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Non-transformed cells respond to fat by inducing glucose metabolism

ABSTRACT: Purpose: To study the effects of high-fat diet feeding in mouse liver tissues with and without the hepatocellular carcinoma-inducing carcinogen DEN. Here, we used RNA sequencing to identify gene expression patterns associated with high-fat diet feeding. Methods: C57BL/6N mice were injected at 2-weeks of age with vehicle control (PBS) or DEN. At 6 weeks, mice were randomized to control diet, or 60% high-fat diet. After 8-weeks of diet exposure, mice underwent a 13C6-glucose labelling protocol, and liver tissues were extracted for anlaysis (metabolomic, transcriptomic). RNA was isolated from mouse liver tissue using Triazol extraction, purified, and libraries generated using KAPA Stranded mRNA Sequencing kit. After cDNA synthesis, adapter ligation, and final cDNA library generation, samples were sequenced on a flow cell (1x50bp single-end reads) and HiSeq4000 (Illumina). Data processing was conducted in an NGS pipeline (Snakemake) and quality control was performed with FastQC. Trimmed data was analyzed for differential expression (DEseq2) and gene set enrichment analysis (GSEA) to look for KEGG pathways and gene ontologies (GO) of interest. Results: Using 13C6-glucose labelling, we determined there was a glycolytic phenotype caused by high-fat diet exposure. Therefore, we focused on a diet effect in our transcriptomcis dataset. By comparing control diet to high-fat diet liver tissue, we found gene sets invloved in peroxisomes and lipid metabolism to be enriched in high-fat diet exposed livers. These findings were in line with in vitro testing of a liver cell line, showing peroxisomal metabolism of palmitate drives ROS production and a glycolytic phenotype. Conclusions: We found high-fat diet exposed liver exhibits a strong metabolic phenotype towards increased glucose metabolism. At the transcriptome level, we found a lipid-reporgramming signature, and not a glucose metabolism signature. The lipid reprogramming signature was in line with in vitro work using liver cell lines, in which exposure to palmitate stimulated a glycolyitic phenotype that was inhibited by targeting peroxisomal-derived ROS species. In combination with metabolic and lipidomic data after long-term exposure to DEN and subsequent tumor formtion, we discovered that high-fat diet can prime liver tissue for carcinogenesis and tumor development by stimulating a Warburg-like metabolism. These findings suggest that fat can induce similar changes in non-transformed liver cells than found in HCC. In conclusion, we show that normal, non-transformed livers respond to fat by inducing glucose metabolism.

ORGANISM(S): Mus musculus

PROVIDER: GSE165752 | GEO | 2021/02/22

REPOSITORIES: GEO

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Project description:Hepatic fat accumulation has been widely associated with diabetes and hepatocellular carcinoma (HCC). Here, we aim to characterize the metabolic response that high fat availability elicits in livers prior to development of these diseases. We find that, after a short term on high fat diet, otherwise healthy mice show elevated hepatic glucose metabolization, activated glucose uptake, glycolysis and glucose contribution to serine as well as elevated pyruvate carboxylase activity compared to control diet mice. To understand other changes in the liver tissue after high fat diet exposure, we conducted untargeted transcriptomics and proteomics. This glucose phenotype occurred independent from transcriptional or proteomic programming, which identified increased peroxisomal and lipid metabolism pathways. Interestingly, we observe that high fat diet fed mice exhibit an increased lactate production when challenged with glucose. This trait seems to find a parallel in a human cohort, where we observe a correlation between waist circumference and lactate secretion after an oral glucose bolus across healthy individuals. In an in vitro model of hepatoma cells, we found physiologically relevant palmitate exposure stimulated production of reactive oxygen species (ROS) and glucose uptake, a similar glycolytic phenotype to the in vivo study. This effect is inhibited upon interference with peroxisomal lipid metabolism and ROS production. Furthermore, we find that with exposure to an HCC-inducing hepatic carcinogen, continuation of high fat diet enhances the formation of HCC (100% with resectable tumors) as compared to control (50% with resectable tumors) in mice. However, regardless of the dietary background, all murine tumors showed similar alterations in glucose metabolism compared to those identified in fat exposed non-transformed mouse livers. Further, the presence of tumors in high fat diet exposed mice normalized glucose tolerance. Lipidomics analysis of tumor tissue and liver tissue from high fat diet exposed mice identified tumor tissue enrichment of diacylglycerol (DG) and phosphatidylcholine (PC) species. Some of these species were also increased in high fat diet liver tissue compared to control diet liver tissue. These findings suggest that fat can induce similar metabolic changes in non-transformed liver cells than found in HCC, and that peroxisomal metabolism of lipids may be a factor in driving a glycolytic metabolism In conclusion, we show that normal, non-transformed livers respond to fat by inducing glucose metabolism.

Project description:Age-related and/or high caloric intake driven changes in visceral adipose tissue contribute to the development of diabetes and cardiovascular disease. The lack of reliable, high-throughput methods to analyze the adipose tissue protein composition has limited our understanding of the protein networks responsible for metabolic regulation of various adipose depots. We have developed a proteomic approach using multiple-dimension liquid chromatography tandem mass spectrometry and multiplexed TMT labeling to analyze protein composition of epididymal adipose tissues isolated from mice fed either low or high fat diet for a short (8 weeks) or a long (18 weeks) term, and from mice that aged (26 weeks old) on low vs. high fat diets. Advancing age and high-fat diet feeding led to graded deterioration, with long-term high fat diet exposure being the worst of all measures of peripheral metabolic health such as body weight, adiposity, plasma fasting glucose, insulin, triglycerides, total cholesterol levels, and glucose and insulin tolerance tests. Epididymal adipose depot proteomic analysis identified >3300 proteins per sample. In response to short-term high fat diet, 43 proteins representing lipid metabolism (e.g., AACS, ACOX1, ACLY) and red-ox pathways (e.g., CPD2, CYP2E, SOD3) were significantly altered (FDR < 10%). Long-term high fat diet significantly altered 55 proteins associated with immune response (e.g., IGTB2, IFIT3, LGALS1) and rennin angiotensin system (e.g. ENPEP, CMA1, CPA3, ANPEP). Age-related changes on low fat diet significantly altered only 18 proteins representing mainly urea cycle (e.g., OTC, ARG1, CPS1), and amino acid biosynthesis (e.g., GMT, AKR1C6). Surprisingly, high fat diet driven age-related changes culminated with alterations in 155 proteins involving primarily the urea cycle (e.g., ARG1, CPS1), immune response/complement activation (e.g., C3, C4b, C8, C9, CFB, CFH, FGA), extracellular remodeling (e.g., EFEMP1, FBN1, FBN2, LTBP4, FERMT2, ECM1, EMILIN2, ITIH3) and apoptosis (e.g., YAP1, HIP1, NDRG1, PRKCD, MUL1) pathways. Our unbiased mass spectrometry method, tailored to adipose tissue, identified both age-related and high fat diet specific proteomic signatures. In addition to well-described immune and extracellular remodeling response to high fat feeding, we have uncovered the pronounced involvement of arginine metabolism in response to advancing age, and branched chain amino acid metabolism in early response to high fat feeding. We were able to uncouple age-related metabolic responses from those associated with caloric intake, a task otherwise difficult to achieve.

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Non-transformed cells respond to fat by inducing glucose metabolism

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