The unforeseen intracellular lifestyle of Enterococcus faecalis in hepatocytes

Enterococcus faecalis is a bacterial species present at a sub-dominant level in the human gut microbiota. This commensal turns into an opportunistic pathogen under specific conditions involving dysbiosis and host immune deficiency. E. faecalis is also the only intestinal pathobiont identified to date as contributing to liver damage in alcoholic liver disease. We have previously observed that E. faecalis is internalized in hepatocytes. Here, the survival and fate of E. faecalis was examined in hepatocytes, the main epithelial cell type in the liver. Although referred to as an extracellular pathogen, we demonstrate that E. faecalis is able to survive and divide in hepatocytes, and form intracellular clusters in two distinct hepatocyte cell lines, in primary mouse hepatocytes, as well as in vivo. This novel process extends to kidney cells. Unravelling the intracellular lifestyle of E. faecalis, our findings contribute to the understanding of pathobiont-driven diseases.

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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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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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Faculty Opinions recommendation of Glycerol induces G6pc in primary mouse hepatocytes and is the preferred substrate for gluconeogenesis both in vitro and in vivo.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.736800444.793568712 ◽

2019 ◽

Author(s):

Michael Roth

Keyword(s):

Primary Mouse Hepatocytes ◽

Primary Mouse ◽

Mouse Hepatocytes

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011033 ◽

2019 ◽

Vol 294 (48) ◽

pp. 18017-18028 ◽

Cited By ~ 7

Author(s):

Katarzyna M. Kalemba ◽

Yujue Wang ◽

Huiting Xu ◽

Eric Chiles ◽

Sara M. McMillin ◽

...

Keyword(s):

Primary Mouse Hepatocytes ◽

Primary Mouse ◽

Mouse Hepatocytes

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Long-Term Engineered Cultures of Primary Mouse Hepatocytes for Strain and Species Comparison Studies During Drug Development

Gene Expression ◽

10.3727/105221619x15638857793317 ◽

2019 ◽

Vol 19 (3) ◽

pp. 199-214

Author(s):

Brenton R. Ware ◽

Grace E. Brown ◽

Valerie Y. Soldatow ◽

Edward L. LeCluyse ◽

Salman R. Khetani

Keyword(s):

Mouse Genetics ◽

Human Populations ◽

Urea Synthesis ◽

Cyp Induction ◽

Compound Libraries ◽

Primary Mouse Hepatocytes ◽

Primary Mouse ◽

Mouse Hepatocytes

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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