Thyroid Hormone at Near Physiologic Concentrations Acutely Increases Oxygen Consumption and Extracellular Acidification in LH86 Hepatoma Cells

Thyroid hormone (T3) has been known to regulate the basal metabolic rate for more than a century, but mechanistic understanding is lacking both at the level of the intact organism and in terms of how T3 alters energy expenditure in individual tissues. The current studies investigate the question of which metabolically relevant genes respond acutely as T3 concentrations increase through the physiologic range in liver cells. Because this has been technically unfeasible historically, we developed a modified protocol for extracellular flux analysis using a 96-well Extracellular Flux Analyzer (Seahorse Bioscience). Using a modified extracellular flux protocol and LH86 human hepatoma cells, we established an experimental system where small but significant changes in O2 consumption could be reproducibly quantified as hypothyroid cells were exposed to near-physiologic final concentrations of T3 approximately 2 orders of magnitude lower than most studies (0.04nM free T3), in only 6–7 hours. Taking advantage of the nondestructive nature of 96-well Extracellular Flux Analyzer measurements, the acute, direct, transcriptional changes that occur were measured in the exact same cells demonstrating increased O2 consumption. An unbiased, genome-wide microarray analysis identified potential candidate genes related to fatty acid oxidation, angiogenesis, nucleotide metabolism, immune signaling, mitochondrial respiration, and cell proliferation. The identified transcriptome is likely enriched in the genes most important for mediating the energetic effects of T3 in hepatoma cells.

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Tri-iodothyronine and cycloheximide enhance insulin-like growth factor-binding protein-1 gene expression in human hepatoma cells

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0100007 ◽

1993 ◽

Vol 10 (1) ◽

pp. 7-13 ◽

Cited By ~ 16

Author(s):

M Angervo ◽

P Leinonen ◽

R Koistinen ◽

M Julkunen ◽

M Seppälä

Keyword(s):

Gene Expression ◽

Thyroid Hormone ◽

Hepatoma Cells ◽

Protein Synthesis Inhibitor ◽

Human Hepatoma ◽

Human Hepatoma Cells ◽

Synthesis Inhibitor ◽

Genes Encoding ◽

Protein Mediator

ABSTRACT The growth-regulating actions of IGFs are modulated by their binding proteins (IGFBPs). The serum concentration of IGFBP-1 is down-regulated by insulin, and in-vitro studies have demonstrated that IGFBP-1 secretion from various tissues and cells can be stimulated by theophylline, forskolin, oestrogen and progesterone. We have studied the effects and mechanisms of thyroid hormone action on IGFBP-1 gene expression and secretion by human hepatoma cells in vitro. Tri-iodothyronine dose-dependently enhanced IGFBP-1 secretion in serum-free HepG2 cell cultures after 24–48 h of exposure, as measured by a specific immunofluorometric assay. This was accompanied by an increase (+ 50%) in the amount of IGFBP-1 mRNA, which could be prevented by cycloheximide, a protein synthesis inhibitor. Cycloheximide transiently enhanced (+ 200%) the accumulation of IGFBP-1 mRNA at 3–12 h of incubation, when no effect of tri-iodothyronine was observed. It is concluded that thyroid hormone stimulates IGFBP-1 secretion slowly by enhancing IGFBP-1 gene expression by a protein mediator. The acute stimulation of IGFBP-1 gene transcription by cycloheximide associates this gene with a number of growth-related genes encoding growth- and tumour-associated peptides.

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Thyroid hormone receptor represses miR-17 expression to enhance tumor metastasis in human hepatoma cells

Oncogene ◽

10.1038/onc.2013.309 ◽

2013 ◽

Vol 32 (38) ◽

pp. 4509-4518 ◽

Cited By ~ 25

Author(s):

Y-H Lin ◽

C-J Liao ◽

Y-H Huang ◽

M-H Wu ◽

H-C Chi ◽

...

Keyword(s):

Thyroid Hormone ◽

Hormone Receptor ◽

Tumor Metastasis ◽

Thyroid Hormone Receptor ◽

Hepatoma Cells ◽

Human Hepatoma ◽

Human Hepatoma Cells

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Thyroid hormone receptors promote metastasis of human hepatoma cells via regulation of TRAIL

Cell Death and Differentiation ◽

10.1038/cdd.2012.58 ◽

2012 ◽

Vol 19 (11) ◽

pp. 1802-1814 ◽

Cited By ~ 26

Author(s):

H-C Chi ◽

S-L Chen ◽

C-J Liao ◽

C-H Liao ◽

M-M Tsai ◽

...

Keyword(s):

Thyroid Hormone ◽

Hormone Receptors ◽

Hepatoma Cells ◽

Thyroid Hormone Receptors ◽

Human Hepatoma ◽

Human Hepatoma Cells

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Mitochondrial Respiration in Female Zucker Rats: Effects of Obesity and Short-Term Metformin Treatment

Current Developments in Nutrition ◽

10.1093/cdn/nzaa063_080 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 1682-1682

Author(s):

Shannon Rose ◽

Eugenia Carvalho ◽

David Irby ◽

Sirish Bennuri ◽

Alexandria Beebe ◽

...

Keyword(s):

Mitochondrial Respiration ◽

Coupling Efficiency ◽

Zucker Rats ◽

Proton Leak ◽

Acid Oxidation ◽

Flux Analysis ◽

Metformin Treatment ◽

Short Term ◽

Funding Sources ◽

Extracellular Flux

Abstract Objectives As childhood obesity rates climb, so has the incidence of type 2 diabetes among children and adolescents. Metformin, an FDA approved anti-hyperglycemic drug, is thought to target the mitochondria, but its effects on mitochondrial function in obesity have not been well studied. We used an obese Zucker rat model to investigate the effects of obesity and short-term metformin treatment on mitochondrial respiration. Methods Five-week old female Zucker rats (n = 16 lean, n = 16 obese) were fed AIN-93 G diet for 8 weeks before equally randomized to receive metformin (mixed in diet at 1 g/kg of feed); thus forming 4 groups with 8 rats each: lean +/– metformin and obese +/– metformin. Rats were sacrificed 10 weeks post-metformin treatment and spleens, perigonadal visceral adipose tissue (VAT) and skeletal muscle (SM; gracilis) collected. Mitochondrial respiration was measured in splenocytes by extracellular flux analysis and in VAT and SM fibers by high resolution respirometry. Results Effects of obesity on mitochondrial respiration were found in VAT and SM, but not splenocytes. In VAT, obese rats exhibited increased OXPHOS capacity over lean rats when octanoylcarnitine and malate were provided as substrates (obese vs lean: 1.33 vs 0.76 pmol O2/s/mg; SEdiff = 0.18, P = .005), and after subsequent additions of pyruvate (P = .012), glutamate (P = .009), and succinate (P = .045). In SM, OXPHOS capacity was increased when octanoylcarnitine and malate were provided as substrates (obese vs lean: 12.18 vs 5.45 pmol O2/s/mg; SEdiff = 2.31, P = .011) in obese vs lean rats. Metformin effects were observed only in splenocytes: coupling efficiency was decreased (metformin vs no metformin; 56.2% vs 69.8%; SEdiff = 4.1%, P = 0.005) and proton leak was increased (P < .001) in metformin treated rats as compared to rats not treated with metformin. Conclusions We found obesity was associated with increased mitochondrial respiration, particularly fatty acid oxidation, in VAT and SM. Short-term metformin treatment did not alter mitochondrial respiration in VAT or SM, but was found to increase proton leak and reduce coupling efficiency in splenocytes. Funding Sources Arkansas Children's Research Institute, Arkansas Biosciences Institute (R.H). S.R. and E.C. are also supported by the National Institute of General Medical Sciences of the National Institutes of Health.

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Abstract 573: Human Microrna-548p Decreases apoB Secretion and Lipid Synthesis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.573 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Liye Zhou ◽

Mahmood Hussain

Keyword(s):

Fatty Acid ◽

Untranslated Region ◽

Lipid Synthesis ◽

Hepatoma Cells ◽

Site Directed Mutagenesis ◽

Acid Oxidation ◽

Human Hepatoma ◽

Human Hepatoma Cells ◽

Apob Mrna ◽

Apob Secretion

Objective: MicroRNAs (miRs) play important regulatory roles in lipid and lipoprotein metabolism. ApoB, as the only essential scaffolding protein in the assembly of very low density lipoproteins, is a target to treat hyperlipidemia and atherosclerosis. We aimed to find out miRs that reduce apoB expression. Approach: Bioinformatics analyses predicted that hsa-miR-548p can interact with apoB mRNA.MiR-548p mimic and control were transfected in human and mouse hepatoma cell lines to test its role in regulating apoB secretion and mRNA expression levels. Site-directed mutagenesis was used to identify the interacting site of miR-548p in human apoB 3′-untranslated region. Fatty acid oxidation and lipid syntheses were examined in miR-548p overexpressing cells to investigate its function in lipid metabolism. Results: Experimentally, we observed that miR-548p significantly reduces apoB secretion from human hepatoma cells in time and dose dependent manner. Mechanistic studies showed that miR-548p interacts with the 3′-untranslated region of human apoB mRNA to enhance posttranscriptional degradation. Bioinformatics algorithms suggested two potential binding sites of miR-548p on human apoB mRNA. Site-directed mutagenesis studies revealed that miR-548p targets site II involving both seed and supplementary sequences. MiR-548p had no effect on fatty acid oxidation but significantly decreased lipid synthesis in human hepatoma cells by reducing the expression of HMGCR and ACSL4 enzymes involved in cholesterol and fatty acid synthesis. In summary, miR-548p reduces lipoprotein production and lipid synthesis by reducing expression of different genes in human hepatoma cells. Conclusion: These studies suggest that miR-548p could be useful in treating atherosclerosis, hyperlipidemia and hepatosteatosis.

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