Reduced levels of cellular energetic metabolites during isolation and cell culture.

<p>Quantification of total intracellular hepatic A) Adenosine, B) AMP, C) ADP, D) ATP and E) ATP/ADP ratio during the isolation procedure and cell culture for a period of up to 48 hours. F) Calculated energy charge values during isolation and <i>in vitro</i> culture. Values are ±SEM of 3 independent experiments. Asterisks indicate significance when compared to the <i>in situ</i> liver <i>(*P</i><0.05, <i>**P<</i>0.01, <i>***P</i><0.001).</p

Primary hepatocyte cultures show a significant decrease in mitochondrial oxygen consumption rate but stable mitochondrial ATP production.

<p>Extracellular flux analysis was used to measure the mitochondrial function of primary hepatocytes cultured for 0 to 48 hours. A) Mitochondrial respiration (OCR levels) after glucose stimulation (10 mM). B) Assessment of ATP-linked respiration following the addition of oligomycin (2 μM). Values are ±SEM of 3 independent experiments <i>(*P<</i>0.05, <i>**P<</i>0.01<i>; # P</i><0.05 when compared to primary hepatocytes cultured for 2,4 and 6 hours).</p

Reduction in TCA metabolites during the isolation procedure and cell culture.

<p>Assessment of intracellular: A) Acetyl-CoA, B) Isocitrate/Citrate ratio, C) Succinate, D) Fumarate and E) Malate during the hepatocyte isolation procedure and cell culture for a period of up to 48 hours. Values are ±SEM of 3 independent experiments. Asterisks indicate significance when compared to the <i>in situ</i> liver <i>(*P</i><0.05, <i>**P</i><0.01, <i>***P</i><0.001).</p

Decrease in antioxidative stress-related metabolites during the isolation procedure and cell culture.

<p>Evaluation of total intracellular: A) NADPH, B) NADP, C) GSH and D) GSSG levels during the hepatocyte isolation procedure from <i>in situ</i> to the washing step in L-15 media and during cell culture over a period of up to 48 hours. Values are ±SEM of 3 independent experiments. Asterisks indicate significance when compared to the <i>in situ</i> liver <i>(*P</i><0.05, <i>**P</i><0.01, <i>***P</i><0.001).</p

Metabolic reprogramming enables hepatocarcinoma cells to efficiently adapt and survive to a nutrient-restricted microenvironment

<p>Hepatocellular carcinoma (HCC) is a metabolically heterogeneous cancer and the use of glucose by HCC cells could impact their tumorigenicity. Dt81Hepa1-6 cells display enhanced tumorigenicity compared to parental Hepa1-6 cells. This increased tumorigenicity could be explained by a metabolic adaptation to more restrictive microenvironments. When cultured at high glucose concentrations, Dt81Hepa1-6 displayed an increased ability to uptake glucose (<i>P</i><0.001), increased expression of 9 glycolytic genes, greater GTP and ATP (<i>P</i><0.001), increased expression of 7 fatty acid synthesis-related genes (<i>P</i><0.01) and higher levels of Acetyl-CoA, Citrate and Malonyl-CoA (<i>P</i><0.05). Under glucose-restricted conditions, Dt81Hepa1-6 used their stored fatty acids with increased expression of fatty acid oxidation-related genes (<i>P</i><0.01), decreased triglyceride content (<i>P</i><0.05) and higher levels of GTP and ATP (<i>P</i><0.01) leading to improved proliferation (<i>P</i><0.05). Inhibition of lactate dehydrogenase and aerobic glycolysis with sodium oxamate led to decreased expression of glycolytic genes, reduced lactate, GTP and ATP levels (<i>P</i><0.01), increased cell doubling time (<i>P</i><0.001) and reduced fatty acid synthesis. When combined with cisplatin, this inhibition led to lower cell viability and proliferation (<i>P</i><0.05). This metabolic-induced tumorigenicity was also reflected in human Huh7 cells by a higher glucose uptake and proliferative capacity compared to HepG2 cells (<i>P</i><0.05). In HCC patients, increased tumoral expression of <i>Glut-1</i>, <i>Hexokinase II</i> and <i>Lactate dehydrogenase</i> correlated with poor survival (<i>P</i> = 2.47E⁻⁵, <i>P</i> = 0.016 and <i>P</i> = 6.58E⁻⁵). In conclusion, HCC tumorigenicity can stem from a metabolic plasticity allowing them to thrive in a broader range of glucose concentrations. In HCC, combining glycolytic inhibitors with conventional chemotherapy could lead to improved treatment efficacy.</p

Dose-dependent tumor development following transfer of Dt81Hepa1-6 cells.

<p>Suspensions of Dt81Hepa1-6 cells [1K to 1M] were injected intrasplenically in C57BL/6 mice and animals were sacrificed after 28 days. (A) Manual count of tumor >0.5mm. (B) <i>AFP</i> (<i>Afp</i>) mRNA expression in liver homogenates by qPCR. (C) Representative microphotographs at 100x magnification of HPS-stained liver slice obtained after injection with 1K and 10K cells. Whole liver photographs of mice injected with 0.1M, 0.5M or 1M cells. Control mice were injected with 1M Hepa1-6 cells. (*<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001).</p

Dt81Hepa1-6 cell transfer leads to rapid tumor development.

<p>A suspension of 1M Dt81Hepa1-6 cells was injected intrasplenically in C57BL/6 mice and animals were sacrificed after 3.5 to 28 days. (A) Manual count of tumor >0.5mm. (B) Alpha-fetoprotein (AFP) (Afp) mRNA expression in liver homogenates as measured by qPCR. (C) Representative microphotographs at 100x magnification of HPS-stained liver slices obtained after 3.5, 7 and 14 days. Whole-liver photographs obtained after 21 or 28 days. Control mice were injected with 1M Hepa1-6 cells and sacrificed at each time point (3.5 to 28 days) and pooled for analysis (Ctrl). (*<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001).</p

Expression of β-Catenin, Cyclin D1 and Integrins by Dt81Hepa1-6 cells.

<p>All experiments were performed on freshly trypsinized cells prior to their injection <i>in vivo</i>. Genes or proteins of interest were analyzed in Hepa1-6, Dt81Hepa1-6, EpCAM+low and EpCAM+high cell lines by qRT-PCR and/or western blot. (A) β-Catenin (<i>Ctnnb1)</i> gene and (B) protein expression shown on immunoblot (C) Cyclin D1 (Ccnd1), (D) Integrin-alpha1 (Itga1) and (E) Integrin-β1 (Itgb1) mRNA expression. (*<i>P</i><0.05; **<i>P</i><0.01).</p

Dt81Hepa1-6 <i>in vitro</i> neoplastic profile.

<p>A cell aggregation assay was performed and (A) the number of cells per aggregate was calculated 24h after seeding both cell lines in non-adherent agar-coated wells. Representative microphotographs are shown. In order to evaluate anchorage-independent growth, a soft-agar colony formation assay was performed; (B) cell lines were seeded at 10K-cell concentration in soft-agar gel [0.3%] in order to form visible colonies and were counted 5 weeks later. Representative microphotographs of cells after 5 weeks are shown. The ability of independent cells to form colonies without cell-to-cell contact was assessed by a proliferative clonal colony assay; (C) cell lines were seeded at 3K/mL concentration to obtain isolated cells and left to grow as colonies over a 4 weeks period and counted. The potential for invasiveness was evaluated with a double-layer droplet cell invasion assay; (D) Cell lines [20K cells] were embedded in COL1 [3mg/mL] to form a double layered droplet and cells penetrating the outer COL1 layer were counted every 12h for 8 days. Area under the curves (AUC) of the time-dependent invasion was calculated and used for statistical analysis. Representative microphotographs are shown. For the wound-healing assays (WHA); (E) normal and COL1-embedded WHA were performed on confluent cells for 24h and length traveled was evaluated using ImageJ. For the cell-spreading assay, (F) Cell lines were seeded for 6h on plastic and COL1 and cells showing an elongated morphology were counted manually. (G) Cell doubling time for Dt81Hepa1-6 and parental Hepa1-6 cells. (H) Viability of Dt81Hepa1-6 and Hepa1-6 cells following <i>in vitro</i> exposure to cisplatin [25ug/mL] for 24h. (*<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001).</p

Upregulation of Krebs cycle and anaerobic glycolysis activity early after onset of liver ischemia

<div><p>The liver is a highly vascularized organ receiving a dual input of oxygenated blood from the hepatic artery and portal vein. The impact of decreased blood flow on glucose metabolism and how hepatocytes could adapt to this restrictive environment are still unclear. Using the left portal vein ligation (LPVL) rat model, we found that cellular injury was delayed after the onset of liver ischemia. We hypothesized that a metabolic adaptation by hepatocytes to maintain energy homeostasis could account for this lag phase. Liver glucose metabolism was characterized by ¹³C- and ¹H-NMR spectroscopy and analysis of high-energy metabolites. ALT levels and caspase 3 activity in LPVL animals remained normal during the first 12 h following surgery (<i>P</i><0.05). Ischemia rapidly led to decreased intrahepatic tissue oxygen tension and blood flow (<i>P</i><0.05) and increased expression of Hypoxia-inducible factor 1-alpha. Intrahepatic glucose uptake, ATP/ADP ratio and energy charge level remained stable for up to 12 h after ligation. Entry of glucose in the Krebs cycle was impaired with lowered incorporation of ¹³C from [U^-13C]glucose into glutamate and succinate from 0.25 to 12 h after LPVL. However, total hepatic succinate and glutamate increased 6 and 12 h after ischemia (<i>P</i><0.05). Glycolysis was initially reduced (<i>P</i><0.05) but reached maximum ¹³C-lactate (<i>P</i><0.001) and ¹³C-alanine (<i>P</i><0.01) enrichments 12 h after LPVL. In conclusion, early liver homeostasis stems from an inherent ability of ischemic hepatocytes to metabolically adapt through increased Krebs cycle and glycolysis activity to preserve bioenergetics and cell viability. This metabolic plasticity of hepatocytes could be harnessed to develop novel metabolic strategies to prevent ischemic liver damage.</p></div