Shona Morrison

Chemerin, a novel adipokine in the regulation of angiogenesis

CONTEXT Chemerin is a new adipokine associated with obesity and the metabolic syndrome. Gene expression levels of chemerin were elevated in the adipose depots of obese compared with lean animals and was markedly elevated during differentiation of fibroblasts into mature adipocytes. OBJECTIVE The objective of the study was to identify factors that affect the regulation and potential function of chemerin using a genetics approach. DESIGN, SETTING, PATIENTS, AND INTERVENTION Plasma chemerin levels were measured in subjects from the San Antonio Family Heart Study, a large family-based genetic epidemiological study including 1354 Mexican-American individuals. Individuals were randomly sampled without regard to phenotype or disease status. MAIN OUTCOME MEASURES A genome-wide association analysis using 542,944 single-nucleotide polymorphisms in a subset of 523 of the same subjects was undertaken. The effect of chemerin on angiogenesis was measured using human endothelial cells and interstitial cells in coculture in a specially formulated medium. RESULTS Serum chemerin levels were found to be highly heritable (h(2) = 0.25; P = 1.4 x 10(-9)). The single-nucleotide polymorphism showing strongest evidence of association (rs347344; P = 1.4 x 10(-6)) was located within the gene encoding epithelial growth factor-like repeats and discoidin I-like domains 3, which has a known role in angiogenesis. Functional angiogenesis assays in human endothelial cells confirmed that chemerin significantly mediated the formation of blood vessels to a similar extent as vascular endothelial growth factor. CONCLUSION Here we demonstrate for the first time that plasma chemerin levels are significantly heritable and identified a novel role for chemerin as a stimulator of angiogenesis.

Rapid Development of Non-Alcoholic Steatohepatitis in Psammomys obesus (Israeli Sand Rat)

Background and Aims A major impediment to establishing new treatments for non-alcoholic steatohepatitis is the lack of suitable animal models that accurately mimic the biochemical and metabolic characteristics of the disease. The aim of this study was to explore a unique polygenic animal model of metabolic disease as a model of non-alcoholic steatohepatitis by determining the effects of 2% dietary cholesterol supplementation on metabolic and liver endpoints in Psammomys obesus (Israeli sand rat). Methods P. obesus were provided ad libitum access to either a standard rodent diet (20% kcal/fat) or a standard rodent diet supplemented with 2% cholesterol (w/w) for 4 weeks. Histological sections of liver from animals on both diets were examined for key features of non-alcoholic steatohepatitis. The expression levels of key genes involved in hepatic lipid metabolism were measured by real-time PCR. Results P. obesus fed a cholesterol-supplemented diet exhibited profound hepatomegaly and steatosis, and higher plasma transaminase levels. Histological analysis identified extensive steatosis, inflammation, hepatocyte injury and fibrosis. Hepatic gene expression profiling revealed decreased expression of genes involved in delivery and uptake of lipids, and fatty acid and triglyceride synthesis, and increased expression of genes involved in very low density lipoprotein cholesterol synthesis, triglyceride and cholesterol export. Conclusions P. obesus rapidly develop non-alcoholic steatohepatitis when fed a cholesterol-supplemented diet that appears to be histologically and mechanistically similar to patients.

Mitochondrial dysfunction has divergent, cell type-dependent effects on insulin action

The contribution of mitochondrial dysfunction to insulin resistance is a contentious issue in metabolic research. Recent evidence implicates mitochondrial dysfunction as contributing to multiple forms of insulin resistance. However, some models of mitochondrial dysfunction fail to induce insulin resistance, suggesting greater complexity describes mitochondrial regulation of insulin action. We report that mitochondrial dysfunction is not necessary for cellular models of insulin resistance. However, impairment of mitochondrial function is sufficient for insulin resistance in a cell type-dependent manner, with impaired mitochondrial function inducing insulin resistance in adipocytes, but having no effect, or insulin sensitising effects in hepatocytes. The mechanism of mitochondrial impairment was important in determining the impact on insulin action, but was independent of mitochondrial ROS production. These data can account for opposing findings on this issue and highlight the complexity of mitochondrial regulation of cell type-specific insulin action, which is not described by current reductionist paradigms.

Compensatory regulation of HDAC5 in muscle maintains metabolic adaptive responses and metabolism in response to energetic stress

Some gene deletions or mutations have little effect on metabolism and metabolic adaptation because of redundancy and/or compensation in metabolic pathways. The mechanisms for redundancy and/ or compensation in metabolic adaptation in mammalian cells are unidentified. Here, we show that in mouse muscle and myogenic cells, compensatory regulation of the histone deacetylase (HDAC5) transcriptional repressor maintains metabolic integrity. HDAC5 phosphorylation regulated the expression of diverse metabolic genes and glucose metabolism in mouse C2C12 myogenic cells. However, loss of AMP‐activated protein kinase (AMPK), a HDAC5 kinase, in muscle did not affect HDAC5 phosphorylation in mouse skeletal muscle during exercise, but resulted in a compensatory increase (32.6%) in the activation of protein kinase D (PKD), an alternate HDAC5 kinase. Constitutive PKD activation in mouse C2C12 myogenic cells regulated metabolic genes and glucose metabolism. Although aspects of this response were HDAC5 phosphorylation dependent, blocking HDAC5 phosphorylation when PKD was active engaged an alternative compensatory adaptive mechanism, which involved post‐transcriptional reductions in HDAC5 mRNA (–93.1%) and protein. This enhanced the expression of a specific subset of metabolic genes and mitochondrial metabolism. These data show that compensatory regulation of HDAC5 maintains metabolic integrity in mammalian cells and reinforces the importance of preserving the cellular metabolic adaptive response.—McGee, S. L., Swinton, C., Morrison, S., Gaur, V., Campbell, D. E., Jorgensen, S. B., Kemp, B. E., Baar, K., Steinberg, G. R., Hargreaves, M. Compensatory regulation of HDAC5 in muscle maintains metabolic adaptive responses and metabolism in response to energetic stress. FASEB J. 28, 3384–3395 (2014). www.fasebj.org

Methazolamide Is a New Hepatic Insulin Sensitizer That Lowers Blood Glucose In Vivo

We previously used Gene Expression Signature technology to identify methazolamide (MTZ) and related compounds with insulin sensitizing activity in vitro. The effects of these compounds were investigated in diabetic db/db mice, insulin-resistant diet-induced obese (DIO) mice, and rats with streptozotocin (STZ)-induced diabetes. MTZ reduced fasting blood glucose and HbA1c levels in db/db mice, improved glucose tolerance in DIO mice, and enhanced the glucose-lowering effects of exogenous insulin administration in rats with STZ-induced diabetes. Hyperinsulinemic-euglycemic clamps in DIO mice revealed that MTZ increased glucose infusion rate and suppressed endogenous glucose production. Whole-body or cellular oxygen consumption rate was not altered, suggesting MTZ may inhibit glucose production by different mechanism(s) to metformin. In support of this, MTZ enhanced the glucose-lowering effects of metformin in db/db mice. MTZ is known to be a carbonic anhydrase inhibitor (CAI); however, CAIs acetazolamide, ethoxyzolamide, dichlorphenamide, chlorthalidone, and furosemide were not effective in vivo. Our results demonstrate that MTZ acts as an insulin sensitizer that suppresses hepatic glucose production in vivo. The antidiabetic effect of MTZ does not appear to be a function of its known activity as a CAI. The additive glucose-lowering effect of MTZ together with metformin highlights the potential utility for the management of type 2 diabetes.

DHA protects against zinc mediated alterations in neuronal cellular bioenergetics.

Zinc accumulation may impair cellular bioenergetics, which is associated with neuronal apoptosis. We simultaneously assessed anaerobic and aerobic metabolism in live cells to characterise this effect and hypothesised that the omega 3 fatty acid docosahexaenoic acid (DHA) would protect against any zinc mediated alterations in bioenergetics. In this study we observed a decrease in cellular oxygen consumption, but not glycolytic rate, following chronic zinc exposure, which was specific for neuronal cells. This was due to impaired ATP turnover, without any other effects on mitochondrial function, and was restored by DHA. DHA had no further effects on bioenergetics. These data suggest that zinc disrupts bioenergetics at a point distal to the respiratory chain, which is restored by DHA.

3T3-L1 adipocytes display phenotypic characteristics of multiple adipocyte lineages

Differentiated 3T3-L1 adipocytes are a widely used in vitro model of white adipocytes. In addition to classical white and brown adipocytes that are derived from different cell lineages, beige adipocytes have also been identified, which have characteristics of both white and brown adipocytes. Here we show that 3T3-L1 adipocytes display features of multiple adipocytes lineages. While the gene expression profile and basal bioenergetics of 3T3-L1 adipocytes was typical of white adipocytes, they responded acutely to catecholamines by increasing oxygen consumption in an UCP1-dependent manner, and by increasing the expression of genes enriched in brown but not beige adipocytes. Chronic exposure to catecholamines exacerbated this phenotype. However, a beige adipocyte differentiation procedure did not induce a beige adipocyte phenotype in 3T3-L1 fibroblasts. These multiple lineage features should be considered when interpreting data from experiments utilizing 3T3-L1 adipocytes.

Zinc finger nuclease mediated knockout of ADP-dependent glucokinase in cancer cell lines: effects on cell survival and mitochondrial oxidative metabolism.

Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.

Lipid Abundance in Zebrafish Embryos Is Regulated by Complementary Actions of the Endocannabinoid System and Retinoic Acid Pathway

The endocannabinoid system (ECS) and retinoic acid (RA) signaling have been associated with influencing lipid metabolism. We hypothesized that modulation of these pathways could modify lipid abundance in developing vertebrates and that these pathways could have a combinatorial effect on lipid levels. Zebrafish embryos were exposed to chemical treatments altering the activity of the ECS and RA pathway. Embryos were stained with the neutral lipid dye Oil-Red-O (ORO) and underwent whole-mount in situ hybridization (WISH). Mouse 3T3-L1 fibroblasts were differentiated under exposure to RA-modulating chemicals and subsequently stained with ORO and analyzed for gene expression by qRT-PCR. ECS activation and RA exposure increased lipid abundance and the expression of lipoprotein lipase. In addition, RA treatment increased expression of CCAAT/enhancer-binding protein alpha. Both ECS receptors and RA receptor subtypes were separately involved in modulating lipid abundance. Finally, increased ECS or RA activity ameliorated the reduced lipid abundance caused by peroxisome proliferator-activated receptor gamma (PPARγ) inhibition. Therefore, the ECS and RA pathway influence lipid abundance in zebrafish embryos and have an additive effect when treated simultaneously. Furthermore, we demonstrated that these pathways act downstream or independently of PPARγ to influence lipid levels. Our study shows for the first time that the RA and ECS pathways have additive function in lipid abundance during vertebrate development.

Scriptaid enhances skeletal muscle insulin action and cardiac function in obese mice

To determine the effect of Scriptaid, a compound that can replicate aspects of the exercise adaptive response through disruption of the class IIa histone deacetylase (HDAC) corepressor complex, on muscle insulin action in obesity.