Effect of an aqueous extract of Chrysobalanus icaco on the adiposity of Wistar rats fed a high-fat diet.

INTRODUCTION: 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.

INTRODUCCIÓN: 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.

Overexpression of acetyl-CoA carboxylase increases fatty acid production in the green alga Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a photosynthetic unicellular model algae with multiple biotechnological advantages, and its fatty acids can be used to produce biofuels. Numerous studies suggest that acetyl-coA carboxylase (ACCa) catalyzes the first committed and rate-limiting step of fatty acid biosynthesis, thereby playing a central role in oil accumulation. Here, we cloned and overexpressed ACCa in C. reinhardtii to directly evaluate its effect on fatty acid synthesis. GC-MS analysis found that the unsaturated FAs contents of the CW15-24 and CW15-85 strains were 55.45% and 56.15%, which were significantly enriched compared to the wild type CW15 (48.39%). Under the optimized conditions, the content of lipid by overexpressed the ACCa gene in the mutant CW15-85 (0.46 g/l) was 1.16-fold greater than control through optimization of N and P sources. Altogether, our data clearly demonstrate that ACCa overexpression in C. reinhardtii can directly increase the synthesis of fatty acids.

mTORC2 Regulates Lipogenic Gene Expression through PPARγ to Control Lipid Synthesis in Bovine Mammary Epithelial Cells.

The mechanistic target of rapamycin complex 2 (mTORC2) primarily functions as an effector of insulin/PI3K signaling to regulate cell proliferation and is associated with cell metabolism. However, the function of mTORC2 in lipid metabolism is not well understood. In the present study, mTORC2 was inactivated by the ATP-competitive mTOR inhibitor AZD8055 or shRNA targeting RICTOR in primary bovine mammary epithelial cells (pBMECs). MTT assay was performed to examine the effect of AZD8055 on cell proliferation. ELISA assay and GC-MS analysis were used to determine the content of lipid. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. We found that cell proliferation, mTORC2 activation, and lipid secretion were inhibited by AZD8055. RICTOR was knocked down and mTORC2 activation was specifically attenuated by the shRNA. Compared to control cells, the expression of the transcription factor gene PPARG and the lipogenic genes LPIN1, DGAT1, ACACA, and FASN was downregulated in RICTOR silencing cells. As a result, the content of intracellular triacylglycerol (TAG), palmitic acid (PA), docosahexaenoic acid (DHA), and other 16 types of fatty acid was decreased in the treated cells; the accumulation of TAG, PA, and DHA in cell culture medium was also reduced. Overall, mTORC2 plays a critical role in regulating lipogenic gene expression, lipid synthesis, and secretion in pBMECs, and this process probably is through PPARγ. This finding provides a model by which lipogenesis is regulated in pBMECs.

The Translational Machinery of Human CD4+ T Cells Is Poised for Activation and Controls the Switch from Quiescence to Metabolic Remodeling.

Naive T cells respond to T cell receptor (TCR) activation by leaving quiescence, remodeling metabolism, initiating expansion, and differentiating toward effector T cells. The molecular mechanisms coordinating the naive to effector transition are central to the functioning of the immune system, but remain elusive. Here, we discover that T cells fulfill this transitional process through translational control. Naive cells accumulate untranslated mRNAs encoding for glycolysis and fatty acid synthesis factors and possess a translational machinery poised for immediate protein synthesis. Upon TCR engagement, activation of the translational machinery leads to synthesis of GLUT1 protein to drive glucose entry. Subsequently, translation of ACC1 mRNA completes metabolic reprogramming toward an effector phenotype. Notably, inhibition of the eIF4F complex abrogates lymphocyte metabolic activation and differentiation, suggesting ACC1 to be a key regulatory node. Thus, our results demonstrate that translation is a direct mediator of T cell metabolism and indicate translation factors as targets for novel immunotherapeutic approaches.

Overexpression of acetyl-CoA carboxylase in Aspergillus terreus to increase lovastatin production.

The present work describes the application of homologous recombination techniques in a wild-type Aspergillus terreus (ATCC 20542) strain to increase the flow of precursors towards the lovastatin biosynthesis pathway. A new strain was generated to overexpress acetyl-CoA carboxylase (ACCase) by replacing the native ACCase promoter with a strong constitutive PadhA promoter from Aspergillus nidulans. Glycerol and a mixture of lactose and glycerol were used independently as the carbon feedstock to determine the degree of response by the A. terreus strains towards the production of acetyl-CoA, and malonyl-CoA. The new strain increased the levels of malonyl-CoA and acetyl-CoA by 240% and 14%, respectively, compared to the wild-type strain. As a result, lovastatin production was increased by 40% and (+)-geodin was decreased by 31% using the new strain. This study shows for the first time that the metabolism of Aspergillus terreus can be manipulated to attain higher levels of precursors and valuable secondary metabolites.

Fatty Acid Synthase and Acetyl-CoA Carboxylase Are Expressed in Nodal Metastatic Melanoma But Not in Benign Intracapsular Nodal Nevi.

BACKGROUND: Melanoma is a potentially lethal form of skin cancer for which the current standard therapy is complete surgical removal of the primary tumor followed by sentinel lymph node biopsy when indicated. Histologic identification of metastatic melanoma in a sentinel node has significant prognostic and therapeutic implications, routinely guiding further surgical management with regional lymphadenectomy. While melanocytes in a lymph node can be identified by routine histopathologic and immunohistochemical examination, the distinction between nodal nevus cells and melanoma can be morphologically problematic. Previous studies have shown that malignant melanoma can over-express metabolic genes such as fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC). This immunohistochemical study aims to compare the utility of FASN and ACC in differentiating sentinel lymph nodes with metastatic melanomas from those with benign nodal nevi in patients with cutaneous melanoma. MATERIALS AND METHODS: Using antibodies against FASN and ACC, 13 sentinel lymph nodes from 13 patients with metastatic melanoma and 14 lymph nodes harboring benign intracapsular nevi from 14 patients with cutaneous malignant melanoma were examined. A diagnosis of nodal melanoma was based on cytologic atypia and histologic comparison with the primary melanoma. All nodal nevi were intracapsular and not trabecular. Immunohistochemistry for Melan-A, S100, human melanoma black 45 (HMB45), FASN, and ACC were performed. The percentage of melanocytes staining with HMB45, FASN, and ACC was determined and graded in 25% increments; staining intensity was graded as weak, moderate, or strong. RESULTS: All metastatic melanomas tested had at least 25% tumor cell staining for both FASN and ACC. Greater than 75% of the tumor cells stained with FAS in 7/13 cases and for ACC in 5/12 cases. Intensity of staining was variable; strong staining for FASN and ACC was observed in 69% and 50% of metastatic melanoma, respectively. HMB45 was negative in 40% of nodal melanoma cases all of which stained with FASN and ACC. Capsular nevi were uniformly negative for FASN, ACC, and HMB45 immunoreactivity. CONCLUSIONS: All metastatic melanoma cases involving sentinel lymph nodes were positive for FASN and ACC while no staining was observed in intracapsular nevi. These findings suggest that FASN and ACC could be used as valuable ancillary stains in the distinction between nodal nevi and metastatic melanoma.

Translational control of lipogenic enzymes in the cell cycle of synchronous, growing yeast cells.

Translational control during cell division determines when cells start a new cell cycle, how fast they complete it, the number of successive divisions, and how cells coordinate proliferation with available nutrients. The translational efficiencies of mRNAs in cells progressing synchronously through the mitotic cell cycle, while preserving the coupling of cell division with cell growth, remain uninvestigated. We now report comprehensive ribosome profiling of a yeast cell size series from the time of cell birth, to identify mRNAs under periodic translational control. The data reveal coordinate translational activation of mRNAs encoding lipogenic enzymes late in the cell cycle including Acc1p, the rate-limiting enzyme acetyl-CoA carboxylase. An upstream open reading frame (uORF) confers the translational control of ACC1 and adjusts Acc1p protein levels in different nutrients. The ACC1 uORF is relevant for cell division because its ablation delays cell cycle progression, reduces cell size, and suppresses the replicative longevity of cells lacking the Sch9p protein kinase regulator of ribosome biogenesis. These findings establish an unexpected relationship between lipogenesis and protein synthesis in mitotic cell divisions.

Hepatic Peroxisome Proliferator-Activated Receptor Gamma Signaling Contributes to Alcohol-Induced Hepatic Steatosis and Inflammation in Mice.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARγ) signaling has been shown to regulate lipogenesis and lipid accumulation. Previous studies have shown that hepatic PPARγ is up-regulated in steatotic liver of both animal and human. However, the effects of hepatic PPARγ signaling on alcoholic liver disease (ALD) remain elusive. METHODS: To determine the role of hepatic PPARγ signaling on ALD, wild-type (WT) and hepatocyte-specific PPARγ knockdown (PPARγ∆Hep) mice were fed a modified Lieber-DeCarli alcohol or isocaloric maltose dextrin control liquid diet for 8 weeks to induce ALD. Blood parameters, hepatic steatosis, and inflammation were measured after 8-week alcohol feeding. RESULTS: Alcohol feeding to WT mice resulted in liver damage (alanine aminotransferase [ALT], 94.68 ± 17.05 U/L; aspartate aminotransferase [AST], 55.87 ± 11.29 U/L), which was significantly alleviated by hepatic PPARγ knockdown (ALT, 57.36 ± 14.98 U/L; AST, 38.06 ± 3.35 U/L). Alcohol feeding led to marked lipid accumulation and up-regulation of lipogenic genes including fatty acid transport protein 1 (FATP1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), lipin1 (LIPIN1), diacylglycerol acyltransferase 1 (DGAT1), and diacylglycerol acyltransferase 2 (DGAT2) in the livers of WT mice. Knockdown of hepatic PPARγ significantly alleviated alcohol-induced lipid accumulation and abolished the up-regulation of FASN, DGAT1, and DGAT2. Silencing of PPARγ in FL83B cells significantly decreased ethanol (EtOH)-, linoleic acid-, and EtOH plus linoleic acid-induced lipid accumulation. Knockdown of hepatic PPARγ also significantly reduced alcohol-induced inflammatory chemokine (monocyte chemotactic protein 1 [MCP1], keratinocyte-derived chemokine [KC], interferon gamma-induced protein 10 [IP-10]) and inflammatory infiltration (lymphocyte antigen 6 complex, locus G [Ly6G], and F4/80). CONCLUSIONS: The results suggest that hepatic PPARγ signaling contributes to alcohol-induced liver injury by promoting hepatic steatosis and inflammation.

Carbon Fixation Driven by Molecular Hydrogen Results in Chemolithoautotrophically Enhanced Growth of Helicobacter pylori.

UNLABELLED: A molecular hydrogen (H2)-stimulated, chemolithoautotrophic growth mode for the gastric pathogen Helicobacter pylori is reported. In a culture medium containing peptides and amino acids, H2-supplied cells consistently achieved 40 to 60% greater growth yield in 16 h and accumulated 3-fold more carbon from [(14)C]bicarbonate (on a per cell basis) in a 10-h period than cells without H2 Global proteomic comparisons of cells supplied with different atmospheric conditions revealed that addition of H2 led to increased amounts of hydrogenase and the biotin carboxylase subunit of acetyl coenzyme A (acetyl-CoA) carboxylase (ACC), as well as other proteins involved in various cellular functions, including amino acid metabolism, heme synthesis, or protein degradation. In agreement with this result, H2-supplied cells contained 3-fold more ACC activity than cells without H2 Other possible carbon dioxide (CO2) fixation enzymes were not up-expressed under the H2-containing atmosphere. As the gastric mucus is limited in carbon and energy sources and the bacterium lacks mucinase, this new growth mode may contribute to the persistence of the pathogen in vivo This is the first time that chemolithoautotrophic growth is described for a pathogen. IMPORTANCE: Many pathogens must survive within host areas that are poorly supplied with carbon and energy sources, and the gastric pathogen Helicobacter pylori resides almost exclusively in the nutritionally stringent mucus barrier of its host. Although this bacterium is already known to be highly adaptable to gastric niches, a new aspect of its metabolic flexibility, whereby molecular hydrogen use (energy) is coupled to carbon dioxide fixation (carbon acquisition) via a described carbon fixation enzyme, is shown here. This growth mode, which supplements heterotrophy, is termed chemolithoautotrophy and has not been previously reported for a pathogen.

Berberine Ameliorates Hepatic Steatosis and Suppresses Liver and Adipose Tissue Inflammation in Mice with Diet-induced Obesity.

Increasing evidence demonstrates that berberine (BBR) is beneficial for obesity-associated non-alcoholic fatty liver disease (NAFLD). However, it remains to be elucidated how BBR improves aspects of NAFLD. Here we revealed an AMP-activated protein kinase (AMPK)-independent mechanism for BBR to suppress obesity-associated inflammation and improve hepatic steatosis. In C57BL/6J mice fed a high-fat diet (HFD), treatment with BBR decreased inflammation in both the liver and adipose tissue as indicated by reduction of the phosphorylation state of JNK1 and the mRNA levels of proinflammatory cytokines. BBR treatment also decreased hepatic steatosis, as well as the expression of acetyl-CoA carboxylase and fatty acid synthase. Interestingly, treatment with BBR did not significantly alter the phosphorylation state of AMPK in both the liver and adipose tissue of HFD-fed mice. Consistently, BBR treatment significantly decreased the phosphorylation state of JNK1 in both hepatoma H4IIE cells and mouse primary hepatocytes in both dose-dependent and time-dependent manners, which was independent of AMPK phosphorylation. BBR treatment also caused a decrease in palmitate-induced fat deposition in primary mouse hepatocytes. Taken together, these results suggest that BBR actions on improving aspects of NAFLD are largely attributable to BBR suppression of inflammation, which is independent of AMPK.

Reciprocal expression of p-AMPKa and p-S6 is strongly associated with the prognosis of gastric cancer.

Activation of AMP-activated protein kinase (AMPK) suppressed mammalian target of rapamycin (mTOR) pathway, resulting in impaired cancer cell proliferation. Two cohorts (50 and 1072 cases) of patients with resected gastric adenocarcinoma were enrolled in the study. Immunohistochemical staining for p-AMPKa, p-ACC, p-mTOR, p-S6, and p-4EBP1 was performed on the 50-patient cohort. Tissue microarray blocks containing samples from 1072 patients of Chinese ethnicity were used for the immunohistochemical detection of p-AMPKa and p-S6 levels. p-AMPK and p-ACC were frequently inactivated in both cohorts of gastric cancer samples, while p-mTOR, p-S6, and p-4EBP1 were frequently activated in the small cohort of gastric cancer. However, only levels of p-AMPKa and p-S6 were associated with the overall survival of gastric cancer patients. In the larger 1072-patient cohort, downregulation of p-AMPKa and upregulation of p-S6 were associated with tumor progression and were independent predictors of survival after resection of primary gastric cancer. Therefore, reciprocal expression of p-AMPKa and p-S6 may be promising prognostic biomarkers in patients with gastric cancer.

Dietary fiber prevents obesity-related liver lipotoxicity by modulating sterol-regulatory element binding protein pathway in C57BL/6J mice fed a high-fat/cholesterol diet.

Adequate intake of dietary fibers has proven metabolic and cardiovascular benefits, molecular mechanisms remain still limited. This study was aimed to investigate the effects of cereal dietary fiber on obesity-related liver lipotoxicity in C57BL/6J mice fed a high-fat/cholesterol (HFC) diet and underlying mechanism. Forty-eight adult male C57BL/6J mice were randomly given a reference chow diet, or a high fat/cholesterol (HFC) diet supplemented with or without oat fiber or wheat bran fiber for 24 weeks. Our results showed mice fed oat or wheat bran fiber exhibited lower weight gain, lipid profiles and insulin resistance, compared with HFC diet. The two cereal dietary fibers potently decreased protein expressions of sterol regulatory element binding protein-1 and key factors involved in lipogenesis, including fatty acid synthase and acetyl-CoA carboxylase in target tissues. At molecular level, the two cereal dietary fibers augmented protein expressions of peroxisome proliferator-activated receptor alpha and gamma, liver X receptor alpha, and ATP-binding cassette transporter A1 in target tissues. Our findings indicated that cereal dietary fiber supplementation abrogated obesity-related liver lipotoxicity and dyslipidemia in C57BL/6J mice fed a HFC diet. In addition, the efficacy of oat fiber is greater than wheat bran fiber in normalizing these metabolic disorders and pathological profiles.

Expression and evolution of the non-canonically translated yeast mitochondrial acetyl-CoA carboxylase Hfa1p.

The Saccharomyces cerevisiae genome encodes two sequence related acetyl-CoA carboxylases, the cytosolic Acc1p and the mitochondrial Hfa1p, required for respiratory function. Several aspects of expression of the HFA1 gene and its evolutionary origin have remained unclear. Here, we determined the HFA1 transcription initiation sites by 5' RACE analysis. Using a novel "Stop codon scanning" approach, we mapped the location of the HFA1 translation initiation site to an upstream AUU codon at position -372 relative to the annotated start codon. This upstream initiation leads to production of a mitochondrial targeting sequence preceding the ACC domains of the protein. In silico analyses of fungal ACC genes revealed conserved "cryptic" upstream mitochondrial targeting sequences in yeast species that have not undergone a whole genome duplication. Our Δhfa1 baker's yeast mutant phenotype rescue studies using the protoploid Kluyveromyces lactis ACC confirmed functionality of the cryptic upstream mitochondrial targeting signal. These results lend strong experimental support to the hypothesis that the mitochondrial and cytosolic acetyl-CoA carboxylases in S. cerevisiae have evolved from a single gene encoding both the mitochondrial and cytosolic isoforms. Leaning on a cursory survey of a group of genes of our interest, we propose that cryptic 5' upstream mitochondrial targeting sequences may be more abundant in eukaryotes than anticipated thus far.

Attenuation by a Vigna nakashimae extract of nonalcoholic fatty liver disease in high-fat diet-fed mice.

A Vigna nakashimae (VN) extract has been shown to have antidiabetic and anti-obesity effects. However, the mechanism underlying the effect of a VN extract on hepatic inflammation and endoplasmic reticulum (ER) stress remains unclear. In the present study, we investigated how a VN extract protects against the development of non-alcoholic fatty liver disease (NAFLD). A VN extract for 12 weeks reduced the body weight, serum metabolic parameters, cytokines, and hepatic steatosis in high-fat diet (HFD)-fed mice. A VN extract decreased HFD-induced hepatic acetyl CoA carboxylase and glucose transporter 4 expressions. In addition to the levels of high-mobility group box 1 and receptor for advanced glycation, the hepatic expression of ATF4 and caspase-3 was also reduced by a VN extract. Thus, these data indicate that a chronic VN extract prevented NAFLD through multiple mechanisms, including inflammation, ER stress, and apoptosis in the liver.

Silybin exerts antioxidant effects and induces mitochondrial biogenesis in liver of rat with secondary biliary cirrhosis.

The accumulation of toxic hydrophobic bile acids in hepatocytes, observed during chronic cholestasis, induces substantial modification in the redox state and in mitochondrial functions. Recent reports have suggested a significant role of impaired lipid metabolism in the progression of chronic cholestasis. In this work we report that changes observed in the expression of the lipogenic enzymes acetyl-CoA carboxylase and fatty acid synthase were associated with a decrease in the activity of citrate carrier (CIC), a protein of the inner mitochondrial membrane closely related to hepatic lipogenesis. We also verified that the impairment of citrate transport was dependent on modification of the phospholipid composition of the mitochondrial membrane and on cardiolipin oxidation. Silybin, an extract of silymarin with antioxidant and anti-inflammatory properties, prevented mitochondrial reactive oxygen species (ROS) production, cardiolipin oxidation, and CIC failure in cirrhotic livers but did not affect the expression of lipogenic enzymes. Moreover, supplementation of silybin was also associated with mitochondrial biogenesis. In conclusion, we demonstrate that chronic cholestasis induces cardiolipin oxidation that in turn impairs mitochondrial function and further promotes ROS production. The capacity of silybin to limit mitochondrial failure is part of its hepatoprotective property.

Overexpression of NgAUREO1, the gene coding for aurechrome 1 from Nannochloropsis gaditana, into Saccharomyces cerevisiae leads to a 1.6-fold increase in lipid accumulation.

Aureochrome-1 (AUREO1) is a transcription factor that is induced by blue light and controls branching of Vaucheria frigida. We have cloned the gene, NgAUREO1, coding for AUREO1 from Nannochloropsis gaditana, and report that the lipid content in recombinant Saccharomyces cerevisiae was 1.6-fold more than in wild-type S. cerevisiae (6.3 % lipid increased to 10 %). Over-expression of AUREO1 in S. cerevisiae up-regulated the expression of acetyl-CoA carboxylase and acyl-CoA:diacylglycerol acyl-transferase but down-regulated the expression of long-chain-acyl CoA synthetase. This enhanced the accumulation of lipid. This study highlights a novel function of AUREO1 and allows a better understanding of the regulation mechanism of fatty acid metabolism.

Effects of short-term refeeding on the expression of genes involved in lipid metabolism in chicks (Gallus gallus).

The aim of this study was to analyze the expression patterns of key genes involved in lipid metabolism in response to feeding in chicks. A total of 18 thirteen day-old male chicks were fasted for 12h. The mRNA levels of the genes in the liver and white adipose tissue were analyzed after 0, 2, and 4h of refeeding. The mRNA levels of sterol regulatory element-binding protein (SREBP) 1, liver X receptor α, peroxisome proliferator-activated receptor (PPAR) γ, acetyl-CoA carboxylase α and fatty acid synthase were significantly increased after 2h of refeeding. In contrast, the mRNA levels of PPARα and carnitine palmitoyltransferase 1a were significantly decreased after 2h of refeeding. The mRNA level of acyl-CoA oxidase was significantly decreased after 4h of refeeding. The mRNA levels of cholesterol metabolism-related genes such as SREBP2 and 3-hydroxy-3-methylglutaryl-CoA reductase were significantly increased after 2h of refeeding. In the white adipose tissue, the mRNA level of PPARγ was significantly increased after 2h of refeeding, whereas the mRNA level of adipose triglyceride lipase was significantly decreased after 4h of refeeding. These results demonstrated that expression of lipid metabolism-related genes is regulated by short-term refeeding in chicks.

Molecular analysis of resistance to acaricidal spirocyclic tetronic acids in Tetranychus urticae: CYP392E10 metabolizes spirodiclofen, but not its corresponding enol.

Spirodiclofen is one of the most recently developed acaricides and belongs to the new family of spirocyclic tetronic acids (ketoenols). This new acaricidal family is an important chemical tool in resistance management strategies providing sustainable control of spider mites such as Tetranychus urticae. Spirodiclofen targets lipid biosynthesis mediated by direct inhibition of acetyl coenzyme A carboxylase (ACCase). In this study, we investigated two genetically distant spider mite strains with high resistance to spirodiclofen. Despite the strong resistance levels to spirodiclofen (up to 680-fold), only limited cross-resistance with other members of this group such as spiromesifen and spirotetramat could be detected. Amplification and sequencing of the ACCase gene from resistant and susceptible strains did not reveal common non-synonymous mutations, and expression levels of ACCase were similar in both resistant and susceptible strains, indicating the absence of target-site resistance. Furthermore, we collected genome-wide expression data of susceptible and resistant T. urticae strains using microarray technology. Analysis of differentially expressed genes revealed a broad response, but within the overlap of two resistant strains, several cytochrome P450s were prominent. Quantitative PCR confirmed the constitutive over-expression of CYP392E7 and CYP392E10 in resistant strains, and CYP392E10 expression was highly induced by spirodiclofen. Furthermore, stage specific expression profiling revealed that expression levels were not significantly different between developing stages, but very low in eggs, matching the age-dependent resistance pattern previously observed. Functional expression of CYP392E7 and CYP392E10 confirmed that CYP392E10 (but not CYP392E7) metabolizes spirodiclofen by hydroxylation as identified by LC-MS/MS, and revealed cooperative substrate binding and a Km of 43 µM spirodiclofen. CYP392E10 also metabolizes spiromesifen, but not spirotetramat. Surprisingly, no metabolism of the hydrolyzed spirodiclofen-enol metabolite could be detected. These findings are discussed in the light of a likely resistance mechanism.

Adaptive metabolic response to 4 weeks of sugar-sweetened beverage consumption in healthy, lightly active individuals and chronic high glucose availability in primary human myotubes.

PURPOSE: Chronic sugar-sweetened beverage (SSB) consumption is associated with obesity and type 2 diabetes mellitus (T2DM). Hyperglycaemia contributes to metabolic alterations observed in T2DM, such as reduced oxidative capacity and elevated glycolytic and lipogenic enzyme expressions in skeletal muscle tissue. We aimed to investigate the metabolic alterations induced by SSB supplementation in healthy individuals and to compare these with the effects of chronic hyperglycaemia on primary muscle cell cultures. METHODS: Lightly active, healthy, lean subjects (n = 11) with sporadic soft drink consumption underwent a 4-week SSB supplementation (140 ± 15 g/day, ~2 g glucose/kg body weight/day, glucose syrup). Before and after the intervention, body composition, respiratory exchange ratio (RER), insulin sensitivity, muscle metabolic gene and protein expression were assessed. Adaptive responses to hyperglycaemia (7 days, 15 mM) were tested in primary human myotubes. RESULTS: SSB supplementation increased fat mass (+1.0 kg, P < 0.05), fasting RER (+0.12, P < 0.05), fasting glucose (+0.3 mmol/L, P < 0.05) and muscle GAPDH mRNA expressions (+0.94 AU, P < 0.05). PGC1α mRNA was reduced (-0.20 AU, P < 0.05). Trends were found for insulin resistance (+0.16 mU/L, P = 0.09), and MondoA protein levels (+1.58 AU, P = 0.08). Primary myotubes showed elevations in GAPDH, ACC, MondoA and TXNIP protein expressions (P < 0.05). CONCLUSION: Four weeks of SSB supplementation in healthy individuals shifted substrate metabolism towards carbohydrates, increasing glycolytic and lipogenic gene expression and reducing mitochondrial markers. Glucose-sensing protein MondoA might contribute to this shift, although further in vivo evidence is needed to corroborate this.

Effect of cholesterol on lipogenesis and VLDL-TG assembly and secretion in goose primary hepatocytes.

To investigate how cholesterol induces hepatocytic steatosis, we investigated the effect of cholesterol on hepatic lipogenesis and the assembly and secretion of very-low-density lipoprotein-triglycerides (VLDL-TGs) in goose primary hepatocytes. We found that cholesterol at 20 µg/ml increased the concentrations of extracellular VLDL, intracellular cholesterol, and intracellular TGs, while cholesterol at 30 µg/ml had a reduced effect (p < 0.05). Additionally, cholesterol at 20 µg/ml, but not at 10 or 30 µg/ml, increased the extracellular TG concentration. Cholesterol increased the fatty acid synthase (FAS) enzyme activity in a dose-dependent manner. Incubation with cholesterol increased the mRNA level of genes involved in lipogenesis, including sterol regulatory element-binding proteins (SREBPs), FAS, acetyl-CoA carboxylase-α (ACCα), and liver X receptors. The mRNA level of the acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1) gene changed in response to cholesterol treatment in a dose-dependent manner. Similar to the regulation of extracellular VLDL and intracellular TG accumulation, the mRNA levels of the microsomal triglyceride transfer protein, forkhead box O1, and DGAT2 increased with treatment with 10 or 20 µg/ml of cholesterol, but decreased with treatment with 30 µg/ml of cholesterol (p < 0.05). Cholesterol had no evident effect on the mRNA level of the apolipoprotein B gene. Incubation with cholesterol at 20 and 30 µg/ml increased the nuclear SREBP-1 protein level (p < 0.05) and the binding affinity of the nuclear SREBP-1 to ACCα SRE probes. In conclusion, cholesterol not only activates the transcription of genes involved in fatty acid synthesis and TG accumulation, but also activates the transcription of genes involved in the assembly and secretion of VLDL-TG in goose primary hepatocytes.