Lipids are important biomolecules that contribute to homeostasis. They can act as energy reserves, are used structurally, and play an important role in metabolic processes including drug metabolism. Lipids complex with proteins resulting in a lipoprotein particle that enables the hydrophobic lipids to be transported throughout the body via the blood stream. Changes in the lipoprotein/lipid profile are associated with diseases such as metabolic dysfunction-associated fatty liver disease (MAFLD), atherosclerosis, hypothyroidism, and cardiovascular disease as well as some genetic disorders. As such, therapeutics are being developed to target dyslipidemia. 

There are distinct differences in the lipoprotein profiles of humans compared with preclinical animal models. Most mouse models do not recapitulate human lipoprotein profiles impacting the translational prediction of studies done in these animals. Chimeric mice with humanized livers offer a solution as the liver plays a crucial role in lipid metabolism and de novo synthesis. Here we will explore the human lipoprotein profile of PXB-mice, our humanized liver chimeric mouse model and PXB-cells, long-term culturable primary human hepatocytes isolated from the PXB-mouse.

Lipoprotein profile differences between mice and humans

The lipoprotein profile of wild type mice, such as C57BL/6, consists of predominately high-density lipoprotein (HDL) with low levels of low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL). Conversely, LDL is the predominate lipoprotein observed in the serum of healthy humans (Figure 1).1 Due to this difference transgenic mouse models have been generated in hopes to mimic the human lipoprotein profile, including apolipoprotein E 3-Leiden (ApoE*3Leiden) and low-density lipoprotein receptor (LDLR) knockout mice (Ldlr-/-Leiden).2 Both models have lower circulating HDL compared to LDL (Figure 2C and D). High-fat diet further enhances the increased LDL levels observed in Ldlr-/-Leiden mice, and as such they are used in diet-induced models of hyperlipidemia, atherosclerosis, and metabolic dysfunction-associated steatohepatitis (MASH) (Figure 2D).2 While these transgenic mice have more similar lipoprotein profiles to humans than wild-type mice, they still do not fully recapitulate the LDL:HDL ratio observed in humans.

Another key limitation of the Ldlr-/- model is that it lacks LDLR, which is a common target for hyperlipidemia therapeutics. Furthermore, there are other important differences between mice and humans that may impact the translational application of these transgenic models. For example, liver expressed cholesteryl ester transfer protein (CETP) is not functional in mice which contributes to the observed difference in the lipoprotein profile between mice and humans. It is well known that mice respond differently to some clinical therapeutics due to differences in drug-metabolizing enzyme expression/activity.3,4 Therefore, it is important to consider these differences when selecting models for pre-clinical studies. Next, we will introduce an alternative model that can be used for lipid-targeting drug discovery and development.

Fig 1_Gordon

Figure 1: Human and mouse (C57BL6) lipoprotein profiles. Total cholesterol was measured by colorimetric assay and plotted versus the fraction number for human plasma (closed circles, n = 3) and mouse plasma (open circles, n = 6). (Gordon SM, et al., 2015)

Fig 2_Olga_v3

Figure 2: Comparison of lipoprotein profiles of humans (A), wild-type mice (B), apolipoprotein E 3-Leiden (ApoE*3Leiden) (C), and low-density lipoprotein receptor (LDLR) knockout mice (Ldlr-/-Leiden) (D). (Olga L, et al., 2022)

PXB-mice have a human lipoprotein profile

The PXB-mouse is a humanized liver model with a high degree of humanization (up to 95% human hepatocyte engraftment). As such, the animals express high levels of liver-specific human genes and proteins, many of which have a similar expression level to human hepatocytes and liver tissue (Figure 3).5

Fig 3_Tateno 2013

Figure 3: Gene expression of PXB-mouse hepatocytes closely resembles human hepatocytes and liver tissue. A cluster analysis of liver signature genes was conducted on isolated PXB-mouse hepatocytes (c-heps), isolated hepatocytes from clinical biopsies (h-heps), as well as several human tissues including liver. 82% of the genes were expressed at similar levels in PXB-mouse hepatocytes and human liver hepatocytes. (Tateno C, et al., 2013)

In the context of the lipoprotein profile, like humans, the predominant lipoprotein is LDL in PXB-mice with lower HDL levels (Figure 4).6 Additionally, the LDL:HDL ratio in PXB-mice is 2.4:1, which is consistent with reported ratios in healthy human controls.6 The expression level of many lipid-related genes in PXB-mice are consistent with isolated human hepatocyte levels. Notably, apolipoproteins are expressed at similar levels as human hepatocytes and the human-specific CETP gene is expressed in PXB-mice albeit at lower levels (Figure 5A).7 Many genes associated with cholesterol synthesis, uptake, storage, and export were also similar between PXB-mice and humans as well as genes associated with triglyceride metabolism (Figure 5A and B).7Fig 4_Papazyan

Figure 4: Serum cholesterol levels of SCID control mice (A) and PXB-mice (B). (Papazyan R. et al., 2018)

Fig 5_Luo

Figure 5: Liver transcription levels of genes associated with cholesterol (a) and triglyceride (b) metabolism pathways. PXB-mice (liver-humanized mice; LHM) were compared to primary human hepatocytes (PHH) from 4 donors. *p<0.05. ∆ human-specific genes. (Luo Y, et al., 2021)

These data demonstrate that PXB-mice express human-specific genes including drug metabolizing enzymes and transporters and have a humanized lipoprotein profile. Additionally, they have normal liver histology and function, making them ideal for pre-clinical studies for a wide range of research applications.

Exploring drug-induced lipid changes in the PXB-mouse model

Here we will give examples of how the PXB-mouse has been used in studies that focus on lipid-targeting therapeutics. Obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist, is known to increase LDL levels in humans.8 FXR activation has been shown to induce lipid homeostasis as well as regulate bile acid signaling. Similarly, in PXB-mice OCA administration increases LDL-C levels (Figure 6).6 Importantly, when PXB-mice were co-treated with atorvastatin the OCA-induced LDL-C increase was significantly reduced (Figure 6).6

Fig 6_PapazyanFigure 6: LDL-C levels were significantly increased in OCA-treated PXB-mice and when mice received co-administration of atorvastatin (ATV) the observed increase was diminished. PXB-mice and SCID animals (control) were treated with OCA and total cholesterol, HDL-C, LDL-C, and VLDL levels were measured by HPLC. The change from baseline levels are shown after treatment with vehicle, OCA (10 mg/kg/day), and/or atorvastatin (10 mg/kg/day). Statistical significance is denoted as follows: * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001; not significant (n.s.). (modified from Papazyan R et al., 2018)

 As mentioned previously, changes in the lipoprotein profile are observed in MAFLD resulting in liver steatosis and triglyceride accumulation. AM095, a selective lipoprotein a receptor (LPAR) antagonist, was tested to see if inhibiting LPAR could reduce liver steatosis and triglyceride levels in the PXB-mouse model. Of note, there is some background steatosis in the PXB-mouse model which is attributed to the inability of mouse growth hormone to bind/activate the human growth hormone receptor present on engrafted hepatocytes.9 After 9 days of treatment with AM095, PXB-mice had a significant reduction in lipid accumulation in the liver compared to vehicle control animals (Figure 7A and B).10 Additionally, they observed a significant reduction in triglyceride levels (Figure 7C).10 They attributed the changes to the reduction of Cd36, which is known to facilitate the uptake of oxidized LDL and free fatty acids (Figure 7D).10 It has been previously demonstrated that mice with reduced liver CD36 expression were protected from high-fat diet-induced hepatic steatosis.11  

Fig 7_Lua

Figure 7: Lipid accumulation was reduced in AM095 treated PXB-mice. Oil Red O (ORO) staining of liver tissue treated with AM095 or vehicle control (DMSO) (A) and quantification of staining (B). Liver triglyceride levels were measured in PXB-mice treated with AM095 or DMSO control (C). QPCR Cd36 gene expression levels were measured in liver tissue samples from control and AM095 treated PXB-mice. *, p<0.05. (Lua I, et al., 2021)

Next, we will show results in PXB-mice for two therapeutic targets that have been used for treating hyperlipidemia, peroxisome proliferator-activated receptor alpha (PPARα) and liver X receptor (LXR). Fenofibrate, a selective PPARα agonist, has been clinically used to reduce LDL levels and triglycerides in patients. While acute fenofibrate treatment (4 days) did not reduce the level of triglycerides in PXB-mice, genes associated with the metabolism of lipoproteins and lipids were significantly increased.12 Importantly, PPARα gene activation in PXB-mice was similar to primary human hepatocytes and human liver slices, demonstrating that this model recapitulates human PPARα activation.12 Like fenofibrate, acute treatment with TLC-2716 (an inverse agonist of LXR) did not significantly reduce liver triglyceride levels, albeit there was a modest decrease in PXB-mice treated for 8 days compared to vehicle control animals (Figure 8A).13 Additionally, LXR inverse agonist treatment reduced the expression of genes associated with LDL uptake and the synthesis of cholesterol and bile acids (Figure 8B).13 Each of these therapeutics were well tolerated in PXB-mice, with no adverse clinical manifestations reported.

Fig 8_Li 2026Figure 8: Liver triglyceride levels (A) and liver expression of genes involved in lipid synthesis and metabolism (B) in PXB-mice treated with TLC-2716 or vehicle control. (modified from Li X et al., 2026)

Unlike traditional mouse models, chimeric mice with humanized livers express human-specific genes, drug-metabolizing enzymes, and transporters, as well as having a human-like lipoprotein profile. Furthermore, OCA treatment in PXB-mice recapitulates observed clinical increases in LDL levels, suggesting that this mouse model is relevant for translational lipid drug discovery and development.

In vitro lipoprotein profile of PXB-cells

PXB-cells are ~95% pure human hepatocytes which are isolated from the PXB-mouse. Like the PXB-mouse, these primary human hepatocytes (PHH) have high human-specific metabolic enzyme expression and transporter activity. Importantly, unlike traditional PHHs, PXB-cells can be cultured long-term with stable expression of phase I and II metabolic enzymes and transporters (up to 30 days or more).

PXB-cells synthesize and secrete both cholesterol and triglycerides, which are primarily classified as VLDL unlike HepG2 and Huh7 cells (Figure 9).14 Lipid droplets are observed immediately after isolation and at 8 days in culture (Figure 10A and B).15 Additionally, when PXB-cells are treated with fenofibrate they secreted significantly less cholesterol and triglycerides, in a dose-dependent manner.14, 16 Cell density is known to affect the expression of genes and as such we have optimized the seeding density of PXB-cells. At our recommended seeding density, the expression of genes involved in PPAR signaling were detected but in lower cell densities these genes are significantly decreased.17 As such, PXB-cells are ideal for in vitro drug discovery programs.Fig 9_Hata 2020

Figure 9: Secreted triglyceride and cholesterol levels were measured for PXB-cells, HepG2 cells, and HuH-7 cells cultured for 48 hours. (Hata K et al., 2020)

Fig 10_Takahasi

Figure 10: Oil red O staining of lipid droplets in PXB-cells after isolation on day 0 (A) and at 8 days post-isolation (B). scale bar: 200 µm (Modified from Takahashi M et al., 2024)

Conclusion

Our validated humanized liver chimeric mouse model (PXB-mice) is stably produced, allowing us to accommodate large-scale animal experiments. Furthermore, the PXB-cells are isolated from the PXB-mouse, demonstrating the versatility of our models for drug discovery programs, both in vitro and in vivo. 

Contact us

Contact us today to learn more about how the PXB-mice and/or their isolated hepatocytes (PXB-cells) can enhance your lipid-targeting research. 

 

References

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  2. Olga L., et al. Lipid profiling analyses from mouse models and human infants. STAR Protocols. (2022) 3(4):101679. doi:10.1016/j.xpro.2022.101679
  3. Karlsson IB, et al., Investigation of biotransformation pathways in a chimeric mouse with a humanized liver. Int J Mol Sci. (2025) 26(3):1141. doi: 10.3390/ijms26031141
  4. Feng B, et al., Utility of chimeric mice with humanized livers for predicting hepatic organic anion-transporting polypeptide 1B-mediated clinical drug-drug interactions. Drug Metab Dispos. (2024) 52(10):1073-1082. doi: 10.1124/dmd.124.001792
  5. Tateno C, et al., Morphological and microarray analyses of human hepatocytes from xenogeneic host livers. Laboratory Investigation (2013) 93(1):54-71. doi: 10.1038/labinvest.2012.158
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  7. Luo Y, et al., Liver-humanized mice: a translational strategy to study metabolic disorders. J Cell Physiol. (2021) 237(1):489-506. doi: 10.1002/jcp.30610
  8. Shadab Siddiqui M et al., Impact of obeticholic acid on the lipidprotein profile in patients with nonalcoholic steatohepatitis. J Hepatol. (2019) 72(1):25-33. doi: 10.1016/j.jhep.2019.10.006
  9. Tateno C and Kojima Y. Characterization and applications of chimeric mice with humanized livers for preclinical drug development. Lab Anim Res. (2020) 36:2. doi: 10.1186/s42826-019-0032-y
  10. Lua I et al., Loss of lysophoshatidic acid receptor 1 in hepatocytes reduces steatosis via down-regulation of CD36. Prostaglandins Other Lipid Mediat. (2021) 156:106577. doi: 10.1016/j.prostaglandins.2021.106577.
  11. Zeng H et al., CD36 promotes de novo lipogenesis in hepatocytes through INSIG2-dependent SREBP1 processing. Mol Metab. (2022) 57:101428. doi: 10.1016/j.molmet.2021.101428.
  12. de la Rosa Rodriguez MA, et al., The whole transcriptome effects of the PPARα agonist fenofibrate on livers of hepatocyte humanized mice. BMC Genomics. (2018) 19(1):443. doi: 10.1186/s12864-018-4834-3
  13. Li X, et al., An oral, liver-restricted LXR inverse agonist for dyslipidemia: preclinical development and phase 1 trial. Nat Med. (2026) Online ahead of print. doi: 10.1038/s41591-025-04169-6
  14. Hata K, et al., Lipoprotein profile and lipid metabolism of PXB-cells, human primary hepatocytes from liver-humanized mice: proposal of novel in vitro system for screening anti-lipidemic drugs. Biomed Res. (2020) 41(1):33-42. doi: 10.2220/biomedres.41.33
  15. Takahashi M, et al., The evaluation of lipid analysis for PXB-cells LA as a human non-fatty liver disease model. BPB Reports. (2024) 7(4):147-156. doi: 10.1248/bpbreports7.4_147
  16. Tomatsu S, et al., High-throughput screening of anti-lipidemic agents using PXB-cells, human hepatocytes from humanized mice livers: Assessment of lipoproteins by an enzyme-linked immunosolvent assay on apolipoproteins. J Biol Macromol. (2021) 21(2):89-92
  17. Yamasaki C, et al., Culture density contributes to hepatic functions of fresh human hepatocytes isolated from chimeric mice with humanized livers: Novel, long-term, functional two-dimensional in vitro tool for developing new drugs. PLoS One. (2020) 15(9):e0237809 doi: 10.1371/journal.pone.0237809

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