Loss of the E3 ubiquitin ligase MARCHF6 alters hepatic lipid metabolism and drives spontaneous hepatosteatosis

Hepatic LDLR abundance was unchanged in Marchf6LKO mice.

“Of note, hepatic LDLR abundance was unchanged in the absence in Marchf6LKO, which is different from what was previously reported in hepatoma cell lines as a consequence of concomitant IDOL induction [30].”

• cites [30] Loregger, 2016, A MARCH6 and IDOL E3 ubiquitin ligase circuit uncouples cholesterol synthesis from Lipoprotein uptake in hepatocytes, Mol Cell Biol sourceSupported

Global or hepatocyte-specific deletion of Marchf6 in mice induces spontaneous accumulation of triglycerides and cholesteryl esters in the liver under chow-fed conditions.

“Global or hepatocyte-specific deletion of Marchf6 induced spontaneous accumulation of triglycerides and cholesteryl esters under chow-fed conditions”

Surviving male Marchf6(-/-) mice show growth retardation.

“A cohort of surviving male Marchf6(−/−) mice, collected over an extended period, was significantly lighter and showed growth retardation (Figure 1B and not shown).”

Silencing of MARCHF6 led to neutral lipid accumulation in PHH.

“Accordingly, silencing of MARCHF6 led to neutral lipid accumulation, as demonstrated by staining with the lipophilic fluorophore BODIPY (Figure 6D).”

After 12 weeks on standard chow diet, Marchf6LKO mice have similar body weight as control counterparts.

“Marchf6LKO mice were born at the expected Mendelian ratio (not shown) and after 12 weeks on standard chow diet had a similar body weight as their control counterparts (Figure 2A), but their livers were enlarged (Figure 2B and Supplementary Fig. 2B).”

The hepatic phenotype of LKO largely phenocopies that of liver-specific transgenic mice that produce a constitutively-active SREBP1c form.

“This could explain why both the precursor and mature (i.e. nuclear) forms of SREBP1 are elevated upon MARCHF6 ablation, and that the hepatic phenotype of LKO largely phenocopies that of liver-specific transgenic mice that produce a constitutively-active SREBP1c form [55].”

• cites [55] Shimano, 1997, Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells, J Clin Investig sourceCheck pending

Hepatic lipid accumulation in Marchf6LKO mice is accompanied by a moderate increase in total cholesterol content.

“Hepatic lipid accumulation in Marchf6LKO mice was characterized by a pronounced increase in hepatic TGs, accompanied by a moderate increase in total cholesterol content (Figure 2I,J).”

A similar SREBP1c-associated gene expression profile was observed in livers of global Marchf6(-/-) mice.

“A similar SREBP1c-associated gene expression profile was observed in livers of global Marchf6(−/−) mice (Supplementary Fig. 5B).”

Lipid droplets in MARCHF6-silenced PHH were bigger.

“Morphometric analysis of LDs in MARCHF6-silenced PHH revealed that these cells contained more LDs and that the droplets were bigger, resulting in a larger fraction of the intracellular space being occupied by LDs compared to control cells (Figure 6E–G).”

Global ablation of Marchf6 in mice results in a marked shift from the expected Mendelian birth distribution.

“In line with it having an important physiological role, global ablation of Marchf6 in mice results in a marked shift from the expected Mendelian birth distribution (this study and [29]).”

• cites [29] Nguyen, 2022, The MARCHF6 E3 ubiquitin ligase acts as an NADPH sensor for the regulation of ferroptosis, Nat Cell Biol sourceNot checked — no full text of the source

Initial results do not support the scenario that SREBP1c itself is a direct degradation target of MARCHF6.

“However, our initial results do not support this scenario (not shown).”

Loss of hepatocyte Marchf6 results in significantly lower circulating TG levels.

“Analysis of plasma lipids revealed that loss of hepatocyte Marchf6 resulted in significant lower circulating TG levels, evident in the VLDL and LDL lipoprotein fractions, and slightly elevated circulating cholesterol levels (Figure 2C–F).”

Neutral TG hydrolase activity is comparable in WT and Marchf6LKO liver samples.

“Additionally, neutral TG hydrolase activity was comparable in WT and Marchf6LKO liver samples, suggesting that TG mobilization from intrahepatic LDs is functional and not impaired (Figure 4B).”

MARCHF6 expression decreases with disease progression in human MASLD/MASH transcriptomic datasets.

“Both datasets revealed that MARCHF6 expression decreased with disease progression, while expression of SREBF1 and its target genes FASN and SCD1 was moderately upregulated (Figure 6H,I).”

• Also stated in: § Discussion · boosted

  “Finally, we provide transcriptional evidence that MARCHF6 expression decreases along the MASLD to MASH progression, accompanied by an increase in SREBP1 targets.”

• cites [50] Kendall, 2023, An integrated gene-to-outcome multimodal database for metabolic dysfunction-associated steatotic liver disease, Nat Med sourceCheck pending
• cites [51] Arendt, 2015, Altered hepatic gene expression in nonalcoholic fatty liver disease is associated with lower hepatic n-3 and n-6 polyunsaturated fatty acids, Hepatology sourceNot checked — no full text of the source

Loss of hepatocyte Marchf6 results in lower TG levels in VLDL and LDL lipoprotein fractions.

“Analysis of plasma lipids revealed that loss of hepatocyte Marchf6 resulted in significant lower circulating TG levels, evident in the VLDL and LDL lipoprotein fractions, and slightly elevated circulating cholesterol levels (Figure 2C–F).”

Bulk mRNA sequencing of Marchf6LKO liver tissue did not reveal obvious affected pathways that could explain the lipid accumulation.

“Bulk mRNA sequencing of Marchf6LKO liver tissue did not reveal obvious affected pathway(s) that could explain the lipid accumulation (Supplementary Fig. 5A).”

SREBP2-regulated genes associated with cholesterol biosynthesis were not affected by the ablation of Marchf6 in the liver.

“Distinct from SREBP1 target genes, SREBP2-regulated genes associated with cholesterol biosynthesis were not affected by the ablation of Marchf6 in the liver (Figure 5C).”

When normalized to body weight, surviving male Marchf6(-/-) mice exhibit a relatively larger liver weight.

“When normalized to body weight, these mice exhibited a relative larger liver weight and lower gonadal fat mass (Figure 1C,D).”

Histological analysis of Marchf6LKO livers shows spatially dispersed lipid droplets rich in neutral lipids.

“Macroscopic examination of Marchf6LKO livers revealed visible fat accumulation, which was corroborated by histological analysis showing spatially dispersed LDs rich in neutral lipids (Figure 2G,H).”

Loss of MARCHF6 enhances sterol pathway flux.

“Loss of MARCHF6 stabilized its substrate squalene epoxidase (SQLE), enhancing sterol pathway flux while concomitantly activating the SREBP1-associated lipogenic transcriptional program and increasing lipoprotein clearance.”

• AssumptionRests on an assumption: Many enzymatic steps in the synthesis of fatty acids and cholesterol require NADPH as a cofactor.

LKO mice develop spontaneous buildup of neutral lipids in lipid droplets.

“A striking observation in LKO mice was the development of spontaneous buildup of neutral lipids in LDs, which we attribute to the combination of cholesteryl-ester and acylglycerol accumulation, increased hepatic de novo FA synthesis and accelerated hepatic lipoprotein uptake.”

The NADP+/NADPH ratio was elevated in the livers of LKO mice.

“However, despite this increase, the NADP+/NADPH ratio was unexpectedly elevated in the livers of LKO mice, suggesting enhanced NADPH consumption in the absence of MARCHF6.”

mRNA expression of DGAT2 was unchanged in PHH upon MARCHF6 silencing.

“The mRNA expression of DGAT1, an enzyme involved in TG synthesis, was only marginally elevated, while expression of DGAT2 was unchanged (Supplementary Fig. 6E).”

Plasma AST and ALT levels are comparable in Marchf6LKO and WT mice.

“Both aspartate aminotransferase (AST) and alanine transaminase (ALT) levels were comparable in Marchf6LKO and WT mice, indicating that liver-specific deletion of Marchf6 is not hepatotoxic (Supplementary Fig. 2E).”

Lipidomic analyses demonstrated remodeling of the hepatic lipidome towards polyunsaturated, long-chain neutral lipids in MARCHF6-deficient liver.

“Accordingly, lipidomic analyses demonstrated remodeling of the hepatic lipidome towards polyunsaturated, long-chain neutral lipids, consistent with increased lipogenesis-driven NADPH consumption.”

Marchf6LKO mice have enlarged livers after 12 weeks on standard chow diet.

“Marchf6LKO mice were born at the expected Mendelian ratio (not shown) and after 12 weeks on standard chow diet had a similar body weight as their control counterparts (Figure 2A), but their livers were enlarged (Figure 2B and Supplementary Fig. 2B).”

In a small cohort of surviving female Marchf6(-/-) mice, there is a trend to increased relative liver mass.

“In a small cohort of surviving female Marchf6(−/−) mice we observed similar growth retardation and a trend to increased relative liver mass (Supplementary Fig. 1B and C).”

Loss of MARCHF6 increases lipoprotein clearance.

“Loss of MARCHF6 stabilized its substrate squalene epoxidase (SQLE), enhancing sterol pathway flux while concomitantly activating the SREBP1-associated lipogenic transcriptional program and increasing lipoprotein clearance.”

A larger fraction of the intracellular space was occupied by lipid droplets in MARCHF6-silenced PHH compared to control cells.

“Morphometric analysis of LDs in MARCHF6-silenced PHH revealed that these cells contained more LDs and that the droplets were bigger, resulting in a larger fraction of the intracellular space being occupied by LDs compared to control cells (Figure 6E–G).”

In the absence of hepatocyte MARCHF6, tracer clearance from the plasma was significantly accelerated.

“In the absence of hepatocyte MARCHF6, tracer clearance from the plasma was significantly accelerated (Figure 4C,D).”

Global loss of Marchf6 results in slightly higher plasma cholesterol levels compared to wildtype controls.

“We also assessed plasma lipid levels and found that global loss of Marchf6 did not alter circulating plasma TG but resulted in slightly higher levels of plasma cholesterol compared to wildtype controls (Figure 1E,F).”

Loss of MARCHF6 activates the SREBP1-associated lipogenic transcriptional program.

“Loss of MARCHF6 stabilized its substrate squalene epoxidase (SQLE), enhancing sterol pathway flux while concomitantly activating the SREBP1-associated lipogenic transcriptional program and increasing lipoprotein clearance.”

• AssumptionRests on an assumption: Many enzymatic steps in the synthesis of fatty acids and cholesterol require NADPH as a cofactor.

Global and hepatocyte-specific loss of MARCHF6 precipitates marked TG accumulation and steatosis in mice, in the absence of caloric overload or apparent metabolic stress.

“Our study establishes MARCHF6 as a pivotal regulator of hepatic lipid metabolism by demonstrating that its global and hepatocyte-specific loss precipitates marked TG accumulation and steatosis, in the absence of caloric overload or apparent metabolic stress.”

Pharmacological inhibition of the oxidative pentose phosphate pathway reduces lipid accumulation in MARCHF6-deficient human hepatocytes.

“In line with this, pharmacological inhibition of the oxidative pentose phosphate pathway reduced lipid accumulation in MARCHF6-deficient human hepatocytes.”

• AssumptionRests on an assumption: Many enzymatic steps in the synthesis of fatty acids and cholesterol require NADPH as a cofactor.

6-AN treatment significantly reduced lipid droplet size and occupancy in MARCHF6-silenced PHHs.

“Indeed, 6-AN treatment significantly reduced LD size and occupancy in PHHs (Figure 6F,G).”

The respiratory exchange ratio (RER) was unchanged in the absence of hepatocyte MARCHF6.

“Similarly, the balance between O2 consumption and CO2 production, and therefore the respiratory exchange ratio (RER) was unchanged in the absence of hepatocyte MARCHF6 (Supplementary Fig. 4F–H).”

Fasting plasma glucose levels are unchanged in Marchf6LKO mice.

“Unlike plasma lipid levels, fasting plasma glucose levels and glucose tolerance were unchanged in Marchf6LKO mice (Supplementary Fig. 2C and D).”

In human hepatocytes, pharmacological interventions targeting sterol synthesis and NADPH availability reduce LD abundance.

“Our study supports the notion that increased DNL is a prominent determinant of the LD phenotype in livers of LKO mice and this is consistent with the phenotype observed in human hepatocytes, where pharmacological interventions targeting sterol synthesis and NADPH availability reduce LD abundance.”

Expression of SREBP1 and its lipogenic target genes was increased in primary human hepatocytes upon MARCHF6 silencing.

“In line with our findings in mouse livers and HepG2 cells, expression of SREBP1 and its lipogenic target genes was increased in PHH upon MARCHF6 silencing (Supplementary Fig. 6E).”

Levels of SREBP1 precursor and mature form and its downstream targets (e.g. ACACA, FASN, SCD1, G6PD) were markedly increased in Marchf6LKO and Marchf6(-/-) livers.

“Consistent with this idea, levels of SREBP1 precursor and mature form and its downstream targets (e.g. ACACA, FASN, SCD1, G6PD) were markedly increased in Marchf6LKO and Marchf6(−/−) livers.”

Macroscopic examination of Marchf6LKO livers revealed visible fat accumulation.

“Macroscopic examination of Marchf6LKO livers revealed visible fat accumulation, which was corroborated by histological analysis showing spatially dispersed LDs rich in neutral lipids (Figure 2G,H).”

Loss of hepatocyte Marchf6 results in slightly elevated circulating cholesterol levels.

“Analysis of plasma lipids revealed that loss of hepatocyte Marchf6 resulted in significant lower circulating TG levels, evident in the VLDL and LDL lipoprotein fractions, and slightly elevated circulating cholesterol levels (Figure 2C–F).”

Global- and hepatocyte-specific loss of MARCHF6 in mice results in elevated abundance of G6PD.

“In line with this heightened NADPH demand, global- and hepatocyte-specific loss of MARCHF6 in mice resulted in elevated abundance of G6PD, the rate-limiting enzyme in the NADPH-producing pentose-phosphate pathway, and an SREBP1-sensitive gene [58,59].”

• cites [58] Shimomura, 1998, Nuclear sterol regulatory element-binding proteins activate genes responsible for the entire program of unsaturated fatty acid biosynthesis in transgenic mouse liver, J Biol Chem sourceCheck pending
• cites [59] Shimano, 1999, Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes, J Biol Chem sourceCheck pending

• AssumptionRests on an assumption: DNL has a high demand for NADPH as a reducing agent.

Surviving male Marchf6(-/-) mice are significantly lighter than controls.

“A cohort of surviving male Marchf6(−/−) mice, collected over an extended period, was significantly lighter and showed growth retardation (Figure 1B and not shown).”

Direct measurement confirmed upregulation of a selection of SREBP1-target genes in Marchf6LKO livers.

“This observation was confirmed by direct measurement of a selection of SREBP1-target genes, while genes associated with TG catabolism and LD formation were unaffected by the loss of hepatic Marchf6 (Figure 5B), which is also consistent with unchanged TGH activity (Figure 4B).”

Hepatic lipid accumulation in Marchf6LKO mice is characterized by a pronounced increase in hepatic TGs.

“Hepatic lipid accumulation in Marchf6LKO mice was characterized by a pronounced increase in hepatic TGs, accompanied by a moderate increase in total cholesterol content (Figure 2I,J).”

Genes associated with TG catabolism and LD formation were unaffected by the loss of hepatic Marchf6.

“This observation was confirmed by direct measurement of a selection of SREBP1-target genes, while genes associated with TG catabolism and LD formation were unaffected by the loss of hepatic Marchf6 (Figure 5B), which is also consistent with unchanged TGH activity (Figure 4B).”

When normalized to body weight, surviving male Marchf6(-/-) mice exhibit lower gonadal fat mass.

“When normalized to body weight, these mice exhibited a relative larger liver weight and lower gonadal fat mass (Figure 1C,D).”

SREBF1 and its target genes FASN and SCD1 are moderately upregulated with disease progression in human MASLD/MASH transcriptomic datasets.

“Both datasets revealed that MARCHF6 expression decreased with disease progression, while expression of SREBF1 and its target genes FASN and SCD1 was moderately upregulated (Figure 6H,I).”

• Also stated in: § Discussion · plain

  “Finally, we provide transcriptional evidence that MARCHF6 expression decreases along the MASLD to MASH progression, accompanied by an increase in SREBP1 targets.”

• cites [50] Kendall, 2023, An integrated gene-to-outcome multimodal database for metabolic dysfunction-associated steatotic liver disease, Nat Med sourceCheck pending
• cites [51] Arendt, 2015, Altered hepatic gene expression in nonalcoholic fatty liver disease is associated with lower hepatic n-3 and n-6 polyunsaturated fatty acids, Hepatology sourceNot checked — no full text of the source

In a small cohort of surviving female Marchf6(-/-) mice, similar growth retardation was observed.

“In a small cohort of surviving female Marchf6(−/−) mice we observed similar growth retardation and a trend to increased relative liver mass (Supplementary Fig. 1B and C).”

Treatment of MARCHF6-silenced PHHs with NB-598 markedly reduced lipid droplet size and occupancy.

“Treatment of MARCHF6-silenced PHHs with the high affinity SQLE inhibitor, NB-598, markedly reduced LD size and occupancy, presumably due to limiting de novo cholesterol synthesis.”

Loss of MARCHF6 in human primary hepatocytes recapitulates the LKO phenotype.

“Importantly, loss of MARCHF6 in human primary hepatocytes recapitulates this phenotype, supporting the translational value of our findings in mice.”

Loss of MARCHF6 stabilizes its substrate squalene epoxidase (SQLE).

“Loss of MARCHF6 stabilized its substrate squalene epoxidase (SQLE)”

Mating of heterozygous Marchf6(+/-) mice resulted in a marked deviation from the anticipated Mendelian distribution of offspring.

“Mating of heterozygote Marchf6(+/−) mice resulted in birth of offspring with a marked deviation from the anticipated Mendelian distribution, most of which did not survive past weaning (Figure 1A).”

Hepatic synthesis of C16:0 and C18:0 fatty acids is markedly increased in Marchf6LKO mice.

“We found a marked increase in hepatic synthesis of C16:0 and C18:0 fatty acids and desaturation (C16:1 and C18:1, respectively) in Marchf6LKO mice (Figure 4E–H).”

Glucose tolerance is unchanged in Marchf6LKO mice.

“Unlike plasma lipid levels, fasting plasma glucose levels and glucose tolerance were unchanged in Marchf6LKO mice (Supplementary Fig. 2C and D).”

There is a modest overall upregulation of SREBP1c-regulated lipogenic genes in Marchf6LKO livers compared to wildtype littermates.

“We therefore analyzed the data in more detail and checked whether known lipogenic genes were differentially expressed in Marchf6LKO livers compared to their wildtype littermates and identified a modest overall upregulation of SREBP1c-regulated lipogenic genes (Figure 5A).”

Principal component analysis showed a clear separation of samples derived from control and Marchf6LKO livers.

“Principal component analysis showed a clear separation of samples derived from control and Marchf6LKO livers suggesting a considerable influence of Marchf6 on the overall hepatic lipid profile (Figure 3A).”

Global loss of Marchf6 does not alter circulating plasma TG levels in mice.

“We also assessed plasma lipid levels and found that global loss of Marchf6 did not alter circulating plasma TG but resulted in slightly higher levels of plasma cholesterol compared to wildtype controls (Figure 1E,F).”

In the absence of MARCHF6, there is a shift toward longer, more unsaturated fatty acids.

“The accompanying shift toward longer, more unsaturated fatty acids points towards changes in the fatty acid elongation and desaturation molecular machinery, likely driven by MARCHF6-regulated metabolic and transcriptional pathways (Figure 3E).”

mRNA expression of DGAT1 was only marginally elevated in PHH upon MARCHF6 silencing.

“The mRNA expression of DGAT1, an enzyme involved in TG synthesis, was only marginally elevated, while expression of DGAT2 was unchanged (Supplementary Fig. 6E).”

MARCHF6-silenced PHH contained more lipid droplets.

“Morphometric analysis of LDs in MARCHF6-silenced PHH revealed that these cells contained more LDs and that the droplets were bigger, resulting in a larger fraction of the intracellular space being occupied by LDs compared to control cells (Figure 6E–G).”

Cholesterol synthesis is increased in the livers of Marchf6LKO mice.

“Likewise, cholesterol synthesis was increased in the livers of Marchf6LKO mice, which is consistent with MARCHF6 being an established regulator of the rate-limiting enzymes HMGCR and SQLE of cholesterol biosynthesis (Figure 4I).”

• AssumptionRests on an assumption: MARCHF6 is an established regulator of the rate-limiting enzymes HMGCR and SQLE of cholesterol biosynthesis.

Histological staining showed marked accumulation of neutral lipids in livers of mice lacking Marchf6.

“Macroscopic examination of male Marchf6(−/−) revealed a pale liver, and histological staining showed marked accumulation of neutral lipids in mice lacking Marchf6 (Figure 1G,H), which was corroborated by elevated hepatic TG levels (Figure 1I).”

No difference in plasma TG levels was observed between control and Marchf6LKO mice upon LPL inhibition.

“No difference in plasma TG levels was observed between control and Marchf6LKO mice upon LPL inhibition, indicating intact VLDL-mediated lipid export in the absence of MARCHF6 (Figure 4A).”

Macroscopic examination of male Marchf6(-/-) mice revealed a pale liver.

“Macroscopic examination of male Marchf6(−/−) revealed a pale liver, and histological staining showed marked accumulation of neutral lipids in mice lacking Marchf6 (Figure 1G,H), which was corroborated by elevated hepatic TG levels (Figure 1I).”

Absence of MARCHF6 led to accumulation of neutral lipids in primary mouse hepatocytes and HepG2 cells.

“Furthermore, ORO staining showed that absence of MARCHF6 led to the accumulation of neutral lipids in primary mouse hepatocytes and HepG2 cells, comparable to what was observed in liver tissue sections (Figure 6B,C and Supplementary Fig. 6C and D).”

Hepatic desaturation to C16:1 and C18:1 is increased in Marchf6LKO mice.

“We found a marked increase in hepatic synthesis of C16:0 and C18:0 fatty acids and desaturation (C16:1 and C18:1, respectively) in Marchf6LKO mice (Figure 4E–H).”

Hepatic TG levels are elevated in male Marchf6(-/-) mice.

“Macroscopic examination of male Marchf6(−/−) revealed a pale liver, and histological staining showed marked accumulation of neutral lipids in mice lacking Marchf6 (Figure 1G,H), which was corroborated by elevated hepatic TG levels (Figure 1I).”

MARCHF6 regulates proteasomal degradation of the LD-associated protein PLIN2.

“In lipid metabolism, MARCHF6 was shown to regulate the proteasomal degradation of the cholesterol biosynthesis enzymes SQLE, DHCR24, LDM, SC4MOL and SC5D [22,23,[25], [26], [27]], as well as of the LD-associated protein PLIN2 [31].”

• cites [31] Nguyen, 2019, N-terminal acetylation and the N-end rule pathway control degradation of the lipid droplet protein PLIN2, J Biol Chem sourceCheck pending

Loss of MARCHF6 is associated with a decrease in anti-ferroptotic SLC7A11, GPX4, and NRF2, likely a secondary effect.

“This was also associated with a decrease in the anti-ferroptotic SLC7A11, GPX4, and NRF2, which is likely a secondary effect, as it cannot be directly explained by the loss of E3 ligase activity.”

• cites [29] Nguyen, 2022, The MARCHF6 E3 ubiquitin ligase acts as an NADPH sensor for the regulation of ferroptosis, Nat Cell Biol sourceNot checked — no full text of the source

Loss of MARCHF6 suggests a disrupted balance between neutral lipid storage and membrane/signaling lipids.

“In the absence of MARCHF6, hepatic lipids are markedly remodeled, with increased di- and tri-TGs and cholesteryl esters alongside decreased phosphatidylcholine, alkyl-diacylglycerols, and ceramide species suggesting a disrupted balance between neutral lipid storage and membrane/signaling lipids (Figure 3B–D).”

Food intake and energy substrate utilization are unlikely to contribute to the lipid phenotype observed in Marchf6LKO mice.

“These findings indicate that food intake and energy substrate utilization are unlikely to contribute to the lipid phenotype observed in Marchf6LKO mice.”

The hepatosteatosis phenotype in MARCHF6 LKO mice reflects a hepatocyte-autonomous defect rather than a consequence of systemic metabolic dysregulation.

“This phenotype reflects a hepatocyte-autonomous defect rather than a consequence of systemic metabolic dysregulation.”

The recapitulation of the phenotype in human primary hepatocytes supports the translational value of the mouse findings.

“Importantly, loss of MARCHF6 in human primary hepatocytes recapitulates this phenotype, supporting the translational value of our findings in mice.”

MARCHF6 is a pivotal regulator of hepatic lipid metabolism.

“Our study establishes MARCHF6 as a pivotal regulator of hepatic lipid metabolism by demonstrating that its global and hepatocyte-specific loss precipitates marked TG accumulation and steatosis, in the absence of caloric overload or apparent metabolic stress.”

MARCHF6 is a multifaceted gatekeeper that integrates sterol turnover, NADPH usage, and lipogenesis to maintain hepatic lipid homeostasis.

“Overall, these data establish MARCHF6 as a multifaceted gatekeeper that integrates sterol turnover, NADPH usage, and lipogenesis to maintain hepatic lipid homeostasis.”

• AssumptionRests on an assumption: Many enzymatic steps in the synthesis of fatty acids and cholesterol require NADPH as a cofactor.
• AssumptionRests on an assumption: The liver is the central organ controlling systemic lipid metabolism.

Enhanced hepatic lipid uptake and synthesis are a primary cause for lipid accumulation in the absence of MARCHF6.

“In aggregate, our results point towards enhanced hepatic lipid uptake and synthesis as a primary cause for lipid accumulation in the absence of MARCHF6.”

The link between DNL and ferroptosis, both regulated by MARCHF6, is emerging as a determinant of human MASLD.

“The link between DNL and ferroptosis, which are both regulated by MARCHF6, is emerging as a determinant of human MASLD [61].”

• cites [61] Cui, 2026, MAFLD: a ferroptotic disease, Trends Mol Med sourceCheck pending

• AssumptionRests on an assumption: One feature shared between MASLD and MASH is accumulation of lipids in the liver.

MARCHF6-regulated lipogenic enzymes require NADPH for their activity.

“Additionally, MARCHF6-regulated lipogenic enzymes require NADPH for their activity [28].”

• cites [28] Fenton, 2024, A paREDOX in the control of cholesterol biosynthesis: does the NADPH sensor and E3 ubiquitin ligase MARCHF6 protect Mammalian cells during oxidative stress by controlling sterol biosynthesis?, Bioessays sourceCheck pending

Marchf6 has a considerable influence on the overall hepatic lipid profile.

“Principal component analysis showed a clear separation of samples derived from control and Marchf6LKO livers suggesting a considerable influence of Marchf6 on the overall hepatic lipid profile (Figure 3A).”

Enhanced hepatic lipid uptake may contribute, at least in part, to the hepatic lipid accumulation observed in Marchf6LKO mice.

“Combined with the markedly faster plasma clearance of the tracers in Marchf6LKO mice, this may contribute, at least in part, to the hepatic lipid accumulation observed in these mice.”

NADPH-dependent de novo lipogenesis is an important driver of lipid accumulation upon MARCHF6 deficiency.

“This result further supports NADPH-dependent de novo lipogenesis as an important driver of lipid accumulation upon MARCHF6 deficiency, yet at this point we cannot rule out other 6-AN-induced pleiotropic effects.”

Lipid accumulation observed in cells with diminished MARCHF6 expression results, at least in part, from increased de novo lipogenesis.

“Taken together these findings in human primary hepatocytes reinforce our observations in the mouse model, demonstrating that the lipid accumulation observed in cells with diminished MARCHF6 expression results, at least in part, from increased de novo lipogenesis.”

Loss of hepatic Marchf6 has dramatic consequences on hepatic lipid metabolism owing to dysregulated clearance of plasma lipoproteins and de novo lipogenesis.

“We show that loss of hepatic Marchf6 has dramatic consequences on hepatic lipid metabolism owing to dysregulated clearance of plasma lipoproteins and de novo lipogenesis (DNL).”

• AssumptionRests on an assumption: The liver is the central organ controlling systemic lipid metabolism.

Loss of MARCHF6 may contribute to development of MASLD.

“Loss of MARCHF6 disrupts this intricate balance, resulting in hepatic neutral-lipid accumulation and hence may contribute to development of MASLD.”

• AssumptionRests on an assumption: One feature shared between MASLD and MASH is accumulation of lipids in the liver.

Liver-specific deletion of Marchf6 is not hepatotoxic.

“Both aspartate aminotransferase (AST) and alanine transaminase (ALT) levels were comparable in Marchf6LKO and WT mice, indicating that liver-specific deletion of Marchf6 is not hepatotoxic (Supplementary Fig. 2E).”

The shift toward longer, more unsaturated fatty acids points towards changes in the fatty acid elongation and desaturation molecular machinery.

“The accompanying shift toward longer, more unsaturated fatty acids points towards changes in the fatty acid elongation and desaturation molecular machinery, likely driven by MARCHF6-regulated metabolic and transcriptional pathways (Figure 3E).”

Prior studies on MARCHF6 were limited to cultured cells.

“These studies were limited to cultured cells, and whether MARCHF6 is a physiological regulator of hepatic and systemic lipid metabolism is unknown at present.”

VLDL-mediated lipid export is intact in the absence of MARCHF6.

“No difference in plasma TG levels was observed between control and Marchf6LKO mice upon LPL inhibition, indicating intact VLDL-mediated lipid export in the absence of MARCHF6 (Figure 4A).”

MARCHF6 regulates proteasomal degradation of the cholesterol biosynthesis enzymes SQLE, DHCR24, LDM, SC4MOL, and SC5D.

“In lipid metabolism, MARCHF6 was shown to regulate the proteasomal degradation of the cholesterol biosynthesis enzymes SQLE, DHCR24, LDM, SC4MOL and SC5D [22,23,[25], [26], [27]], as well as of the LD-associated protein PLIN2 [31].”

• cites [22,23,[25], [26], [27]]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

A common feature of these MARCHF6 target enzymes is the consumption of NADPH as part of their enzymatic action.

“Notably, a common feature of these enzymes is the consumption of NADPH as part of their enzymatic action [28].”

• cites [28] Fenton, 2024, A paREDOX in the control of cholesterol biosynthesis: does the NADPH sensor and E3 ubiquitin ligase MARCHF6 protect Mammalian cells during oxidative stress by controlling sterol biosynthesis?, Bioessays sourceCheck pending

MARCHF6 sits at the interface between lipid synthesis, hepatic lipid clearance, and redox homeostasis.

“Mechanistically, MARCHF6 sits at the interface between lipid synthesis, hepatic lipid clearance, and redox homeostasis.”

The elevated NADP+/NADPH ratio suggests enhanced NADPH consumption in the absence of MARCHF6.

“However, despite this increase, the NADP+/NADPH ratio was unexpectedly elevated in the livers of LKO mice, suggesting enhanced NADPH consumption in the absence of MARCHF6.”

• AssumptionRests on an assumption: DNL has a high demand for NADPH as a reducing agent.

Increased DNL is a prominent determinant of the LD phenotype in livers of LKO mice.

“Our study supports the notion that increased DNL is a prominent determinant of the LD phenotype in livers of LKO mice and this is consistent with the phenotype observed in human hepatocytes, where pharmacological interventions targeting sterol synthesis and NADPH availability reduce LD abundance.”

The lipidomic remodeling is consistent with increased lipogenesis-driven NADPH consumption.

“Accordingly, lipidomic analyses demonstrated remodeling of the hepatic lipidome towards polyunsaturated, long-chain neutral lipids, consistent with increased lipogenesis-driven NADPH consumption.”

• AssumptionRests on an assumption: Many enzymatic steps in the synthesis of fatty acids and cholesterol require NADPH as a cofactor.

The hepatic lipid accumulation caused by Marchf6 deletion is a cell-autonomous hepatic defect independent of caloric excess.

“revealing a cell-autonomous hepatic defect independent of caloric excess”

MARCHF6 controls cholesterol biosynthesis by coupling the level of cholesterol to ubiquitylation and subsequent degradation of SQLE.

“Working together with its cognate E2 conjugating enzyme UBE2J2 [24], MARCHF6 controls cholesterol biosynthesis by coupling the level of the end-product, cholesterol, to ubiquitylation and subsequent degradation of SQLE.”

• cites [24] Tan, 2019, Differential use of E2 ubiquitin conjugating enzymes for regulated degradation of the rate-limiting enzymes HMGCR and SQLE in cholesterol biosynthesis, Atherosclerosis sourceCheck pending

MARCHF6 is a key regulator of hepatic lipid homeostasis.

“Collectively, these findings identify MARCHF6 as a key regulator of hepatic lipid homeostasis. Its loss triggers a coordinated lipidome shift that enhances neutral lipid storage and promotes the accumulation of longer, more unsaturated fatty acids characteristic of hepatosteatosis.”