Erratum: Lee et al. Ginseng Extracts, GS-KG9 and GS-E3D, Prevent Blood-Brain Barrier Disruption and Thereby Inhibit Apoptotic Cell Death of Hippocampal Neurons in Streptozotocin-Induced Diabetic Rats. Nutrients 2020, 12, 2383.

The authors have requested that the following changes be made to their paper [...].

The beneficial effect of glycerophosphocholine to local fat accumulation: a comparative study with phosphatidylcholine and aminophylline.

Injection lipolysis or mesotherapy gained popularity for local fat dissolve as an alternative to surgical liposuction. Phosphatidylcholine (PPC) and aminophylline (AMPL) are commonly used compounds for mesotherapy, but their efficacy and safety as lipolytic agents have been controversial. Glycerophosphocholine (GPC) is a choline precursor structurally similar to PPC, and thus introduced in aesthetics as an alternative for PPC. This study aimed to evaluate the effects of GPC on adipocytes differentiation and lipolysis and compared those effects with PPC and AMPL using in vitro and in vivo models. Adipogenesis in 3T3-L1 was measured by Oil Red O staining. Lipolysis was assessed by measuring the amount of glycerol released in the culture media. To evaluate the lipolytic activity of GPC on a physiological condition, GPC was subcutaneously injected to one side of inguinal fat pads for 3 days. Lipolytic activity of GPC was assessed by hematoxylin and eosin staining in adipose tissue. GPC significantly suppressed adipocyte differentiation of 3T3-L1 in a concentration-dependent manner (22.3% inhibition at 4 mM of GPC compared to control). Moreover, when lipolysis was assessed by glycerol release in 3T3-L1 adipocytes, 6 mM of GPC stimulated glycerol release by two-fold over control. Subcutaneous injection of GPC into the inguinal fat pad of mice significantly reduced the mass of fat pad and the size of adipocytes of injected site, and these effects of GPC were more prominent over PPC and AMPL. Taken together, these results suggest that GPC is the potential therapeutic agent as a local fat reducer.

Pectin lyase-modified red ginseng extract improves glucose homeostasis in high fat diet-fed mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Red ginseng has long been used as a traditional folk medicine for various diseases including diabetes. Recently, a preparation of red ginseng extract by pectin lyase modification has been developed and named as GS-E3D. AIM OF THE STUDY: The aim of this study is to evaluate the preventive effect of GS-E3D on hyperglycemia induced by feeding a high fat diet (HFD) in mice. MATERIALS AND METHODS: GS-E3D was orally administered to C57BL/6J mice at different doses (250, 500, or 1000 mg/kg/day) for 6 weeks while on a HFD. Body weight and blood glucose were monitored weekly, and oral glucose tolerance test (OGTT) was performed at 5th week of the experiment. Glycemic indications and metabolic parameters were further measured in serum. RESULTS: Six weeks of GS-E3D treatment to mice significantly inhibited HFD-induced body weight gain, hyperglycemia, hyperinsulinemia and hypertriglyceridemia. Notably, GS-E3D treated mice at doses of 250, 500 and 1000 mg/kg showed 41.8%, 45.0% and 55.1% reduction in insulin resistance index, respectively, compared to HFD control mice. OGTT revealed that GS-E3D markedly prevented steep rise of blood glucose and insulin levels after glucose challenge and ameliorated HFD-induced glucose and insulin intolerance. The histological analysis showed enlarged adipocytes in HFD-fed mice whereas the adipocyte hypertrophy was prevented in GS-E3D treated mice in a dose-dependent manner. Furthermore, when peripheral glucose uptake level was assessed by total and membranous glucose transporter type 4 (GLUT4) protein contents, GS-E3D restored GLUT4 protein expression to the levels of regular diet fed mice, and dose-dependently translocated them to the plasma membrane. CONCLUSION: The results collectively show that GS-E3D ameliorates obesity-related impaired glucose tolerance by improving insulin sensitivity in the epidydimal adipose tissue.

GS-KG9 ameliorates diabetic neuropathic pain induced by streptozotocin in rats.

BACKGROUND: Diabetic neuropathy is one of the most devastating ailments of the peripheral nervous system. Neuropathic pain develops in ∼30% of diabetics. Here, we examined the suppressive effect of GS-KG9 on neuropathic pain induced by streptozotocin (STZ). METHODS: Hyperglycemia was induced by intraperitoneal injection of STZ. Rats showing blood glucose level > 250 mg/dL were divided into five groups, and treatment groups received oral saline containing GS-KG9 (50 mg/kg, 150 mg/kg, or 300 mg/kg) twice daily for 4 wk. The effects of GS-KG9 on pain behavior, microglia activation in the lumbar spinal cord and ventral posterolateral (VPL) nucleus of the thalamus, and c-Fos expression in the dorsal horn of the lumbar spinal cord were examined. RESULTS: The development of neuropathic pain began at Day 5 and peaked at Week 4 after STZ injection. Mechanical and thermal pains were both significantly attenuated in GS-KG9-treated groups from 10 d after STZ injection as compared to those in the STZ control. GS-KG9 also repressed microglia activation in L4 dorsal horn and VPL region of the thalamus. In addition, increase in c-Fos-positive cells within L4 dorsal horn lamina I and II of the STZ control group was markedly alleviated by GS-KG9. CONCLUSION: These results suggest that GS-KG9 effectively relieves STZ-induced neuropathic pain by inhibiting microglial activation in the spinal cord dorsal horn and VPL region of the thalamus.

Targets of monoclonal antibodies for immunological diseases.

Immunological disorders such as allergy, autoimmune diseases, auto-inflammatory syndromes and immunological deficiency syndromes are difficult to treat with chemical drugs. Recently, many monoclonal antibodies targeting various molecules including interleukin, tumor necrosis factor-α, cluster of differentiation, integrins, complement C5 and B lymphocyte stimulator are clinically available and give a hope to patients suffering from these intractable diseases. Here, we selected twenty-seven monoclonal antibodies approved by US FDA since 1997 and they are classified according to their target molecules. Although these biomedicines possessed some restrictions such as high cost and susceptible to infectious disease, these drawbacks can be overcome through cost-cutting innovations including biosimilars and careful monitoring. New targets are emerging rapidly and more effective biomedicines with acceptable side effects are in the pipeline for next decade.

Ginseng seed oil ameliorates hepatic lipid accumulation in vitro and in vivo.

BACKGROUND: Despite the large number of studies on ginseng, pharmacological activities of ginseng seed oil (GSO) have not been established. GSO is rich in unsaturated fatty acids, mostly oleic and linoleic acids. Unsaturated fatty acids are known to exert a therapeutic effect in nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the protective effect and underlying mechanisms of GSO against NAFLD using in vitro and in vivo models. METHODS: In vitro lipid accumulation was induced by free fatty acid mixture in HepG2 cells and by 3 wk of high fat diet (HFD)-feeding in Sprague-Dawley rats prior to hepatocyte isolation. The effects of GSO against diet-induced hepatic steatosis were further examined in C57BL/6J mice fed a HFD for 12 wk. RESULTS: Oil Red O staining and intracellular triglyceride levels showed marked accumulation of lipid droplets in both HepG2 cells and rat hepatocytes, and these were attenuated by GSO treatment. In HFD-fed mice, GSO improved HFD-induced dyslipidemia and hepatic insulin resistance. Increased hepatic lipid contents were observed in HFD-fed mice and it was lowered in GSO (500 mg/kg)-treated mice by 26.4% which was evident in histological analysis. Pathway analysis of hepatic global gene expression indicated that GSO increased the expression of genes associated with ß-oxidation (Ppara, Ppargc1a, Sirt1, and Cpt1a) and decreased the expression of lipogenic genes (Srebf1 and Mlxipl), and these were confirmed with reverse transcription and quantitative polymerase-chain reaction. CONCLUSION: These findings suggest that GSO has a beneficial effect on NAFLD through the suppression of lipogenesis and stimulation of fatty acid degradation pathway.

Meta- and cross-species analyses of insulin resistance based on gene expression datasets in human white adipose tissues.

Ample evidence indicates that insulin resistance (IR) is closely related to white adipose tissue (WAT), but the underlying mechanisms of IR pathogenesis are still unclear. Using 352 microarray datasets from seven independent studies, we identified a meta-signature which comprised of 1,413 genes. Our meta-signature was also enriched in overall WAT in in vitro and in vivo IR models. Only 12 core enrichment genes were consistently enriched across all IR models. Among the meta-signature, we identified a drug signature made up of 211 genes with expression levels that were co-regulated by thiazolidinediones and metformin using cross-species analysis. To confirm the clinical relevance of our drug signature, we found that the expression levels of 195 genes in the drug signature were significantly correlated with both homeostasis model assessment 2-IR score and body mass index. Finally, 18 genes from the drug signature were identified by protein-protein interaction network cluster. Four core enrichment genes were included in 18 genes and the expression levels of selected 8 genes were validated by quantitative PCR. These findings suggest that our signatures provide a robust set of genetic markers which can be used to provide a starting point for developing potential therapeutic targets in improving IR in WAT.

Compound K attenuates glucose intolerance and hepatic steatosis through AMPK-dependent pathways in type 2 diabetic OLETF rats.

BACKGROUND/AIMS: Non-alcoholic fatty liver disease is associated with insulin resistance. Compound K (CK) is the final metabolite of panaxadiol ginsenosides that have been shown to exert antidiabetic effects. However, the molecular mechanism of the antidiabetic effects in the liver have not been elucidated; further, whether CK has beneficial effects in hepatosteatosis remains unclear. Therefore, we evaluated the effect of CK on hepatosteatosis as well as its mechanism in high-fat diet (HFD)-fed type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. METHODS: Twenty-four-week-old male OLETF rats were assigned to four groups: control (saline), CK 10 mg/kg, CK 25 mg/kg, or metformin 300 mg/kg (positive control); all treatments were administered orally for 12 weeks. RESULTS: Fasting glucose levels of the CK25 group were significantly lower than those of the control group during the 12 weeks. The results of the oral glucose tolerance test showed that both the glucose concentration after glucose loading and the fasting insulin levels of the CK25 group were significantly lower than those of the control. Hepatosteatosis was significantly improved by CK25. CK25 and metformin significantly increased the phosphorylation of hepatic adenosine monophosphate-activated protein kinase (AMPK). CK25 significantly inhibited the expression of sterol regulatory element-binding protein-1c and fatty acid synthase, while upregulating that of peroxisome proliferator-activated receptor-α and carnitine palmitoyltransferase-1. CONCLUSIONS: CK improved glucose intolerance and hepatosteatosis in HFD-fed OLETF rats through AMPK activation, which has dual mode of action that involves decreasing the synthesis of fatty acids and increasing fatty acid oxidation.

IL-6 inhibitors for treatment of rheumatoid arthritis: past, present, and future.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by polyarthritis. Numerous agents with varying mechanisms are used in the treatment of RA, including non-steroidal anti-inflammatory drugs, disease-modifying anti-rheumatic drugs, and some biological agents. Studies to uncover the cause of RA have recently ended up scrutinizing the importance of pro-inflammatory cytokine such as tumor necrosis factor α (TNF-α) and interleukin (IL)-6 in the pathogenesis of RA. TNF-α inhibitors are increasingly used to treat RA patients who are non-responsive to conventional anti-arthritis drugs. Despite its effectiveness in a large patient population, up to two thirds of RA patients are found to be partially responsive to anti-TNF therapy. Therefore, agents targeting IL-6 such as tocilizumab (TCZ) attracted significant attention as a promising agent in RA treatment. In this article, we review the mechanism of anti-IL-6 in the treatment of RA, provide the key efficacy and safety data from clinical trials of approved anti-IL-6, TCZ, as well as six candidate IL-6 blockers including sarilumab, ALX-0061, sirukumab, MEDI5117, clazakizumab, and olokizumab, and their future perspectives in the treatment of RA.

Eugenol ameliorates hepatic steatosis and fibrosis by down-regulating SREBP1 gene expression via AMPK-mTOR-p70S6K signaling pathway.

Beneficial effect of eugenol on fatty liver was examined in hepatocytes and liver tissue of high fat diet (HFD)-fed C57BL/6J mice. To induce a fatty liver, palmitic acid or isolated hepatocytes from HFD-fed Sprague-Dawley (SD) rats were used in vitro studies, and C57BL/6J mice were fed HFD for 10 weeks. Lipid contents were markedly decreased when hepatocytes were treated with eugenol for up to 24 h. Gene expressions of sterol regulatory element binding protein 1 (SREBP1) and its target enzymes were suppressed but those of lipolysis-related proteins were increased. As a regulatory kinase for lipogenic transcriptional factors, the AMP-activated protein kinase (AMPK) signaling pathway was examined. Protein expressions of phosphorylated Ca(2+)-calmodulin dependent protein kinase kinase (CAMKK), AMPK and acetyl-CoA carboxylase (ACC) were significantly increased and those of phosphorylated mammalian target of rapamycin (mTOR) and p70S6K were suppressed when the hepatocytes were treated with eugenol at up to 100 µM. These effects were all reversed in the presence of specific inhibitors of CAMKK, AMPK or mTOR. In vivo studies, hepatic triglyceride (TG) levels and steatosis score were decreased by 45% and 72%, respectively, in eugenol-treated mice. Gene expressions of fibrosis marker protein such as α-smooth muscle actin (α-SMA), collagen type I (Col-I) and plasminogen activator inhibitor-1 (PAI-1) were also significantly reduced by 36%, 63% and 40% in eugenol-treated mice. In summary, eugenol may represent a potential intervention in populations at high risk for fatty liver.

Clinical implication of SGLT2 inhibitors in type 2 diabetes.

Treatment of type 2 diabetes mellitus (T2DM) continues to present challenges, with many patients failing to achieve glycemic targets. Despite the availability of many oral and injectable anti-diabetic agents, therapeutic efficacy is often offset by undesirable side effects such as hypoglycemia, weight gain and cardiovascular complications. Therefore, the search for new therapeutic agents with an improved benefit-risk profile continues. Recent research has focused on the kidney as a potential therapeutic target, especially because maximal renal glucose reabsorption is increased in T2DM. Under normal physiological conditions, nearly all filtered glucose is reabsorbed in the proximal tubule of the nephron via the sodium/glucose co-transporter 2 (SGLT2). SGLT2-inhibitors are a new class of oral anti-diabetes, which reduce hyperglycemia by increasing urinary glucose excretion independently of insulin secretion or action. Canagliflozin and dapagliflozin in US market, and ipragliflozin and luseogliflozin in Japan market are now available for glycemic control in type 2 diabetics. There are several phase III clinical ongoing trials involving this new class of medications. This review examines some of the key efficacy and safety data from clinical trials of the SGLT2 inhibitors approved, and their future perspectives in the treatment of T2DM.

AMP-activated protein kinase: An emerging target for ginseng.

The adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a key sensor of cellular energy. Once activated, it switches on catabolic pathways generating adenosine triphosphate (ATP), while switching off biosynthetic pathways consuming ATP. Pharmacological activation of AMPK by metformin holds a therapeutic potential to reverse metabolic abnormalities such as type 2 diabetes and nonalcoholic fatty liver disease. In addition, altered metabolism of tumor cells is widely recognized and AMPK is a potential target for cancer prevention and/or treatment. Panax ginseng is known to be useful for treatment and/or prevention of cancer and metabolic diseases including diabetes, hyperlipidemia, and obesity. In this review, we discuss the ginseng extracts and ginsenosides that activate AMPK, we clarify the various mechanisms by which they achieve this, and we discuss the evidence that shows that ginseng or ginsenosides might be useful in the treatment and/or prevention of metabolic diseases and cancer.

Effects of eugenol on hepatic glucose production and AMPK signaling pathway in hepatocytes and C57BL/6J mice.

Eugenol is a phenylpropanoid with many pharmacological activities, but its anti-hyperglycemic activity is not yet fully explored. For in vitro study, HepG2 cells and primary rat hepatocytes were used, and glucose production was induced by adding 100 nM of glucagon in the presence of gluconeogenic substrates. In animal study, hyperglycemia was induced by high fat diet (HFD) in male C57BL/6J mice, and eugenol was orally administered at 20 or 40 mg per kg (E20, E40) for 15 weeks. Eugenol significantly inhibited glucagon-induced glucose production and phosphorylated AMPK in the HepG2 and primary rat hepatocytes, and these effects were reversed in the presence of compound C (an AMPK inhibitor) or STO-609 (a CAMKK inhibitor). In addition, the protein and gene expression levels of CREB, CRTC2·CREB complex, PGC-1α, PEPCK and G6Pase were all significantly suppressed. Moreover, inhibition of AMPK by over-expression of dominant negative AMPK prevented eugenol from suppressions of gluconeogenic gene expression and hepatic glucose production. In animal study, plasma glucose and insulin levels of the E40 group were decreased by 31% and 63%, respectively, when compared to those of HFD control. In pyruvate tolerance tests, pyruvate-induced glucose excursions were decreased, indicating that the anti-hyperglycemic activity of eugenol is primarily due to the suppression of hepatic gluconeogenesis. In summary, eugenol effectively ameliorates hyperglycemia through inhibition of hepatic gluconeogenesis via modulating CAMKK-AMPK-CREB signaling pathway. Eugenol or eugenol-containing medicinal plants could represent a promising therapeutic agent to prevent type 2 diabetes.

Beneficial effect of betulinic acid on hyperglycemia via suppression of hepatic glucose production.

The inhibitory effect of betulinic acid (BA) on hepatic glucose production was examined in HepG2 cells and high fat diet (HFD)-fed ICR mice. BA significantly inhibited the hepatic glucose production (HGP) and gene expression levels of PGC-1α, PEPCK, and G6Pase. BA activated AMPK and suppressed the expression level of phosphorylated CREB. These effects were all abolished in the presence of compound C (an AMPK inhibitor). Moreover, inhibition of AMPK by overexpression of dominant negative AMPK prevented BA from suppression of HGP, indicating that the inhibitory effect of BA on HGP is AMPK-dependent. In addition, BA markedly phosphorylated CAMKK, and phosphorylation of AMPK and ACC, and suppression of HGP were all reversed in the presence of STO-609 (a CAMKK inhibitor), suggesting that CAMKK is an upstream kinase for AMPK. In an animal study, HFD-fed ICR mice were orally administered with 5 or 10 mg of BA per kg (B5 and B10) for three weeks. Plasma glucose, triglyceride, and the insulin resistance index of the B10 group were decreased by 34%, 59%, and 38%, respectively. In a pyruvate tolerance test, pyruvate-induced glucose excursion was decreased by 27% when mice were pretreated with 10 mg/kg of BA. In summary, BA effectively ameliorates hyperglycemia through inhibition of hepatic gluconeogenesis via modulating the CAMKK-AMPK-CREB signaling pathway.

Nectandrin A Enhances the BMP-Induced Osteoblastic Differentiation and Mineralization by Activation of p38 MAPK-Smad Signaling Pathway.

Osteoblastic activity of nectandrin A was examined in C2C12 cells. Nectandrin A enhances the BMP-induced osteoblastic differentiation and mineralization, manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and increased calcium contents. In C2C12 cells co-transfected with expression vector encoding Smad4 and Id1-Luc reporter, nectandrin A increased Id1 luciferase activity in a concentration-dependent manner, when compared to that in BMP-2 treated cells, indicating that Smad signaling pathway is associated with nectandrin A-enhanced osteoblastic differentiation in C2C12 cells. In addition, nectandrin A activated p38 mitogen-activated protein kinase (MAPK) in time- and concentration-dependent manners, and phosphorylated form of pSmad1/5/8 and alkaline phosphatase activity were both decreased when the cells were pretreated with SB203580, a p38 MAPK inhibitor, suggesting that p38 MAPK might be an upstream kinase for Smad signaling pathway. Taken together, nectandrin A enhances the BMP-induced osteoblastic differentiation and mineralization of C2C12 cells via activation of p38 MAPK-Smad signaling pathway, and it has a therapeutic potential for osteoporosis by promoting bone formation.

Korean red ginseng extract alleviates advanced glycation end product-mediated renal injury.

The effect of Korean red ginseng (KRG) on diabetic renal damage was investigated using streptozotocin (STZ)-induced diabetic rats. The diabetic rats showed loss of body weight gain, and increases in kidney weight and urine volume, whereas the oral administration of KRG at a dose of 100 or 250 mg/kg of body weight per day for 28 d prevented these diabetes-induced physiological abnormalities. Among the kidney function parameters, elevated plasma levels of urea nitrogen and creatinine in diabetic control rats tended to be lowered in KRG-treated rats. In addition, administration of KRG at a dose of 100 mg/kg body weight in the diabetic rats showed significant decreases in serum glucose and tumor necrosis factor-α (TNF-α), implying that KRG might prevent the pathogenesis of diabetic complications caused by impaired glucose metabolism and oxidative stress. KRG also significantly reduced advanced glycation end product (AGE) formation and secretion from kidney of diabetic rats. Furthermore, KRG decreased the levels of N-(carboxymethyl) lysine and expression of AGE receptor. KRG also reduced the overexpression of cyclooxygenase-2 and inducible nitric oxide synthase in the kidney via deactivation of nuclear factor-kappa B. We also found that KRG prevented STZ-induced destruction of glomerular structure and significantly suppressed high glucose-induced fibronectin production. Taken together, KRG ameliorates abnormalities associated with diabetic nephropathy through suppression of inflammatory pathways activated by TNF-α and AGEs. These findings indicate that KRG has a beneficial effect on pathological conditions associated with diabetic nephropathy.

Caffeine attenuates lipid accumulation via activation of AMP-activated protein kinase signaling pathway in HepG2 cells.

The main purpose of this study is to examine the effect of caffeine on lipid accumulation in human hepatoma HepG2 cells. Significant decreases in the accumulation of hepatic lipids, such as triglyceride (TG), and cholesterol were observed when HepG2 cells were treated with caffeine as indicated. Caffeine decreased the mRNA level of lipogenesis-associated genes (SREBP1c, SREBP2, FAS, SCD1, HMGR and LDLR). In contrast, mRNA level of CD36, which is responsible for lipid uptake and catabolism, was increased. Next, the effect of caffeine on AMP-activated protein kinase (AMPK) signaling pathway was examined. Phosphorylation of AMPK and acetyl-CoA carboxylase were evidently increased when the cells were treated with caffeine as indicated for 24 h. These effects were all reversed in the presence of compound C, an AMPK inhibitor. In summary, these data indicate that caffeine effectively depleted TG and cholesterol levels by inhibition of lipogenesis and stimulation of lipolysis through modulating AMPK-SREBP signaling pathways.

Licochalcone A regulates hepatic lipid metabolism through activation of AMP-activated protein kinase.

Licochalcone A (LA) is a major phenolic ingredient of Glycyrrhiza plant. Although multiple pharmacological activities of LA have been reported, effect on hepatic lipid metabolism is unknown yet. The present study showed LA to suppress the hepatic triglyceride accumulation in HepG2 cells and ICR mice fed on a high fat diet (HFD). LA inhibited lipogenesis via suppression of sterol regulatory element-binding protein 1c (SREBP1c) and its target enzymes (stearoyl-CoA desaturase 1, fatty acid synthase and glycerol-3-phosphate acyltransferase) transcription. In addition, LA up-regulated gene expression of proteins such as peroxisome proliferator-activated receptor α (PPARα) and fatty acid transporter (FAT/CD36), which are responsible for lipolysis and fatty acid transport, respectively. These effects were mediated through activation of AMP-activated protein kinase (AMPK), and were abrogated when HepG2 cells were treated with an AMPK inhibitor, compound C. To explore how LA activates AMPK, oxygen consumption rate and ATP levels were measured in HepG2 cells. LA significantly inhibited the mitochondrial respiration and ATP levels, suggesting that LA activated AMPK indirectly. In animal study, LA (5 and 10mg/kg) was orally administered to six-week-old mice once a day for 3 weeks. In vitro results were likely to hold true in vivo experiment, as LA markedly lowered the triglyceride levels and activated AMPK signaling pathway in the liver of ICR mice fed on a HFD. In conclusion, the current study suggests that LA suppressed hepatic triglyceride accumulation through modulation of AMPK-SREBP signaling pathway and thus LA may be a potential therapeutic agent for treating fatty liver disease.

Betulinic acid alleviates non-alcoholic fatty liver by inhibiting SREBP1 activity via the AMPK-mTOR-SREBP signaling pathway.

Non-alcoholic fatty liver disease (NAFLD) is emerging as the most common liver disease in industrialized countries. The discovery of food components that can ameliorate NAFLD is therefore of interest. Betulinic acid (BA) is a triterpenoid with many pharmacological activities, but the effect of BA on fatty liver is as yet unknown. To explore the possible anti-fatty liver effects and their underlying mechanisms, we used insulin-resistant HepG2 cells, primary rat hepatocytes and liver tissue from ICR mice fed a high-fat diet (HFD). Oil Red O staining revealed that BA significantly suppressed excessive triglyceride accumulation in HepG2 cells and in the livers of mice fed a HFD. Ca(+2)-calmodulin dependent protein kinase kinase (CAMKK) and AMP-activated protein kinase (AMPK) were both activated by BA treatment. In contrast, the protein levels of sterol regulatory element-binding protein 1 (SREBP1), mammalian target of rapamycin (mTOR) and S6 kinase (S6K) were all reduced when hepatocytes were treated with BA for up to 24h. We found that BA activates AMPK via phosphorylation, suppresses SREBP1 mRNA expression, nuclear translocation and repressed SREBP1 target gene expression in HepG2 cells and primary hepatocytes, leading to reduced lipogenesis and lipid accumulation. These effects were completely abolished in the presence of STO-609 (a CAMKK inhibitor) or compound C (an AMPK inhibitor), indicating that the BA-induced reduction in hepatic steatosis was mediated via the CAMKK-AMPK-SREBP1 signaling pathway. Taken together, our results suggest that BA effectively ameliorates intracellular lipid accumulation in liver cells and thus is a potential therapeutic agent for the prevention of fatty liver disease.

Quercetin enhances hypoxia-mediated apoptosis via direct inhibition of AMPK activity in HCT116 colon cancer.

Tumor hypoxia is considered the best validated target in clinical oncology because of its significant contribution to chemotherapy failure and drug resistance. As an approach to target hypoxia, we assessed the potential of quercetin, a flavonoid widely distributed in plants, as a anticancer agent under hypoxic conditions and examined its pharmacological mechanisms by primarily focusing on the role of AMP-activated protein kinase (AMPK). Quercetin significantly attenuated tumor growth in an HCT116 cancer xenograft in vivo model with a substantial reduction of AMPK activity. In a cell culture system, quercetin more dramatically induced apoptosis of HCT116 cancer cells under hypoxic conditions than normoxic conditions, and this was tightly associated with inhibition of hypoxia-induced AMPK activity. An in vitro kinase assay demonstrated that quercetin directly inhibits AMPK activity. Inhibition of AMPK by expressing a dominant-negative form resulted in an increase of apoptosis under hypoxia, and a constitutively active form of AMPK effectively blocked quercetin-induced apoptosis under hypoxia. Collectively, our data suggest that quercetin directly inhibits hypoxia-induced AMPK, which plays a protective role against hypoxia. Quercetin also reduced the activity of hypoxia-inducible factor-1 (HIF-1), a major transcription factor for adaptive cellular response to hypoxia. Moreover, quercetin sensitized HCT116 cancer cells to the anticancer drugs cisplatin and etoposide under hypoxic conditions. Our findings suggest that AMPK may serve as a novel target for overcoming tumor hypoxia-associated negative aspects.