Virginia McDonough

Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in Schizosaccharomyces pombe

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 °C and 30 °C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 °C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 °C compared to supplemented cells at 30 °C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.

Mutations in erg4 affect the sensitivity of Saccharomyces cerevisiae to medium-chain fatty acids

We have found that the medium-chain fatty acids (MCFAs) undecanoic acid (11:0), 10-undecenoic acid (11:1 Delta 10), and lauric acid (12:0) can affect the growth of Saccharomyces cerevisiae in a dose-dependent manner. The principal effect was a longer lag phase in MCFA-containing medium, although higher concentrations of 11:1 Delta 10 inhibited growth. Their relative order of inhibitory action was 11:1 Delta 10>11:0>12:0. Cellular content with MCFA supplementation was dependent on the concentration and the particular species of fatty acid, with 12:0 showing the highest relative accumulation and 11:1 Delta 10 the lowest at all concentrations. We have isolated and characterized a mutant that is hypersensitive to MCFA supplementation and is unable to grow at the normally permissive condition of 1 mM 11:1 Delta 10. However, it does not appear to accumulate higher relative levels of the fed MCFA compared to wild-type cells. Complementation of the mutant revealed that the ERG4 gene, encoding the enzyme that catalyzes the last step in ergosterol biosynthesis, had been mutated. The fatty acid composition of the erg4 Delta mutant differs only slightly from wild-type cells, mainly involving an increase in the relative amount of 12:0. These results indicate that yeast require ergosterol for optimal growth on certain MCFAs. We discuss the role ergosterol may have in cells responding to exogenous MCFAs and in supporting optimal cell growth.

Improving Journal Club: Increasing Student Discussion and Understanding of Primary Literature in Molecular Biology through the Use of Dialectical Notes.

Reading the primary literature in a journal club format is an excellent practice where undergraduate students can develop their abilities in experimental data analysis and critical thinking, learn about new ideas and methods, and gain a foothold in scientific discourse. However, students are not familiar with the format, writing style, and depth of knowledge assumed when first reading journal articles. This can inhibit class discussion. To alleviate this problem, the author has instituted the use of dialectical notes in journal club, so that students fully engage the article. This novel use of a strategy borrowed from humanities has improved class participation.

A role for MGA2, but not SPT23, in activation of transcription of ERG1 in Saccharomyces cerevisiae.

The SaccharomycescerevisiaeMGA2 gene encodes an important regulator of unsaturated fatty acid production, by controlling transcription and mRNA stability of OLE1, the gene encoding the Δ9 fatty acid desaturase. Lipid composition studies indicated that the mga2Δ strain contains elevated relative amounts of squalene when compared to wild-type cells. The deletion of the MGA2 homologue SPT23 did not impact squalene levels. To explore the role of MGA2 in the regulation of sterol synthesis, the transcription of the ERG1 gene, which encodes squalene epoxidase, was studied using an ERG1 promoter-lacZ reporter gene construct. We report here that in addition to MGA2s role in regulation of unsaturated fatty acids, MGA2 is required for full basal expression of ERG1. Mga2p was found to be controlled by a novel regulator in its activation of ERG1, as neither unsaturated fatty acids nor cobalt affected ERG1 expression, as had previously been shown for Mga2ps regulation of OLE1. Further, response to miconazole treatment, which inhibits production of ergosterol at a later step in the sterol biosynthetic pathway and results in up-regulation of several genes in ergosterol synthesis, was not affected in the mga2Δ mutant. In each case, the spt23Δ mutant strain shows similar ERG1 expression to wild-type cells, while the mga2Δ/spt23Δ strain shows reduced ERG1 expression, comparable to the mga2Δ, suggesting that the role of regulation of ERG1 transcription is unique to Mga2p.

Mice lacking ARV1 have reduced signs of metabolic syndrome and non-alcoholic fatty liver disease

Metabolic syndrome (MetS) is a term used to characterize individuals having at least three of the following diseases: obesity, dyslipidemia, hyperglycemia, insulin resistance, hypertension, and nonalcoholic fatty liver disease (NAFLD). It is widespread, and the number of individuals with MetS is increasing. However, the events leading to the manifestation of MetS are not well-understood. Here, we show that loss of murine ARV1 (mARV1) results in resistance to acquiring diseases associated with MetS. Arv1−/− animals fed a high-fat diet were resistant to diet-induced obesity, had lower blood cholesterol and triglyceride levels, and retained glucose tolerance and insulin sensitivity. Livers showed no gross morphological changes, contained lower levels of cholesterol, triglycerides, and fatty acids, and showed fewer signs of NAFLD. Knockout animals had elevated levels of liver farnesol X receptor (FXR) protein and its target, small heterodimer protein (SHP). They also had decreased levels of CYP7α1, CYP8β1, and mature SREBP1 protein, evidence suggesting that liver FXR signaling was activated. Strengthening this hypothesis was the fact that peroxisome proliferator-activating receptor α (PPARα) protein was elevated, along with its target, fibroblast growth factor 21 (FGF21). Arv1−/− animals excreted more fecal cholesterol, free fatty acids, and bile acids. Their small intestines had 1) changes in bile acid composition, 2) an increase in the level of the intestinal FXR antagonist, tauromuricholic acid, and 3) showed signs of attenuated FXR signaling. Overall, we believe that ARV1 function is deleterious when consuming a high-fat diet. We further hypothesize that ARV1 is critical for initiating events required for the progression of diseases associated with MetS and NAFLD.