Cinnamon extract improves abnormalities in glucose tolerance by decreasing Acyl-CoA synthetase long-chain family 1 expression in adipocytes.

We previously demonstrated that cinnamon extract (CE) alleviates streptozotocin-induced type 1 diabetes in rats. The present study aimed to elucidate the detailed molecular target of cinnamon in cultured adipocytes and epididymal adipose tissue of type 2 diabetes model mice. Two-dimensional gel electrophoresis was employed to determine the molecular target of cinnamon in adipocytes. The function of Acyl-CoA synthetase long-chain family-1 (ACSL1), a molecular target of cinnamon that was identified in this study, was further investigated in 3T3-L1 adipocytes using specific inhibitors. Type 2 diabetes model mice (KK-Ay/TaJcl) were used to investigate the effect of CE on glucose tolerance, ACSL1 expression, and related signal molecules in vivo. CE decreased ACSL1 mRNA and protein expression in 3T3-L1 adipocytes but increased glucose uptake and AMPK signaling activation; moreover, a similar effect was observed with an ACSL1 inhibitor. CE improved glucose tolerance and downregulated ACSL1 in mice adipose tissue in vivo. ACSL1 was demonstrated as a molecular target of CE in type 2 diabetes both in a cell culture system and diabetic mouse model.

Identification of molecular target of diallyl trisulfide in leukemic cells.

To identify the molecular target of diallyl trisulfide (DATS) in human leukemic cell line U937, we examined modification of thiol group(s) of cellular proteins by the redox 2D PAGE. A unique protein spot appeared by DATS treatment was identified to be heat shock protein 27 (HSP27). Hsp27 is suggested to be one of the molecular target of DATS in U937.

Diallyl trisulfide induces apoptosis in Jurkat cells by the modification of cysteine residues in thioredoxin.

We reported the regulation of protein function by oxidative modification of the specific cysteine residue(s) by diallyl trisulfide (DATS). In this study, we examined if DATS modifies the cysteine residue of thioredoxin (Trx) by urea-polyacryl amide gel electrophoresis. DATS modified two specific cysteine residues in Trx and this oxidative modification of cysteine residues would be sole causative of the apoptosis induced by DATS in leukemic cells.

Sulphur-containing compounds of durian activate the thermogenesis-inducing receptors TRPA1 and TRPV1.

Durian (Durio zibethinus Murr.) is classified as a body-warming food in Indian herbalism, and its hyperthermic effect is empirically known in Southeast Asia. To investigate the mechanism underlying this effect, we focused on the thermogenesis-inducing receptors, TRPA1 and TRPV1. Durian contains sulphides similar to the TRPA1 and TRPV1 agonists of garlic. Accordingly, we hypothesized that the thermogenic effect of durian is driven by sulphide-induced TRP channel activation. To investigate our hypothesis, we measured the TRPA1 and TRPV1 activity of the sulphur-containing components of durian and quantified their content in durian pulp. These sulphur-containing components had a stronger effect on TRPA1 than TRPV1. Furthermore, sulphide content in the durian pulp was sufficient to evoke TRP channel activation and the main agonist was diethyl disulphide. From these results, we consider that the body-warming effect of durian is elicited by TRPA1 activation with its sulphides, as can be seen in spices.

Cinnamon extract enhances glucose uptake in 3T3-L1 adipocytes and C2C12 myocytes by inducing LKB1-AMP-activated protein kinase signaling.

We previously demonstrated that cinnamon extract (CE) ameliorates type 1 diabetes induced by streptozotocin in rats through the up-regulation of glucose transporter 4 (GLUT4) translocation in both muscle and adipose tissues. This present study was aimed at clarifying the detailed mechanism(s) with which CE increases the glucose uptake in vivo and in cell culture systems using 3T3-L1 adipocytes and C2C12 myotubes in vitro. Specific inhibitors of key enzymes in insulin signaling and AMP-activated protein kinase (AMPK) signaling pathways, as well as small interference RNA, were used to examine the role of these kinases in the CE-induced glucose uptake. The results showed that CE stimulated the phosphorylation of AMPK and acetyl-CoA carboxylase. An AMPK inhibitor and LKB1 siRNA blocked the CE-induced glucose uptake. We also found for the first time that insulin suppressed AMPK activation in the adipocyte. To investigate the effect of CE on type 2 diabetes in vivo, we further performed oral glucose tolerance tests and insulin tolerance tests in type 2 diabetes model rats administered with CE. The CE improved glucose tolerance in oral glucose tolerance tests, but not insulin sensitivity in insulin tolerance test. In summary, these results indicate that CE ameliorates type 2 diabetes by inducing GLUT4 translocation via the AMPK signaling pathway. We also found insulin antagonistically regulates the activation of AMPK.

Role of p16(INK4a) in the inhibition of DNA synthesis stimulated by HGF or EGF in primary cultured rat hepatocytes.

In the present study, we investigated the role of p16(INK4a) in the inhibition of DNA synthesis stimulated by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) using RNA interference in primary cultured rat hepatocytes. The transfection of small interfering RNAs targeting p16(INK4a) reduced the corresponding mRNA and protein expression by more than approximately 90% and 50%, respectively, at 24 h after transfection. In the cells transfected with p16(INK4a) small interfering RNA, control, HGF, and EGF-stimulated DNA synthesis as assessed by (3)H-thymidine incorporation increased by approximately 1.5-fold, 1.6-fold, and 1.7-fold, respectively, compared with that in the control small interfering RNA-transfected cells. These findings indicate that p16(INK4a) plays a significant role in the inhibition of DNA synthesis.

Advanced glycation endproducts stimulate renal epithelial cells to release chemokines that recruit macrophages, leading to renal fibrosis.

Diabetic nephropathy is a major complication of diabetes and tubulointerstitial fibrosis is one of its manifestations. This study aimed to clarify the pathogenicity of advanced glycation endproducts (AGEs) toward NRK-52E, a tubular epithelial cell line. The AGE-exposed cells significantly increased gene expression of transforming growth factor beta, plasminogen activator inhibitor-1, and tissue transglutaminase, and a medium conditioned by them showed strong potential to recruit macrophages, partly through a chemokine, monocyte chemoattractant protein-1. Albumin denatured by maintenance at 37 °C for 120 d exhibited similar activities, but they were lower than those of the AGEs. Thus, AGEs generated in diabetic patients might exacerbate fibrosis in the kidneys directly through renal epithelial cell stimulation, and indirectly by recruitment of macrophages.

Annexin A3 as a negative regulator of adipocyte differentiation.

Annexin A3 is a protein belonging to the annexin family, and it is mainly present in cellular membranes as a phospholipid-binding protein that binds via the calcium ion. However, its physiological function remains to be clarified. We examined the expression of annexin A3 in mouse tissues and found for the first time that annexin A3 mRNA and its protein were expressed more strongly in adipose tissues than in other tissues. In adipose tissues, annexin A3-expressing cells were present in the stromal vascular fraction, and precisely identical to Pref-1-positive preadipocytes, Pref-1 being an epidermal growth factor repeat-containing transmembrane protein that inhibits adipogenesis. In 3T3-L1 cells, used as a model of adipogenesis, annexin A3 was down-regulated at an early phase of adipocyte differentiation, and this pattern paralleled that of Pref-1. Suppression of annexin A3 in these cells with siRNA caused elevation of the PPARγ2 mRNA level and lipid droplet accumulation. In conclusion, our data suggest that annexin A3 is a negative regulator of adipocyte differentiation.

Beneficial effects of cinnamon on the metabolic syndrome, inflammation, and pain, and mechanisms underlying these effects - a review.

Cinnamon is one of the most important herbal drugs and has been widely used in Asia for more than 4000 years. As a folk medicine, cinnamon has been traditionally applied to the treatment of inflammatory disorders and gastric diseases. After chemical profiling of cinnamon's components, their biological activities including antimicrobial, antiviral, antioxidant, antitumor, antihypertension, antilipemic, antidiabetes, gastroprotective and immunomodulatory were reported by many investigators. As a result, current studies have been performed mostly focusing on the bioactivity of cinnamon toward the recently generalized metabolic syndrome involving diabetes. In this review article, we provide an overview of the recent literature describing cinnamon's potential for preventing the metabolic syndrome.

Verification of the antidiabetic effects of cinnamon (Cinnamomum zeylanicum) using insulin-uncontrolled type 1 diabetic rats and cultured adipocytes.

It has long been believed that an intake of cinnamon (Cinnamomum zeylanicum) alleviates diabetic pathological conditions. However, it is still controversial whether the beneficial effect is insulin-dependent or insulin-mimetic. This study was aimed at determining the insulin-independent effect of cinnamon. Streptozotocin-induced diabetic rats were divided into four groups and orally administered with an aqueous cinnamon extract (CE) for 22 d. The diabetic rats that had taken CE at a dose of more than 30 mg/kg/d were rescued from their hyperglycemia and nephropathy, and these rats were found to have upregulation of uncoupling protein-1 (UCP-1) and glucose transporter 4 (GLUT4) in their brown adipose tissues as well as in their muscles. This was verified by using 3T3-L1 adipocytes in which CE upregulates GLUT4 translocation and increases the glucose uptake. CE exhibited its anti-diabetic effect independently from insulin by at least two mechanisms: i) upregulation of mitochondrial UCP-1, and ii) enhanced translocation of GLUT4 in the muscle and adipose tissues.

Relationship between lipophilicity and inhibitory activity against cancer cell growth of nine kinds of alk(en)yl trisulfides with different side chains.

Of the compounds contained in or derived from garlic (Allium sativum L.), alk(en)yl sulfides are known to be responsible for most of the physiological or neutraceutical functions of garlic. We previously found that diallyl trisulfide (DATS) is a potent inhibitor of tubulin polymerization and cancer cell growth, and an effective stimulator of the hepatic detoxification system. Here, we synthesized nine trisulfides having different aliphatic side chains, and determined their log P, a parameter for lipophilicity of nonionized solutes, and inhibitory activities, IC50 (microM), toward cancer cell growth. The log P values of these trisulfides ranged from the lowest, 2.72, for dimethyl trisulfide, to the highest, 7.62, for dipentyl trisulfide. The relationship between the IC50 and log P of the nine trisulfides was parabolic in nature, in which dibutenyl- and dipropyl-compounds, determined to have a log P of approximately 5, were located at the minimum point of the parabola, indicating the maximum potency. The reason why DATS, having a log P of about 4, was excessively stronger than diethyl trisulfide, with a similar log P, is not fully understood; but perhaps it can be explained by a higher reactivity of the diallyl compound in nucleophilic substitution. The compounds with 3-carbon chains were stronger in terms of growth inhibition than the others; but weaker compounds, those with 4- or 5-carbon chains, showed higher activity if a double bond was introduced into them to reduce their log P to the effective range. In this study, we confirmed the superiority of trisulfides with 3-carbon chains [i.e., DATS and dipropyl trisulfide (DPTS)]. In addition, we observed for the first time that dibutenyl trisulfide, a compound not found in garlic, is one of the potent structures among alk(en)yl trisulfides.

Replacing white rice with pre-germinated brown rice mildly ameliorates hyperglycemia and imbalance of adipocytokine levels in type 2 diabetes model rats.

Pre-germinated brown rice (PR) has been developed industrially in order to enhance the nutritional functions of its source material, brown rice (BR). The present study was aimed at clarifying the effect of PR on the type 2 diabetes mellitus. We employed Otsuka Long-Evans Tokushima Fatty (OLETF) rats as a model of type-2 diabetes mellitus. OLETF rats were fed on either PR or white rice (WR) from the age of 4 to 35 wk. Age-matched male Long-Evans Tokushima Otsuka (LETO) rats as a non-diabetic control were also fed on WR. The HbA(1c) level in OLETF rats was significantly higher than that in LETO rats. However, the level was lower in PR-fed OLETF rats than in WR-fed OLETF rats. The plasma concentrations of TNF-α and PAI-1 in OLETF rats were higher than those in LETO rats. However, both elevated levels were decreased by the PR-feeding, but not by the WR-feeding. On the other hand, the plasma adiponectin concentration in OLETF rats was lower than that in LETO rats. The decrease in adiponectin level of OLETF rats was ameliorated by PR-feeding. The size of adipocytes in PR-fed OLETF rats was smaller than that in WR-fed OLETF rats. In summary, intake of PR instead of WR ameliorates both insulin resistance and imbalance of the levels of plasma adipocytokines leading to diabetic complications.

Diallyl trisulfide protects rats from carbon tetrachloride-induced liver injury.

Alk(en)yl sulfides have been found to be responsible for the anticancer, antithrombotic, and antioxidant effects of garlic. We sought to identify the most potent structure of sulfides that exhibits a hepatoprotective effect against carbon tetrachloride (CCl(4))-induced acute liver injury in rats. Rats were pretreated with diallyl trisulfide (DATS) i.g. at a dose of 500 micromol/kg body weight for 5 d. On d 6, CCl(4) was administered i.g. at a dose of 2.5 mL/kg body weight. Twenty-four hours after CCl(4) administration, rats were killed and plasma and liver samples collected. DATS pretreatment significantly suppressed the CCl(4)-induced elevation of plasma aspartate aminotransferase and alanine aminotransferase activities (P < 0.05). Histological observations supported the hepatoprotective effects. Western blot and spectrophotometric analyses indicated that DATS suppressed cytochrome P450 2E1 activity and its protein level and elevated those of glutathione S-transferase. Dipropyl trisulfide (DPTS), which is a saturated alkyl chain analogue of DATS, did not affect CCl(4)-induced liver toxicity or drug-metabolizing enzymes. These results suggest that hepatoprotective activity of trisulfides is due to their regulation of drug-metabolizing enzymes. Furthermore, the effects of 6 kinds of alk(en)yl trisulfides, including DATS and DPTS, on phase II enzyme activity were examined in rats. Alk(en)yl trisulfides were administered i.g. (500 micromol/kg body weight) to rats for 5 d. Only the allyl group-containing DATS and allyl methyl trisulfide enhanced these activities.

Antibacterial potential of garlic-derived allicin and its cancellation by sulfhydryl compounds.

Allicin (allyl 2-propenethiosulfinate), an antibacterial principle of garlic, has drawn much attention, since it has potent antimicrobial activity against a range of microorganisms, including methicillin-resistant Staphylococcus aureus. There have been many reports on the antibacterial properties of allicin, but no quantitative comparison of antibacterial activities between freshly prepared garlic extract and clinically useful antibiotics has been performed. To verify the substantial antibacterial effect of aqueous garlic extract, we compared it with those of allicin and several clinically useful antibiotics using two representative bacteria commonly found in the human environment, Gram-positive S. aureus and Gram-negative Escherichia coli. The garlic extract had more potent anti-staphylococcal activity than an equal amount of allicin. In terms of antibiotic potency against Gram-positive and Gram-negative bacteria, authentic allicin had roughly 1-2% of the potency of streptomycin (vs. S. aureus), 8% of that of vancomycin (vs. S. aureus), and only 0.2% of that of colistin (vs. E. coli). The antibacterial activity of allicin was completely abolished by cysteine, glutathione and coenzyme A, but not by non-SH-compounds. The oxygen in the structure (-S(=O)-S-) of allicin therefore functions to liberate the S-allyl moiety, which might be an offensive tool against bacteria.

A novel function of thrombin-activatable fibrinolysis inhibitor during rat liver regeneration and in growth-promoted hepatocytes in primary culture.

Thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits anti-fibrinolytic activity by removing C-terminal lysine residues from fibrin or plasminogen receptor proteins on the cellular surface, and plays an important role in the regulation of fibrinolysis. In this study, we examined the regulation of TAFI in hepatocytes during liver regeneration, and revealed its pivotal role in hepatocyte proliferation. In rat models, partial hepatectomy or carbon tetrachloride (CCl4)-induced acute liver injury suppressed the levels of plasma TAFI activity and hepatic TAFI mRNA, whereas this operation markedly increased both the hepatic plasmin activity and the level of proliferating cell nuclear antigen. In primary cultures of rat hepatocytes, the TAFI mRNA level was decreased under growth-promoting culture conditions. Treatment of the hepatocytes with TAFI siRNA increased the amount of plasmin on the hepatocytes and promoted hepatocyte proliferation. We concluded that TAFI regulates plasmin activity through its enzymatic activity whereby it reduces the plasminogen-binding capacity of the hepatocytes. The TAFI gene expression is down-regulated in hepatocyte proliferation for producing a fibrinolytic microenvironment suitable for cell growth. This is the first report on the role of TAFI in the pericellular fibrinolysis necessary for cellular proliferation.

Diallyl sulfides in garlic activate both TRPA1 and TRPV1.

We searched for novel agonists of TRP receptors especially for TRPA1 and TRPV1 in foods. We focused attention on garlic compounds, diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS). In TRPA1 or TRPV1 heterogeneously expressed CHO cells, all of those compounds increased [Ca(2+)](i) in concentration-dependent manner. The EC(50) values of DADS and DATS were similar to that of allyl isothiocyanate (AITC) and that of DAS was 170-fold larger than that of AITC. Maximum responses of these sulfides were equal to that of AITC. The EC(50) values of these compounds for TRPV1 were around 100 microM against that of capsaicin (CAP), 25.6 nM and maximum responses of garlic compounds were half to that of CAP. The Ca(2+) responses were significantly suppressed by co-application of antagonist. We conclude that DAS, DADS, and DATS are agonist of both TRPA1 and TRPV1 but with high affinity for TRPA1.

Thermostability of allicin determined by chemical and biological assays.

The garlic-derived antibacterial principle, alk(en)yl sulfinate compounds, has long been considered as very short-lived substance. However, there are some data showing a rather more stable nature of allicin. We determined here the thermostability of allicin by a systematic analyses employing chemical quantification and an antibacterial activity assay. Allicin in an aqueous extract of garlic was degraded stoichiometrically in proportion to the temperature; we estimated the half-life of allicin to be about a year at 4 degrees C (from 1.8 mg/ml to 0.9 mg/ml) and 32 d at 15 degrees C, but only 1 d at 37 degrees C (from 2.0 mg/ml to 1.0 mg/ml). The half-life values for antibacterial activity showed a similar trend in results: 63 d or more at 4 degrees C for both antibacterial activities, 14 d for anti-staphylococcal activity, and 26 d for anti-escherichia activity at 15 degrees C, but only 1.2 d and 1.9 d for the respective activities at 37 degrees C. Such antibacterial activities were attributable to the major allicin, allyl 2-propenylthiosulfinate. Surprisingly, the decline in the quantity of allicin was not accompanied by its degradation; instead, allicin became a larger molecule, ajoene, which was 3-times larger than allicin.

Binding of plasminogen to hepatocytes isolated from injured mouse liver and nonparenchymal-cell-dependent proliferation of hepatocytes.

Plasmin is an essential enzyme located in the pericellular microenvironment of liver cells during liver regeneration. Previously, we reported that liver regeneration ability was significantly increased in alpha2-antiplasmin gene knockout mice as compared to wild-type mice, but it was significantly decreased in plasminogen knockout mice, or Plg/alpha2-antiplasmin gene knockout mice. The present study aimed to demonstrate direct interaction between plasminogen and mouse hepatocytes in the process of liver regeneration. Using the isolated hepatocytes from mice we analyzed following subjects: binding capacity of plasminogen to hepatocytes, plasminogen activation in the presence of hepatocytes, and proliferation ability of hepatocytes cocultured with liver nonparenchymal cells. The isolated hepatocytes from plasminogen wild-type mice bound to immobilized plasminogen. The mouse hepatocytes enhanced plasminogen activation, and impaired the inhibitory effect of alpha2-antiplasmin. The proliferation ability of hepatocytes after liver injury was studied. In plasminogen wild-type and plasminogen knockout mice, the hepatocytes cocultured with nonparenchymal cells, which were obtained from mice without CCl4 injection, showed similar proliferation abilities. On the contrary, the proliferation ability of hepatocytes cocultured with nonparenchymal cells, which were obtained from CCl4-treated plasminogen knockout mice, was significantly impaired as compared to wild-type mice. These results indicate that the plasminogen-plasmin system on the surface of mouse hepatocytes plays an important role in liver regeneration.

Alkenyl group is responsible for the disruption of microtubule network formation in human colon cancer cell line HT-29 cells.

Alk(en)yl trisulfides (R-SSS-R') are organosulfur compounds produced by crushed garlic and other Allium vegetables. We found that these compounds exhibit potent anticancer effects through the reaction with microtubules, causing cell cycle arrest. Nine alk(en)yl trisulfides including dimethyl trisulfide, diethyl trisulfide, dipropyl trisulfide (DPTS), dibutyl trisulfide, dipentyl trisulfide, diallyl trisulfide (DATS), dibutenyl trisulfide, dipentenyl trisulfide and allyl methyl trisulfide were synthesized and added to cultures of HT-29 human colon cancer cells at a concentration of 10 muM. The trisulfides with alkenyl groups such as DATS, but not those with alkyl groups, induced rapid microtubule disassembly at 30-60 min as well as cell cycle arrest during the mitotic phase approximately at 4 h after the treatment. Both DATS-induced microtubule disassembly and the cell cycle arrest were cancelled by the simultaneous treatment of the cancer cells with 2 mM L-cysteine, glutathione (GSH) or N-acetyl-L-cysteine. Reciprocally, L-buthionine-(S,R)-sulfoximine (500 muM), an inhibitor of GSH synthesis, enhanced the power of DATS in inducing the cell cycle arrest. These results indicate that alk(en)yl trisulfide react with sulfhydryl groups in cysteine residues of cellular proteins such as microtubule proteins. Thus, the present study provides evidence that trisulfides with alkenyl groups have potent anticancer activities, at least in part, directed toward microtubules. These findings suggest that alkenyl trisulfides and their structurally related compounds may provide novel and effective anticancer agents.

Anticancer effects of diallyl trisulfide derived from garlic.

Alk(en)yl sulfides are characteristic flavor components of garlic. Several lines of epidemiological study indicate that the risk of a certain cancer can be prevented by consumption of garlic. In this manuscript, we examined the anticancer property of garlic-derived alk(en)yl sulfides, and the molecular basis especially for diallyl trisulfide which is a major constituent of the garlic oil. Alk(en)yl sulfides with different numbers of sulfur atom (i.e., mono-, di-, and trisulfide) were synthesized and purified (>99%). The anticancer activity of the alk(en)yl sulfides was primarily examined using human colon cancer cells HCT-15 and DLD-1. The growth of the cells was significantly suppressed by diallyl trisulfide, but neither diallyl monosulfide nor diallyl disulfide showed such an effect. The number of cells arrested at G2/M phase, the cells with a sub-G1 DNA content, and the cells with caspase-3 activity were dramatically increased by diallyl trisulfide treatment. Diallyl trisulfide disrupted microtubule network formation of the cells, and microtubule fragments could be seen at the interphase. There was a specific oxidative modification of cysteine residues Cys12 beta and Cys354 beta, forming S-allylmercaptocysteines in the tubulin molecule. These results suggest that diallyl trisulfide is responsible, at least in part, for the epidemiologically proven anticancer effect for garlic eaters.