Blueberry-derived exosomes-like nanoparticles ameliorate nonalcoholic fatty liver disease by attenuating mitochondrial oxidative stress.

Accumulating evidence indicates that mitochondrial dysfunction and oxidative stress play a pivotal role in the initiation and progression of nonalcoholic fatty liver disease (NAFLD). In this study, we found that blueberry-derived exosomes-like nanoparticles (BELNs) could ameliorate oxidative stress in rotenone-induced HepG2 cells and high-fat diet (HFD)-fed C57BL/6 mice. Preincubation with BELNs decreased the level of reactive oxygen species (ROS), increased the mitochondrial membrane potential, and prevented cell apoptosis by inducing the expression of Bcl-2 and heme oxygenase-1 (HO-1) and decreasing the content of Bax in rotenone-treated HepG2 cells. We also found that preincubation with BELNs accelerated the translocation of Nrf2, an important transcription factor of antioxidative proteins, from the cytoplasm to the nucleus in rotenone-treated HepG2 cells. Moreover, administration of BELNs improved insulin resistance, ameliorated the dysfunction of hepatocytes, and regulated the expression of detoxifying/antioxidant genes by affecting the distribution of Nrf2 in the cytoplasm and nucleus of hepatocytes of HFD-fed mice. Furthermore, BELNs supplementation prevented the formation of vacuoles and attenuated the accumulation of lipid droplets by inhibiting the expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase 1 (ACC1), the two key transcription factors for de novo lipogenesis in the liver of HFD-fed mice. These findings suggested that BELNs can be used for the treatment of NAFLD because of their antioxidative activity.

Differential cytotoxicity, ER/oxidative stress, dysregulated AMPKα signaling, and mitochondrial stress by ethanol and its metabolites in human pancreatic acinar cells.

BACKGROUND: Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disorder of the exocrine pancreatic gland. A previous study from this laboratory showed that ethanol (EtOH) causes cytotoxicity, dysregulates AMPKα and ER/oxidative stress signaling, and induces inflammatory responses in primary human pancreatic acinar cells (hPACs). Here we examined the differential cytotoxicity of EtOH and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters; FAEEs) metabolites in hPACs was examined to understand the metabolic basis and mechanism of ACP. METHODS: We evaluated concentration-dependent cytotoxicity, AMPKα inactivation, ER/oxidative stress, and inflammatory responses in hPACs by incubating them for 6 h with EtOH, acetaldehyde, or FAEEs at clinically relevant concentrations reported in alcoholic subjects using conventional methods. Cellular bioenergetics (mitochondrial stress and a real-time ATP production rate) were determined using Seahorse XFp Extracellular Flux Analyzer in AR42J cells treated with acetaldehyde or FAEEs. RESULTS: We observed concentration-dependent increases in LDH release, inactivation of AMPKα along with upregulation of ACC1 and FAS (key lipogenic proteins), downregulation of p-LKB1 (an oxidative stress-sensitive upstream kinase regulating AMPKα) and CPT1A (involved in ß-oxidation of fatty acids) in hPACs treated with EtOH, acetaldehyde, or FAEEs. Concentration-dependent increases in oxidative stress and ER stress as measured by GRP78, unspliced XBP1, p-eIF2α, and CHOP along with activation of p-JNK1/2, p-ERK1/2, and p-P38MAPK were present in cells treated with EtOH, acetaldehyde, or FAEEs, respectively. Furthermore, a significant decrease was observed in the total ATP production rate with subsequent mitochondrial stress in AR42J cells treated with acetaldehyde and FAEEs. CONCLUSIONS: EtOH and its metabolites, acetaldehyde and FAEEs, caused cytotoxicity, ER/oxidative and mitochondrial stress, and dysregulated AMPKα signaling, suggesting a key role of EtOH metabolism in the etiopathogenesis of ACP. Because oxidative EtOH metabolism is negligible in the exocrine pancreas, the pathogenesis of ACP could be attributable to the formation of FAEEs and related pancreatic acinar cell injury.

Effect of an aqueous extract of Chrysobalanus icaco on the adiposity of Wistar rats fed a high-fat diet / Efecto del extracto acuoso de Chrysobalanus icaco sobre la adiposidad de ratas Wistar alimentadas con una dieta alta en grasas

OBJECTIVE: the purpose of this study was to investigate the effects of Chrysobalanus icaco on adiposity and its mechanism of action in the gene and protein expression of acetyl-CoA carboxylase (ACC), a key enzyme in lipogenesis. METHOD: Wistar rats were divided into a regular or control group (CG) and a high-fat diet (HFD) group. HFD was treated with saline or aqueous extract of Chrysobalanus icaco (AECI) for four weeks. Body weight and food intake were assessed. Subcutaneous, retroperitoneal and periepididymal adipose tissue samples were collected and weighed. Adipocytes from periepididymal tissue were isolated and analyzed. The gene and protein expression of ACC in subcutaneous tissue was determined. RESULTS: AECI showed no effect on intake or body weight. However, the weight of the fat pads and the gene and protein expression of ACC were lower, and glucose tolerance was improved. CONCLUSION: the aqueous extract of Chrysobalanus icaco proved beneficial for the treatment of obesity, preventing fat storage and improving glycemic homeostasis

OBJETIVO: el objetivo de este estudio fue investigar los efectos del extracto acuoso de Chrysobalanus icaco (AECI) en la adiposidad y su mecanismo de acción en la expresión génica y proteica de la acetil-CoA-carboxilasa (ACC), una enzima clave para la lipogénesis. MÉTODOS: se usaron ratones macho Wistar que se asignaron a una dieta estándar de control (CG) o a una rica en grasa (HFD). La HFD se trató con solución salina o con extracto acuoso de Chrysobalanus icaco (AECI) durante cuatro semanas. Se evaluaron el peso corporal y el consumo alimentario. Se aislaron y analizaron muestras de tejido adiposo subcutáneo, retroperitoneal y periepididímico. Se determinó la expresión génica y proteica de ACC en el tejido subcutáneo. RESULTADOS: el AECI no mostró ningún efecto sobre la ingesta de alimento y tampoco sobre el peso corporal. Sin embargo, el tratamiento con AECI redujo el peso de los tejidos adiposos y la expresión génica y proteica de ACC, y mejoró también la tolerancia a la glucosa. CONCLUSIÓN: Chrysobalanus icaco (AECI) resultó ser beneficioso para el tratamiento de la obesidad, previniendo el almacenamiento de grasa y mejorando la homeostasis glucémica

Sirtuin 3 improves fatty acid metabolism in response to high nonesterified fatty acids in calf hepatocytes by modulating gene expression.

Sirtuin 3 (SIRT3), a mitochondrial deacetylase, is a key regulator of energy metabolism in the liver. In nonruminants, the hepatic abundance of SIRT3 is decreased in individuals with nonalcoholic fatty liver diseases, and recovery of SIRT3 alleviates hepatic triacylglycerol (TG) deposition. However, the level of SIRT3 expression and its effects on lipid metabolism in dairy cows have not been characterized. Here we studied the hepatic expression of SIRT3 in cows with fatty liver and the role of SIRT3 in fatty acid metabolism in bovine hepatocytes. This in vivo study involved 10 healthy cows and 10 cows with fatty liver, from which we collected samples of liver tissue and blood. Primary hepatocytes were isolated from Holstein calves and treated with 0, 0.5, or 1.0 mM nonesterified fatty acids (NEFA) for 24 h or transinfected with SIRT3 overexpression adenovirus (Ad-SIRT3)/SIRT3-short interfering (si)RNA for 48 h. Cows with fatty liver displayed lower serum glucose concentrations but higher serum NEFA and ß-hydroxybutyrate concentrations relative to healthy cows. Cows with fatty liver also had significant lower mRNA and protein abundance of hepatic SIRT3. Incubation of primary hepatocytes with NEFA reduced SIRT3 abundance in primary hepatocytes in a dose-dependent manner. Fatty acid (1 mM) treatment also markedly increased the abundance of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS) but significantly decreased the abundance of carnitine palmitoyltransferase I (CPT1A), carnitine palmitoyltransferase II (CPT2), and acyl-CoA oxidase (ACO). Knockdown of SIRT3 by SIRT3-siRNA downregulated the mRNA abundance of CPT1A, CPT2, and ACO. In contrast, overexpression of SIRT3 by Ad-SIRT3 upregulated the mRNA abundance of CPT1A, CPT2, and ACO; downregulated the mRNA abundance of ACC1 and FAS; and consequently, decreased intracellular TG concentrations. Overexpression of SIRT3 ameliorated exogenous NEFA-induced TG accumulation by downregulating the abundance of ACC1 and FAS and upregulating the abundance of CPT1A, CPT2, and ACO in calf hepatocytes. Our data demonstrated that cows with fatty liver had lower hepatic SIRT3 contents, and an increase in SIRT3 abundance by overexpression mitigated TG deposition by modulating the expression of lipid metabolism genes in bovine hepatocytes. These data suggest a possible role of SIRT3 as a therapeutic target for fatty liver disease prevention in periparturient dairy cattle.

Black tea affects obesity by reducing nutrient intake and activating AMP-activated protein kinase in mice.

The effects of certain tea components on the prevention of obesity in humans have been reported recently. However, whether Yinghong NO. 9 black tea consumption has beneficial effects on obesity are not known. Here, we obtained a Yinghong NO. 9 black tea infusion (Y9 BTI) and examined the anti-obesity effects of its oral administration. ICR mice were fed a standard diet supplemented with Y9 BTI at 0.5, 1.0, or 2.0 g/kg body weight for two weeks, and the body weight were recorded. HE staining was used to evaluate the effect of Y9 BTI on mice liver. Western blot analysis was used to detect the expression levels of related proteins in the mice liver and adipose. We found that the body weights of the mice in the control group were significantly higher than those of the mice in the middle and high dose groups. The results of western blot showed that Y9 BTI up-regulated the expression of liver kinase B1 (LKB1) and adenosine monophosphate-activated protein kinase (AMPK) and also increased in AMPK phosphorylation (p-AMPK) and LKB1 phosphorylation (p-LKB1). Y9 BTI significantly down-regulated Fas Cell Surface Death Receptor(FAS) and activated the phosphorylation of acetyl-CoA carboxylase (ACC). Furthermore, Y9 BTI (2.0 g/kg BW) down-regulated the expression of three factors (IL-1ß, Cox-2, and iNOS). Altogether, Y9 BTI supplementation reduced the feed intake of mice and may prevent obesity by inhibiting lipid absorption. These results suggest that Y9 BTI may regulate adipogenic processes through the LKB1/AMPK pathway.

Butyrate Regulates Liver Mitochondrial Function, Efficiency, and Dynamics in Insulin-Resistant Obese Mice.

Fatty liver, oxidative stress, and mitochondrial dysfunction are key pathophysiological features of insulin resistance and obesity. Butyrate, produced by fermentation in the large intestine by gut microbiota, and its synthetic derivative, the N-(1-carbamoyl-2-phenyl-ethyl) butyramide, FBA, have been demonstrated to be protective against insulin resistance and fatty liver. Here, hepatic mitochondria were identified as the main target of the beneficial effect of both butyrate-based compounds in reverting insulin resistance and fat accumulation in diet-induced obese mice. In particular, butyrate and FBA improved respiratory capacity and fatty acid oxidation, activated the AMPK-acetyl-CoA carboxylase pathway, and promoted inefficient metabolism, as shown by the increase in proton leak. Both treatments consistently increased utilization of substrates, especially fatty acids, leading to the reduction of intracellular lipid accumulation and oxidative stress. Finally, the shift of the mitochondrial dynamic toward fusion by butyrate and FBA resulted in the improvement not only of mitochondrial cell energy metabolism but also of glucose homeostasis. In conclusion, butyrate and its more palatable synthetic derivative, FBA, modulating mitochondrial function, efficiency, and dynamics, can be considered a new therapeutic strategy to counteract obesity and insulin resistance.

Effects of dietary phosphate on glucose and lipid metabolism.

Recent epidemiological and animal studies have suggested that excess intake of phosphate (Pi) is a risk factor for the progression of chronic kidney disease and its cardiovascular complications. However, little is known about the impact of dietary high Pi intake on the development of metabolic disorders such as obesity and type 2 diabetes. In this study, we investigated the effects of dietary Pi on glucose and lipid metabolism in healthy rats. Male 8-wk-old Sprague-Dawley rats were divided into three groups and given experimental diets containing varying amounts of Pi, i.e., 0.2 [low Pi(LP)], 0.6 [control Pi(CP)], and 1.2% [high Pi(HP)]. After 4 wk, the HP group showed lower visceral fat accumulation compared with other groups, accompanied by a low respiratory exchange ratio (V̇CO2/V̇O2) without alteration of locomotive activity. The HP group had lower levels of plasma insulin and nonesterified fatty acids. In addition, the HP group also showed suppressed expression of hepatic lipogenic genes, including sterol regulatory element-binding protein-1c, fatty acid synthase, and acetyl-CoA carboxylase, whereas there was no difference in hepatic fat oxidation among the groups. On the other hand, uncoupling protein (UCP) 1 and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression were significantly increased in the brown adipose tissue (BAT) of the HP group. Our data demonstrated that a high-Pi diet can negatively regulate lipid synthesis in the liver and increase mRNA expression related to lipid oxidation and UCP1 in BAT, thereby preventing visceral fat accumulation. Thus, dietary Pi is a novel metabolic regulator.

Thymoquinone, a bioactive component of Nigella sativa, normalizes insulin secretion from pancreatic ß-cells under glucose overload via regulation of malonyl-CoA.

Thymoquinone (2-isopropyl-5-methylbenzo-1,4-quinone) is a major bioactive component of Nigella sativa, a plant used in traditional medicine to treat a variety of symptoms, including elevated blood glucose levels in type 2 diabetic patients. Normalization of elevated blood glucose depends on both glucose disposal by peripheral tissues and glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells. We employed clonal ß-cells and rodent islets to investigate the effects of thymoquinone (TQ) and Nigella sativa extracts (NSEs) on GSIS and cataplerotic metabolic pathways implicated in the regulation of GSIS. TQ and NSE regulated NAD(P)H/NAD(P)(+) ratios via a quinone-dependent redox cycling mechanism. TQ content was positively correlated with the degree of redox cycling activity of NSE extracts, suggesting that TQ is a major component engaged in mediating NSE-dependent redox cycling. Both acute and chronic exposure to TQ and NSE enhanced GSIS and were associated with the ability of TQ and NSE to increase the ATP/ADP ratio. Furthermore, TQ ameliorated the impairment of GSIS following chronic exposure of ß-cells to glucose overload. This protective action was associated with the TQ-dependent normalization of chronic accumulation of malonyl-CoA, elevation of acetyl-CoA carboxylase (ACC), fatty acid synthase, and fatty acid-binding proteins following chronic glucose overload. Together, these data suggest that TQ modulates the ß-cell redox circuitry and enhances the sensitivity of ß-cell metabolic pathways to glucose and GSIS under normal conditions as well as under hyperglycemia. This action is associated with the ability of TQ to regulate carbohydrate-to-lipid flux via downregulation of ACC and malonyl-CoA.

Pyruvate modifies metabolic flux and nutrient sensing during extracorporeal membrane oxygenation in an immature swine model.

Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support for infants and children with postoperative cardiopulmonary failure. Nutritional support is mandatory during ECMO although specific actions for substrates on the heart have not been delineated. Prior work shows that enhancing pyruvate oxidation promotes successful weaning from ECMO. Accordingly, we tested the hypothesis that prolonged systemic pyruvate supplementation activates pyruvate oxidation in an immature swine model in vivo. Twelve male mixed-breed Yorkshire piglets (age 30-49 days) received systemic infusion of either normal saline (group C) or pyruvate (group P) during the final 6 h of 8 h of ECMO. Over the final hour, piglets received [2-(13)C] pyruvate, as a reference substrate for oxidation, and [(13)C6]-l-leucine, as an indicator for amino acid oxidation and protein synthesis. A significant increase in lactate and pyruvate concentrations occurred, along with an increase in the absolute concentration of the citric acid cycle intermediates. An increase in anaplerotic flux through pyruvate carboxylation in group P occurred compared with no change in pyruvate oxidation. Additionally, pyruvate promoted an increase in the phosphorylation state of several nutrient-sensitive enzymes, like AMP-activated protein kinase and acetyl CoA carboxylase, suggesting activation for fatty acid oxidation. Pyruvate also promoted O-GlcNAcylation through the hexosamine biosynthetic pathway. In conclusion, although prolonged pyruvate supplementation did not alter pyruvate oxidation, it did elicit changes in nutrient- and energy-sensitive pathways. Therefore, the observed results support the further study of pyruvate and its downstream effect on cardiac function.

Vigna nakashimae extract prevents hepatic steatosis in obese mice fed high-fat diets.

Vigna species are important food resources and are traditionally used for the treatment of various diseases. In this study, we examined the inhibitory effects of Vigna nakashimae (VN) extract on high-fat diet (HFD)-induced hepatic steatosis and elucidated the molecular mechanisms. C57BL/6J mice were fed an HFD with or without VN extract for 16 weeks. VN extract decreased HFD-induced body weight, liver weight, hepatic lipid accumulation, and plasma alanine aminotransferase, and suppressed oxidative stress and inflammation associated with hepatitis. VN extract decreased plasma lipid levels and the expression of lipogenic genes in the livers of HFD-fed mice. VN extract significantly increased phosphorylation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and acetyl-CoA carboxylase, and expression of fatty acid oxidation genes in the liver of VN-treated HFD mice and HepG2 cells. Further, VN extract inhibited insulin or glucose-stimulated lipogenesis in HepG2 cells. In conclusion, VN extract prevents HFD-induced hepatic steatosis and lipotoxicity through AMPK activation.

Beneficial effects of mangiferin on hyperlipidemia in high-fat-fed hamsters.

SCOPE: Mangiferin, a natural polyphenol, has been shown to have hypolipidemic effect in rat and mouse. However, the mechanism of action is not well understood. This study was conducted to determine the effect and mechanism of action of mangiferin on hyperlipidemia induced in hamsters by a high-fat diet. METHODS AND RESULTS: Forty male hamsters were randomly assigned to normal control, high-fat control, and high fat with mangiferin (50 and 150 mg/kg BW) groups. Mangiferin treatment significantly decreased final body weight, liver weight and visceral fat-pad weight, serum triglyceride (TG) and total free fatty acid (FFA) concentrations, hepatic TG levels and hepatic and muscle total FFA contents. Mangiferin upregulated mRNA expression of peroxisome proliferator-activated receptor-α (PPAR-α), fatty acid translocase (CD36) and carnitine palmitoyltransferase 1 (CPT-1), but downregulated mRNA expression of sterol regulatory element-binding protein 1c (SREBP-1c), acetyl CoA carboxylase (ACC), acyl-CoA:diacylglycerol acyltransferase 2 (DGAT-2) and microsomal triglyceride transfer protein (MTP) in liver. Mangiferin also stimulated mRNA expression of PPAR-α, CD36, CPT-1 and lipoprotein lipase (LPL) in muscle. CONCLUSIONS: The results suggest that mangiferin may ameliorate hypertriglyceridemia partly by modulating the expression levels of genes involved in lipid oxidation and lipogenesis.

Effects of dietary biotin and avidin on growth, survival, feed conversion, biotin status and gene expression of zebrafish Danio rerio.

A study was conducted to investigate the effects of dietary avidin on growth, survival, food conversion, biotin status and gene expression of zebrafish (Danio rerio Hamilton-Buchanan) juveniles (average wet mass 0.178 g) fed 7 purified diets for 12 weeks. Experimental diets were formulated to provide 0×, 1×, 15×, 30×, 60× and 120× excess avidin versus biotin kg(-1) diet, on a molar basis; a control diet contained neither supplemental biotin nor avidin. Fish fed the control diet had the lowest percentage weight gain and the highest mortality, while the highest percentage weight gain and the lowest mortality was observed with the 0× diet (P<0.05). A linear relationship was observed between feed conversion ratio (FCR) and dietary avidin (r=0.876; P<0.0001). Fish fed diets with 120× more avidin than biotin had the highest whole-body biotin content, while the lowest value was obtained with the control and avidin-free diets (P<0.05). Elevated levels of acetyl CoA carboxylase-A (acca), methylcrotonyl CoA carboxylase (mcc) and propionyl CoA carboxylase-A (pcca) transcripts were recorded in fish fed the control diet, in comparison to the other diets. A broken-line analysis indicated that feeding zebrafish a diet with 60 times more avidin than the dietary biotin requirement level will cause biotin deficiency signs.

Rhein, an inhibitor of adipocyte differentiation and adipogenesis.

Rhein (RH), a compound purified from Radix et Rhizoma Rhei, has been used to alleviate liver and kidney damage. It is found that RH inhibited the differentiation of 3T3-L1 preadipocytes induced by differentiation medium in a time- and dose-dependent manner. It was revealed that RH downregulated the expression of adipogenesis-specific transcription factors PPARγ and C/EBPα, as well as their upstream regulator, C/EBPß. Furthermore, the PPARγ target genes that are involved in adipocyte differentiation, such as CD36, aP2, acyl CoA oxidase, uncoupled protein 2, acetyl-CoA carboxylase, and fatty acid synthase, were reduced after to RH. In addition, high-fat diet-induced weight gain and adiposity were reversed by RH in C57BL/6 mice. Consistent with the cells' results, RH downregulated the mRNA levels of PPARγ and C/EBPα, and their downstream target genes in C57BL/6 mice. Taken together, adipocyte differentiation and adipogenesis were inhibited by RH in cultured cells and in rodent models of obesity. The evidence implied that RH was a potential candidate for preventing metabolic disorders.

AICAR and Compound C regulate food intake independently of AMP-activated protein kinase in lines of chickens selected for high or low body weight.

AMP-activated protein kinase (AMPK) functions to maintain cellular and body energy balance. Our aim was to investigate the effect of intracerebroventricular (ICV) administration of AMPK stimulator AICAR and AMPK inhibitor Compound C on food intake in lines of chickens that had undergone long-term selection from a common founder population for high (HWS) or low (LWS) body weight. AICAR caused a quadratic dose-dependent decrease in food intake in LWS but not HWS chicks. Compound C caused a quadratic dose-dependent increase in food intake in HWS but not in LWS chicks. Key aspects of the AMPK pathway, including upstream kinases mRNA expression, AMPK subunit α mRNA expression and phosphorylation, and a downstream target acetyl CoA carboxylase (ACC) phosphorylation were not affected by either AICAR or Compound C in either line. The exception was a significant inhibitory effect of AICAR on ACC phosphorylation ratio due to increased total ACC protein content without changing phosphorylated ACC protein levels. Thus, the anorexigenic effect of AICAR in LWS chicks and orexigenic effect of Compound C in HWS chicks resulted from activation or inhibition of other kinase pathways separate from AMPK. These results suggest genetic variation in feeding response for central AICAR and Compound C in chickens, which may contribute to the different body weights between the HWS and LWS lines.

Clozapine activates AMP-activated protein kinase (AMPK) in C2C12 myotube cells and stimulates glucose uptake.

AIMS: Clozapine has previously been implicated in the dysregulation of energy balance and glucose metabolism in the central nervous system, but its effects in the periphery have yet to be thoroughly elucidated. The objective of this study was to characterize the effects of clozapine on AMP-activated protein kinase (AMPK) activity in the skeletal muscles. MAIN METHODS: Myotube C2C12 cells were incubated under control conditions, or with clozapine. Expression levels of phosphorylation status of AMPK and its direct downstream Acetyl-CoA carboxylase (ACC) were analyzed by Western blot. Intracellular calcium concentration was measured with calcium indicator dye, fluo-3AM. 2-deoxyglucose uptake was assessed via the scintillation count. KEY FINDINGS: We reported that clozapine activated AMPK in mouse C2C12 myotubes and also stimulated glucose uptake. Clozapine also increased intracellular calcium concentrations of C2C12 cells, and pretreatment with either ethylenediaminetetraacetic acid (EDTA), an extracellular calcium chelator, or 1.8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), a Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, blocked clozapine-induced AMPK activation. SIGNIFICANCE: These results demonstrate that clozapine increases glucose uptake through CaMKK-AMPK pathway in myotube C2C12 cells.

Quercetin suppresses HeLa cell viability via AMPK-induced HSP70 and EGFR down-regulation.

Quercetin, an anti-oxidant flavonoid that is widely distributed in the plant kingdom, has been suggested to have chemopreventive effects on cancer cells, although the mechanism is not completely understood. In this study, we found that quercetin increased the phosphorylation of AMP-activated protein kinase (AMPK) and downstream acetyl-CoA carboxylase (ACC) and suppressed the viability of HeLa cells. AICAR, an AMPK activator, and quercetin down-regulated heat shock protein (HSP)70 and increased the activity of the pro-apoptotic effector, caspase 3. Knock-down of AMPK blocked quercetin-mediated HSP70 down-regulation. Moreover, knock-down of HSP70 enhanced quercetin-mediated caspase 3 activation. Furthermore, quercetin sustained epidermal growth factor receptor (EGFR) activation by suppressing the phosphatases, PP2a and SHP-2. Finally, quercetin increased the interaction between EGFR and Cbl, and also induced the tyrosine phosphorylation of Cbl. Together, these results suggest that quercetin may have anti-tumor effects on HeLa cells via AMPK-induced HSP70 and down-regulation of EGFR.

Effects of metformin on bovine granulosa cells steroidogenesis: possible involvement of adenosine 5' monophosphate-activated protein kinase (AMPK).

In mammals, IGFs are important for the proliferation and steroidogenesis of ovarian cells. Metformin is an insulin sensitizer molecule used for the treatment of the infertility of women with polycystic ovary syndrome. It is, however, unclear whether metformin acts on ovarian cells. Adenosine 5' monophosphate-activated protein kinase (AMPK) is involved in metformin action in various cell types. We investigated the effects of metformin on bovine granulosa cell steroidogenesis in response to IGF1 and FSH, and studied AMPK in bovine ovaries. In granulosa cells from small follicles, metformin (10 mM) reduced production of both progesterone and estradiol and decreased the abundance of HSD3B, CYP11A1, and STAR proteins in presence or absence of FSH (10(-8) M) and IGF1 (10(-8) M). In cows, the different subunits of AMPK are expressed in various ovarian cells including granulosa and theca cells, corpus luteum, and oocytes. In bovine granulosa cells from small follicles, metformin, like AICAR (1 mM) a pharmaceutical activator of AMPK, increased phosphorylation of both Thr172 of AMPK alpha and Ser 79 of ACACA (Acetyl-CoA Carboxylase). Both metformin and AICAR treatment reduced progesterone and estradiol secretion in presence or absence of FSH and IGF1. Metformin decreased phosphorylation levels of MAPK3/MAPK1 and MAPK14 in a dose- and time-dependent manner. The adenovirus-mediated production of dominant negative AMPK abolished the effects of metformin on secretion of progesterone and estradiol and on MAPK3/MAPK1 phosphorylation but not on MAPK14 phosphorylation. Thus, in bovine granulosa cells, metformin decreases steroidogenesis and MAPK3/MAPK1 phosphorylation through AMPK activation.

Increased Akt protein expression is associated with decreased ceramide content in skeletal muscle of troglitazone-treated mice.

Although it is generally believed that thiazolidinediones ameliorate insulin resistance by lowering circulating free fatty acids, direct effects of these drugs in skeletal muscle may also contribute to their antidiabetic action. We report that troglitazone administration to mice for 1 day increased the protein expression of Akt (two-fold induction, P<0.001) in skeletal muscle without significant changes in the levels of free fatty acids in plasma. Increased Akt protein expression was associated with reduced phospho-AMP-activated protein kinase abundance and with a fall in the phosphorylation of acetyl-CoA carboxylase, which in turn resulted in an increase in the content of muscular malonyl-CoA (2.4-fold, P<0.05) and lactate (1.4-fold, P<0.05). Troglitazone treatment did not affect the mRNA levels of either Akt1 or Akt2, suggesting that a transcriptional mechanism was not involved, but caused a dramatic reduction in the content of muscular ceramides (76%, P<0.001), lipid-derived second messengers known to increase Akt degradation. Our data indicate that troglitazone treatment inhibited de novo ceramide synthesis, since the content of its precursor, palmitoyl-CoA, was reduced (55%, P=0.05). These results were confirmed in C2C12 myotubes, where troglitazone treatment increased Akt protein expression and prevented the reduction of this protein and the increase in ceramide levels caused by palmitate. These findings implicate ceramide as an important intermediate in the regulation of Akt after troglitazone treatment.

Hepatic lipid and carbohydrate metabolism in rats fed a commercial mixture of conjugated linoleic acids (Clarinol G-80).

BACKGROUND: Conjugated linoleic acids (CLAs) exert numerous effects in animal models as well as in humans. Among other things, CLAs decrease plasma lipid levels and bring about hepatic steatosis. The latter effects are attributed to an agonistic action of CLAs on the peroxisome-proliferator-activated receptor family primarily responsible for activating genes involved in lipid metabolism and are related to changes in mRNA levels. Such changes are not necessarily reflected in changes in activity of controlling enzymes. AIM OF THE STUDY: To investigate the effects of CLAs treatment on lipid metabolism, we determined lipid concentrations in plasma, lipoproteins and liver and measured the activity of a number of key enzymes in hepatic lipid metabolism as differences in lipid concentrations should be related to changes in enzyme activities. These variables were determined with the rat as a model. METHODS: Rats were fed a control diet or a diet containing 1.15% trans-10, cis-12 isomer and 1.11% cis-9, trans-11 isomer as part of a commercial mixture of CLAs. After 2 w the animals were killed, and plasma and liver fractions isolated. Subsequently, lipid concentrations of cholesterol, triacylglycerols and phospholipids were determined in the isolated lipoproteins. In livers homogenates, the concentrations of glycogen, cholesterol, triacylglycerol and phospholipids and the activities of enzymes catalyzing pacesetting steps of metabolism were determined, i. e. acetyl-CoA carboxylase, fatty acid synthase, diacylglycerol acyltransferase, 3-hydroxy- acyl-CoA dehydrogenase, citrate synthase and phosphofructokinase. RESULTS: CLAs induced a lowering of the cholesterol levels in total plasma and in LDL and HDL lipoproteins and of phospholipid concentrations in LDL and HDL. CLAs treatment decreased the hepatic activity of diacylglycerol acyltransferase and had no effect on any of the other enzyme activities. CONCLUSIONS: In other studies enhanced specific activities of ACC and FAS were found in livers of mice using the same or similar methods and experimental protocol as in the present study. The lack of effect of CLAs treatment on hepatic key enzymes of fatty acid synthesis and oxidation in Wistar rats questions the use of this strain for studying the mechanism(s) underlying CLA's effects on these parameters. However, in the rat model we observed reduced levels of cholesterol in total plasma and in LDL and HDL. Therefore, some aspects like loss of body fat are better studied in mice; for other aspects like reduction in serum cholesterol level the rat may be the model of choice.

The effects of a high-fat or high-sucrose diet on serum lipid profiles, hepatic acyl-CoA synthetase, carnitine palmitoyltransferase-I, and the acetyl-CoA carboxylase mRNA levels in rats.

The purpose of this study was to investigate the effects of altering relative intakes of fat and carbohydrates on serum lipid profiles, hepatic acyl-CoA synthetase (ACS), carnitine palmitoyltransferase-I (CPT-I), and the acetyl-CoA carboxlyase (ACC) mRNA level in Sprague-Dawley rats. For four weeks the rats were fed either an AIN-76 diet or one of its modified diets that were supplemented with 20% beef tallow (high-fat diet, HF) and 66.3% sucrose (high-sucrose diet, HS). The HS group had significantly higher serum triglyceride and total cholesterol concentrations when compared with the other groups. Serum LDL-cholesterol concentrations in the HS and HF groups were significantly higher when compared to the normal diet (ND) group. Serum HDL-cholesterol levels of the ND and HS groups were significantly higher than those of the HF group. The hepatic total lipid level of the HF group was significantly higher than those of other groups; triglyceride levels of the HS and HF groups were significantly higher than those of the ND group. Hepatic ACS mRNA levels of the HF group were significantly higher than those of the ND group. Hepatic CPT-I mRNA levels were higher in the HF group than other groups. Also, ACC mRNA levels in the liver increased in the HF group. In conclusion, changes in the composition of dietary fat and carbohydrates could affect the hepatic ACS, CPT-I, and ACC mRNA levels. These results facilitate our understanding of the coordinated regulation of the ACS, CPT-I, and ACC mRNA levels and will serve to enhance our understanding of the molecular mechanisms that underlie the regulation of fatty acid metabolism.