Persistent mTORC1 activation underlies sex dimorphic progression of MASLD in mice with hepatocyte prohibitin-1 deficiency

Persistent mTORC1 activation underlies sex dimorphic progression of MASLD in mice with hepatocyte prohibitin-1 deficiency | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Persistent mTORC1 activation underlies sex dimorphic progression of MASLD in mice with hepatocyte prohibitin-1 deficiency Amany A. Alowaisi, Jolonda C. Mahoney, Ran Huo, Islam A. Berdaweel, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7481614/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Prohibitins (PHB1,2) are highly conserved lipid-raft associated proteins that physically interact to form a multimeric ring supercomplex in mitochondrial and plasma membranes where they are intimately involved in regulating cellular metabolism. Prior studies in disparate cell models have implicated PHB1 as a mediator of insulin signaling and its downstream effector, the mechanistic target of rapamycin complex 1 (mTORC1), but the mechanisms and physiological implications of these interactions are unclear. Here, we examined the role of PHB1 in regulating insulin and nutrient mediated activation of mTORC1 in liver using genetic and pharmacological approaches in mice and hepatocyte culture. Interestingly, male mice with hepatocyte-specific PHB1 haploinsufficiency (hPHB1-KD) at 6 months displayed features consistent with metabolic dysfunction-associated steatotic liver disease (MASLD), characterized by liver steatosis and impaired glucose tolerance with hyperinsulinemia, while these parameters were unaffected or even mildly improved in age-matched hPHB1-KD females. Both sexes of hPHB1-KD mice displayed increased basal phosphorylation of mTORC1 and its downstream targets (S6, 4EBP1) in liver compared with WT in fasted state, with minimal responsiveness to insulin. Transcriptomic data revealed strong upregulation of Lpin1 gene in male hPHB1-KD mice, a phosphatidic acid phosphatase regulated by mTORC1 that critically regulates hepatic lipid metabolism. Integrated transcript-/metabolomic analysis showed enriched glycerolipid metabolism and upregulation of MASLD pathway in the liver of hPHB1-KD males. Parallel experiments in AML12 hepatocytes confirmed that PHB1 knockdown causes hyper-activation of mTORC1 signaling, increased cytoplasmic lipin-1 expression and localization, and increased lipid droplet formation. Furthermore, one week of treatment with mTORC1 inhibitor Torin1 reduced hepatic triglycerides and normalized mTORC1 signaling in hPHB1-KD males to levels comparable with WT. Collectively, these findings demonstrate that PHB1 is essential for maintaining metabolic homeostasis in liver via control of mTORC1-lipin1 axis, and further confirm that metabolic effects of PHB1 deficiency in liver are sexually dimorphic. Biological sciences/Physiology/Metabolism/Metabolic diseases/Metabolic syndrome Biological sciences/Physiology/Metabolism/Fat metabolism Biological sciences/Cell biology/Mechanisms of disease Biological sciences/Cell biology/Cell signalling/Insulin signalling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Among the most highly conserved proteins in nature are the prohibitin domain containing family of proteins ( 1 , 2 ). Prohibitin-1 (PHB1) is a pleiotropic, evolutionarily conserved protein ubiquitously expressed in all eukaryotic cells that, depending on cell type and intracellular localization, participates in a variety of cellular functions including proliferation, metabolism, and apoptosis ( 3 , 4 ). Owing to these diverse functions, PHB1 has gained significant attention as a potential therapeutic target in chronic diseases such as obesity, type 2 diabetes, neurodegenerative diseases and cancer ( 5 , 6 ). PHB1 associates with its closely related homolog prohibitin 2 (PHB2) in mitochondrial membranes, forming a large ring-like complex that functions as a mitochondrial chaperone to preserve mitochondria structure and function ( 7 , 8 ). Loss of either PHB1 or PHB2 via genetic ablation results in destabilization and equivalent ablation of the other, highlighting PHB1/2 interdependence within the mitochondria at the protein level. However, their functional roles in extra-mitochondrial compartments appear to be independent and non-redundant ( 9 ). PHB1 has an essential role in preserving liver integrity and function, and its deficiency is linked to liver injury ( 3 , 10 , 11 ). Complete ablation of hepatocyte PHB1 quickly leads to liver cholestatic injury, steatohepatitis, and carcinogenesis ( 3 ). Moreover, reduced PHB1 levels have also been reported in livers of obese mice and in obese patients who are at increased risk of metabolic associated steatohepatitis (MASH) ( 12 ). Recent studies have implicated PHB1 as a potential mediator of two major nutrient-sensing kinases in the liver, AMPK and mTORC1 ( 13 , 14 ). Specifically, mechanistic target of rapamycin complexes 1 and 2 (mTORC1,2) signaling coordinates hepatocellular growth and metabolism, and loss of mTORC1 regulation contributes to pathogenesis of metabolic dysfunction associated steatotic liver disease (MASLD) ( 15 , 16 ). Through its activation by nutrients, insulin and growth factor signaling ( 17 ), mTORC1 enhances protein synthesis via phosphorylation of key downstream targets such as ribosomal S6 kinase (S6) and eukaryotic initiation factor 4E-binding protein (4EBP) ( 18 ). Activated mTORC1 also promotes lipogenesis through activation of sterol regulatory element-binding protein 1 (SREBP1), which regulates lipogenic gene expression, either via S6 ( 19 ) or phosphorylation and cellular localization of its downstream target, lipin-1 ( 17 , 20 ). Further understanding of the molecular mediators and regulators within the mTORC1 axis in liver is therefore essential for identifying new therapeutic targets in metabolic diseases. Interestingly, PHB1 ablation in Schwann cells was found to cause constitutive mTORC1 activation, leading to demyelination and peripheral neuropathy in mice ( 21 ). Another report showed that PHB2 knockdown in podocytes also led to mTORC1 hyper-activation and breakdown of the kidney filtration barrier ( 22 ). Despite the drastic difference between the cell models used in those studies and hepatocytes, it seemed plausible that a link between PHB’s and mTORC1 might underlie insulin responsiveness and control of metabolic homeostasis by the liver. In this study, we generated a constitutive hepatocyte-specific PHB1 haploinsufficient (i.e. heterozygous) mouse model to investigate these questions. The rationale for using a partial PHB1 knockdown model is two-fold: first, homozygous PHB1 knockout in hepatocytes is severely toxic and rapidly leads to hepatocellular carcinoma as previously described ( 11 ), and second, prior studies have reported reduced liver PHB1 levels in obese patients at risk of MASH ( 12 ), thereby increasing translational significance of a PHB1 deficient model. Our findings reveal an important role for PHB1 in regulating mTORC1 activity and lipogenesis in hepatocytes, in part via control of lipin-1 expression, and further show that loss of PHB1 levels leads to MASLD in a sex-specific manner. Results Hepatocyte PHB1 deficiency does not significantly affect liver mitochondrial function but promotes steatosis in male mice To determine the role of PHB1 in liver metabolic regulation, we generated hepatocyte-specific PHB1 deficient mice (hPHB1-KD) by crossing Phb1 fl/fl mice with mice expressing Cre recombinase under control of the albumin promoter (Figure S1 A, B). Only heterozygous mice (i.e. deletion of one Phb1 allele) were included in this study. We confirmed the knockdown of PHB1 in liver tissues at both the mRNA and protein levels (Figure S1 C, D). Consistent with previous findings, the reduction in PHB1 was accompanied by a decrease in PHB2, highlighting their interdependence for protein stability. Both male and female mice were included in our study as previous reports have found that PHB1 has metabolic effects that are sex specific ( 23 ). Mice were maintained on a regular chow diet for 16 weeks, and final testing performed at 5–6 months of age. Male hPHB1-KD mice had a significantly higher body weight compared with WT mice at end of the study (P < 0.05); an effect not observed in female hPHB1-KD mice (Fig. 1 A). This increased body weight was accompanied by greater % body fat and lower lean mass in male hPHB1-KD mice compared with WT, whereas no comparable differences were observed in female hPHB1-KD mice (Fig. 1 B, C). Triglyceride levels in liver and plasma were also increased in hPHB1-KD males compared with WT (Fig. 1 D, E). Histological analysis with oil red O staining revealed larger lipid droplets in hPHB1-KD males, but no significant liver structural injury or fibrosis as shown in hematoxylin and eosin (H & E) and picrosirius red (PSR) staining, respectively (Fig. 1 F). No lipid droplet accumulation was observed in livers of female hPHB1-KD mice (Fig. 1 G). As steatosis is frequently a consequence of decreased mitochondrial content and/or fat oxidation capacity, mitochondrial respiration was assessed in fresh liver samples from WT and hPHB1-KD mice using a variety of substrates. No significant differences in maximal ADP-stimulated respiration were observed with any substrate in either male or female hPHB1-KD mice, although males had lower basal (i.e., no ADP, state 2) respiration with palmitoyl-l-carnitine (Figure S2 A). Further assessment of mitochondrial respiratory complex expression in liver lysates revealed no differences between WT and hPHB1-KD mice (Figure S2 B). Systemic glucose metabolism is perturbed in male mice with hepatocyte PHB1 deficiency Given the established association between hepatic steatosis and loss of glycemic control ( 24 ), we next assessed glucose and insulin tolerance in hPHB1-KD mice. Glucose (GTT) and insulin tolerance tests (ITT) revealed that male hPHB1-KD mice exhibited impaired glucose tolerance and reduced insulin sensitivity as indicated by a significant increase in the area under the curve (AUC) for both tests (Fig. 2 A, C). Surprisingly, hPHB1-KD females had improved glucose tolerance and no change in insulin responsiveness (Fig. 2 B, D). These alterations in glycemic control were not due to changes in gluconeogenesis, as no significant differences were observed with pyruvate tolerance tests in hPHB1-KD mice compared with WT in either sex (Figure S3 ). To evaluate insulin signaling at the molecular level, a subset of mice was fasted for 6 hours and administered either i.p. insulin (10 U/kg) or saline and euthanized 15 minutes later. Insulin-stimulated Ser473 (S473) AKT phosphorylation was unchanged in livers of hPHB1-KD mice from both sexes compared with WT (Fig. 2 E). In gastrocnemius muscle, however, insulin-stimulated phosphorylation was significantly lower in male hPHB1-KD but was unexpectedly enhanced in female hPHB1-KD muscle relative to WT (Fig. 2 F). We further assessed glucose-stimulated insulin release by fasting mice for 6 hours followed by i.p. administration of either saline or 50% dextrose (1 g/kg), then euthanized after 30 minutes. Male hPHB1-KD mice displayed elevated basal plasma insulin concentrations, even in the absence of glucose stimulation, while no significant differences in plasma insulin levels were observed among female hPHB1-KD mice (Fig. 2 G). Transcript-/metabolomic analysis reveals Lpin1 as a novel regulator of lipogenesis and MASLD pathogenesis in male mice with hepatocyte PHB1 deficiency To gather mechanistic insights on changes at the molecular level in the hPHB1-KD mice, total RNA sequencing and unbiased metabolomic analysis was performed on bulk liver tissue samples which had been immediately clamped in liquid N 2 – cooled aluminum tongs after rapid dissection to minimize any hypoxia-related changes during tissue harvest ( 25 ). Differential gene expression (DEG) analysis identified approximately 20 genes with significantly altered expression between WT and hPHB1-KD male mice, based on adjusted p-value ( 1 or 0.35 or < -0.35) (Table S1 ). Intriguingly, of the DEG’s, a ~ 4-fold up-regulation of Lpin1 was observed in male hPHB1-KD mice compared with WT (adjusted p-value < 0.0005, beta value = 1.623); Fig. 3 A; Table S1 ). Lpin1 encodes lipin-1, a phosphatidic acid phosphatase that regulates hepatic lipid metabolism at multiple levels and compartmentalized manner in hepatocytes. In the cytoplasm, Lipin1 promotes triglyceride synthesis through its phosphatidic acid phosphatase function, while in the nucleus, it acts as a transcriptional co-regulator to enhance fatty acid oxidation ( 26 ). Notably, Lpin1 up-regulation was not observed in females (Table S2 ). Subsequent KEGG pathway enrichment analysis, performed using the BioJupies platform, also revealed significant upregulation of the MASLD pathway in male hPHB1-KD mice (Fig. 3 B, Table S3 ), while in female hPHB1-KD mice this pathway was downregulated (Table S4). For deeper insight, we conducted unbiased metabolomic profiling of the liver samples (Table S5), followed by integrative pathway analysis using MetaboAnalyst 5.0 software. DEG’s with p-value < 0.05 in male mice were analyzed in conjunction with metabolites showing a p-value < 0.1 using KEGG pathway. This integrative analysis revealed glycerolipid metabolism as one of the most significantly enriched pathways in hPHB1-KD males compared with WT (Fig. 3 C). PHB1 knockdown increases cytoplasmic lipin-1 and triglyceride accumulation in hepatocytes We next examined lipin-1 content in liver samples from hPHB1-KD and WT mice but found no significant differences in either sex (Figure S4). However, bulk liver tissue is a dense and heterogenous mix of many cell types, so to determine whether PHB1 deficiency specifically affects lipin-1 expression and localization in hepatocytes, we performed targeted silencing of PHB1 in AML12 hepatocytes using siRNA targeting Phb1 mRNA, or a scrambled non-targeting control siRNA (scrRNA). Immunoblot confirmed Phb1 siRNA enabled ≥ 50% knockdown of PHB1 compared with scrRNA, consistent with our hPHB1-KD mouse model (Figure S5). Importantly, immunofluorescent analysis revealed that PHB1-deficient AML12 cells had a significant increase in cytoplasmic lipin-1 compared with controls, while nuclear fluorescence levels remained unchanged (Fig. 4 A left panels, B-C). Furthermore, lipid droplets stained with Nile Red were substantially increased in the AML12 cells following PHB1 knockdown (Fig. 4 A right panels, D). Hyper-active mTORC1 signaling underlies pathogenesis of liver steatosis in male mice with hepatocyte PHB1 deficiency Prior studies have reported that mTORC1 regulates lipin-1 expression and function in multiple cell types ( 20 , 27 , 28 ), and given the link between PHB’s and mTORC1 implicated in the studies described above, we next tested whether mTORC1 signaling is perturbed in hepatocytes with PHB1 deficiency. Immunoblot analysis of both AML12 (Figure S5) and liver tissues from hPHB1-KD mice clearly show that PHB1 knockdown leads to increased phosphorylation of the well-known mTORC1 targets S6 at Serine 235/236, and 4EBP1 at serine 65 (Fig. 5 A). Notably, this activation of mTORC1 signaling in hPHB1-KD liver was not significantly augmented by insulin stimulation in either sex. To determine whether persistent mTORC1 activation plays a mechanistic role in the pathogenesis of hepatic steatosis, a cohort of 22-week-old hPHB1-KD male mice was treated with daily injections of Torin1 (20 mg/kg, i.p.), a potent mTORC1 inhibitor, or vehicle, for seven consecutive days. Torin1 treatment suppressed mTORC1 signaling, corresponding with decreased S6 and 4EBP1 phosphorylation in the liver (Fig. 5 B) and effectively reversed hepatic triglyceride accumulation in hPHB1-KD male mice to levels comparable to those of vehicle-treated WT controls (Fig. 5 C). Discussion MASLD has become the most prevalent chronic liver disease globally, affecting more than 30% of adolescents and adults ( 29 ). It encompasses a pathological continuum ranging from benign hepatic steatosis to progressive stages such as MASH, fibrosis, cirrhosis, and ultimately hepatocellular carcinoma, all of which contribute to considerable clinical and economic burdens ( 30 ). Additionally, MASLD, especially MASH with fibrosis, increases the risk of type 2 diabetes, cardiovascular disease, and certain extrahepatic cancers ( 31 , 32 ). Current therapies for MASLD primarily aim to reduce hepatic lipid burden or enhance their clearance, with several agents showing promise in improving steatosis and inflammation; however, most are limited by side effects and do not address the underlying metabolic dysregulation ( 33 – 36 ). Thus, new targets that drive MASLD pathogenesis are urgently needed for therapeutic investigation. A comprehensive proteomic study published > 20 years ago reported that PHB1 is downregulated in livers of ob/ob mice and obese humans, and in a mouse model of MASH ( 12 ). Despite over two decades of work since then, the link between PHB1 downregulation and liver disease pathogenesis has remained largely obscure. In this study, we demonstrated that hepatocyte-specific knockdown (but not complete ablation) of PHB1 leads to hepatic steatosis and impaired glucose tolerance in male mice by 6 months of age, characteristic of MASLD. Mechanistically, hepatocyte PHB1 deficiency induced constitutive mTORC1 activation and upregulated expression and cytoplasmic localization of lipin-1, a key regulator of lipid metabolism in hepatocyte. Importantly, we further demonstrate that the pathological lipid accumulation in hPHB1-KD male mice is, at least in part, mTORC1-dependent, as pharmacological inhibition by Torin1 mitigated hepatic steatosis. A role for PHB1 in regulating mitochondrial function has been well-characterized ( 37 ), and evidence implicating its broader role in metabolic regulation by the liver has emerged in recent years ( 3 ). Disruptions in PHB1 expression have been observed during the onset of MASH in a MAT1A −/− mouse model as previously described ( 3 , 12 ), as well as methionine-choline-deficient diet models ( 10 , 38 ), where steatosis emerged in PHB1 deficient mice as early as 3 days after onset of the diet ( 10 ). Our study is the first to report a mechanism by which PHB1 down-regulation in hepatocytes even in absence of injury leads to MASLD development, and document a clear role for aberrant mTORC1 signaling as a major pathogenic driver of the disease. Importantly, hepatocyte PHB1 deficiency causes steatosis and loss of glycemic control in male mice even without a high fat diet or increased caloric intake, underscoring its pivotal role in controlling metabolic homeostasis in liver. In a previous study, full ablation of PHB1 in hepatocytes caused severe liver injury as early as 3 weeks of age, associated with necrosis, inflammation, and mitochondrial dysfunction, with progression to hepatocellular carcinoma by 8 months. Of note, the mice in that study did not exhibit significant triglyceride accumulation at 14 weeks, despite increased plasma cholesterol ( 11 ). Our model of hepatocyte PHB1 haploinsufficiency maintained liver mitochondrial integrity and is consistent with a prior report which demonstrated that partial PHB1 loss in hepatocytes does not disrupt mitochondrial gene expression ( 39 ). Furthermore, transcriptomic profiling of both mouse and human liver tissues across various stages of MASLD revealed a weak association between MASLD progression and mitochondrial dysfunction ( 40 ). A strength of the partial PHB1 knockdown approach is that it enabled us to investigate the mechanisms by which PHB1 regulates hepatic lipid metabolism independently of liver injury or mitochondrial impairments, both of which are severe confounders. A key finding of our study is the upregulation of lpin1 gene in male hPHB1-KD mice. Lipin-1 is a member of the lipin family of proteins, Mg²⁺ -dependent enzymes that function as phosphatidic acid phosphatases in mammalian cells to regulate lipid metabolism via triglyceride synthesis and transcriptional regulation of lipid handling genes. In liver, the function of lipin-1 depends on its cellular localization. In nucleus, it acts as a transcriptional coactivator of PPARα and PGC1α to induce the expression of genes associated with mitochondrial fatty acid oxidation, including suppression of SREBP1 transcriptional activity, thereby decreasing lipogenesis. In cytoplasm, lipin-1 catalyzes the dephosphorylation of phosphatidic acid to produce diacylglycerol, a key step in TG synthesis, and VLDL secretion ( 28 , 41 ). It was shown that mice with liver lpin1 ablation had decreased body weight, adiposity, and liver and plasma triglyceride content ( 41 ). Our study revealed a sex-specific upregulation of lpin1 gene expression in livers of hPHB1-KD males, although we did not observe an increase in lipin-1 protein expression in liver lysates from these mice, suggesting that lipin-1 may be regulated at the post-transcriptional level and possibly cell-type specific within the heterogenous tissue of the liver. Since lipin-1 functions as a downstream effector of mTORC1, the increased Lpin1 gene expression observed in hPHB1-KD males may be mediated through PHB1 effect on mTORC1 signaling specifically in hepatocytes. Prior studies have shown that activated mTORC1 phosphorylates lipin-1 and enhances its cytoplasmic localization ( 20 , 28 ), an essential control node for sustaining lipin-1 phosphatidic acid phosphatase activity in response to changes in intracellular pH, membrane structure, or fatty acid accumulation. Also, mTORC-1 phosphorylation of lipin1 promotes the nuclear entry of activated SREBP1 and expression of its target lipogenic genes ( 26 ). Conversely, treatment with mTORC1 inhibitor, Torin1, promotes the nuclear localization of Lipin-1. This nuclear shift plays a critical role in regulating the SREPB1c transcriptional activity ( 20 , 28 , 42 ). Our transcriptomic analysis showed modest downregulation of Srebpf1 expression (Ln FC= -0.52; ~2-fold change) in the hPHB1-KD male liver, possibly an adaptation to the steatosis. A noteworthy finding of our study is that the transcriptomic and metabolic alterations seen with mTORC1 activation in the hPHB1-KD male mice were absent in females, indicating a sex-specific effect of hepatic PHB1 deficiency. Previous studies have reported crosstalk between estrogen and insulin signaling in the regulation of Lpin1 in liver ( 43 ). It is therefore plausible that estrogen signaling in females mitigates the upregulation of Lpin1 associated with PHB1 deficiency, thereby conferring protection against MASLD pathogenesis in female mice. Our data highlight a male-specific susceptibility to steatosis and systemic insulin resistance associated with PHB1 deficiency, aligning with previous reports that PHB1 interacts with sex hormone signaling pathways ( 44 ) and results in sex specific metabolic phenotypes in white adipose tissue and liver ( 45 ). In an in vitro model, estradiol treatment of 3T3-L1 and C9 cell lines resulted in a dose-dependent increase in both Phb1 mRNA and protein levels. Furthermore, PHB1 expression was significantly reduced when those cells were co-incubated with the estrogen receptor antagonist fulvestrant, indicating that up-regulation of PHB1 was mediated by estrogen receptor ( 46 ). Together with our findings, the evidence points to a potential role for sex steroid signaling in protecting female mice against metabolic dysfunction, warranting further investigation. mTOR is a serine/threonine kinase that mediates its regulatory functions through two structurally and functionally distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), each characterized by specific components, downstream effectors, and biological functions. mTORC1 drives cellular anabolic processes such as protein and lipid biosynthesis, while mTORC2 is primarily involved in regulating cell survival and cytoskeletal organization ( 18 ).Although this study is the first to link PHB1 to mTORC1 signaling in liver, PHB1 has been implicated in regulation of mTORC1 in a few prior studies with disparate cell models, most of which are proliferating cells. In an ovarian cancer cell line, PHB1 was found to inhibit mTORC1 through its interaction with FKBP8 ( 14 ). Two other studies have shown that PHB1 deletion leads to persistent upregulation of mTORC1 signaling, which are aligned more closely with our findings ( 21 , 22 ) which show persistent mTORC1 activation in PHB1-deficient livers even in the absence of insulin stimulation and under fasting conditions. The pathogenic effect of this persistent mTORC1 signaling was revealed by the determination that one week of Torin1 treatment significantly suppressed mTORC1 signaling in liver and attenuated hepatic lipid accumulation in male PHB1 KD mice, suggesting that in normal condition, PHB1 negatively regulates mTORC1 to maintain lipid homeostasis in liver. Taken together, our findings support a model in which PHB1 regulates hepatic lipid metabolism by modulating mTORC1-dependent control of lipin-1 localization, promoting its nuclear functions and preventing excessive triglyceride synthesis in the cytoplasm. An important consideration from our model is that PHB1 knockdown caused depletion of PHB2 (Figure S1 C), consistent with what is known about the interdependence of these proteins. It is intriguing that hepatocyte-specific PHB2 knockout mice have a number of metabolic features that are directly opposite those of hPHB1-KD mice. Specifically, the PHB2 knockout mice exhibit severe hypoglycemia, impaired insulin signaling, reduced plasma insulin levels, impaired gluconeogenesis, and reduced hepatic glycogen content. Moreover, the hepatocyte-specific PHB2 knockout mice develop hypolipidemia in parallel with liver steatosis, along with a sharp reduction in white adipose tissue mass, indicative of increased lipolysis and fat redistribution to the liver ( 47 ). Unfortunately, mTORC1 signaling was not evaluated in the liver from these mice, but it is clear from the divergence in phenotype that although PHB1 and PHB2 are interdependent in their cellular expression, they govern hepatic metabolism through distinct regulatory mechanisms. To conclude, our study illustrates a clear pathological mechanism linking decreased hepatocyte PHB1 to MASLD via unrestrained and persistent mTORC1 signaling, ultimately leading to increased cytoplasmic lipin-1 expression, steatosis and loss of glycemic control in male mice. Furthermore, the sex-specific nature of this phenotype unmasks PHB’s as potential regulatory nodes of sex signaling, particularly in metabolic disorders and endocrinology. They also reveal PHB’s as provocative therapeutic targets to mitigate MASLD and MASH. Materials and Methods Animal Model All animal experiments were approved by the University of Iowa Institutional Animal Care and Use Committee (IACUC). Mice with hepatocyte-specific PHB1-deficienecy were generated by crossing homozygous Phb1 floxxed ( Phb1 fl/fl ) mice, in which exon 2 of the Phb gene is flanked by loxP sites, with Albumin-Cre mice (Alb-Cre). Beginning at 8 weeks of age, male and female WT and hPHB1-KD mice weremaintained on regular chow diet (Research Diets (D20122207), NJ, USA; 12.7% kcal from fat, 72.3% kcal from carbohydrates, and 15% kcal from protein) for a total duration of 16 weeks. Study personnel remained blinded regarding mice genotypes until group assignments were revealed at time of analysis. Metabolic testing and phenotyping Mice body weights were continuously monitored throughout duration of the study. At ~ 5 months of age, mice were subjected to glucose (GTT), pyruvate (PTT), and insulin tolerance (ITT) tests separated by week-long intervals. On the day of each test, mice were singly housed and subjected to a 6-hour food restriction period. For GTT, mice were administered an i.p. injection of dextrose (1 g/kg of a 50% dextrose/saline solution (Nova Tech Inc., NE, USA)). For PTT, mice were administered an i.p. injection of a 1:10 pyruvate: lactate solution (Sigma, WI, USA) equivalent to 2 g/kg pyruvate. For ITT, mice were administered an i.p. injection of 0.75 units/kg of insulin (Eli Lilly, IN, USA). Blood glucose levels were measured from tail vein at 0-, 30-, 60-, and 120-minutes post-injection using a OneTouch Verio Flex glucometer (Lifescan, Switzerland). Body fat and lean mass were noninvasively measured using NMR within 24 hours prior to euthanasia using the Time Domain NMR Analyzer (minispec LF50, Bruker, MA, USA). Mice were euthanized under isoflurane anesthesia, and tissues were rapidly dissected and freeze clamped using aluminum tongs pre-cooled in liquid nitrogen. For the Torin1 experiment, male mice at age of 22-weeks received either Torin1 (20 mg/kg) (MedChemExpress, NJ, USA) or vehicle (20% N-methyl-2-pyrrolidone, NMP, 40% PEG400 in water) once daily via i.p. administration for 7 consecutive days. Following the treatment period, mice were euthanized and liver samples were collected for analysis as described above. Plasma and liver triglycerides Plasma and liver triglyceride content were measured using a commercial kit (Cayman Chemical, MI, USA) according to the manufacturer's protocol. Histology Liver tissues were fixed in 10% formalin, embedded in paraffin for subsequent staining with hematoxylin-eosin (H&E) and picrosirius red. For lipid droplets staining, formalin fixed liver tissue was embedded in OCT and frozen, sectioned then stained with Oil Red O. Images were captured using Olympus BX63 microscope. Plasma insulin Mice were fasted for 6 hours and euthanized 30 minutes after saline or 50% dextrose injection (1g/kg i.p). Blood was collected in EDTA coated tubes via exsanguination at time of euthanasia and centrifuged at 6000xg for 6 minutes to isolate plasma. Plasma insulin was measured using a commercial insulin rodent chemiluminescence ELISA kit (Alpco, NH, USA). Metabolomics Unbiased metabolomic profiling of bulk liver tissue samples from WT and hPHB1-KD mice was performed using LC-MS, analyzed by MetaboAnalyst 5.0 web-based platform. A more detailed description of the method can be found in the Supplemental Material online. RNA Sequencing RNA was isolated from liver tissue samples (n = 5/group) using the RNeasy Fibrous Tissue Kit (QIAGEN, Maryland, USA). RNA library preparation and sequencing was performed by the genomics division of the University of Iowa Institute of Human Genetics using standardized protocols and Illumina NovaSeq 6000 genome sequencer. Reads were acquired with 200 b.p. paired-end reads with an average depth of ~ 50M reads per sample. Adapter trimmed fastq were aligned using Kallisto ( 48 ) to reference transcriptome (“ ftp://ftp.ensembl.org/pub/release98/fasta/mus_musculus/cdna/Mus_musculus.GRCm38.cdna.all.fa.gz ”). Kallisto index and Kallisto quant were run using default settings, with specific input parameters of 100 bootstraps and 16 threads. Differential expression analysis was performed using Sleuth version 0.30.0 aggregating abundance on Ensemble genes, with a two-step Likelihood Ratio Test and Wald Test ( 49 ) Volcano plots were generated using R package VolcanoseR ( 50 ). Reads were also aligned with STAR version XX to an index built of “Mus_musculus.GRCm38.98.chr.gtf”. Gene counts were extracted from BAM files using subread version 1.5.2 and FeatureCounts. This count matrix was uploaded to Biojupies ( 51 ) for secondary analyses to calculate DEG’s and enrichR pathway analyses. Raw sequencing reads are deposited into GEO archive ###. Genes were considered differentially expressed between WT and hPHB1-KD mice based on beta value (> 0.35 or 1 or < -1, and adjusted p value < 0.05. Immunoblot analysis Liver tissue protein extracts were prepared using lysis buffer (50mM HEPES, 150mM NaCl, 1% Triton X-100, 2mM EGTA. 1mM MgCl2, 0.5mM DTT) supplemented with protease and phosphatase inhibitor (Sigma, WI, USA). AML 12 lysates were prepared using commercial RIPA buffer with the same inhibitors. Protein extracts were loaded onto 4–20% polyacrylamide gel (BioRad, CA, USA) for electrophoresis, then transferred to PVDF or nitrocellulose membrane, blocked in 5% bovine serum albumin (BSA) in 1X Tris-based saline with Tween (TBST). The membrane was incubated in a solution of primary antibodies (Table S6) contained in 0.5% BSA in TBST overnight at 4ºC. Following wash steps, membranes were incubated in solution of secondary antibodies (HRP conjugated goat anti-rabbit or anti mouse, Jackson Immunoresearch, PA, USA) for 2 hours at room temperature. After another set of wash steps, membranes were incubated in a chemiluminescent substrate (ThermoFisher Scientific Inc, MA, USA), and images were captured using an iBright FL1000 Imaging System (ThermoFisher Scientific Inc., MA, USA). Densitometry analysis was performed using Image J ( https://imagej.net/ij/ ). Liver mitochondria isolation and respiratory flux analysis Liver mitochondria were isolated as previously described ( 52 ). Briefly, liver tissue was dissected and rinsed in cold mitochondrial isolation medium (MIM) (300 mM sucrose, 10 mM Na-HEPES, 0.2 mM EDTA, pH 7.2), then thoroughly minced on ice in MIM supplemented with 0.5% BSA and 1mM EGTA. The tissue suspension was homogenized using a Dounce apparatus, then centrifuged at 600 g for 10 minutes at 4°C. The supernatant was collected and centrifuged at 8000 g for 15 minutes at 4°C. The pellet was resuspended in the same buffer and centrifuged again at 8000 g for 15 minutes at 4°C. The final pellet was resuspended in MIM buffer supplemented with 0.5% BSA and 1mM EGTA. Protein concentration was measured using Pierce BCA protein assay (ThermoFisher Scientific Inc, MA, USA). Mitochondrial respiration was assessed using the Oroboros O 2 K as follows: Briefly, 250 µg of isolated mitochondria were added to Buffer Z-lite (105 mM K-MES, 30 mM KCl, 10 mM KH 2 PO 4 , 5 mM MgCl 2 -6H 2 O, 0.05% BSA, pH 7.4), supplemented with 1 mM EGTA, 4 mM D-glucose, 1.7 U/mL glucose-6-phosphate dehydrogenase and 3.4 U/mL hexokinase (Roche, Sigma, WI, USA). After stabilization of oxygen concentration, substrates were added to measure oxygen consumption in the following order and concentrations, 25 µM palmitoyl carnitine with 2 mM malate, 650 µM ADP, 5 mM glutamate, 5 mM succinate. Data was analyzed using Oroboros DatLab software. AML12 culture and Phb1 silencing AML12 cells were purchased from ATCC (CRL-2254, VA, USA) and cultured in DMEM-F12 supplemented with 10% FBS, 1X insulin transferrin selenium (ITS) and 40 ng/ml dexamethasone and incubated at 37°C with humidified air containing 5% CO2. The siRNA targeting mouse Phb1 (ON-TARGETplus mouse Phb smart pool, L-060391-01-0005) and the nontargeting siRNA (ON-TARGETplus non-targeting pool, D-001810-10-05) were purchased from Horizon Discovery (Horizon Discovery, CO, USA). The non-targeting scrambled siRNA was used as a negative control. AML12 cells were grown in a 24 well plate and reverse transfected with 10nM siRNA using Lipofectamine RNAimax and opti-mem in the complete cell growth medium for 72 hours prior to biochemical analysis and/or imaging. Nile red staining of AML12 cells AML12 cells were washed with PBS twice, fixed in 4% PFA at room temperature for 30 minutes, permeabilized with 0.1% Triton-x100 for 5 minutes, then stained with Nile Red (1 µg/ml; Thermo-Fisher Scientific Inc, MA, USA) for 20 minutes at 37°C. Nuclei were counterstained using VECTASHIELD® anti-fade mounting medium plus DAPI. Images were captured using a Zeiss LSM 980 confocal microscope. Mean fluorescence intensity was analyzed using Zen Blue 3.3 software. Immunofluorescence staining of AML12 cells AML12 cells were washed with PBS, fixed with 4% PFA for 30 minutes at room temperature, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA, and 5% normal goat serum in PBS for 1 hour at room temperature. Subsequently, cells were incubated overnight at 4°C with an anti-lipin1 antibody (Abcam, MA, USA; dilution 1:200). After thorough washing with 0.04% TBST, cells were incubated with Alexa Fluor 488 conjugated secondary antibody (1:200 dilution) for 2 hours at room temperature in the dark. Finally, the cells were mounted using Vectashield antifade mounting medium containing DAPI. Images were captured using a Zeiss LSM 980 confocal microscope. Mean fluorescence intensity was analyzed using Zen blue 3.3 software. Statistical analysis Statistical analysis was performed in GraphPad Prism version 10. Data are presented as mean ± S.D. Unpaired t-test was used for two group comparisons, and one-way ANOVA was used for multiple group comparisons. Alpha-level of significance for main effects between groups was set at P < 0.05. Declarations Conflict of Interest The authors have no conflicts of interest to declare. Ethics Statement This study was approved by the Institution of Animal care and use committee at the University of Iowa. All animal experiments were performed in accordance with institutional and legal ethical standards. Supplementary information Additional supplementary information for this article is available online at Funding This work was supported by funding from the American Heart Association Strategically Focused Research Network award 20SFRN35200003, and National Heart Lung Blood Institute award R01HL167087. Acknowledgements Authors would like to thank Dr. William Paradee and the staff at the University of Iowa Viral Vector core facility for generating Phb1 fl/fl mice, and staff at the University of Iowa Genomics, Metabolomics, Central Microscopy, Histology and Metabolic Phenotyping core facilities for their resources and expertise. Special thanks to Maggie Cullum for assisting with Torin1 administration and other mouse model tasks. References Nuell MJ, Stewart DA, Walker L, Friedman V, Wood CM, Owens GA, et al. Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells. Mol Cell Biol. 1991;11(3):1372–81. Ande SR, Mishra S. Nuclear coded mitochondrial protein prohibitin is an iron regulated iron binding protein. Mitochondrion. 2011;11(1):40–7. Barbier-Torres L, Lu SC. Prohibitin 1 in liver injury and cancer. Exp Biol Med (Maywood). 2020;245(5):385–94. Mishra S, Murphy LC, Murphy LJ. The Prohibitins: emerging roles in diverse functions. J Cell Mol Med. 2006;10(2):353–63. Signorile A, Sgaramella G, Bellomo F, De Rasmo D. Prohibitins: A Critical Role in Mitochondrial Functions and Implication in Diseases. Cells. 2019;8(1). Theiss AL, Sitaraman SV. The role and therapeutic potential of prohibitin in disease. Biochim Biophys Acta. 2011;1813(6):1137–43. Steglich G, Neupert W, Langer T. Prohibitins regulate membrane protein degradation by the m-AAA protease in mitochondria. Mol Cell Biol. 1999;19(5):3435–42. Nijtmans LG, de Jong L, Artal Sanz M, Coates PJ, Berden JA, Back JW, et al. Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins. EMBO J. 2000;19(11):2444–51. Mishra S. Phb1:Phb2 heterodimers in the mitochondria-beyond functional interdependence. J Biol Chem. 2019;294(40):14836. Sanchez-Quiles V, Segura V, Bigaud E, He B, O'Malley BW, Santamaria E, et al. Prohibitin-1 deficiency promotes inflammation and increases sensitivity to liver injury. J Proteomics. 2012;75(18):5783–92. Ko KS, Tomasi ML, Iglesias-Ara A, French BA, French SW, Ramani K, et al. Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice. Hepatology. 2010;52(6):2096–108. Santamaria E, Avila MA, Latasa MU, Rubio A, Martin-Duce A, Lu SC, et al. Functional proteomics of nonalcoholic steatohepatitis: mitochondrial proteins as targets of S-adenosylmethionine. Proc Natl Acad Sci U S A. 2003;100(6):3065–70. Kanagaki S, Tsutsui Y, Kobayashi N, Komine T, Ito M, Akasaka Y, et al. Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins. iScience. 2023;26(4):106293. Zhang J, Yin Y, Wang J, Zhang J, Liu H, Feng W, et al. Prohibitin regulates mTOR pathway via interaction with FKBP8. Front Med. 2021;15(3):448–59. Panwar V, Singh A, Bhatt M, Tonk RK, Azizov S, Raza AS, et al. Multifaceted role of mTOR (mammalian target of rapamycin) signaling pathway in human health and disease. Signal Transduct Target Ther. 2023;8(1):375. Marcondes-de-Castro IA, Reis-Barbosa PH, Marinho TS, Aguila MB, Mandarim-de-Lacerda CA. AMPK/mTOR pathway significance in healthy liver and non-alcoholic fatty liver disease and its progression. J Gastroenterol Hepatol. 2023;38(11):1868–76. Mao Z, Zhang W. Role of mTOR in Glucose and Lipid Metabolism. Int J Mol Sci. 2018;19(7). Saxton RA, Sabatini DM. mTOR Signaling in Growth, Metabolism, and Disease. Cell. 2017;169(2):361–71. Duvel K, Yecies JL, Menon S, Raman P, Lipovsky AI, Souza AL, et al. Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol Cell. 2010;39(2):171–83. Peterson TR, Sengupta SS, Harris TE, Carmack AE, Kang SA, Balderas E, et al. mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway. Cell. 2011;146(3):408–20. Della-Flora Nunes G, Wilson ER, Hurley E, He B, O'Malley BW, Poitelon Y, et al. Activation of mTORC1 and c-Jun by Prohibitin1 loss in Schwann cells may link mitochondrial dysfunction to demyelination. Elife. 2021;10. Ising C, Koehler S, Brahler S, Merkwirth C, Hohne M, Baris OR, et al. Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure. EMBO Mol Med. 2015;7(3):275–87. Xu YXZ, Ande SR, Mishra S. Gonadectomy in Mito-Ob mice revealed a sex-dimorphic relationship between prohibitin and sex steroids in adipose tissue biology and glucose homeostasis. Biol Sex Differ. 2018;9(1):37. Watt MJ, Miotto PM, De Nardo W, Montgomery MK. The Liver as an Endocrine Organ-Linking NAFLD and Insulin Resistance. Endocr Rev. 2019;40(5):1367–93. Rauckhorst AJ, Borcherding N, Pape DJ, Kraus AS, Scerbo DA, Taylor EB. Mouse tissue harvest-induced hypoxia rapidly alters the in vivo metabolome, between-genotype metabolite level differences, and (13)C-tracing enrichments. Mol Metab. 2022;66:101596. Bi L, Jiang Z, Zhou J. The role of lipin-1 in the pathogenesis of alcoholic fatty liver. Alcohol Alcohol. 2015;50(2):146–51. Shimizu K, Fukushima H, Ogura K, Lien EC, Nihira NT, Zhang J, et al. The SCFbeta-TRCP E3 ubiquitin ligase complex targets Lipin1 for ubiquitination and degradation to promote hepatic lipogenesis. Sci Signal. 2017;10(460). Eaton JM, Mullins GR, Brindley DN, Harris TE. Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association. J Biol Chem. 2013;288(14):9933–45. Younossi ZM, Golabi P, Price JK, Owrangi S, Gundu-Rao N, Satchi R, et al. The Global Epidemiology of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis Among Patients With Type 2 Diabetes. Clin Gastroenterol Hepatol. 2024;22(10):1999–2010 e8. Younossi ZM, Paik JM, Henry L, Yang J, Fernandes G, Stepanova M, et al. The Growing Economic and Clinical Burden of Nonalcoholic Steatohepatitis (NASH) in the United States. J Clin Exp Hepatol. 2023;13(3):454–67. Targher G, Corey KE, Byrne CD, Roden M. The complex link between NAFLD and type 2 diabetes mellitus - mechanisms and treatments. Nat Rev Gastroenterol Hepatol. 2021;18(9):599–612. Targher G, Byrne CD, Tilg H. MASLD: a systemic metabolic disorder with cardiovascular and malignant complications. Gut. 2024;73(4):691–702. Mazhar IJ, Yasir M, Sarfraz S, Shlaghya G, Narayana SH, Mushtaq U, et al. Vitamin E and Pioglitazone: A Comprehensive Systematic Review of Their Efficacy in Non-alcoholic Fatty Liver Disease. Cureus. 2023;15(8):e43635. Mudaliar S, Henry RR, Sanyal AJ, Morrow L, Marschall HU, Kipnes M, et al. Efficacy and safety of the farnesoid X receptor agonist obeticholic acid in patients with type 2 diabetes and nonalcoholic fatty liver disease. Gastroenterology. 2013;145(3):574–82 e1. Harris E. FDA Okays First Drug for Scarring From Fatty Liver Disease. JAMA. 2024;331(17):1439. Pirola L. Elafibranor, a dual PPARalpha and PPARdelta agonist, reduces alcohol-associated liver disease: Lessons from a mouse model. World J Gastroenterol. 2025;31(4):99312. Oyang L, Li J, Jiang X, Lin J, Xia L, Yang L, et al. The function of prohibitins in mitochondria and the clinical potentials. Cancer Cell Int. 2022;22(1):343. Tomita K, Tamiya G, Ando S, Ohsumi K, Chiyo T, Mizutani A, et al. Tumour necrosis factor alpha signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice. Gut. 2006;55(3):415–24. Lee K, Yu H, Shouse S, Kong B, Lee J, Lee SH, et al. RNA-Seq Reveals Different Gene Expression in Liver-Specific Prohibitin 1 Knock-Out Mice. Front Physiol. 2021;12:717911. Zhao QY, Ge LH, Zhang K, Chen HF, Zhan XX, Yang Y, et al. Assessment of mitochondrial function in metabolic dysfunction-associated fatty liver disease using obese mouse models. Zool Res. 2020;41(5):539–51. Kajimoto K, Suemitsu E, Sato Y, Sakurai Y, Harashima H. Liver-Specific Silencing of Lipin1 Reduces Fat Mass as Well as Hepatic Triglyceride Biosynthesis in Mice. Biol Pharm Bull. 2016;39(10):1653–61. Peterfy M, Harris TE, Fujita N, Reue K. Insulin-stimulated interaction with 14-3-3 promotes cytoplasmic localization of lipin-1 in adipocytes. J Biol Chem. 2010;285(6):3857–64. Gowri PM, Sengupta S, Bertera S, Katzenellenbogen BS. Lipin1 regulation by estrogen in uterus and liver: implications for diabetes and fertility. Endocrinology. 2007;148(8):3685–93. Xu YXZ, Bassi G, Mishra S. Prohibitin: a prime candidate for a pleiotropic effector that mediates sex differences in obesity, insulin resistance, and metabolic dysregulation. Biol Sex Differ. 2019;10(1):25. Ande SR, Nguyen KH, Padilla-Meier GP, Wahida W, Nyomba BL, Mishra S. Prohibitin overexpression in adipocytes induces mitochondrial biogenesis, leads to obesity development, and affects glucose homeostasis in a sex-specific manner. Diabetes. 2014;63(11):3734–41. Choi M, Chaudhari HN, Ji YR, Ryoo ZY, Kim SW, Yun JW. Effect of estrogen on expression of prohibitin in white adipose tissue and liver of diet-induced obese rats. Mol Cell Biochem. 2015;407(1–2):181–96. Li L, Martin-Levilain J, Jimenez-Sanchez C, Karaca M, Foti M, Martinou JC, et al. In vivo stabilization of OPA1 in hepatocytes potentiates mitochondrial respiration and gluconeogenesis in a prohibitin-dependent way. J Biol Chem. 2019;294(34):12581–98. Bray NL, Pimentel H, Melsted P, Pachter L. Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016;34(5):525–7. Pimentel H, Bray NL, Puente S, Melsted P, Pachter L. Differential analysis of RNA-seq incorporating quantification uncertainty. Nat Methods. 2017;14(7):687–90. Goedhart J, Luijsterburg MS. VolcaNoseR is a web app for creating, exploring, labeling and sharing volcano plots. Sci Rep. 2020;10(1):20560. Torre D, Lachmann A, Ma'ayan A. BioJupies: Automated Generation of Interactive Notebooks for RNA-Seq Data Analysis in the Cloud. Cell Syst. 2018;7(5):556–61 e3. Katunga LA, Gudimella P, Efird JT, Abernathy S, Mattox TA, Beatty C, et al. Obesity in a model of gpx4 haploinsufficiency uncovers a causal role for lipid-derived aldehydes in human metabolic disease and cardiomyopathy. Mol Metab. 2015;4(6):493–506. Additional Declarations (Not answered) Supplementary Files SupplementalmethodsDetailedmetabolomics.docx Detailed metabolomic methodology OriginalFullLengthWesternBlots.pptx Uncropped original western blots SupplementaryFiguresFINAL.pptx All supplementary Figures Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7481614","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":515565256,"identity":"143a5cc1-3b63-43c6-8bbf-9e3485374d82","order_by":0,"name":"Amany A. 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1","display":"","copyAsset":false,"role":"figure","size":135603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of hepatocyte PHB1 deficiency on body composition and triglyceride metabolism. A. \u003c/strong\u003eMice body weight changes over the duration of the study (n=12/group) in WT and hPHB1-KD male (top panel) and female (lower panel) mice. \u003cstrong\u003eB.\u003c/strong\u003e Body fat and lean mass \u003cstrong\u003e(C)\u003c/strong\u003e percents of WT and hPHB1-KD male and female mice determined by NMR analysis at 6 months age (n=6-12/group). \u003cstrong\u003eD. \u003c/strong\u003eTriglycerides level in Liver tissue homogenates and plasma \u003cstrong\u003e(E)\u003c/strong\u003eof WT and hPHB1-KD male and female mice (n=6-10/group). \u003cstrong\u003eF.\u003c/strong\u003eRepresentative images showing hematoxylin and eosin (H\u0026amp;E), picrosirius red (PSR) and oil red O staining of histological sections of liver tissues from WT and hPHB1-KD male (left panel) and \u003cstrong\u003e(G)\u003c/strong\u003e female (right panel) mice (scale bar 50 µm), n=2-4/group, images are representative of 10 image fields captured per mouse under X20 magnification. All data are presented as mean SD. *P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7481614/v1/90dff9227fa97b27632c1c4e.jpg"},{"id":92178899,"identity":"58d0988f-ae51-41c8-b968-8d976d0a89ee","added_by":"auto","created_at":"2025-09-25 13:20:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":113457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlycemic control and metabolic parameters of hepatocyte PHB1 deficient mice at 6 months of age. (A-B) \u003c/strong\u003eBlood glucose levels during glucose tolerance test (GTT) of WT and hPHB1-KD male \u003cstrong\u003e(A)\u003c/strong\u003e and female \u003cstrong\u003e(B) \u003c/strong\u003emice with total AUC analysis for each test (n = 6 –12/group). \u003cstrong\u003e(C-D).\u003c/strong\u003e Blood glucose levels during insulin tolerance test (ITT) of WT and hPHB1-KD male \u003cstrong\u003e(C)\u003c/strong\u003eand female \u003cstrong\u003e(D)\u003c/strong\u003e mice with total AUC analysis for each test (n = 6 –12/group). \u003cstrong\u003e(E-F).\u003c/strong\u003e Representative immunoblots of phosphorylated AKT at Serin 473 (S473) in liver \u003cstrong\u003e(E) \u003c/strong\u003eand gastrocnemius muscle lysates \u003cstrong\u003e(F)\u003c/strong\u003efrom WT and hPHB1-KD male and female mice after fasting for 6 hours followed by i.p. insulin stimulation (10 units/kg) (n = 3/group). \u003cstrong\u003eG.\u003c/strong\u003e Plasma insulin levels in WT and hPHB1-KD male and female mice after 6 hours fasting followed by i.p. dextrose administration (1g/kg) (n = 5/group). Unpaired \u003cem\u003et\u003c/em\u003e-test was used for comparison between genotypes within the same sex group. All data are presented as mean ± SD. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Slide2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7481614/v1/edd5290401d3be550118a319.jpg"},{"id":92180255,"identity":"8d96d64b-e8a5-46e7-b9e2-047b13cab5f0","added_by":"auto","created_at":"2025-09-25 13:28:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLiver transcript-/metabolomic analysis. A. \u003c/strong\u003eVolcano plot representation of DEG’s in liver of hPHB1-KD males compared with WT. Annotated genes on the plot represent DEG’s that are significantly up- (red) or downregulated (blue) in hPHB1-KD mice. The Y-axis shows statistical significance, expressed as –log10 p-value while the X-axis denotes the beta value (natural log of fold change) between groups. \u003cstrong\u003eB.\u003c/strong\u003e KEGG pathway enrichment analysis performed using BioJupies software. Bar graph was prepared using GraphPad prism 10 software to show upregulated pathways involved in liver metabolism \u003cstrong\u003eC.\u003c/strong\u003e Joint pathway analysis by integrating transcriptomic (DEGs with p value \u0026lt;0.05 and beta value \u0026gt; 0.35 (one-fold change)) and metabolomic (metabolites with p-value \u0026lt;0.1) data using KEGG pathway on MetaboAnalyst software version 5. The plot represents the pathway impact with its significance presented as -1og10 p-value.\u003c/p\u003e","description":"","filename":"Slide3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7481614/v1/265847596d00ec05a2ff07bb.jpg"},{"id":92178902,"identity":"7214439d-e989-49b0-bbcb-a54544092eb0","added_by":"auto","created_at":"2025-09-25 13:20:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":153716,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of acute PHB1 knockdown in cultured AML12 hepatocytes. A. \u003c/strong\u003eThe left panels depict representative confocal microscopy images of lipin-1 (green) and its overlay with nuclear DAPI stain. The right panels depict representative images for Nile red staining of lipid droplets (red) and the overlay with DAPI. Images were captured at 63X magnification. Scale bar= 10µm. \u003cstrong\u003e(B-C)\u003c/strong\u003e. Lipin-1 mean fluorescence intensity (MFI) in the nucleus \u003cstrong\u003e(B)\u003c/strong\u003e and cytoplasm \u003cstrong\u003e(C)\u003c/strong\u003eexpressed in arbitrary units (AU), n=20-24 images/group. \u003cstrong\u003eD. \u003c/strong\u003eQuantification of mean fluorescence intensity (MFI) of Nile red expressed in arbitrary units (AU), n= 14-16 images/group. Analysis was performed using Zen blue software. Each data point represents one image field captured for that particular group. All data are presented as mean SD. One way ANOVA for three group comparison. **P\u0026lt;0.005, ***P\u0026lt;0.0005, ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Slide4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7481614/v1/166ae49e2761aa26bcea8107.jpg"},{"id":92178905,"identity":"12d92b95-1297-449b-a8f8-c0a1d802a3d8","added_by":"auto","created_at":"2025-09-25 13:20:33","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":83741,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePersistent mTORC1 signaling in hPHB1-KD liver and the effect of Torin1 treatment. A. \u003c/strong\u003eRepresentative immunoblots for phosphorylated mTORC1 and its downstream targets 4-EBP1 (at Serine 65, S65) and S6 (at Serine 235/236, S235/236) in liver lysates of WT and hPHB1-KD male and female mice (n= 3/group), at baseline and following insulin stimulation (10 units/kg), with corresponding densitometry analysis shown in panels to the right. *p\u0026lt;0.05, **P\u0026lt;0.005. \u003cstrong\u003eB.\u003c/strong\u003e Representative immunoblots of phospho-4EBP1 and phospho-S6 after 7 days of daily Torin1 (20mg/kg, i.p.) or vehicle treatment in male WT and hPHB1-KD mice (n= 3/group), with corresponding densitometry analysis shown in panels to the right. *p\u0026lt;0.05. \u003cstrong\u003eC.\u003c/strong\u003e Quantification of liver triglyceride (TG) content after Torin1 or vehicle treatment in male WT and hPHB1-KD mice. Data are normalized to liver tissue weight (mg/g). N=5-8/group. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **P\u0026lt;0.01. All data are presented as mean ± SD. Unpaired \u003cem\u003et\u003c/em\u003e-test for comparisons.\u003c/p\u003e","description":"","filename":"Slide5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7481614/v1/b3787d12c0225640fdd2120b.jpg"},{"id":97673021,"identity":"1d4f3707-77d8-491c-91a4-888eab9dcd3b","added_by":"auto","created_at":"2025-12-08 09:39:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1619514,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7481614/v1/97b427e9-cf15-4032-82f4-050d47a99a5f.pdf"},{"id":92178901,"identity":"8e7d1768-3960-4cdf-b4ab-76d92e250d70","added_by":"auto","created_at":"2025-09-25 13:20:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14435,"visible":true,"origin":"","legend":"Detailed metabolomic methodology","description":"","filename":"SupplementalmethodsDetailedmetabolomics.docx","url":"https://assets-eu.researchsquare.com/files/rs-7481614/v1/14c6b3f9be78c819dea7d4e0.docx"},{"id":92178911,"identity":"add0bf7e-5258-40f7-a540-95993aa22e49","added_by":"auto","created_at":"2025-09-25 13:20:33","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6495293,"visible":true,"origin":"","legend":"Uncropped original western blots","description":"","filename":"OriginalFullLengthWesternBlots.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7481614/v1/b3e869abb5a370e6728818b7.pptx"},{"id":92180258,"identity":"89e3ea16-9bf4-41a0-b368-1cfbde593b96","added_by":"auto","created_at":"2025-09-25 13:28:33","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":7296303,"visible":true,"origin":"","legend":"All supplementary Figures","description":"","filename":"SupplementaryFiguresFINAL.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7481614/v1/a67a48300962efa4d21cfc39.pptx"}],"financialInterests":"(Not answered)","formattedTitle":"Persistent mTORC1 activation underlies sex dimorphic progression of MASLD in mice with hepatocyte prohibitin-1 deficiency","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAmong the most highly conserved proteins in nature are the prohibitin domain containing family of proteins (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Prohibitin-1 (PHB1) is a pleiotropic, evolutionarily conserved protein ubiquitously expressed in all eukaryotic cells that, depending on cell type and intracellular localization, participates in a variety of cellular functions including proliferation, metabolism, and apoptosis (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Owing to these diverse functions, PHB1 has gained significant attention as a potential therapeutic target in chronic diseases such as obesity, type 2 diabetes, neurodegenerative diseases and cancer (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). PHB1 associates with its closely related homolog prohibitin 2 (PHB2) in mitochondrial membranes, forming a large ring-like complex that functions as a mitochondrial chaperone to preserve mitochondria structure and function (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Loss of either PHB1 or PHB2 via genetic ablation results in destabilization and equivalent ablation of the other, highlighting PHB1/2 interdependence within the mitochondria at the protein level. However, their functional roles in extra-mitochondrial compartments appear to be independent and non-redundant (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePHB1 has an essential role in preserving liver integrity and function, and its deficiency is linked to liver injury (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Complete ablation of hepatocyte PHB1 quickly leads to liver cholestatic injury, steatohepatitis, and carcinogenesis (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Moreover, reduced PHB1 levels have also been reported in livers of obese mice and in obese patients who are at increased risk of metabolic associated steatohepatitis (MASH) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Recent studies have implicated PHB1 as a potential mediator of two major nutrient-sensing kinases in the liver, AMPK and mTORC1 (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Specifically, mechanistic target of rapamycin complexes 1 and 2 (mTORC1,2) signaling coordinates hepatocellular growth and metabolism, and loss of mTORC1 regulation contributes to pathogenesis of metabolic dysfunction associated steatotic liver disease (MASLD) (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Through its activation by nutrients, insulin and growth factor signaling (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), mTORC1 enhances protein synthesis via phosphorylation of key downstream targets such as ribosomal S6 kinase (S6) and eukaryotic initiation factor 4E-binding protein (4EBP) (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Activated mTORC1 also promotes lipogenesis through activation of sterol regulatory element-binding protein 1 (SREBP1), which regulates lipogenic gene expression, either via S6 (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) or phosphorylation and cellular localization of its downstream target, lipin-1 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Further understanding of the molecular mediators and regulators within the mTORC1 axis in liver is therefore essential for identifying new therapeutic targets in metabolic diseases.\u003c/p\u003e\u003cp\u003eInterestingly, PHB1 ablation in Schwann cells was found to cause constitutive mTORC1 activation, leading to demyelination and peripheral neuropathy in mice (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Another report showed that PHB2 knockdown in podocytes also led to mTORC1 hyper-activation and breakdown of the kidney filtration barrier (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Despite the drastic difference between the cell models used in those studies and hepatocytes, it seemed plausible that a link between PHB\u0026rsquo;s and mTORC1 might underlie insulin responsiveness and control of metabolic homeostasis by the liver. In this study, we generated a constitutive hepatocyte-specific PHB1 haploinsufficient (i.e. heterozygous) mouse model to investigate these questions. The rationale for using a partial PHB1 knockdown model is two-fold: first, homozygous PHB1 knockout in hepatocytes is severely toxic and rapidly leads to hepatocellular carcinoma as previously described (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), and second, prior studies have reported reduced liver PHB1 levels in obese patients at risk of MASH (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), thereby increasing translational significance of a PHB1 deficient model.\u003c/p\u003e\u003cp\u003eOur findings reveal an important role for PHB1 in regulating mTORC1 activity and lipogenesis in hepatocytes, in part via control of lipin-1 expression, and further show that loss of PHB1 levels leads to MASLD in a sex-specific manner.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eHepatocyte PHB1 deficiency does not significantly affect liver mitochondrial function but promotes steatosis in male mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the role of PHB1 in liver metabolic regulation, we generated hepatocyte-specific PHB1 deficient mice (hPHB1-KD) by crossing \u003cem\u003ePhb1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice with mice expressing Cre recombinase under control of the albumin promoter (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, B). Only heterozygous mice (i.e. deletion of one \u003cem\u003ePhb1\u003c/em\u003e allele) were included in this study. We confirmed the knockdown of PHB1 in liver tissues at both the mRNA and protein levels (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC, D). Consistent with previous findings, the reduction in PHB1 was accompanied by a decrease in PHB2, highlighting their interdependence for protein stability. Both male and female mice were included in our study as previous reports have found that PHB1 has metabolic effects that are sex specific (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Mice were maintained on a regular chow diet for 16 weeks, and final testing performed at 5\u0026ndash;6 months of age. Male hPHB1-KD mice had a significantly higher body weight compared with WT mice at end of the study (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); an effect not observed in female hPHB1-KD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This increased body weight was accompanied by greater % body fat and lower lean mass in male hPHB1-KD mice compared with WT, whereas no comparable differences were observed in female hPHB1-KD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). Triglyceride levels in liver and plasma were also increased in hPHB1-KD males compared with WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). Histological analysis with oil red O staining revealed larger lipid droplets in hPHB1-KD males, but no significant liver structural injury or fibrosis as shown in hematoxylin and eosin (H \u0026amp; E) and picrosirius red (PSR) staining, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). No lipid droplet accumulation was observed in livers of female hPHB1-KD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs steatosis is frequently a consequence of decreased mitochondrial content and/or fat oxidation capacity, mitochondrial respiration was assessed in fresh liver samples from WT and hPHB1-KD mice using a variety of substrates. No significant differences in maximal ADP-stimulated respiration were observed with any substrate in either male or female hPHB1-KD mice, although males had lower basal (i.e., no ADP, state 2) respiration with palmitoyl-l-carnitine (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). Further assessment of mitochondrial respiratory complex expression in liver lysates revealed no differences between WT and hPHB1-KD mice (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSystemic glucose metabolism is perturbed in male mice with hepatocyte PHB1 deficiency\u003c/h2\u003e\u003cp\u003eGiven the established association between hepatic steatosis and loss of glycemic control (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), we next assessed glucose and insulin tolerance in hPHB1-KD mice. Glucose (GTT) and insulin tolerance tests (ITT) revealed that male hPHB1-KD mice exhibited impaired glucose tolerance and reduced insulin sensitivity as indicated by a significant increase in the area under the curve (AUC) for both tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C). Surprisingly, hPHB1-KD females had improved glucose tolerance and no change in insulin responsiveness (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, D). These alterations in glycemic control were not due to changes in gluconeogenesis, as no significant differences were observed with pyruvate tolerance tests in hPHB1-KD mice compared with WT in either sex (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). To evaluate insulin signaling at the molecular level, a subset of mice was fasted for 6 hours and administered either i.p. insulin (10 U/kg) or saline and euthanized 15 minutes later. Insulin-stimulated Ser473 (S473) AKT phosphorylation was unchanged in livers of hPHB1-KD mice from both sexes compared with WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In gastrocnemius muscle, however, insulin-stimulated phosphorylation was significantly lower in male hPHB1-KD but was unexpectedly enhanced in female hPHB1-KD muscle relative to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). We further assessed glucose-stimulated insulin release by fasting mice for 6 hours followed by i.p. administration of either saline or 50% dextrose (1 g/kg), then euthanized after 30 minutes. Male hPHB1-KD mice displayed elevated basal plasma insulin concentrations, even in the absence of glucose stimulation, while no significant differences in plasma insulin levels were observed among female hPHB1-KD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscript-/metabolomic analysis reveals\u003c/b\u003e \u003cb\u003eLpin1\u003c/b\u003e \u003cb\u003eas a novel regulator of lipogenesis and MASLD pathogenesis in male mice with hepatocyte PHB1 deficiency\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo gather mechanistic insights on changes at the molecular level in the hPHB1-KD mice, total RNA sequencing and unbiased metabolomic analysis was performed on bulk liver tissue samples which had been immediately clamped in liquid N\u003csub\u003e2\u003c/sub\u003e \u0026ndash; cooled aluminum tongs after rapid dissection to minimize any hypoxia-related changes during tissue harvest (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Differential gene expression (DEG) analysis identified approximately 20 genes with significantly altered expression between WT and hPHB1-KD male mice, based on adjusted p-value (\u0026lt;\u0026thinsp;0.05) and fold change\u0026thinsp;\u0026gt;\u0026thinsp;1 or \u0026lt; -1 (corresponds to beta value (Ln FC)\u0026thinsp;\u0026gt;\u0026thinsp;0.35 or \u0026lt; -0.35) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Intriguingly, of the DEG\u0026rsquo;s, a\u0026thinsp;~\u0026thinsp;4-fold up-regulation of \u003cem\u003eLpin1\u003c/em\u003e was observed in male hPHB1-KD mice compared with WT (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0005, beta value\u0026thinsp;=\u0026thinsp;1.623); Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). \u003cem\u003eLpin1\u003c/em\u003e encodes lipin-1, a phosphatidic acid phosphatase that regulates hepatic lipid metabolism at multiple levels and compartmentalized manner in hepatocytes. In the cytoplasm, Lipin1 promotes triglyceride synthesis through its phosphatidic acid phosphatase function, while in the nucleus, it acts as a transcriptional co-regulator to enhance fatty acid oxidation (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Notably, \u003cem\u003eLpin1\u003c/em\u003e up-regulation was not observed in females (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Subsequent KEGG pathway enrichment analysis, performed using the BioJupies platform, also revealed significant upregulation of the MASLD pathway in male hPHB1-KD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), while in female hPHB1-KD mice this pathway was downregulated (Table S4). For deeper insight, we conducted unbiased metabolomic profiling of the liver samples (Table S5), followed by integrative pathway analysis using MetaboAnalyst 5.0 software. DEG\u0026rsquo;s with p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 in male mice were analyzed in conjunction with metabolites showing a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.1 using KEGG pathway. This integrative analysis revealed glycerolipid metabolism as one of the most significantly enriched pathways in hPHB1-KD males compared with WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePHB1 knockdown increases cytoplasmic lipin-1 and triglyceride accumulation in hepatocytes\u003c/h3\u003e\n\u003cp\u003eWe next examined lipin-1 content in liver samples from hPHB1-KD and WT mice but found no significant differences in either sex (Figure S4). However, bulk liver tissue is a dense and heterogenous mix of many cell types, so to determine whether PHB1 deficiency specifically affects lipin-1 expression and localization in hepatocytes, we performed targeted silencing of PHB1 in AML12 hepatocytes using siRNA targeting \u003cem\u003ePhb1\u003c/em\u003e mRNA, or a scrambled non-targeting control siRNA (scrRNA). Immunoblot confirmed \u003cem\u003ePhb1\u003c/em\u003e siRNA enabled\u0026thinsp;\u0026ge;\u0026thinsp;50% knockdown of PHB1 compared with scrRNA, consistent with our hPHB1-KD mouse model (Figure S5). Importantly, immunofluorescent analysis revealed that PHB1-deficient AML12 cells had a significant increase in cytoplasmic lipin-1 compared with controls, while nuclear fluorescence levels remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA left panels, B-C). Furthermore, lipid droplets stained with Nile Red were substantially increased in the AML12 cells following PHB1 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA right panels, D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eHyper-active mTORC1 signaling underlies pathogenesis of liver steatosis in male mice with hepatocyte PHB1 deficiency\u003c/h3\u003e\n\u003cp\u003ePrior studies have reported that mTORC1 regulates lipin-1 expression and function in multiple cell types (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), and given the link between PHB\u0026rsquo;s and mTORC1 implicated in the studies described above, we next tested whether mTORC1 signaling is perturbed in hepatocytes with PHB1 deficiency. Immunoblot analysis of both AML12 (Figure S5) and liver tissues from hPHB1-KD mice clearly show that PHB1 knockdown leads to increased phosphorylation of the well-known mTORC1 targets S6 at Serine 235/236, and 4EBP1 at serine 65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Notably, this activation of mTORC1 signaling in hPHB1-KD liver was not significantly augmented by insulin stimulation in either sex. To determine whether persistent mTORC1 activation plays a mechanistic role in the pathogenesis of hepatic steatosis, a cohort of 22-week-old hPHB1-KD male mice was treated with daily injections of Torin1 (20 mg/kg, i.p.), a potent mTORC1 inhibitor, or vehicle, for seven consecutive days. Torin1 treatment suppressed mTORC1 signaling, corresponding with decreased S6 and 4EBP1 phosphorylation in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and effectively reversed hepatic triglyceride accumulation in hPHB1-KD male mice to levels comparable to those of vehicle-treated WT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMASLD has become the most prevalent chronic liver disease globally, affecting more than 30% of adolescents and adults (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). It encompasses a pathological continuum ranging from benign hepatic steatosis to progressive stages such as MASH, fibrosis, cirrhosis, and ultimately hepatocellular carcinoma, all of which contribute to considerable clinical and economic burdens (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Additionally, MASLD, especially MASH with fibrosis, increases the risk of type 2 diabetes, cardiovascular disease, and certain extrahepatic cancers (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Current therapies for MASLD primarily aim to reduce hepatic lipid burden or enhance their clearance, with several agents showing promise in improving steatosis and inflammation; however, most are limited by side effects and do not address the underlying metabolic dysregulation (\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Thus, new targets that drive MASLD pathogenesis are urgently needed for therapeutic investigation.\u003c/p\u003e\u003cp\u003eA comprehensive proteomic study published\u0026thinsp;\u0026gt;\u0026thinsp;20 years ago reported that PHB1 is downregulated in livers of \u003cem\u003eob/ob\u003c/em\u003e mice and obese humans, and in a mouse model of MASH (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Despite over two decades of work since then, the link between PHB1 downregulation and liver disease pathogenesis has remained largely obscure. In this study, we demonstrated that hepatocyte-specific knockdown (but not complete ablation) of PHB1 leads to hepatic steatosis and impaired glucose tolerance in male mice by 6 months of age, characteristic of MASLD. Mechanistically, hepatocyte PHB1 deficiency induced constitutive mTORC1 activation and upregulated expression and cytoplasmic localization of lipin-1, a key regulator of lipid metabolism in hepatocyte. Importantly, we further demonstrate that the pathological lipid accumulation in hPHB1-KD male mice is, at least in part, mTORC1-dependent, as pharmacological inhibition by Torin1 mitigated hepatic steatosis.\u003c/p\u003e\u003cp\u003eA role for PHB1 in regulating mitochondrial function has been well-characterized (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), and evidence implicating its broader role in metabolic regulation by the liver has emerged in recent years (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Disruptions in PHB1 expression have been observed during the onset of MASH in a \u003cem\u003eMAT1A\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mouse model as previously described (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), as well as methionine-choline-deficient diet models (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), where steatosis emerged in PHB1 deficient mice as early as 3 days after onset of the diet (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Our study is the first to report a mechanism by which PHB1 down-regulation in hepatocytes even in absence of injury leads to MASLD development, and document a clear role for aberrant mTORC1 signaling as a major pathogenic driver of the disease. Importantly, hepatocyte PHB1 deficiency causes steatosis and loss of glycemic control in male mice even without a high fat diet or increased caloric intake, underscoring its pivotal role in controlling metabolic homeostasis in liver.\u003c/p\u003e\u003cp\u003eIn a previous study, full ablation of PHB1 in hepatocytes caused severe liver injury as early as 3 weeks of age, associated with necrosis, inflammation, and mitochondrial dysfunction, with progression to hepatocellular carcinoma by 8 months. Of note, the mice in that study did not exhibit significant triglyceride accumulation at 14 weeks, despite increased plasma cholesterol (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Our model of hepatocyte PHB1 haploinsufficiency maintained liver mitochondrial integrity and is consistent with a prior report which demonstrated that partial PHB1 loss in hepatocytes does not disrupt mitochondrial gene expression (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Furthermore, transcriptomic profiling of both mouse and human liver tissues across various stages of MASLD revealed a weak association between MASLD progression and mitochondrial dysfunction (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). A strength of the partial PHB1 knockdown approach is that it enabled us to investigate the mechanisms by which PHB1 regulates hepatic lipid metabolism independently of liver injury or mitochondrial impairments, both of which are severe confounders.\u003c/p\u003e\u003cp\u003eA key finding of our study is the upregulation of \u003cem\u003elpin1\u003c/em\u003e gene in male hPHB1-KD mice. Lipin-1 is a member of the lipin family of proteins, Mg\u0026sup2;⁺ -dependent enzymes that function as phosphatidic acid phosphatases in mammalian cells to regulate lipid metabolism via triglyceride synthesis and transcriptional regulation of lipid handling genes. In liver, the function of lipin-1 depends on its cellular localization. In nucleus, it acts as a transcriptional coactivator of PPARα and PGC1α to induce the expression of genes associated with mitochondrial fatty acid oxidation, including suppression of SREBP1 transcriptional activity, thereby decreasing lipogenesis. In cytoplasm, lipin-1 catalyzes the dephosphorylation of phosphatidic acid to produce diacylglycerol, a key step in TG synthesis, and VLDL secretion (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). It was shown that mice with liver \u003cem\u003elpin1\u003c/em\u003e ablation had decreased body weight, adiposity, and liver and plasma triglyceride content (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Our study revealed a sex-specific upregulation of \u003cem\u003elpin1\u003c/em\u003e gene expression in livers of hPHB1-KD males, although we did not observe an increase in lipin-1 protein expression in liver lysates from these mice, suggesting that lipin-1 may be regulated at the post-transcriptional level and possibly cell-type specific within the heterogenous tissue of the liver. Since lipin-1 functions as a downstream effector of mTORC1, the increased \u003cem\u003eLpin1\u003c/em\u003e gene expression observed in hPHB1-KD males may be mediated through PHB1 effect on mTORC1 signaling specifically in hepatocytes. Prior studies have shown that activated mTORC1 phosphorylates lipin-1 and enhances its cytoplasmic localization (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), an essential control node for sustaining lipin-1 phosphatidic acid phosphatase activity in response to changes in intracellular pH, membrane structure, or fatty acid accumulation. Also, mTORC-1 phosphorylation of lipin1 promotes the nuclear entry of activated SREBP1 and expression of its target lipogenic genes (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Conversely, treatment with mTORC1 inhibitor, Torin1, promotes the nuclear localization of Lipin-1. This nuclear shift plays a critical role in regulating the SREPB1c transcriptional activity (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Our transcriptomic analysis showed modest downregulation of \u003cem\u003eSrebpf1\u003c/em\u003e expression (Ln FC= -0.52; ~2-fold change) in the hPHB1-KD male liver, possibly an adaptation to the steatosis.\u003c/p\u003e\u003cp\u003eA noteworthy finding of our study is that the transcriptomic and metabolic alterations seen with mTORC1 activation in the hPHB1-KD male mice were absent in females, indicating a sex-specific effect of hepatic PHB1 deficiency. Previous studies have reported crosstalk between estrogen and insulin signaling in the regulation of \u003cem\u003eLpin1\u003c/em\u003e in liver (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). It is therefore plausible that estrogen signaling in females mitigates the upregulation of \u003cem\u003eLpin1\u003c/em\u003e associated with PHB1 deficiency, thereby conferring protection against MASLD pathogenesis in female mice.\u003c/p\u003e\u003cp\u003eOur data highlight a male-specific susceptibility to steatosis and systemic insulin resistance associated with PHB1 deficiency, aligning with previous reports that PHB1 interacts with sex hormone signaling pathways (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) and results in sex specific metabolic phenotypes in white adipose tissue and liver (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). In an \u003cem\u003ein vitro\u003c/em\u003e model, estradiol treatment of 3T3-L1 and C9 cell lines resulted in a dose-dependent increase in both \u003cem\u003ePhb1\u003c/em\u003e mRNA and protein levels. Furthermore, PHB1 expression was significantly reduced when those cells were co-incubated with the estrogen receptor antagonist fulvestrant, indicating that up-regulation of PHB1 was mediated by estrogen receptor (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Together with our findings, the evidence points to a potential role for sex steroid signaling in protecting female mice against metabolic dysfunction, warranting further investigation.\u003c/p\u003e\u003cp\u003emTOR is a serine/threonine kinase that mediates its regulatory functions through two structurally and functionally distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), each characterized by specific components, downstream effectors, and biological functions. mTORC1 drives cellular anabolic processes such as protein and lipid biosynthesis, while mTORC2 is primarily involved in regulating cell survival and cytoskeletal organization (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).Although this study is the first to link PHB1 to mTORC1 signaling in liver, PHB1 has been implicated in regulation of mTORC1 in a few prior studies with disparate cell models, most of which are proliferating cells. In an ovarian cancer cell line, PHB1 was found to inhibit mTORC1 through its interaction with FKBP8 (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Two other studies have shown that PHB1 deletion leads to persistent upregulation of mTORC1 signaling, which are aligned more closely with our findings (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) which show persistent mTORC1 activation in PHB1-deficient livers even in the absence of insulin stimulation and under fasting conditions. The pathogenic effect of this persistent mTORC1 signaling was revealed by the determination that one week of Torin1 treatment significantly suppressed mTORC1 signaling in liver and attenuated hepatic lipid accumulation in male PHB1 KD mice, suggesting that in normal condition, PHB1 negatively regulates mTORC1 to maintain lipid homeostasis in liver.\u003c/p\u003e\u003cp\u003eTaken together, our findings support a model in which PHB1 regulates hepatic lipid metabolism by modulating mTORC1-dependent control of lipin-1 localization, promoting its nuclear functions and preventing excessive triglyceride synthesis in the cytoplasm.\u003c/p\u003e\u003cp\u003eAn important consideration from our model is that PHB1 knockdown caused depletion of PHB2 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC), consistent with what is known about the interdependence of these proteins. It is intriguing that hepatocyte-specific PHB2 knockout mice have a number of metabolic features that are directly opposite those of hPHB1-KD mice. Specifically, the PHB2 knockout mice exhibit severe hypoglycemia, impaired insulin signaling, reduced plasma insulin levels, impaired gluconeogenesis, and reduced hepatic glycogen content. Moreover, the hepatocyte-specific PHB2 knockout mice develop hypolipidemia in parallel with liver steatosis, along with a sharp reduction in white adipose tissue mass, indicative of increased lipolysis and fat redistribution to the liver (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Unfortunately, mTORC1 signaling was not evaluated in the liver from these mice, but it is clear from the divergence in phenotype that although PHB1 and PHB2 are interdependent in their cellular expression, they govern hepatic metabolism through distinct regulatory mechanisms.\u003c/p\u003e\u003cp\u003eTo conclude, our study illustrates a clear pathological mechanism linking decreased hepatocyte PHB1 to MASLD via unrestrained and persistent mTORC1 signaling, ultimately leading to increased cytoplasmic lipin-1 expression, steatosis and loss of glycemic control in male mice. Furthermore, the sex-specific nature of this phenotype unmasks PHB\u0026rsquo;s as potential regulatory nodes of sex signaling, particularly in metabolic disorders and endocrinology. They also reveal PHB\u0026rsquo;s as provocative therapeutic targets to mitigate MASLD and MASH.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAnimal Model\u003c/h2\u003e\u003cp\u003e All animal experiments were approved by the University of Iowa Institutional Animal Care and Use Committee (IACUC). Mice with hepatocyte-specific PHB1-deficienecy were generated by crossing homozygous \u003cem\u003ePhb1\u003c/em\u003e floxxed (\u003cem\u003ePhb1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e) mice, in which exon 2 of the \u003cem\u003ePhb\u003c/em\u003e gene is flanked by loxP sites, with Albumin-Cre mice (Alb-Cre). Beginning at 8 weeks of age, male and female WT and hPHB1-KD mice weremaintained on regular chow diet (Research Diets (D20122207), NJ, USA; 12.7% kcal from fat, 72.3% kcal from carbohydrates, and 15% kcal from protein) for a total duration of 16 weeks. Study personnel remained blinded regarding mice genotypes until group assignments were revealed at time of analysis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMetabolic testing and phenotyping\u003c/h3\u003e\n\u003cp\u003eMice body weights were continuously monitored throughout duration of the study. At ~\u0026thinsp;5 months of age, mice were subjected to glucose (GTT), pyruvate (PTT), and insulin tolerance (ITT) tests separated by week-long intervals. On the day of each test, mice were singly housed and subjected to a 6-hour food restriction period. For GTT, mice were administered an i.p. injection of dextrose (1 g/kg of a 50% dextrose/saline solution (Nova Tech Inc., NE, USA)). For PTT, mice were administered an i.p. injection of a 1:10 pyruvate: lactate solution (Sigma, WI, USA) equivalent to 2 g/kg pyruvate. For ITT, mice were administered an i.p. injection of 0.75 units/kg of insulin (Eli Lilly, IN, USA). Blood glucose levels were measured from tail vein at 0-, 30-, 60-, and 120-minutes post-injection using a OneTouch Verio Flex glucometer (Lifescan, Switzerland).\u003c/p\u003e\u003cp\u003eBody fat and lean mass were noninvasively measured using NMR within 24 hours prior to euthanasia using the Time Domain NMR Analyzer (minispec LF50, Bruker, MA, USA). Mice were euthanized under isoflurane anesthesia, and tissues were rapidly dissected and freeze clamped using aluminum tongs pre-cooled in liquid nitrogen.\u003c/p\u003e\u003cp\u003eFor the Torin1 experiment, male mice at age of 22-weeks received either Torin1 (20 mg/kg) (MedChemExpress, NJ, USA) or vehicle (20% N-methyl-2-pyrrolidone, NMP, 40% PEG400 in water) once daily via i.p. administration for 7 consecutive days. Following the treatment period, mice were euthanized and liver samples were collected for analysis as described above.\u003c/p\u003e\n\u003ch3\u003ePlasma and liver triglycerides\u003c/h3\u003e\n\u003cp\u003ePlasma and liver triglyceride content were measured using a commercial kit (Cayman Chemical, MI, USA) according to the manufacturer's protocol.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eHistology\u003c/h2\u003e\u003cp\u003eLiver tissues were fixed in 10% formalin, embedded in paraffin for subsequent staining with hematoxylin-eosin (H\u0026amp;E) and picrosirius red. For lipid droplets staining, formalin fixed liver tissue was embedded in OCT and frozen, sectioned then stained with Oil Red O. Images were captured using Olympus BX63 microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePlasma insulin\u003c/h2\u003e\u003cp\u003eMice were fasted for 6 hours and euthanized 30 minutes after saline or 50% dextrose injection (1g/kg i.p). Blood was collected in EDTA coated tubes via exsanguination at time of euthanasia and centrifuged at 6000xg for 6 minutes to isolate plasma. Plasma insulin was measured using a commercial insulin rodent chemiluminescence ELISA kit (Alpco, NH, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eMetabolomics\u003c/h2\u003e\u003cp\u003eUnbiased metabolomic profiling of bulk liver tissue samples from WT and hPHB1-KD mice was performed using LC-MS, analyzed by MetaboAnalyst 5.0 web-based platform. A more detailed description of the method can be found in the Supplemental Material online.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eRNA Sequencing\u003c/h2\u003e\u003cp\u003eRNA was isolated from liver tissue samples (n\u0026thinsp;=\u0026thinsp;5/group) using the RNeasy Fibrous Tissue Kit (QIAGEN, Maryland, USA). RNA library preparation and sequencing was performed by the genomics division of the University of Iowa Institute of Human Genetics using standardized protocols and Illumina NovaSeq 6000 genome sequencer. Reads were acquired with 200 b.p. paired-end reads with an average depth of ~\u0026thinsp;50M reads per sample. Adapter trimmed fastq were aligned using Kallisto (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) to reference transcriptome (\u0026ldquo;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eftp://ftp.ensembl.org/pub/release98/fasta/mus_musculus/cdna/Mus_musculus.GRCm38.cdna.all.fa.gz\u003c/span\u003e\u003cspan address=\"http://ftp://ftp.ensembl.org/pub/release98/fasta/mus_musculus/cdna/Mus_musculus.GRCm38.cdna.all.fa.gz\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u0026rdquo;). Kallisto index and Kallisto quant were run using default settings, with specific input parameters of 100 bootstraps and 16 threads. Differential expression analysis was performed using Sleuth version 0.30.0 aggregating abundance on Ensemble genes, with a two-step Likelihood Ratio Test and Wald Test (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) Volcano plots were generated using R package VolcanoseR (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Reads were also aligned with STAR version XX to an index built of \u0026ldquo;Mus_musculus.GRCm38.98.chr.gtf\u0026rdquo;. Gene counts were extracted from BAM files using subread version 1.5.2 and FeatureCounts. This count matrix was uploaded to Biojupies (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) for secondary analyses to calculate DEG\u0026rsquo;s and enrichR pathway analyses.\u003c/p\u003e\u003cp\u003eRaw sequencing reads are deposited into GEO archive ###. Genes were considered differentially expressed between WT and hPHB1-KD mice based on beta value (\u0026gt;\u0026thinsp;0.35 or \u0026lt;\u0026thinsp;\u0026minus;\u0026thinsp;0.35) which corresponds to FC\u0026thinsp;\u0026gt;\u0026thinsp;1 or \u0026lt; -1, and adjusted p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eImmunoblot analysis\u003c/h2\u003e\u003cp\u003eLiver tissue protein extracts were prepared using lysis buffer (50mM HEPES, 150mM NaCl, 1% Triton X-100, 2mM EGTA. 1mM MgCl2, 0.5mM DTT) supplemented with protease and phosphatase inhibitor (Sigma, WI, USA). AML 12 lysates were prepared using commercial RIPA buffer with the same inhibitors. Protein extracts were loaded onto 4\u0026ndash;20% polyacrylamide gel (BioRad, CA, USA) for electrophoresis, then transferred to PVDF or nitrocellulose membrane, blocked in 5% bovine serum albumin (BSA) in 1X Tris-based saline with Tween (TBST). The membrane was incubated in a solution of primary antibodies (Table S6) contained in 0.5% BSA in TBST overnight at 4\u0026ordm;C. Following wash steps, membranes were incubated in solution of secondary antibodies (HRP conjugated goat anti-rabbit or anti mouse, Jackson Immunoresearch, PA, USA) for 2 hours at room temperature. After another set of wash steps, membranes were incubated in a chemiluminescent substrate (ThermoFisher Scientific Inc, MA, USA), and images were captured using an iBright FL1000 Imaging System (ThermoFisher Scientific Inc., MA, USA). Densitometry analysis was performed using Image J (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.net/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eLiver mitochondria isolation and respiratory flux analysis\u003c/h2\u003e\u003cp\u003eLiver mitochondria were isolated as previously described (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Briefly, liver tissue was dissected and rinsed in cold mitochondrial isolation medium (MIM) (300 mM sucrose, 10 mM Na-HEPES, 0.2 mM EDTA, pH 7.2), then thoroughly minced on ice in MIM supplemented with 0.5% BSA and 1mM EGTA. The tissue suspension was homogenized using a Dounce apparatus, then centrifuged at 600\u003cem\u003eg\u003c/em\u003e for 10 minutes at 4\u0026deg;C. The supernatant was collected and centrifuged at 8000\u003cem\u003eg\u003c/em\u003e for 15 minutes at 4\u0026deg;C. The pellet was resuspended in the same buffer and centrifuged again at 8000\u003cem\u003eg\u003c/em\u003e for 15 minutes at 4\u0026deg;C. The final pellet was resuspended in MIM buffer supplemented with 0.5% BSA and 1mM EGTA. Protein concentration was measured using Pierce BCA protein assay (ThermoFisher Scientific Inc, MA, USA). Mitochondrial respiration was assessed using the Oroboros O\u003csub\u003e2\u003c/sub\u003eK as follows: Briefly, 250 \u0026micro;g of isolated mitochondria were added to Buffer Z-lite (105 mM K-MES, 30 mM KCl, 10 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e-6H\u003csub\u003e2\u003c/sub\u003eO, 0.05% BSA, pH 7.4), supplemented with 1 mM EGTA, 4 mM D-glucose, 1.7 U/mL glucose-6-phosphate dehydrogenase and 3.4 U/mL hexokinase (Roche, Sigma, WI, USA). After stabilization of oxygen concentration, substrates were added to measure oxygen consumption in the following order and concentrations, 25 \u0026micro;M palmitoyl carnitine with 2 mM malate, 650 \u0026micro;M ADP, 5 mM glutamate, 5 mM succinate. Data was analyzed using Oroboros DatLab software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAML12 culture and\u003c/b\u003e \u003cb\u003ePhb1\u003c/b\u003e \u003cb\u003esilencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAML12 cells were purchased from ATCC (CRL-2254, VA, USA) and cultured in DMEM-F12 supplemented with 10% FBS, 1X insulin transferrin selenium (ITS) and 40 ng/ml dexamethasone and incubated at 37\u0026deg;C with humidified air containing 5% CO2. The siRNA targeting mouse \u003cem\u003ePhb1\u003c/em\u003e (ON-TARGETplus mouse \u003cem\u003ePhb\u003c/em\u003e smart pool, L-060391-01-0005) and the nontargeting siRNA (ON-TARGETplus non-targeting pool, D-001810-10-05) were purchased from Horizon Discovery (Horizon Discovery, CO, USA). The non-targeting scrambled siRNA was used as a negative control. AML12 cells were grown in a 24 well plate and reverse transfected with 10nM siRNA using Lipofectamine RNAimax and opti-mem in the complete cell growth medium for 72 hours prior to biochemical analysis and/or imaging.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eNile red staining of AML12 cells\u003c/h2\u003e\u003cp\u003eAML12 cells were washed with PBS twice, fixed in 4% PFA at room temperature for 30 minutes, permeabilized with 0.1% Triton-x100 for 5 minutes, then stained with Nile Red (1 \u0026micro;g/ml; Thermo-Fisher Scientific Inc, MA, USA) for 20 minutes at 37\u0026deg;C. Nuclei were counterstained using VECTASHIELD\u0026reg; anti-fade mounting medium plus DAPI. Images were captured using a Zeiss LSM 980 confocal microscope. Mean fluorescence intensity was analyzed using Zen Blue 3.3 software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence staining of AML12 cells\u003c/h2\u003e\u003cp\u003eAML12 cells were washed with PBS, fixed with 4% PFA for 30 minutes at room temperature, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA, and 5% normal goat serum in PBS for 1 hour at room temperature. Subsequently, cells were incubated overnight at 4\u0026deg;C with an anti-lipin1 antibody (Abcam, MA, USA; dilution 1:200). After thorough washing with 0.04% TBST, cells were incubated with Alexa Fluor 488 conjugated secondary antibody (1:200 dilution) for 2 hours at room temperature in the dark. Finally, the cells were mounted using Vectashield antifade mounting medium containing DAPI. Images were captured using a Zeiss LSM 980 confocal microscope. Mean fluorescence intensity was analyzed using Zen blue 3.3 software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed in GraphPad Prism version 10. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. Unpaired t-test was used for two group comparisons, and one-way ANOVA was used for multiple group comparisons. Alpha-level of significance for main effects between groups was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of Interest\u003c/h2\u003e\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics Statement\u003c/h2\u003e\u003cp\u003e This study was approved by the Institution of Animal care and use committee at the University of Iowa. All animal experiments were performed in accordance with institutional and legal ethical standards.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003cp\u003eAdditional supplementary information for this article is available online at\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by funding from the American Heart Association Strategically Focused Research Network award 20SFRN35200003, and National Heart Lung Blood Institute award R01HL167087.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eAuthors would like to thank Dr. William Paradee and the staff at the University of Iowa Viral Vector core facility for generating \u003cem\u003ePhb1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice, and staff at the University of Iowa Genomics, Metabolomics, Central Microscopy, Histology and Metabolic Phenotyping core facilities for their resources and expertise. Special thanks to Maggie Cullum for assisting with Torin1 administration and other mouse model tasks.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNuell MJ, Stewart DA, Walker L, Friedman V, Wood CM, Owens GA, et al. Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells. Mol Cell Biol. 1991;11(3):1372\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnde SR, Mishra S. Nuclear coded mitochondrial protein prohibitin is an iron regulated iron binding protein. Mitochondrion. 2011;11(1):40\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarbier-Torres L, Lu SC. Prohibitin 1 in liver injury and cancer. Exp Biol Med (Maywood). 2020;245(5):385\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMishra S, Murphy LC, Murphy LJ. The Prohibitins: emerging roles in diverse functions. J Cell Mol Med. 2006;10(2):353\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSignorile A, Sgaramella G, Bellomo F, De Rasmo D. Prohibitins: A Critical Role in Mitochondrial Functions and Implication in Diseases. Cells. 2019;8(1).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTheiss AL, Sitaraman SV. The role and therapeutic potential of prohibitin in disease. Biochim Biophys Acta. 2011;1813(6):1137\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSteglich G, Neupert W, Langer T. Prohibitins regulate membrane protein degradation by the m-AAA protease in mitochondria. Mol Cell Biol. 1999;19(5):3435\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNijtmans LG, de Jong L, Artal Sanz M, Coates PJ, Berden JA, Back JW, et al. Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins. EMBO J. 2000;19(11):2444\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMishra S. Phb1:Phb2 heterodimers in the mitochondria-beyond functional interdependence. J Biol Chem. 2019;294(40):14836.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSanchez-Quiles V, Segura V, Bigaud E, He B, O'Malley BW, Santamaria E, et al. Prohibitin-1 deficiency promotes inflammation and increases sensitivity to liver injury. J Proteomics. 2012;75(18):5783\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKo KS, Tomasi ML, Iglesias-Ara A, French BA, French SW, Ramani K, et al. Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice. Hepatology. 2010;52(6):2096\u0026ndash;108.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSantamaria E, Avila MA, Latasa MU, Rubio A, Martin-Duce A, Lu SC, et al. Functional proteomics of nonalcoholic steatohepatitis: mitochondrial proteins as targets of S-adenosylmethionine. Proc Natl Acad Sci U S A. 2003;100(6):3065\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKanagaki S, Tsutsui Y, Kobayashi N, Komine T, Ito M, Akasaka Y, et al. Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins. iScience. 2023;26(4):106293.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang J, Yin Y, Wang J, Zhang J, Liu H, Feng W, et al. Prohibitin regulates mTOR pathway via interaction with FKBP8. Front Med. 2021;15(3):448\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePanwar V, Singh A, Bhatt M, Tonk RK, Azizov S, Raza AS, et al. Multifaceted role of mTOR (mammalian target of rapamycin) signaling pathway in human health and disease. Signal Transduct Target Ther. 2023;8(1):375.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarcondes-de-Castro IA, Reis-Barbosa PH, Marinho TS, Aguila MB, Mandarim-de-Lacerda CA. AMPK/mTOR pathway significance in healthy liver and non-alcoholic fatty liver disease and its progression. J Gastroenterol Hepatol. 2023;38(11):1868\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMao Z, Zhang W. Role of mTOR in Glucose and Lipid Metabolism. Int J Mol Sci. 2018;19(7).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaxton RA, Sabatini DM. mTOR Signaling in Growth, Metabolism, and Disease. Cell. 2017;169(2):361\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDuvel K, Yecies JL, Menon S, Raman P, Lipovsky AI, Souza AL, et al. Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol Cell. 2010;39(2):171\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeterson TR, Sengupta SS, Harris TE, Carmack AE, Kang SA, Balderas E, et al. mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway. Cell. 2011;146(3):408\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDella-Flora Nunes G, Wilson ER, Hurley E, He B, O'Malley BW, Poitelon Y, et al. Activation of mTORC1 and c-Jun by Prohibitin1 loss in Schwann cells may link mitochondrial dysfunction to demyelination. Elife. 2021;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIsing C, Koehler S, Brahler S, Merkwirth C, Hohne M, Baris OR, et al. Inhibition of insulin/IGF-1 receptor signaling protects from mitochondria-mediated kidney failure. EMBO Mol Med. 2015;7(3):275\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu YXZ, Ande SR, Mishra S. Gonadectomy in Mito-Ob mice revealed a sex-dimorphic relationship between prohibitin and sex steroids in adipose tissue biology and glucose homeostasis. Biol Sex Differ. 2018;9(1):37.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWatt MJ, Miotto PM, De Nardo W, Montgomery MK. The Liver as an Endocrine Organ-Linking NAFLD and Insulin Resistance. Endocr Rev. 2019;40(5):1367\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRauckhorst AJ, Borcherding N, Pape DJ, Kraus AS, Scerbo DA, Taylor EB. Mouse tissue harvest-induced hypoxia rapidly alters the in vivo metabolome, between-genotype metabolite level differences, and (13)C-tracing enrichments. Mol Metab. 2022;66:101596.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBi L, Jiang Z, Zhou J. The role of lipin-1 in the pathogenesis of alcoholic fatty liver. Alcohol Alcohol. 2015;50(2):146\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShimizu K, Fukushima H, Ogura K, Lien EC, Nihira NT, Zhang J, et al. The SCFbeta-TRCP E3 ubiquitin ligase complex targets Lipin1 for ubiquitination and degradation to promote hepatic lipogenesis. Sci Signal. 2017;10(460).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEaton JM, Mullins GR, Brindley DN, Harris TE. Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association. J Biol Chem. 2013;288(14):9933\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYounossi ZM, Golabi P, Price JK, Owrangi S, Gundu-Rao N, Satchi R, et al. The Global Epidemiology of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis Among Patients With Type 2 Diabetes. Clin Gastroenterol Hepatol. 2024;22(10):1999\u0026ndash;2010 e8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYounossi ZM, Paik JM, Henry L, Yang J, Fernandes G, Stepanova M, et al. The Growing Economic and Clinical Burden of Nonalcoholic Steatohepatitis (NASH) in the United States. J Clin Exp Hepatol. 2023;13(3):454\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTargher G, Corey KE, Byrne CD, Roden M. The complex link between NAFLD and type 2 diabetes mellitus - mechanisms and treatments. Nat Rev Gastroenterol Hepatol. 2021;18(9):599\u0026ndash;612.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTargher G, Byrne CD, Tilg H. MASLD: a systemic metabolic disorder with cardiovascular and malignant complications. Gut. 2024;73(4):691\u0026ndash;702.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMazhar IJ, Yasir M, Sarfraz S, Shlaghya G, Narayana SH, Mushtaq U, et al. Vitamin E and Pioglitazone: A Comprehensive Systematic Review of Their Efficacy in Non-alcoholic Fatty Liver Disease. Cureus. 2023;15(8):e43635.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMudaliar S, Henry RR, Sanyal AJ, Morrow L, Marschall HU, Kipnes M, et al. Efficacy and safety of the farnesoid X receptor agonist obeticholic acid in patients with type 2 diabetes and nonalcoholic fatty liver disease. Gastroenterology. 2013;145(3):574\u0026ndash;82 e1.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHarris E. FDA Okays First Drug for Scarring From Fatty Liver Disease. JAMA. 2024;331(17):1439.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePirola L. Elafibranor, a dual PPARalpha and PPARdelta agonist, reduces alcohol-associated liver disease: Lessons from a mouse model. World J Gastroenterol. 2025;31(4):99312.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOyang L, Li J, Jiang X, Lin J, Xia L, Yang L, et al. The function of prohibitins in mitochondria and the clinical potentials. Cancer Cell Int. 2022;22(1):343.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTomita K, Tamiya G, Ando S, Ohsumi K, Chiyo T, Mizutani A, et al. Tumour necrosis factor alpha signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice. Gut. 2006;55(3):415\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee K, Yu H, Shouse S, Kong B, Lee J, Lee SH, et al. RNA-Seq Reveals Different Gene Expression in Liver-Specific Prohibitin 1 Knock-Out Mice. Front Physiol. 2021;12:717911.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao QY, Ge LH, Zhang K, Chen HF, Zhan XX, Yang Y, et al. Assessment of mitochondrial function in metabolic dysfunction-associated fatty liver disease using obese mouse models. Zool Res. 2020;41(5):539\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKajimoto K, Suemitsu E, Sato Y, Sakurai Y, Harashima H. Liver-Specific Silencing of Lipin1 Reduces Fat Mass as Well as Hepatic Triglyceride Biosynthesis in Mice. Biol Pharm Bull. 2016;39(10):1653\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeterfy M, Harris TE, Fujita N, Reue K. Insulin-stimulated interaction with 14-3-3 promotes cytoplasmic localization of lipin-1 in adipocytes. J Biol Chem. 2010;285(6):3857\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGowri PM, Sengupta S, Bertera S, Katzenellenbogen BS. Lipin1 regulation by estrogen in uterus and liver: implications for diabetes and fertility. Endocrinology. 2007;148(8):3685\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu YXZ, Bassi G, Mishra S. Prohibitin: a prime candidate for a pleiotropic effector that mediates sex differences in obesity, insulin resistance, and metabolic dysregulation. Biol Sex Differ. 2019;10(1):25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnde SR, Nguyen KH, Padilla-Meier GP, Wahida W, Nyomba BL, Mishra S. Prohibitin overexpression in adipocytes induces mitochondrial biogenesis, leads to obesity development, and affects glucose homeostasis in a sex-specific manner. Diabetes. 2014;63(11):3734\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChoi M, Chaudhari HN, Ji YR, Ryoo ZY, Kim SW, Yun JW. Effect of estrogen on expression of prohibitin in white adipose tissue and liver of diet-induced obese rats. Mol Cell Biochem. 2015;407(1\u0026ndash;2):181\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi L, Martin-Levilain J, Jimenez-Sanchez C, Karaca M, Foti M, Martinou JC, et al. In vivo stabilization of OPA1 in hepatocytes potentiates mitochondrial respiration and gluconeogenesis in a prohibitin-dependent way. J Biol Chem. 2019;294(34):12581\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBray NL, Pimentel H, Melsted P, Pachter L. Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016;34(5):525\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePimentel H, Bray NL, Puente S, Melsted P, Pachter L. Differential analysis of RNA-seq incorporating quantification uncertainty. Nat Methods. 2017;14(7):687\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGoedhart J, Luijsterburg MS. VolcaNoseR is a web app for creating, exploring, labeling and sharing volcano plots. Sci Rep. 2020;10(1):20560.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTorre D, Lachmann A, Ma'ayan A. BioJupies: Automated Generation of Interactive Notebooks for RNA-Seq Data Analysis in the Cloud. Cell Syst. 2018;7(5):556\u0026ndash;61 e3.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKatunga LA, Gudimella P, Efird JT, Abernathy S, Mattox TA, Beatty C, et al. Obesity in a model of gpx4 haploinsufficiency uncovers a causal role for lipid-derived aldehydes in human metabolic disease and cardiomyopathy. Mol Metab. 2015;4(6):493\u0026ndash;506.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7481614/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7481614/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProhibitins (PHB1,2) are highly conserved lipid-raft associated proteins that physically interact to form a multimeric ring supercomplex in mitochondrial and plasma membranes where they are intimately involved in regulating cellular metabolism. Prior studies in disparate cell models have implicated PHB1 as a mediator of insulin signaling and its downstream effector, the mechanistic target of rapamycin complex 1 (mTORC1), but the mechanisms and physiological implications of these interactions are unclear. Here, we examined the role of PHB1 in regulating insulin and nutrient mediated activation of mTORC1 in liver using genetic and pharmacological approaches in mice and hepatocyte culture. Interestingly, male mice with hepatocyte-specific PHB1 haploinsufficiency (hPHB1-KD) at 6 months displayed features consistent with metabolic dysfunction-associated steatotic liver disease (MASLD), characterized by liver steatosis and impaired glucose tolerance with hyperinsulinemia, while these parameters were unaffected or even mildly improved in age-matched hPHB1-KD females. Both sexes of hPHB1-KD mice displayed increased basal phosphorylation of mTORC1 and its downstream targets (S6, 4EBP1) in liver compared with WT in fasted state, with minimal responsiveness to insulin. Transcriptomic data revealed strong upregulation of \u003cem\u003eLpin1\u003c/em\u003e gene in male hPHB1-KD mice, a phosphatidic acid phosphatase regulated by mTORC1 that critically regulates hepatic lipid metabolism. Integrated transcript-/metabolomic analysis showed enriched glycerolipid metabolism and upregulation of MASLD pathway in the liver of hPHB1-KD males. Parallel experiments in AML12 hepatocytes confirmed that PHB1 knockdown causes hyper-activation of mTORC1 signaling, increased cytoplasmic lipin-1 expression and localization, and increased lipid droplet formation. Furthermore, one week of treatment with mTORC1 inhibitor Torin1 reduced hepatic triglycerides and normalized mTORC1 signaling in hPHB1-KD males to levels comparable with WT. Collectively, these findings demonstrate that PHB1 is essential for maintaining metabolic homeostasis in liver via control of mTORC1-lipin1 axis, and further confirm that metabolic effects of PHB1 deficiency in liver are sexually dimorphic.\u003c/p\u003e","manuscriptTitle":"Persistent mTORC1 activation underlies sex dimorphic progression of MASLD in mice with hepatocyte prohibitin-1 deficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 13:20:28","doi":"10.21203/rs.3.rs-7481614/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a97b3ccc-47a2-442b-a402-5fe6fe4a3f97","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54767340,"name":"Biological sciences/Physiology/Metabolism/Metabolic diseases/Metabolic syndrome"},{"id":54767341,"name":"Biological sciences/Physiology/Metabolism/Fat metabolism"},{"id":54767342,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":54767343,"name":"Biological sciences/Cell biology/Cell signalling/Insulin signalling"}],"tags":[],"updatedAt":"2025-12-05T15:24:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-25 13:20:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7481614","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7481614","identity":"rs-7481614","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}