mcd diet  (Valiant Co Ltd)

Journal: The Journal of Biological Chemistry

Article Title: HuR-driven reversible mitochondrial shuttling buffers cytosolic miRNA levels in hepatic cells

doi: 10.1016/j.jbc.2026.111255

Figure Lengend Snippet: The starvation-induced enrichment of mito-miRs in hepatic cell mitochondria. A , fold change in cellular miRNA content after 16 h of amino acid starvation of Huh7 cells. Relative levels of various miRNAs were quantified using qRT–PCR. Data were normalized to U6 snRNA (n = 3). B , miRNA-specific Ct values are plotted to show the changes in their relative abundance in purified mitochondria after amino acid starvation of Huh7 cells. The analysis indicates a significant decrease in Ct values for miRNAs let-7b, let-7g, and miR-181a in starved cell mitochondria, reflecting their enrichment in the mitochondrial fraction on starvation. A bar diagram displays the fold enrichment of let-7b and let-7g mito-miRs in purified mitochondria compared with let-7a (n = 3). C , Western blot analysis done with fed and starved Huh7 extracts exhibited increased levels of expression of the stress marker protein phosphorylated–eukaryotic initiation factor 2 alpha and cleaved PARP, accompanied by decreased 4EBP1 (eukaryotic initiation factor 4E–binding protein 1) phosphorylation. β-actin served as the loading control for all samples. Relative levels, quantified from band intensity, are plotted at the bottom of Western blot panels against the beta-actin normalized value of Fed Huh7 cells as a unit. D , cellular miRNA levels change in the liver of mice under a normal diet (fed) and after 12h of starvation, as estimated by qRT–PCR and normalization against U6 snRNA levels (n = 3). E , the fold changes of let-7b and let-7g in the isolated pure mitochondrial fraction obtained from the liver of mice having a normal diet (fed) or starved for 12 h. Levels of each miRNA in the fed condition are considered as units (n = 3). F and G , the relative enrichment of mito-miRs with the outer membrane and intermembrane space (OM + IMS) ( F ) and inner membrane and mitochondrial matrix (IM + matrix) ( G ) of mitochondria isolated from control (fed) and 16 h amino acid–starved Huh7 cells (starved). Ct values are plotted (n = 6). H , fold change in mitochondrial miR-122 and let-7g levels in control and methionine/choline-deficient (MCD) diet–exposed animal’s liver mitochondria (n = 4). I , Western blot analysis ( left panel ) and relative quantification ( right panel ) of different proteins present in the mitochondrial fractions isolated from the control and MCD diet–exposed animal liver (n = 3). J , effect of thapsigargin (TG) on the expression of stress-responsive proteins in Huh7 cells determined by Western blot analysis. β-actin was used as a loading control. K – L , effect of TG treatment (2.5 micromolar;16 h) on cellular ( K ) and mitochondrial ( L ) miRNA content. Relative levels of miRNAs were measured and plotted. For cellular and mitochondrial samples, U6 and let-7a levels were used for normalization. Relative change in let-7a in mitochondrial fraction was determined from Ct values and plotted in L . Values at the control set were used as a unit (n = 3). M , cellular miR-122 levels increased, whereas its mitochondrial levels remained unchanged when cells were treated (10 micromolar, 24 h) with a pharmacological blocker of extracellular vesicle production, GW4869, a powerful, selective, noncompetitive inhibitor of neutral sphingomyelinase (N-SMase), an enzyme crucial for generating ceramide, which drives the formation and release of exosomes (extracellular vesicles) from cells . Normalization was done with U6 snRNA and let-7a for cellular and mitochondrial miR-122, respectively (n = 3). Statistically analyzed data are indicated as mean ± SD of three experiments, with statistical significance marked as ns (nonsignificant), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, calculated using two-tailed Student’s t test. PARP, poly(ADP-ribose) polymerase; qRT–PCR, quantitative RT–PCR.

Article Snippet: They were randomly assigned to two groups and fed either a standard chow or an MCD diet (MP Biomedicals; catalog no.: 0296043910) for 4 weeks.

Techniques: Quantitative RT-PCR, Purification, Western Blot, Expressing, Marker, Binding Assay, Phospho-proteomics, Control, Isolation, Membrane, Quantitative Proteomics, Two Tailed Test

Journal: bioRxiv

Article Title: Flexibility of systemic one-carbon metabolism partially buffers dietary methyl donor deficiency

doi: 10.64898/2026.02.05.703880

Figure Lengend Snippet: (a) Schematic showing intravenous infusion of [trimethyl- H 9 ]choline and [methyl- C 1 ]methionine in mice fed a methionine and choline deficient diet or fed a paired control diet. (b) Rate of appearance of choline calculated from m+9 serum enrichments from 8 h intravenous infusion of [trimethyl- H 9 ]choline. (c) Rate of appearance of methionine calculated from m+1 serum enrichments from 8 h intravenous infusion of [methyl- C 1 ]methionine. (d) Normalized labeling of tissue SAM from 8 hour intravenous infusion of [methyl- C 1 ]methionine. (e) Normalized labeling of tissue SAM from 8 hour intravenous infusion of [trimethyl- H 9 ]choline. The red line represents the fraction of serum methionine that is labeled in control conditions and the green line represents the fraction of serum methionine that is labeled in MCD diet. Labeling below these lines indicates that labeling is potentially sourced from serum methionine pools rather than tissue autonomous production. (f) Normalized labeling of pooled PCs in the liver from 8 hour intravenous infusion of [trimethyl- H 9 ]choline. (g) Ratio of ion counts of pooled PC species to pooled PE species. Bars represent mean ± S.D. Each data point represents data from an independent mouse. P-values were calculated with two-way ANOVA with Šídák correction for multiple comparisons. All experiments were performed in male ad lib fed C57Bl/6N mice during the light cycle. PC, phosphatidylcholine; PE, phosphatidylethanoline; PEMT, phosphatidylethanolamine methyltransferase

Article Snippet: Methyl deficiency was induced by transitioning mice from standard chow to a methionine and choline-deficient (MCD) diet (MP Biomedicals, MP296043910), while age-matched controls received a matched methionine and choline replete control diet (MP Biomedicals, MP296044110).

Techniques: Control, Labeling

Journal: bioRxiv

Article Title: Flexibility of systemic one-carbon metabolism partially buffers dietary methyl donor deficiency

doi: 10.64898/2026.02.05.703880

Figure Lengend Snippet: (a) Normalized labeling of serum and tissue serine from 8 hour intravenous infusion of [trimethyl- H 9 ]choline.The red line represents the fraction of serum serine that is labeled in control conditions and the green line represents the fraction of serum serine that is labeled in MCD diet. Labeling below these lines indicates that labeling is potentially sourced from serum serine pools rather than tissue autonomous production. (b) Relative ion counts for glycine across tissues in MCD diet and control diet fed mice. Bars represent mean ± S.D. Each data point represents data from an independent mouse. P-values were calculated with two-way ANOVA with Šídák correction for multiple comparisons (a) or Mixed-effects analysis with Šídák correction for multiple comparisons (b). All experiments were performed in male ad lib fed C57Bl/6N mice during the light cycle. MCD, methionine and choline deficient

Article Snippet: Methyl deficiency was induced by transitioning mice from standard chow to a methionine and choline-deficient (MCD) diet (MP Biomedicals, MP296043910), while age-matched controls received a matched methionine and choline replete control diet (MP Biomedicals, MP296044110).

Techniques: Labeling, Control

Journal: bioRxiv

Article Title: Flexibility of systemic one-carbon metabolism partially buffers dietary methyl donor deficiency

doi: 10.64898/2026.02.05.703880

Figure Lengend Snippet: (a) Schematic of the folate cycle highlighting reactions that support production of 5,10-methylene-THF and SAM. (b) Schematic showing intravenous infusion of [2,3,3- H 3 ]serine and [U- C ]glycine in mice fed a methionine and choline deficient diet or fed a paired control diet. (c) Rate of appearance of serine calculated from m+3 serum enrichments from 8 h intravenous infusion of [2,3,3- H 3 ]serine. (d) Rate of appearance of glycine calculated from m+2 serum enrichments from 8 h intravenous infusion of [U- C 2 ]glycine. (e) Schematic of use of [2,3,3- H 3 ]serine tracing to measure the contribution of the folate cycle to SAM synthesis. (f) Normalized labeling of tissue SAM from 8 hour intravenous infusion of [2,3,3- H 3 ]serine. The red line represents the fraction of serum methionine that is labeled in control conditions and the green line represents the fraction of serum methionine that is labeled in MCD diet. Labeling below these lines indicates that labeling is potentially sourced from serum methionine pools rather than tissue autonomous production. Bars represent mean ± S.D. Each data point represents data from an independent mouse. P-values were calculated with two tailed T-test (c,d) or two-way ANOVA with Šídák correction for multiple comparisons (f). All experiments were performed in male ad lib fed C57Bl/6N mice during the light cycle. MCD, methionine and choline deficient; SHMT, serine hydroxymethyltransferase, MTR, 5-methyltetrahyrofolate homocysteine methyltransferase; SAM, s-adenosylmethionine

Article Snippet: Methyl deficiency was induced by transitioning mice from standard chow to a methionine and choline-deficient (MCD) diet (MP Biomedicals, MP296043910), while age-matched controls received a matched methionine and choline replete control diet (MP Biomedicals, MP296044110).

Techniques: Control, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: Flexibility of systemic one-carbon metabolism partially buffers dietary methyl donor deficiency

doi: 10.64898/2026.02.05.703880

Figure Lengend Snippet: (a) Schematic showing potential sources of circulating serine. (b) Normalized labeled fraction of serum serine from infusion of [U- C 2 ]glycine. (c) Normalized labeled fraction of serum serine from infusion of [U- C 6 ]glucose. (d) Rate of appearance of essential amino acid phenylalanine from intravenous infusion of [ N]phenylalanine. (e) Serine production fluxes from different sources measured using the serine rate of appearance and infusions of U- C 2 ]glycine, U- C 6 ]glucose, and [ N]phenylalanine. (f) Relative ion counts for serine across tissues in MCD diet and control diet fed mice. (g) Schematic of proposed mechanism. When methionine and choline are deficient in the diet, there is increased release of serine from the kidney that supports maintained methionine, PC, and choline synthesis in the liver. Thus, methionine and choline fluxes are maintained by one-carbon metabolism buffering. Bars represent mean ± S.D. Each data point represents data from an independent mouse. P-values were calculated with two tailed T-test (b), two-way ANOVA with Šídák correction for multiple comparisons (e), or mixed-effects analysis with Šídák correction for multiple comparisons (f). All experiments were performed in male ad lib fed C57Bl/6N mice during the light cycle. SAM, s-adenosylmethionine; MCD, methionine and choline deficient

Article Snippet: Methyl deficiency was induced by transitioning mice from standard chow to a methionine and choline-deficient (MCD) diet (MP Biomedicals, MP296043910), while age-matched controls received a matched methionine and choline replete control diet (MP Biomedicals, MP296044110).

Techniques: Labeling, Control, Two Tailed Test

Journal: JHEP Reports

Article Title: Hepatic immune environment differences among common mouse strains in models of MASH and liver cancer

doi: 10.1016/j.jhepr.2025.101380

Figure Lengend Snippet: Changes of liver immune subsets under MASH in BALB/c, C57BL/6, and FVB/N mice fed with MCD diet. Female BALB/c, C57BL/6, and FVB/N mice were fed with MCD diet ( vs. regular diet) to induce MASH. (A) The development of MASH was confirmed by H&E staining. Liver immune cells from MASH mice or control mice were prepared and immune subsets were measured by flow cytometry analysis. The comparison between MCD (green) and control (gray) was performed in each liver immune subsets of the three mouse strains (B–G). The overall changes of various liver immune subsets from three mouse strains are shown (H). The size of circle represents the log 10 transformed fold changes of each immune subset. The color gradient represents the relative frequences of each immune subset. The distribution of fold changes of each type of immune cell is also shown; n = 4 per group, two-way ANOVA with Bonferroni correction, ∗ p <0.05. MASH, metabolic dysfunction-associated steatohepatitis; MCD diet, methionine- and choline-deficient diet; Tregs, regulatory T cells.

Article Snippet: MASH was induced in mice by feeding a methionine- and choline-deficient (MCD) diet (Research diets inc., New Brunswick, NJ, USA Ref. A02082002BR) for a period of 3 weeks, or a Western diet (Envigo, Indianapolis, IN, USA, Ref. TD.120528) with high sugar solution (23.1 g/L d-fructose and 18.9 g/L d-glucose) with weekly intraperitoneal injections of carbon tetrachloride (CCl 4 ) (Sigma, St. Louis, MO, USA, Cat# 289116) at a dose of 0.32 mg/g of body weight for 12 weeks as previously reported.

Techniques: Staining, Control, Flow Cytometry, Comparison, Transformation Assay

Journal: JHEP Reports

Article Title: Hepatic immune environment differences among common mouse strains in models of MASH and liver cancer

doi: 10.1016/j.jhepr.2025.101380

Figure Lengend Snippet: Cross-species comparison of liver immune changes by MASH between mice and humans. The published human scRNA-seq dataset GSE159977 of CD45+ cells from either MASH or healthy livers were processed using Seurat (5.1.0). (A) shows the UMAP split based on healthy or MASH. (B) shows the dot plot of marker genes for each annotated cell clusters. (C) Liver CD45 + cell compositions were measured in naïve BALB/c, C57BL/6, and FVB/N strains by flow cytometry as described in Fig. 1 A. CD45 + cell composition of healthy human liver or human HCC adjacent liver tissues were calculated based on the scRNA-seq datasets of GSE159977 or phs003279.v1.p1, respectively. (D,E) CD45 + or CD4 + T cell compositions of MASH or healthy human livers were calculated from GSE159977 . (F) In total liver CD45 + cells, the frequencies of shared major liver immune subsets between mice and human, including CD8+ T cell, CD4+ T cell, B cells, γδT cells and NK cells, were calculated in MASH or healthy human livers ( GSE159977 ) and MASH or control mice of three strains under either MCD diet or Western + CCl 4 diet MASH model. CCl 4 , carbon tetrachloride; HCC, hepatocellular carcinoma; MASH, metabolic dysfunction-associated steatohepatitis; MAIT cells, mucosal-associated invariant T cells; MCD diet, methionine- and choline-deficient diet; NK, natural killer; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: MASH was induced in mice by feeding a methionine- and choline-deficient (MCD) diet (Research diets inc., New Brunswick, NJ, USA Ref. A02082002BR) for a period of 3 weeks, or a Western diet (Envigo, Indianapolis, IN, USA, Ref. TD.120528) with high sugar solution (23.1 g/L d-fructose and 18.9 g/L d-glucose) with weekly intraperitoneal injections of carbon tetrachloride (CCl 4 ) (Sigma, St. Louis, MO, USA, Cat# 289116) at a dose of 0.32 mg/g of body weight for 12 weeks as previously reported.

Techniques: Comparison, Marker, Flow Cytometry, Control, Western Blot