Glycerol induces G6pc in primary mouse hepatocytes and is the preferred substrate for gluconeogenesis both in vitro and in vivo

Testing drugs in isogenic rodent strains to satisfy regulatory requirements is insufficient for derisking organ toxicity in genetically diverse human populations; in contrast, advances in mouse genetics can help mitigate these limitations. Compared to the expensive and slower in vivo testing, in vitro cultures enable the testing of large compound libraries toward prioritizing lead compounds and selecting an animal model with human-like response to a compound. In the case of the liver, a leading cause of drug attrition, isolated primary mouse hepatocytes (PMHs) rapidly decline in function within current culture platforms, which restricts their use for assessing the effects of longer-term compound exposure. Here we addressed this challenge by fabricating mouse micropatterned cocultures (mMPCC) containing PMHs and 3T3-J2 murine embryonic fibroblasts that displayed 4 weeks of functions; mMPCCs created from either C57Bl/6J or CD-1 PMHs outperformed collagen/Matrigel™ sandwich-cultured hepatocyte monocultures by ∼143-fold, 413-fold, and 10-fold for albumin secretion, urea synthesis, and cytochrome P450 activities, respectively. Such functional longevity of mMPCCs enabled in vivo relevant comparisons across strains for CYP induction and hepatotoxicity following exposure to 14 compounds with subsequent comparison to responses in primary human hepatocytes (PHHs). In conclusion, mMPCCs display high levels of major liver functions for several weeks and can be used to assess strain- and species-specific compound effects when used in conjunction with responses in PHHs. Ultimately, mMPCCs can be used to leverage the power of mouse genetics for characterizing subpopulations sensitive to compounds, characterizing the degree of interindividual variability, and elucidating genetic determinants of severe hepatotoxicity in humans.

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Lipid and glucose metabolism in hepatocyte cell lines and primary mouse hepatocytes: a comprehensive resource for in vitro studies of hepatic metabolism

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00365.2018 ◽

2019 ◽

Vol 316 (4) ◽

pp. E578-E589 ◽

Cited By ~ 15

Author(s):

Shilpa R. Nagarajan ◽

Moumita Paul-Heng ◽

James R. Krycer ◽

Daniel J. Fazakerley ◽

Alexandra F. Sharland ◽

...

Keyword(s):

Fatty Acid ◽

Glucose Metabolism ◽

Cell Lines ◽

Fatty Acid Metabolism ◽

Acid Metabolism ◽

Primary Hepatocytes ◽

Primary Mouse Hepatocytes ◽

Primary Mouse ◽

Mouse Hepatocytes

The liver is a critical tissue for maintaining glucose, fatty acid, and cholesterol homeostasis. Primary hepatocytes represent the gold standard for studying the mechanisms controlling hepatic glucose, lipid, and cholesterol metabolism in vitro. However, access to primary hepatocytes can be limiting, and therefore, other immortalized hepatocyte models are commonly used. Here, we describe substrate metabolism of cultured AML12, IHH, and PH5CH8 cells, hepatocellular carcinoma-derived HepG2s, and primary mouse hepatocytes (PMH) to identify which of these cell lines most accurately phenocopy PMH basal and insulin-stimulated metabolism. Insulin-stimulated glucose metabolism in PH5CH8 cells, and to a lesser extent AML12 cells, responded most similarly to PMH. Notably, glucose incorporation in HepG2 cells were 14-fold greater than PMH. The differences in glucose metabolic activity were not explained by differential protein expression of key regulators of these pathways, for example glycogen synthase and glycogen content. In contrast, fatty acid metabolism in IHH cells was the closest to PMHs, yet insulin-responsive fatty acid metabolism in AML12 and HepG2 cells was most similar to PMH. Finally, incorporation of acetate into intracellular-free cholesterol was comparable for all cells to PMH; however, insulin-stimulated glucose conversion into lipids and the incorporation of acetate into intracellular cholesterol esters were strikingly different between PMHs and all tested cell lines. In general, AML12 cells most closely phenocopied PMH in vitro energy metabolism. However, the cell line most representative of PMHs differed depending on the mode of metabolism being investigated, and so careful consideration is needed in model selection.

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miR-222 regulates proliferation of primary mouse hepatocytes in vitro

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2019.02.093 ◽

2019 ◽

Vol 511 (3) ◽

pp. 644-649 ◽

Cited By ~ 2

Author(s):

Miki Higashi ◽

Mitsuhiro Yoneda ◽

Takeya Nakagawa ◽

Masaaki Ikeda ◽

Takashi Ito

Keyword(s):

Primary Mouse Hepatocytes ◽

Primary Mouse ◽

Mouse Hepatocytes

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Novel liver-specific TORC2 siRNA corrects hyperglycemia in rodent models of type 2 diabetes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00158.2009 ◽

2009 ◽

Vol 297 (5) ◽

pp. E1137-E1146 ◽

Cited By ~ 49

Author(s):

Maziyar Saberi ◽

David Bjelica ◽

Simon Schenk ◽

Takeshi Imamura ◽

Gautam Bandyopadhyay ◽

...

Keyword(s):

Type 2 Diabetes ◽

Hepatic Glucose Production ◽

Glucose Production ◽

Primary Mouse Hepatocytes ◽

Primary Mouse ◽

Hepatic Glucose ◽

Zdf Rats ◽

Mouse Hepatocytes

The transcription factor TORC2 [transducer of regulated cAMP-responsive element-binding protein (CREB) activity 2] is a major regulator of hepatic gluconeogenesis and is increased in hyperglycemic rodent models. Because chronic hyperglycemia and increased hepatic glucose production, via increased gluconeogenesis, is a key feature of type 2 diabetes, an effective in vivo method to efficiently knock down TORC2 could provide a potential therapy for treating hyperglycemia and type 2 diabetes. To assess this, primary mouse hepatocytes, high-fat diet (HFD)-fed mice, and Zucker diabetic fatty (ZDF) rats were treated with a siRNA against TORC2 (siTORC2), which was delivered via a novel lipid nanoparticle system, or control siRNA (siCON). Compared with siCON, administration of siTORC2 resulted in highly efficient, sustained (1–3 wk) knockdown of TORC2 and its gluconeogenic target genes phospho enolpyruvate carboxykinase and glucose-6-phophatase in primary mouse hepatocytes and in the livers of HFD-fed mice. In mice, this knockdown was specific to the liver and did not occur in kidney, skeletal muscle, or adipose tissue. In HFD-fed mice, siTORC2 reduced in vivo gluconeogenic capacity, fasting hepatic glucose production, and hyperglycemia, and led to improved hepatic and skeletal muscle insulin sensitivity. siTORC2 treatment also improved systemic hyperglycemia in ZDF rats. In conclusion, these results demonstrate the importance of TORC2 in modulating HGP in vivo and highlight a novel, liver-specific siRNA approach for the potential treatment of hyperglycemia and type 2 diabetes.

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Role of the Thrombin-PAR-1 Pathway in Coxsackievirus Induced Hepatitis

Blood ◽

10.1182/blood.v124.21.1470.1470 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1470-1470

Author(s):

Kohei Tatsumi ◽

Silvio Antoniak ◽

Nigel Mackman

Keyword(s):

Bone Marrow ◽

Immune Response ◽

Innate Immune Response ◽

Innate Immune ◽

Primary Mouse Hepatocytes ◽

Primary Mouse ◽

Mouse Hepatocytes ◽

Transplantation Experiments

Abstract Objective: Coxsackievirus B3 (CVB3) can infect different tissues including the heart and liver. Recently, we found that activation of the coagulation cascade and protease-activated receptor 1 (PAR-1) enhances toll-like receptor-3 (TLR3) mediated interferon-β (IFN-β) expression and protects mice from CVB3-induced myocarditis. Here, we investigated the role of PAR-1 in early anti-viral responses in mice and isolated hepatocytes. Methods: Wild-type (WT) and PAR-1 deficient (PAR-1-/-) mice were infected with CVB3 intraperitoneally. The innate immune response, viral load, liver enzyme plasma levels, and inflammation levels were analyzed. Bone-marrow transplantation experiments with the combination of WT mice PAR-1-/- mice were performed to identify the cellular source of PAR-1 contributing to the innate immune response to CVB3. We also analyzed the effect of the direct thrombin inhibition with dabigatran etexilate on CVB3 hepatitis. In addition, we analyzed the effect of PAR-1 activation on TLR3-dependent interferon (IFN)-β expression in primary mouse hepatocytes and the human hepatocyte cell line PH5CH8 in vitro. Results: PAR-1-/- mice exhibited a reduced early innate immune response in the liver at day 4 after infection, which was associated at later times (day 8) to higher viral titers in the liver, increased alanine transaminase plasma levels and more remarkable inflammation compared to control WT mice. Bone marrow transplantation experiments demonstrated that PAR-1 on non-hematopoietic played the major role in the innate immune response of CVB3 hepatitis. Stimulation of PAR-1 with either thrombin or agonist peptide on primary mouse hepatocytes and human PH5CH8 cells in vitro enhanced the antiviral response to dsRNA by increasing IFN-β and C-X-C motif chemokine 10 (CXCL10) expressions, supporting the results of in vivo experiments. Conclusion: Our results suggest that activation of PAR-1 on hepatocytes enhances the innate immune response to CVB3 in the liver. Disclosures No relevant conflicts of interest to declare.

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Abstract 563: Serum Amyloid A3 is a High Density Lipoprotein-associated Acute Phase Protein

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.563 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Maria C De Beer ◽

Ailing Ji ◽

Victoria P Noffsinger ◽

Preetha Schridas ◽

Frederick C De Beer ◽

...

Keyword(s):

Acute Phase ◽

Density Lipoprotein ◽

Serum Amyloid ◽

Primary Mouse Hepatocytes ◽

Primary Mouse ◽

Evolutionarily Conserved ◽

Rapid Clearance ◽

Mouse Hepatocytes ◽

Extrahepatic Tissues

Acute phase serum amyloid (SAA) is a family of evolutionarily conserved, secreted proteins that exerts innate functions relevant to vascular disease. In humans, two SAA isoforms (SAA1 and SAA2) are highly induced in the liver and extrahepatic tissues under the regulation of inflammatory cytokines. During severe inflammation, SAA1/2 levels can increase ≥1000-fold in plasma, where it is found associated with HDL. Mice produce an additional acute phase SAA, SAA3, which is thought to be produced mainly by adipocytes and macrophages and has not previously been found circulating on HDL. The goal of this study was to investigate whether SAA3 serves as a third liver-derived, HDL-associated acute phase SAA in mice. Using isoform-specific oligonucleotide primers for qRT-PCR, we determined that SAA3 is transcriptionally induced to a similar extent (~2500-fold) compared to SAA1.1/2.1 (~6000-fold) in livers of C57BL/6 mice 19 hr after lipopolysaccharide (LPS) injection (100 μg/mouse). SAAs were also robustly induced in fat tissue (SAA1/2~100-fold; SAA3~400-fold). The analysis of primary mouse hepatocytes and in situ hybridization of mouse liver sections indicated that liver-derived SAAs are produced by hepatocytes and not other stromal cells, including Kupffer cells. All 3 SAA isoforms were detected in plasma of LPS-injected mice, although SAA3 levels were ~20% of SAA1/2. After separation by FPLC, virtually all of plasma SAA1/2 eluted with the HDL fraction, whereas ~15% of plasma SAA3 appeared to be lipid poor/free. HDL isolated from acute phase mouse plasma by density gradient ultracentrifugation was subjected to isoelectric focusing to determine the relative recovery of the various SAA isoforms. Whereas the bulk of plasma SAA1.1 was found in the d=1.063-1.21 fraction, only ~50% of SAA2.1 and ~10% of SAA3 was recovered after ultracentrifugation. These findings suggest that SAA3 may be more loosely associated with HDL compared to SAA1.1/2.1, which may give rise to lipid poor/free SAA3 that is susceptible to more rapid clearance in vivo. We conclude that SAA3 is a major hepatic acute phase SAA in mice that may produce systemic effects during inflammation. Future studies investigating SAA pathobiology in mice must take into account the previously under-studied SAA3.

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PI3-kinase activity modulates apo B available for hepatic VLDL production in apobec-1−/− mice

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00472.2005 ◽

2006 ◽

Vol 291 (3) ◽

pp. G382-G388 ◽

Cited By ~ 32

Author(s):

Doru V. Chirieac ◽

Nicholas O. Davidson ◽

Charles E. Sparks ◽

Janet D. Sparks

Keyword(s):

Insulin Resistance ◽

Kinase Activity ◽

Pi3 Kinase ◽

Apo B ◽

Glucose Injection ◽

Primary Mouse ◽

Mouse Hepatocytes ◽

Vldl Triglyceride

Insulin regulates hepatic VLDL production by activation of phosphatidylinositide 3-kinase (PI3-kinase) which decreases apo B available for lipid assembly. The current study evaluated the dependence of the VLDL apolipoprotein B (apo B) pathway on PI3-kinase activity in vivo. VLDL production was examined in B100 only, apo B mRNA editing catalytic subunit 1 ( apobec-1 −/−) mice, using the Triton WR 1339 method. Glucose injection suppressed VLDL triglyceride production by 28% in male and by 32% in female mice compared with saline-injected controls. When wortmannin was injected to inhibit PI3-kinase, VLDL triglyceride production was increased by 52% in males and by 89% in females, and VLDL B100 levels paralleled triglyceride changes. Pulse-chase experiments in primary mouse hepatocytes showed that wortmannin increased net freshly synthesized B100 availability by >35%. To test whether physiological insulin resistance produced equivalent effects to wortmannin, we studied male apobec-1 −/− mice who became hyperlipidemic on being fed a fructose-enriched diet. Fructose-fed apobec-1 −/− mice had significantly higher VLDL triglyceride and B100 production rates compared with chow-fed mice, and rates were refractile to glucose or wortmannin. Hepatic VLDL triglyceride and B100 production in wortmannin-injected chow-fed mice equaled that observed in fructose-fed mice. Together, results suggest in vivo and in vitro that wortmannin-sensitive PI3-kinases maintain a basal level of VLDL suppression that is sensitive to changes in activation and that can increase VLDL production when PI3-kinase is inhibited to levels similar to those induced by insulin resistance.

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