Team:Hong Kong HKUST/Project/module2

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<a href=#1>Fatty Acid Quantification</a>
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Revision as of 19:51, 27 September 2013


Fatty Acid Sensing Mechanism

Overview

In 2009, Prof. James Liao's research group at UCLA published their findings that mice expressing synthetic glyoxylate shunt had increased resistance to diet-induced obesity. To engineer this behavior in mice, they introduced glyoxylate shunt genes to mouse liver cells, employing a constitutive promoter for expression of the said genes. The aim of this module is to introduce an inducible system that allows tunable fatty acid uptake by sensing fatty acid concentrations. Such a system would reduce the risk of fatty acid deficiency when fatty acid concentration is below normal.

Four different fatty acid induced promoters were investigated, namely: Liver Fatty Acid Binding Protein 1 (FABP1) promoter, Peroxisome Proliferator-Activated Receptor-alpha (PPAR-alpha) promoter, Glucose Regulated Protein (GRP78) promoter, Fatty Acid Metabolism Regulator Protein (FadR) and pFadBA promoter.

Cell Viability

We worked to introduce an inducible system that allows tunable fatty acid uptake regulated by fatty acid concentrations. Fatty acid uptake was to be quantified to compare the activity of wild type cells with the activity of our engineered cells expressing inducible glyoxylate shunt. High fatty acid levels are known to lead to apoptosis, so we conducted cell viability tests using MTT assay at different sodium palmitate concentrations. The objective was to determine the range of fatty acid concentrations to be introduced into our cells that would allow more than 60% viability after 24 hours incubation and/or more than 50% in 48 hours.

Fatty Acid Quantification

Two fatty acid quantification methods were investigated to measure fatty acid uptake rate of our constitutive and inducible glyoxylate systems: 1) Gas Chromatography-Mass Spectrophotometry (GC-MS), and 2) fatty acid quantification kit (Sigma-Aldrich; St. Louis, MO). While we managed to measure the fatty acid quantity in cell culture medium using GC-MS, we were not able to use the fatty acid quantification kit due to time limitations.

Liver Fatty Acid Binding Protein 1 (FABP1) promoter

Fatty acid binding proteins (FABPs) are lipid-binding proteins that regulate fatty acid uptake and transfer between extra-and intracellular membranes. There are 9 different FABPs identified with tissue-specific distribution, including FABP1 in liver. Some, such as PPAR, are believed to transport fatty acids from the plasma membrane to intracellular receptors, and as such have a selective cooperation with the receptor. The FABP1 promoter was designed to sense fatty acid concentration and drive expression of glyoxylate genes.

Peroxisome Proliferator-Activated Receptor-alpha (PPAR-alpha) Promoter

The peroxisome proliferator – activated receptors (PPARs) function s transcription factors to regulate expression of genes. The expression of PPAR-alpha can be up-regulated by increased fatty acid concentration in mammalian liver cells. The promoter of PPAR-alpha is constitutive, white elevated extracellular palmitate amount to 150uM increases expression of genes by over 4 times in 48 hours. In our project, PPAR-alpha promoter was used to sense fatty acid and perform inducible glyoxylate shunt.

Glucose Regulated Protein (GRP78) Promoter

GRP78 (HSPA5) is involved in the folding and assembly of proteins in the endoplasmic reticulum (ER). High concentration of fatty acids disrupts cell homeostasis, causing endoplasmic reticulum stress (ERS). This in turn, activates the unfolded protein response (UPR) that consists of three transmembrane proteins: IRE1, PERK and ATF6. Three signals constitutively activate the GRP78 promoter with the help of other factors, such as NF-Y, ERSF, YY1 and cleaved ATF6, acquired from the normal stress response followed by UPR. The activated GRP78 promoter by high fatty acid concentration is used to drive increased expression of glyoxylate genes.

Fatty Acid Metabolism Regulator Protein (FadR) and pFadBA

FadR is a bacterial transcription factor that regulates lipid metabolism of fatty acid biosynthesis and beta-oxidation. The binding of FadR is inhibited by fatty acyl-CoA compounds, which are intermediates of fatty acid degradation. In the absence of fatty acid, a constitutively expressed fatty acid metabolism regulator protein FadR binds to Pfad promoter (pFadBA) and inhibits the expression of glyoxylate genes.

References

1 Guzman, Carla et al. "The human liver fatty acid binding protein (FABP1) gene is activated by FOXA1 and PPARα; and repressed by C/EBPα: Implications in FABP1 down-regulation in nonalcoholic fatty liver disease." Biochemica et Biophysica Acta (BBA) - Molecular and Cell Biology. 1831.4 (April 2013): 803-818. Web. 23 Sep. 2013. .

2 Ines Pineda Torra et al. “Characterization of the human PPARalpha promoter: Identification of a functional Nuclear Receptor Response Element.”