Non-alcoholic fatty liver disease in mice with heterozygous mutation in TMED2

Abstract

The transmembrane emp24 domain/p24 (TMED) family are essential components of the vesicular transport machinery. Members of the TMED family serve as cargo receptors implicated in selection and packaging of endoplasmic reticulum (ER) luminal proteins into coatomer (COP) II coated vesicles for anterograde transport to the Golgi. Deletion or mutations of Tmed genes in yeast and Drosophila results in ER-stress and activation of the unfolded protein response (UPR). The UPR leads to expression of genes and proteins important for expanding the folding capacity of the ER, degrading misfolded proteins, and reducing the load of new proteins entering the ER. The UPR is activated in non-alcoholic fatty liver disease (NAFLD) in human and mouse and may contribute to the development and the progression of NAFLD. Tmed2, the sole member of the vertebrate Tmed β subfamily, exhibits tissue and temporal specific patterns of expression in embryos and developing placenta but is ubiquitously expressed in all adult organs. We previously identified a single point mutation, the 99J mutation, in the signal sequence of Tmed2 in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Histological and molecular analysis of livers from heterozygous mice carrying the 99J mutation, Tmed299J/+, revealed a requirement for TMED2 in liver health. We show that Tmed299J/+ mice had decreased levels of TMED2 and TMED10, dilated endoplasmic reticulum membrane, and increased phosphorylation of eIF2α, indicating ER-stress and activation of the UPR. Increased expression of Srebp1a and 2 at the newborn stage and increased incidence of NAFLD were also found in Tmed299J/+ mice. Our data establishes Tmed299J/+ mice as a novel mouse model for NAFLD and supports a role for TMED2 in liver health.

Fig 1. TMED2 level in livers of wildtype and Tmed2^99J/+ mice at P5, 1–2 months and 3–6 months.

A. RT-qPCR shows no difference in Tmed2 in livers. B. Western blot analysis showed significantly reduced TMED2 in livers of P5 Tmed2^99J/+mice compared to wildtype littermates C. Representative images of Western blot showing expression of TMED2 and β-ACTIN, used as a loading control. D. Reduced TMED2 in livers of 1–2 months and 3–6 months Tmed2^99J/+mice compared to age-matched wildtype. E. Representative images of Western blot showing expression of TMED2 and total protein used as loading control. 3 animals of each genotype were analyzed per age group. **P<0.01 by t-test. WT = wildtype.

Fig 2. TMED10 level in livers of wildtype and Tmed2^99J/+ mice at P5, 1–2 months and 3–6 months.

A. RT-qPCR shows no difference in Tmed2 in livers. B. Western blot analysis revealed significantly decreased TMED10 in livers of Tmed2^99J/+mice compared to wildtype littermates at P5 and 3-6months. C. Representative images of Western blot gel showing expression of TMED10 and β-ACTIN, used as a loading control. *P<0.05 by t-test. WT = wildtype.

Fig 3. Tmed2^99J/+ livers exhibit dilated ER and increased level of the UPR marker phosphorylated-eIF2α.

A-D. Transmission Electron Microscopy (TEM) pictures showing dilated ER (arrows) in hepatocytes of Tmed2^99J/+ mice (B, D) when compared to wildtype littermates (A, C; scale bar = 500nm). E. Phosphorylated eIF2α (p-eIF2α) was significantly increased in livers of 3–6 months Tmed2^99J/+ mice when compared to age-matched wildtype controls. F. No significant difference was found in levels of total eIF2α when Tmed2^99J/+ mice were compared to age-matched wildtype controls. G. Representative images of Western blot gel showing expression of p-eIF2α, total eIF2α, and total protein loading control. H. RT-qPCR indicate no significant difference in levels of Gadd34, Chop, and Atf4 in livers of 3–6 months Tmed2^99J/+ mice when compared to age-matched wildtype controls. 3 animals of each genotype were analyzed per age group, *P<0.05 by t-test. N = nucleus, ER = Endoplasmic Reticulum, WT = Wildtype. Arrows indicate dilated ER.

Fig 4. Increased NAFLD in Tmed2^99J/+ mice.

Representative images of Hematoxylin & Eosin stained liver sections showing A). a healthy liver section; and phenotypes scored for on Table 1; B). macrosteatosis; C). microsteatosis D). lobular inflammation; E). portal inflammation; and F). ballooning. G. Significantly more Tmed2^99J/+mice had NAFLD scores of ≥ 4 when compared to age-matched wildtype controls. **P<0.01 using Fisher exact t-test. Arrows indicate inflammatory cells. Scale bar = 50um. WT = Wildtype.

Fig 5. Representative images of liver samples stained with Oil Red O and Sudan Black B.

A). wildtype liver with score of 1 had no Oil Red O staining. B). Tmed2^99J/+ liver with score of 5 had intense Oil Red O staining. C). Same wildtype sample as in A had no Sudan Black B staining. D). Same Tmed2^99J/+ sample as in B had intense Sudan Black B staining. Scale bar = 50um. WT = Wildtype, n = 4 for each genotype.

Fig 6. Expression of lipid biosynthesis regulators-SREBPs in wildtype and Tmed2^99J/+ livers.

A). Srebp1α level was increased in Tmed2^99J/+ increased in P5 Tmed2^99J/+ mice compared to age-matched wildtype controls. B). Srebp1c level was comparable in Tmed2^99J/+ and age-matched wildtype control mice at all stages. C). Srebp2 level was increased in P5 Tmed2^99J/+ mice compared to age-matched wildtype controls. D). Levels of activated SREBP1C was comparable in Tmed2^99J/+ and age-matched wildtype control at 1–2 and 3–6 months. E). Levels of activated SREBP2 was comparable in Tmed2^99J/+ and age-matched wildtype control at 1–2 and 3–6 months. F). Representative images of Western blot showing expression of full SREBP1C, cleaved SREBP1C (active form), SREBP2, and total protein loading control. 3 animals of each genotype were analyzed per age group. WT = wildtype. *P<0.05 by t-test.