), repeated oral administration of DBT (2.5 g kg(-1) per day) for 14 days was found to significantly alleviate the hyperglycemia but made no influence on plasma lipid profiles nor weight gain in fructose chow-fed rats. Also, the higher degree of insulin resistance as measured by homeostasis model assessment of basal insulin resistance in fructose chow-fed rats was significantly decreased by repeated DBT treatment. DBT displays the characteristic of rosiglitazone by increasing the whole-body insulin sensitivity in fructose chow-fed rats after 2-week treatment, as evidenced by the marked elevation of composite whole-body insulin sensitivity index during the oral glucose tolerance test. DBT improves insulin sensitivity through increased post-receptor insulin signaling mediated by enhancements in insulin receptor substrate-1-associated phosphatidylinositol 3-kinase step and glucose transporter subtype 4 translocation in soleus muscles of animals exhibiting insulin resistance. DBT is therefore proposed as potentially useful adjuvant therapy for patients with insulin resistance and/or the patients who wish to increase insulin sensitivity.

The present study is designed to investigate the role of atypical protein kinase C (PKC) in the signaling of mu-opioid receptors (MOR) for glucose uptake in myoblast C(2)C(12) cells. Loperamide enhanced the uptake of radioactive deoxyglucose into C(2)C(12) cells in a concentration-dependent manner that was abolished in cells pre-incubated with GF109203X at concentrations sufficient to block PKC. Inhibition of the atypical zeta (zeta) isoform of PKC using myristoylated PKC pseudosubstrate resulted in a concentration-dependent decrease of loperamide-stimulated glucose uptake into C(2)C(12) cells. In addition, loperamide elicited the phosphorylation of PKC-zeta in C(2)C(12) cells in a concentration-dependent manner that was abolished by pretreatment with naloxonazine at concentrations sufficient to block MOR. These results suggest the mediation of PKC-zeta in MOR signaling for glucose uptake in C(2)C(12) cells. Activation of PKC-zeta by MOR stimulation is highly relevant to the search for therapeutic targets for glucose transport in insulin-sensitive tissues.

The role of hyperglycaemia in the pathogenesis of hypotension in diabetic disorders was investigated using the changes in cardiac M(2)-muscarinic receptor (M(2)-mAChR) gene expression in type-1-like diabetic rats and cultured cardiomyocytes. Blood pressure was markedly decreased in diabetic rats following the intravenous injection of streptozotocin (STZ) for 8 weeks. Also, the baroreflex sensitivity (Delta HR/Delta BP), as measured by the changes in heart rate (Delta HR) and mean blood pressure (Delta BP) 1 min after the intravenous injection of phenylephrine (10 microg/kg), was significantly increased. Arecaidine propargyl ester (APE), a M(2)-mAChR agonist produced a marked reduction in heart rate in these diabetic rats. Normalization of plasma glucose in diabetic rats using insulin (0.5 IU) or phlorizin (1 mg/kg) injection attenuated the blood pressure reduction and reversed the mRNA and protein levels of cardiac M(2)-mAChR. A high concentration of glucose (20 mmol/l) directly influenced the increase in gene expression of M(2)-mAChR in the H9c2 cardiac cell line. Hyperglycaemia induced an increase in cardiac M(2)-mAChR gene expression, suggesting a role in the pathogenesis of hypotension in diabetic disorders.

The present study was conducted to explore the effects of myricetin on insulin resistance in rats fed for 6 weeks with a diet containing 60% fructose. Repeated intravenous (i.v.) injection of myricetin (1 mg/kg per injection, 3 times daily) for 14 days was found to significantly decrease the high glucose and triglyceride levels in plasma of fructose chow-fed rats. Also, the higher degree of insulin resistance in fructose chow-fed rats as measured by homeostasis model assessment of basal insulin resistance was significantly decreased by myricetin treatment. Myricetin increased the whole-body insulin sensitivity in fructose chow-fed rats, as evidenced by the marked elevation of composite whole-body insulin sensitivity index during the oral glucose tolerance test. Myricetin was found to reverse the defect in expression of insulin receptor substrate-1 (IRS-1) and the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) in soleus muscle of fructose chow-fed rats under the basal state, despite the protein expression of insulin receptor (IR). Increased basal phosphorylation of IR and IRS-1 as well as Akt was observed in parallel. The reduced level of insulin action on phosphorylation of IR, IRS-1 and Akt in soleus muscle of fructose chow-fed rats was reversed by myricetin treatment. Furthermore, myricetin treatment improved the defective insulin action on the translocation of glucose transporter subtype 4 (GLUT 4) in insulin-resistant soleus muscle. These findings indicate that myricetin improves insulin sensitivity through the enhancement of insulin action on IRS-1-associated PI 3-kinase and GLUT 4 activity in soleus muscles of animals exhibiting insulin resistance.

1. Sinomenine is an alkaloid with a wide range of pharmacological actions. In the present study, we investigated the effect of sinomenine on blood pressure and its possible mechanisms of action. 2. Spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were given intraperitoneal injections of sinomenine. At 30 min, 2.5-10 mg/kg sinomenine decreased systolic blood pressure (SBP) in a dose-dependent manner in SHR, but had no effect on the SBP in WKY rats. 3. The vascular effect of sinomenine was then examined in aortic rings isolated from Wistar rats. Sinomenine (0.1-10 micromol/L) produced concentration-dependent relaxation in aortic rings precontracted with phenylephrine (10 nmol/L) or KCl (40 mmol/L). Glibenclamide (1-100 micromol/L), a specific inhibitor of ATP-sensitive K(+) channels attenuated the sinomenine-induced relaxation, but this effect was not observed when inhibitors of other types of K(+) channels were used. 4. We further investigated the effects of sinomenine on changes in intracellular Ca(2+) concentrations ([Ca(2+)](i)) in cultured aortic smooth muscle (A7r5) cells by using the Ca(2+)-sensitive dye fura-2 as an indicator. Sinomenine, over the concentration range 0.1-10 micromol/L, decreased the increases in [Ca(2+)](i) elicited by phenylephrine (1 micromol/L) or KCl (40 mmol/L) in a concentration-dependent manner. Glibenclamide (1-100 micromol/L) abolished the effects of sinomenine. 5. In conclusion, sinomenine causes vascular relaxation by opening ATP-sensitive K(+) channels, thus decreasing [Ca(2+)](i).

Peroxisome proliferator-activated receptors (PPARs) play key roles in the regulation of energy homeostasis and inflammation while the agonists of PPARalpha and PPARgamma are recently used for therapy in clinic. However, functions of PPARdelta are still unclear. In the present study, we investigated the changes of PPARdelta gene expression in the lung of diabetic rats. The mean level of mRNA transcripts encoding PPARdelta was increased in the lung isolated from streptozotocin-induced diabetic rats (STZ-diabetic rats) to about 1.6-fold of that in normal rats. Exogenous insulin at the dose sufficient to normalize the plasma glucose of STZ-diabetic rats reversed the mRNA level of PPARdelta in lungs after a 4-day treatment. Similar results were also observed in STZ-diabetic rats that received the treatment of phlorizin to reverse the plasma glucose level for 4 days. Otherwise, the protein level of PPARdelta was higher in the lung of STZ-diabetic rats than that in normal rats. Treatment with exogenous insulin or phlorizin reversed this elevated protein level of PPARdelta in the lung of STZ-diabetic rats to near the normal level. The obtained results suggest that increase of plasma glucose is responsible for the higher gene expression of PPARdelta in the lung of STZ-diabetic rats.

In an attempt to probe a new target to improve insulin resistance, miracle fruit (Synsepalum dulcificum) was employed to investigate the effect on insulin resistance induced by fructose-rich chow in rats. Single oral administration of the powder of this miracle fruit decreased the plasma glucose in a dose-dependent manner for 150 min in rats fed fructose-rich chow for 4 weeks. Insulin action on the glucose disposal rate was measured using the glucose-insulin index, the value of the areas under the curve of glucose and insulin during the intraperitoneal glucose tolerance test. Oral administration of miracle fruit (0.2 mg/kg) to fructose-rich chow fed rats, three times daily for 3 days, reversed the raised value of the glucose-insulin index, indicating that miracle fruit has the ability to improve insulin sensitivity. The plasma glucose lowering action of tolbutamide, induced by secretion of endogenous insulin, is widely used to characterize the formation of insulin resistance. The time for the loss of the plasma glucose lowering response to tolbutamide (10.0 mg/kg, i.p.) in fructose-rich chow fed rats was markedly delayed after treatment with miracle fruit compared with the vehicle-treated group. Thus providing supportive data that oral administration of miracle fruit could delay the development of insulin resistance in rats. Also, the in vivo insulin sensitivity was markedly raised by miracle fruit. In conclusion, the results suggest that miracle fruit may be used as an adjuvant for treating diabetic patients with insulin resistance because this fruit has the ability to improve insulin sensitivity., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Streptozotocin-induced diabetic (STZ-diabetic) rats were employed to investigate the mechanism(s) whereby myricetin, the active principle of Abelmoschus moschatus (Malvaceae), exerts its glucose-lowering effects. Myricetin was purified from the aerial portion of the plant and administered intravenously. A dose-dependent decrease in plasma glucose concentration was observed 30 min following injection, in parallel with increased plasma beta-endorphin-like immunoreactivity (BER). Myricetin enhanced BER release similarly from isolated adrenal medulla. Plasma glucose-lowering and BER-elevating effects of myricetin were both eliminated after bilateral adrenalectomy. Myricetin failed to lower plasma glucose after treatment with opioid mu-receptor antagonists and in opioid mu-receptor knockout diabetic mice. Injection of myricetin three times daily for three consecutive days resulted in increased expression of the glucose transporter subtype 4 (GLUT 4) in soleus muscle and in reduced expression of phosphoenolpyruvate carboxykinase (PEPCK) in liver; these inductions were preventable by opioid mu-receptor blockade. Findings support the conclusion that the plasma glucose-lowering action of myricetin in insulin-deficient animals is mediated by activation of opioid mu-receptors of peripheral tissues in response to increased beta-endorphin secretion. Opioid mu-receptor activation is held responsible for the enhancement of muscle GLUT 4 gene expression and the attenuation of hepatic PEPCK gene expression observed in these myricetin-treated diabetic animals.

We investigated the plasma glucose-lowering mechanism(s) of Rh2, a ginsenoside derived from Panax ginseng, in rats with streptozotocin-induced diabetes (STZ-diabetic rats). After intravenous injection over 120 min into fasting STZ-diabetic rats, Rh2 decreased plasma glucose in a dose-dependent manner. In parallel to the lowering of plasma glucose, an increase of plasma beta-endorphin-like immunoreactivity was observed. In addition, naloxone and naloxonazine at doses sufficient to block opioid mu-receptors inhibited the plasma glucose-lowering action of Rh2 in genetically wild-type, diabetic mice. In contrast, Rh2 failed to lower plasma glucose in opioid mu-receptor knockout diabetic mice. An increase in gene expression at both the mRNA and protein levels of glucose transporter subtype 4 (GLUT 4) was observed in soleus muscle obtained from STZ-diabetic rats treated with Rh2 three times daily for one day; this increase in expression was absent when opioid mu-receptors were blocked. In conclusion, our results suggest that ginsenoside Rh2 may lower plasma glucose in STZ-diabetic rats based on an increase in beta-endorphin secretion that activates opioid mu-receptors thereby resulting in an increased expression of GLUT 4.

1. The aim of the present study was to clarify the role of ginsenoside Rh2 as the active compound in Panax ginseng root for lowering plasma glucose in animals. 2. Plasma glucose was assessed using the glucose oxidase method. Changes in the levels of insulin and C-peptide in plasma were measured by ELISA using commercially available kits. 3. After intravenous injection into fasting Wistar rats for 60 min, ginsenoside Rh2 (0.1-1.0 mg/kg) decreased plasma glucose in a dose-dependent manner. In parallel with the decrease in plasma glucose, increases in plasma insulin levels, as well as plasma C-peptide, were observed in rats receiving the same treatment. These effects of Rh2 were reversed by atropine (0.1-1.0 mg/kg), but not affected by the ganglionic nicotinic antagonists pentolinium or hexamethonium (both at 7.5 mg/kg). 4. Disruption of synaptically available acetylcholine (ACh) using an inhibitor of choline uptake (hemicholinium-3; 1-10 microg/kg) or an inhibitor of vesicular ACh transport (vesamicol; 1.5-3.5 mg/kg) abolished the actions of Rh2. In addition, physostigmine (0.1-0.5 mg/kg), at a concentration sufficient to inhibit acetylcholinesterase, enhanced the actions of the ginsenoside Rh2. Thus, mediation of the effects of Rh2 to enhance insulin secretion by ACh released from nerve terminals can be considered. 5. Blockade of the increase in plasma insulin and the plasma glucose-lowering action of Rh2 by 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP; 5-10 microg/kg) indicates the participation of muscarinic M(3) receptors. Increases in plasma C-peptide level induced by Rh2 were also sensitive to 4-DAMP. 6. The results of the present study suggest that ginsenoside Rh2 has the ability to increase insulin secretion as a result of the release of ACh from nerve terminals that then stimulates muscarinic M(3) receptors in pancreatic cells. This finding shows the mechanism for the plasma glucose-lowering action of ginsenoside Rh2, that is one of the major principles contained in P. ginseng root. Thus, ginsenoside Rh2 may be applied as an adjuvant for the management of diabetes.

1. In an attempt to develop new substances for handling insulin resistance, an aqueous extract of the root of Acanthopanax senticosus (Araliaceae) was used to screen the effect on insulin resistance induced by fructose-rich chow in rats. 2. Insulin action on glucose disposal rate was measured using the glucose-insulin index, the product from areas under the curve of glucose and insulin during the intraperitoneal glucose tolerance test. In addition to the modification of feeding behaviour and a marked decrease in bodyweight, oral administration (three times daily for 3 days) of the aqueous extract of A. senticosus root to rats that had received fructose-rich chow for 4 weeks reversed the elevated value of the glucose--insulin index, indicating that this herb has the ability to improve insulin sensitivity. 3. Time for the loss of the plasma glucose-lowering response to tolbutamide (10.0 mg/kg, i.p.) in fructose-rich chow-fed rats was markedly delayed by repeated treatment with the aqueous extract of A. senticosus root compared with the vehicle (saline) -treated group. Thus, an improving effect of A. senticosus root on insulin resistance can be considered. 4. An increase in insulin sensitivity following the administration of this herb was further identified using the plasma glucose-lowering action of exogenous insulin in streptozotocin (STZ)-diabetic rats. Oral administration of the aqueous extract of A. senticosus root at a dose of 150.0 mg/kg three times daily to STZ-diabetic rats increased the responses to exogenous insulin 10 days later. 5. The results obtained suggest that oral administration of the aqueous extract from A. senticosus root has the ability to improve insulin sensitivity and delay the development of insulin resistance in rats and, thus, may be used as an adjuvant therapy for patients with insulin resistance.

The role of beta-endorphin in the plasma glucose-lowering action of tetrandrine in streptozotocin-induced diabetic rats (STZ-diabetic rats) was investigated. The plasma glucose concentration was assessed by the glucose oxidase method. The enzyme-linked immunosorbent assay was used to determine the plasma level of beta-endorphin-like immunoreactivity (BER). The mRNA levels of glucose transporter subtype 4 (GLUT4) in soleus muscle and phosphoenolpyruvate carboxykinase (PEPCK) in the liver of STZ-diabetic rats were detected by Northern blotting analysis. The expressed protein of GLUT4 or PEPCK was characterized by Western blotting analysis. Tetrandrine dose-dependently increased plasma BER in a manner parallel to the decrease of plasma glucose in STZ-diabetic rats. Moreover, the plasma glucose-lowering effect of tetrandrine was inhibited by naloxone and naloxonazine at doses sufficient to block opioid μ-receptors. Further, tetrandrine failed to produce plasma glucose-lowering action in opioid μ-receptor knockout diabetic mice. Bilateral adrenalectomy eliminated the plasma glucose-lowering effect and plasma BER-elevating effect of tetrandrine in STZ-diabetic rats. Both effects were abolished by treatment with hexamethonium or pentolinium at doses sufficient to block nicotinic receptors. Tetrandrine enhanced BER release directly from the isolated adrenal medulla of STZ-diabetic rats and this action was abolished by the blockade of nicotinic receptors. Repeated intravenous administration of tetrandrine (1.0 mg/kg) to STZ-diabetic rats for 3 days resulted in an increase in the mRNA and protein levels of the GLUT4 in soleus muscle, in addition to the lowering of plasma glucose. Similar treatment with tetrandrine reversed the elevated mRNA and protein levels of PEPCK in the liver of STZ-diabetic rats. The obtained results suggest that tetrandrine may induce the activation of nicotinic receptors in adrenal medulla to enhance the secretion of beta-endorphin, which could stimulate opioid μ-receptors to increase glucose utilization or/and reduce hepatic gluconeogenesis to lower plasma glucose levels in STZ-diabetic rats.

1. The effects of endurance training on the anti-oxidant status in diabetes were studied using obese Zucker rats. 2. We used a moderate exercise programme consisting of treadmill running at 20 m/min and 0% incline for 1 h/day, 7 days/week, for 8 weeks. At the end of the experimental period, changes in hepatic anti-oxidant enzymes in terms of protein content and mRNA levels were detected using western blotting analysis and northern blotting analysis, respectively. In addition, anti-oxidant enzyme activity was determined. 2. A significant reduction in mRNA levels and the protein content of hepatic Mn-superoxide dismutase (SOD) and glutathione peroxidase (GPx) were observed in non-exercise obese groups, but the mRNA and protein levels of these enzymes were markedly increased after exercise training. In addition, exercise training reversed the decreased enzyme activities of Mn-SOD and GPx in obese Zucker rats. 3. The diabetes-related lowering of the glutathione (GSH) concentration was elevated in exercised obese Zucker rats, indicating a marked effect of regular moderate exercise on the endogenous anti-oxidant system. 4. There were no marked changes in hepatic Cu/Zn-SOD in terms of mRNA levels, protein content and activity in sedentary obese Zucker rats compared with their lean littermates. Endurance training did not modify the gene expression and activity of hepatic Cu/Zn-SOD. 5. The results of the present study suggest that regular moderate exercise could improve the anti-oxidant defence function of Mn-SOD, GPx and GSH in obese Zucker rats.

Genistein, an isoflavonoid natural product, is widely used to inhibit protein tyrosine kinase (PTK). In the present study, we investigated the possible influence of genistein on alpha (1)-adrenoceptors (AR) in cultured C2C12 cells. Genistein enhanced the uptake of radioactive glucose into C2C12 cells in a concentration-dependent manner. Similar results were also observed in samples treated with daidzein, the inactive congener for PTK inhibition. The effect of genistein on alpha (1)-AR was further characterized using the displacement of [ (3)H]prazosin binding in C2C12 cells. The increase in radioactive glucose uptake by genistein was abolished by RS17053 at a concentration sufficient to block alpha (1A)-AR. The pharmacological inhibition of phospholipase C (PLC) by U73122 resulted in a concentration-dependent reduction of genistein-stimulated glucose uptake in C2C12 cells. This inhibition by U73122 was specific because the inactive congener, U73343, failed to modify the action of genistein. Moreover, genistein can activate alpha (1A)-AR at a concentration (1 micromol/L) lower than that (50 micromol/L) needed to abolish the insulin-stimulated phosphorylation of PTK. The obtained data indicate an activation of alpha (1A)-AR by genistein to increase the glucose uptake into C2C12 cells and this supports the application of genistein as a TK inhibitor.

We investigate the mechanism(s) of plasma glucose lowering action of puerarin in streptozotocin-induced diabetic rats (STZ-diabetic rats). Puerarin at the effective dosage to lower higher plasma glucose increased plasma beta-endorphin-like immunoreactivity (BER) in STZ-diabetic rats. Both effects of puerarin were abolished by the pretreatment with prazosin. Also, puerarin enhanced BER release from isolated rat adrenal medulla in a concentration-dependent manner that can be abolished by prazosin. Moreover, bilateral adrenalectomy in STZ-diabetic rats eliminated the actions of puerarin including the plasma glucose lowering effect and plasma BER elevating effect. In addition, naloxone and naloxonazine inhibited the plasma glucose lowering action of puerarin. Unlike in wild-type diabetic mice, puerarin failed to lower the plasma glucose in opioid micro-receptor knockout diabetic mice. In conclusion, our results suggest that puerarin may activate alpha (1)-adrenoceptors on the adrenal gland to enhance the secretion of beta-endorphin to result in a decrease of plasma glucose in STZ-diabetic rats.

We investigated the mechanism(s) by which isoferulic acid lowers plasma glucose levels in streptozotocin-induced diabetic rats (STZ-diabetic rats). In STZ-diabetic rats, isoferulic acid dose dependently lowered plasma glucose concentrations and increased plasma beta-endorphin-like immunoreactivity (BER). Both of these effects of isoferulic acid were abolished by pretreatment of rats with tamsulosin or 2-[2,6-dimethoxyphenoxyethyl]aminomethyl-1,4-benzodioxane hydrochloride (WB 4101) at doses sufficient to block alpha1-adrenoceptors. Also, isoferulic acid enhanced BER release from isolated rat adrenal medulla in a concentration-dependent manner that could be abolished by treatment with alpha1-adrenoceptor antagonists. Moreover, bilateral adrenalectomy in STZ-diabetic rats eliminated the activities of isoferulic acid, including the plasma glucose-lowering effect and the plasma BER-elevating effect. Naloxone and naloxonazine inhibited the plasma glucose-lowering activity of isoferulic acid at doses sufficient to block opioid mu-receptors. In contrast with the effect in wild-type diabetic mice, isoferulic acid failed to lower plasma glucose levels in opioid mu-receptor knockout diabetic mice. Treatment of STZ-diabetic rats with isoferulic acid three times in 1 day resulted in an increase in the expression of the glucose transporter subtype 4 form in soleus muscle. This effect was blocked by alpha1-adrenoceptor or opioid mu-receptor antagonists. The reduction of elevated mRNA or protein level of hepatic phosphoenolpyruvate carboxykinase was also impeded in the same groups of STZ-diabetic rats. In conclusion, our results suggest that isoferulic acid may activate alpha1-adrenoceptors to enhance the secretion of beta-endorphin, which can stimulate the opioid mu-receptors to increase glucose use or/and reduce hepatic gluconeogenesis, resulting in a decrease of plasma glucose in STZ-diabetic rats.

1. Tetramethylpyrazine (TMP) is one of the active principles contained in Ligusticum chuanxiong Hort. (Umbelliferae), a herb that has been used widely in China to treat vascular disorders. 2. In an attempt to elucidate the possible mechanisms of action of TMP, the effect of TMP on intracellular calcium concentrations ([Ca2+]i) was investigated in cultured vascular smooth muscle (A7r5) cells using the Ca(2+)-sensitive dye Fura-2 as an indicator. 3. The increase in [Ca2+]i in A7r5 cells produced by vasopressin (1 micromol/L) or phenylephrine (1 micromol/L) was attenuated by TMP in a concentration-dependent manner. Only inhibitors specific to ATP-sensitive potassium (KATP) channels or small conductance calcium-activated potassium (SKCa) channels attenuated the action of TMP (10 micromol/L) on [Ca2+]i. However, blockers of other K+ channels failed to modify the inhibitory action of TMP (10 micromol/L) on [Ca2+]i. 4. The action of TMP on membrane potential in A7r5 cells was monitored by the fluorescence of bisoxonol. Tetramethylpyrazine caused a concentration-dependent inhibition of changes in membrane potential elicited by KCl (20 mmol/L) or phenylephrine (1 micro mol/L), an effect that was totally reversed by glibenclamide (100 micromol/L) and apamin (100 nmol/L) in combination. 5. The results obtained indicate that the decrease in [Ca2+]i in A7r5 cells produced by TMP is mediated mainly by opening of KATP and/or SKCa channels.

Diabetes-associated alterations in prostate alpha(1A)-adrenoceptor (alpha(1A)-AR) gene expression were studied using a streptozotocin (STZ) induced diabetic rat model. Male Wistar rats were divided into four groups: group I animals were vehicle-treated normal rats; group II consisted of vehicle-treated, STZ-diabetic rats; group III represented insulin-treated, STZ-diabetic rats (0.5 IU/kg three times daily for 4 days), and group IV animals were phlorizin-treated, STZ-diabetic rats (1 mg/kg three times daily for 4 days). The expression of mRNA that encoded protein of alpha(1A)-AR in the rat prostate was studied using reverse transcription combined with polymerase chain reaction. The alpha(1A)-AR protein expression in the prostate was studied by Western blotting analysis with a polyclonal antiserum. A 2.51 +/- 0.21-fold increase in the mRNA level of alpha(1A)-AR was observed in the prostate of diabetic rats (n = 8, p < 0.05). Similarly, there was a 2.23 +/- 0.10-fold increase in the alpha(1A)-AR protein level (n = 8, p < 0.05). Both insulin and phlorizin treatments restored the normal levels of mRNA and protein expression. In conclusion, the gene expression of alpha(1A)-AR is increased in the prostate of diabetic rats. Hyperglycemia plays a major role in this alteration., (Copyright 2003 S. Karger AG, Basel)

We have characterized the effects of cinnamic acid and its derivatives on alpha(1)-adrenoceptor subtypes. The cinnamic acid with a methoxyl group and/or a hydroxyl group showed the ability to stimulate radioactive glucose uptake into C(2)C(12) cells, a cell line that specifically expresses the alpha(1A)-adrenoceptor subtype of alpha(1)-adrenoceptors. However, cinnamic acid without chemical modification diminished the glucose uptake into C(2)C(12) cells. It was shown that methoxylation and/or hydroxylation of cinnamic acid had higher affinities for alpha(1A)-adrenoceptors investigated using [(3)H]prazosin binding experiments in C(2)C(12) cells. The effect of these derivatives on alpha(1A)-adrenoceptors was further characterized using the displacement of [(3)H]prazosin binding in rat prostate. We found that 3,5-dimethoxy-4- hydroxycinnamic acid, the cinnamic acid derivative with two methoxyl groups and hydroxylation at the fourth carbon on the benzene ring, had a higher affinity for the alpha(1A)-adrenoceptor subtype, showing a smaller IC50 value (the concentration for production of 50% inhibition) to displace [(3)H]prazosin binding in rat prostate. Affinity of these compounds for alpha(1B)-adrenoceptors was identified using [(3)H]prazosin-binding experiments in rat spleen. However, we found no marked differences in the IC50 values between these cinnamic acid analogues to displace the [(3)H]prazosin binding in rat spleen. In conclusion, our data indicated that methoxylation and/or hydroxylation of cinnamic acid might raise the affinity for alpha(1A)-adrenoceptors.

The antihyperglycemic action of puerarin, purified from the roots of Pueraria lobata, was investigated in streptozotocin-induced diabetic rats (STZ-diabetic rats). Bolus intravenous injection of puerarin decreased the plasma glucose concentrations in a dose-dependent manner in STZ-diabetic rats. Similar treatment with puerarin also decreased the plasma glucose in normal rats, although the effect was not as great as that in STZ-diabetic rats. Puerarin at the effective dose (15.0 mg/kg) significantly attenuated the increase of plasma glucose induced by an intravenous glucose challenge test in normal rats. In the isolated soleus muscle of STZ-diabetic rats, puerarin enhanced the uptake of radioactive glucose in a concentration-dependent manner. Moreover, the mRNA and protein levels of the subtype 4 form of glucose transporter (GLUT4) in soleus muscle were increased after repeated intravenous administration of puerarin in STZ-diabetic rats for 3 days. These results suggest that puerarin can increase the glucose utilization to lower plasma glucose in diabetic rats lacking insulin.

Role of opioid mu-receptor that has been showed to involve in the insulin-dependent diabetic rats in the induction of insulin resistance remains unclear. The present study is performed to clarify this point. The wild-type mice or opioid mu-receptor knockout mice were employed to induce insulin resistance by feeding with fructose-rich chow or by the repeated intraperitoneal injections of long-acting form of human insulin at 0.5 IU/kg three times daily. The basal plasma glucose concentration was not markedly changed in fructose-fed mice regardless the presence of opioid mu-receptor or not. However, the plasma glucose lowering activity of tolbutamide (10.0 mg/kg) disappeared rapidly in opioid mu-receptor knockout mice receiving fructose-rich chow as compared to that in wild type group. In opioid mu-receptor knockout mice, the elevation of plasma glucose concentration and the loss of plasma glucose lowering activity of tolbutamide were observed at 15 days after insulin injection. However, similar change was obtained at 21 days later of insulin injection in wild-type mice, showing that decrease of insulin action was more markedly in opioid mu-receptor knockout mice. Our results indicated that opioid mu-receptor is related to the delay of insulin resistance induced by fructose-fed method or insulin repeated injection.

Aim: Effects on insulin sensitivity of die-huang-wan, the herbal mixture widely used to treat diabetic disorder in Chinese traditional medicine, were investigated in vivo., Methods: The obese Zucker rats were employed as insulin-resistant animal model. Also, insulin-resistance was induced by the repeated intraperitoneal injections of long-acting human insulin at 0.5 U/kg three times daily into adult male Wistar rats. Insulin resistance was identified using the loss of tolbutamide (10 mg/kg) or electroacupuncture (EA)-induced plasma glucose lowering action. The plasma glucose concentration was examined by glucose oxidase assay., Results: The plasma glucose-lowering action induced by tolbutamide was significantly enhanced in obese Zucker rats receiving the repeated administration of die-huang-wan at dosage of 26 mg/kg for 3 d. Furthermore, administration of die-huang-wan delayed the formation of insulin resistance in rats that were induced by the daily repeated injection of human long-acting insulin at 0.5 U/kg three times daily and identified by the loss of tolbutamide- or EA-induced hypoglycemia. In streptozotocin-induced diabetic rats, oral administration of metformin at 320 mg/kg once daily made an increase of the response to exogenous short-acting human insulin 15 d later. This is consistent with the view that metformin can increase insulin sensitivity. Similar treatment with die-huang-wan at an effective dose (26.0 mg/kg) also increased the plasma glucose lowering action of exogenous insulin at 10 d later. The effect of die-huang-wan on insulin sensitivity seems to produce more rapidly than that of metformin., Conclusion: The present study found that oral administration of die-huang-wan increased insulin sensitivity and delayed the development of insulin resistance in rats.

Effects of puerarin, an active principle contained in the roots of Pueraria lobata (Leguminosae), on the regulation of glucose metabolism in an insulin deficient state were investigated in cultured myoblast C 2 C 12 cells using glucose uptake as indicator. Puerarin enhanced the uptake of radioactive glucose into C 2 C 12 cells in a concentration-dependent manner, which was abolished by prazosin pretreatment. Activation of alpha 1 -adrenoceptors by puerarin was further indicated by the displacement of [ 3H]prazosin binding in C 2 C 12 cells. The stimulatory action of puerarin on glucose uptake was also reduced in C 2 C 12 cells pre-incubated with the antagonists, both WB 4101 and RS 17 056, at concentrations sufficient to block alpha 1A -adrenoceptor (alpha 1A -AR). An activation of alpha 1A -AR seems responsible for the action of puerarin in C 2 C 12 cells. Pharmacological inhibition of phospholipase C (PLC) by U73312 resulted a concentration-dependent decrease of puerarin-stimulated glucose uptake in C 2 C 12 cells. This inhibition of glucose uptake by U73122 was specific because the inactive congener, U73343, failed to block puerarin-stimulated glucose uptake. Moreover, both chelerythrine and GF 109203X diminished the action of puerarin at concentration sufficient to inhibit protein kinase C (PKC). The obtained data suggest that an activation of alpha 1A -AR by puerarin in C 2 C 12 cells may increase the glucose uptake via the PLC-PKC pathway.

We have recently observed that Die-Huang-Wan has an ability to stimulate the secretion of insulin to decrease the plasma glucose levels in normal rats. In the present study, this effect of Die-Huang-Wan was reversed by the general muscarinic antagonists atropine and scopolamine, but not affected by the ganglionic nicotinic antagonist pentolinium or hexamethonium. Moreover, disruption of synaptically available acetylcholine using an inhibitor of choline uptake, hemicholinium-3, or vesicular acetylcholine transport, vesamicol, abolished the actions induced by Die-Huang-Wan. Mediation of acetylcholine released from nerve terminals by this product can thus be considered. Also, physostigmine at concentration sufficient to inhibit acetylcholinesterase enhanced the effect of Die-Huang-Wan. Blockade of the increase of plasma insulin and plasma glucose lowering action of Die-Huang-Wan by 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP) indicated the mediation of muscarinic M3 receptors. The results suggest that Die-Huang-Wan may enhance the release of acetylcholine from nerve terminals to stimulate the muscarinic M3 receptors for augmenting insulin release to produce plasma glucose lowering action.

Opioid plays an important role in the regulation of glucose homeostasis in diabetic rats lacking insulin. The present study investigated the changes of beta-endorphin biosynthesis in the adrenal medulla of streptozotocin-induced diabetic rats (STZ-diabetic rats) by determination of the gene expression of pro-opiomelanocortin (POMC) and the amount of beta-endorphin. Expression of the distinct mRNA that encodes proteins of POMC was studied using reverse transcription combined with polymerase chain reaction (RT-PCR). Results of RT-PCR demonstrated that the mRNA level of POMC in the adrenal gland markedly increased in STZ-diabetic rats as compared with that in normal rats. The content of beta-endorphin-like immunoreactivity (BER) in the adrenal medulla, determined by enzyme-linked immunosorbent assay, was actually higher in diabetic rats with insulin deficiency. Normalization of the plasma glucose concentration in STZ-diabetic rats with exogenous insulin or phlorizin, an inhibitor of the renal tubular glucose transport, reversed the mRNA level of POMC in the adrenal gland after 4 days of treatment. A similar decrease of BER amount also observed in the adrenal medulla of STZ-diabetic rats received the same treatment with exogenous insulin or phlorizin. Therefore, correction of hyperglycemia in STZ-diabetic rats could reverse the higher gene expression of POMC in the adrenal gland. These results suggest that hyperglycemia is responsible for the increase of POMC gene expression to enhance beta-endorphin biosynthesis in the adrenal gland of STZ-diabetic rats.