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Pcyt2 deficiency causes age-dependant development of nonalcoholic steatohepatitis and insulin resistance that could be attenuated with phosphoethanolamine

The mechanisms of NASH development in the context of age and genetics are not fully elucidated. This study investigates the age-dependent liver defects during NASH development in mice with heterozygous deletion of Pcyt2 (Pcyt2+/−), the rate limiting enzyme in phosphatidylethanolamine (PE) synthesis. Further, the therapeutic potential of Pcyt2 substrate, phosphoethanolamine (PEtn), is examined. Pcyt2+/− were investigated at 2 and 6–8 months (mo) of age and in addition, 6-mo old Pcyt2+/− with developed NASH were supplemented with PEtn for 8 weeks and glucose and fatty acid metabolism, insulin signaling, and inflammation were examined. Heterozygous ablation of Pcyt2 causes changes in liver metabolic regulators from young age, prior to the development of liver disease which does not occur until adulthood. Only older Pcyt2+/− experiences perturbed glucose and fatty acid metabolism. Older Pcyt2+/− liver develops NASH characterized by increased glucose production, accumulation of TAG and glycogen, and increased inflammation. Supplementation with PEtn reverses Pcyt2+/− steatosis, inflammation, and other aspects of NASH, showing that was directly caused by Pcyt2 deficiency. Pcyt2 deficiency is a novel mechanism of metabolic dysregulation due to reduced membrane ethanolamine phospholipid synthesis, and the metabolite PEtn offers therapeutic potential for NASH reversion.


Introduction
The progression of liver disease from simple steatosis to a dangerous state that includes in ammation and brosis, known as nonalcoholic steatohepatitis (NASH), is incompletely understood. NASH is tightly associated with obesity, diabetes and metabolic syndrome and can further advance to cirrhosis, hepatocellular carcinoma, and death 1 . It is predicted that by year 2030 NASH prevalence will have increased by 63% and liver related deaths by 178% from 2015 2 . Currently, therapy is restricted to lifestyle interventions as there is no pharmacological treatment for NASH 1 , highlighting the urgency for improved insight into NASH development.

Phosphatidylethanolamine (PE) is important lipid component of cellular membranes where it is involved
with essential processes including cell division, cell signalling, membrane fusion, autophagy, and apoptosis 3 . The major pathway for PE synthesis is via the CDP-ethanolamine Kennedy pathway 4 . First, ethanolamine kinase (EK) phosphorylates ethanolamine (Etn) to phosphoethanolamine (PEtn). In the second and rate limiting step of the Kennedy pathway, CTP-phosphoethanolamine cytidylyltransferase (Pcyt2) transfers CTP to PEtn to form CDP-Etn and pyrophosphate. Lastly, CDP-ethanolamine:1,2diacylglycerol ethanolamine-phosphotransferase (EPT) catalyzes the condensation of CDP-Etn and diacylglycerol (DAG) to produce PE and CMP 4 . The production of PE can be modulated by availability of substrates ethanolamine and DAG 5 , however under most physiological conditions Pcyt2 is the predominant regulator of PE synthesis 3 . Pcyt2 protein exists as three isoforms encoded by a single gene: the catalytically active Pcyt2α and Pcyt2β, generated via exon skipping mechanism, and the third isoform, intron retention variant Pcyt2γ which functions as a negative regulator of enzyme activity 6 .
Human PCYT 2 and mouse Pcyt2 genes are similarly regulated with LXR, EGR1, and NFkB transcription factors showing their conserved role in lipid metabolism, cell growth, and in ammation 7 . In addition, Pcyt2 is activity is stimulated by nutrient de ciency and PKCα/β phosphorylation at αSer 215 and αSer 223 / βSer 205 residues 8 .
Mounting evidence suggests that disruptions in PE homeostasis are linked to obesity and the development of diabetes and fatty liver diseases [9][10][11][12] . Recent human studies have negatively correlated the expression of PCYT2 with BMI in obese/overweight cancer patients 10 and in an obese cohort 11 . On the other hand, the expression of PE degrading genes, PE methyltransferase (PEMT) and phosphatidylserine (PS) synthase 2 (PSS2), positively correlate with BMI 11 . Moreover, comparison of adipocytes to human visceral adipose tissue from obese subjects have suggested the negative association of PCYT2 expression with type 2 diabetes and insulin resistance 12 . Previously, we established that the enzymatic activity of Pcyt2 is drastically reduced through the induction of mutations at PKC phosphorylation sites 8 and two catalytic sites 13 . Severe consequences of PCYT2 mutation and resultant diminished enzymatic activity we recently demonstrated in patients with multiple mutations that caused autosomal spastic paraplegia and profound lipid abnormalities 14,15 . According to dbSNP database PCYT2 is heavily mutated, with over 4400 single nucleotide polymorphisms identi ed and 60 allele mutations within the gene coding region 16 . Together this suggests the candidacy of PCYT2 gene and importance of establishing the signi cance of reduced PCYT2 activity in metabolic diseases.
The Pcyt2 heterozygous ETKO mouse is an attractive model to study the contribution of membrane phospholipid metabolism to NASH as it exhibits similar features to humans, importantly, the association of gradual disease onset and decreased PE synthesis with age [17][18][19] , since early disease pathologies are not well established.
We have shown that complete deletion of Pcyt2 gene in mice is embryonically lethal 20 and that heterozygous ETKO show reduced PE turnover, and increased DAG and TAG accumulation leading to the development of obesity and insulin resistance at an older age 13,20,21 . Decreased PE content associates with age in the brain and mitochondria of liver and muscle 17,18 , and TAG accumulation in muscle 18 , suggesting the potential for age-related alterations in Pcyt2 activity and PE homeostasis. Previous mouse models used to study NASH have induced the disease via choline/methionine de cient and high-fat diets, however these models exhibit meaningful differences in their etiology and pathogenic changes 22 , whereas ETKO develop NASH on a normal diet. Moreover, ETKO mice are a unique model for representing NASH in humans because, unlike previous models 23,24 , they exhibit both histological and in ammatory features of NASH as well as associated metabolic physiology 19,20 . In this study, we characterize the agedependent development of liver defects with a focus on the main pathways that contribute to the development and sustainment of ETKO NASH. We further hypothesize that supplementation of the Pcyt2 substrate, PEtn, will reverse the liver pathological state in ETKO.

Animals and Treatments
Heterozygous Pcyt2 mice (ETKO) were generated and genotyped as previously described 20 . All procedures were approved by the University of Guelph's Animal Care Committee and were in accordance with guidelines of the Canadian Council on Animal Care (CCAC). We also followed the ARRIVE guidelines for reporting results. Mice were housed in a temperature-controlled facticity and exposed to a 12 h light/12 h dark cycle beginning with light at 7:00 a.m. Mice were fed a standardized chow diet (Harlan Teklad S-2335) and had free access to water. Mice that were supplemented with PEtn (PEA, Sigma-Aldrich 268674) were done so through free access to water containing 1 mg/mL of PEtn. Dosage of PEtn was calculated based on physiological levels of Etn (10-75 µM) 25 and previously determined water intake 26 . Wild type littermates (WT) and ETKO mice (n = 6-10) were euthanized under both fasted and fed conditions at 2 and 6-8 mo. ETKO mice that were supplemented with PEtn (ETKO-PEtn) were euthanized under fed conditions at 6-8 mo (n = 6-12 per group). The treatment period lasted 8 weeks. No differences were observed between age-matched males and females and thus, both sexes were used for nal analysis.

Immunoprecipitation
Liver homogenates (n = 4) were thawed on ice prior to being pre-cleared with protein G agarose beads (Promega) for 2 h at 4°C. Liver protein of 125 µg was pre-cleared with 25 µl of agarose beads. Supernatants were thereafter removed and incubated with a 1:100 dilution of speci c antibodies overnight at 4°C. Following the overnight incubation at 4°C, 30 and 60 µl of agarose beads were added to the sample. All samples were incubated for 2 h at 4°C and centrifuged at 4°C for 10 min at 14,000 rpm.
Following centrifugation for 10 min at 14,000 rpm, the supernatant was removed, divided into aliquots, and stored at -80°C until immunoblotting experiments were conducted using IR, IRS-1, pTyrIRS1 (Millipore) and p85PI3K (Cell Signaling) antibodies.

Gene Expression Analysis
Frozen liver tissue weighing between 50-100 mg (n = 3-4 per group), was homogenized in 1 mL of TRIzol Reagent (Thermo Scienti c) according to the manufacturer's protocol to isolate mRNA. cDNA was synthesized from 2 µg of total mRNA using a poly(dT) primer and Superscript III reverse transcriptase (Invitrogen). Expression of various genes involved in lipid metabolism were determined by polymerase chain reaction (PCR) using the primer sequences and conditions listed in Supplementary Table 1. PCR reactions were carried out using the following cycle parameters: 30 s at 94°C, 30 s at respective melting temperatures (Tm) in Supplementary Table 1, and 30s at 72°C for 32 cycles. PCR products were resolved on a 1.5% agarose gel and quanti ed using ImageJ. Reactions were standardized to glyceraldehyde 3phosphate dehydrogenase (GAPDH).

Glucose and Pyruvate Tolerance Tests
For glucose tolerance test (GTT), mice (n = 4 per group) were fasted for 6 h before intraperitoneal injection (I.P.) of 2mg/kg of glucose in 0.9% saline. For pyruvate tolerance test (PTT), mice (n = 4 per group) were fasted overnight then administrated 2 g/kg of body weight of sodium pyruvate by I.P. injection. Blood glucose level was measured by glucometer immediately before injection and 30, 60 and 120 mins after glucose injection and 15, 30, 60, 120 mins after pyruvate injection.

Hepatocyte Glucose Production
Primary hepatocytes were isolated as previously described 19 . Brie y, livers from 8-mo WT and ETKO mice were perfused with 0.5% collagenase and hepatocytes resuspended in PBS containing 0.5% BSA, 5 mM glucose and 3.3 mM pyruvate. Hepatocytes were plated on 6-well collagen-coated plates and allowed to attach for 2 to 4 h in Williams medium E. Floating cells were removed and media replaced with Williams medium E containing 10% FBS and 1% antibiotic-antimycotic solution. To measure glucose release, hepatocytes were incubated 3 h in a glucose-free DMEM (no phenol red, pH 7.4) supplemented with 20 mM sodium lactate and 2 mM sodium pyruvate. Glucose present in the media was determined with a glucose assay kit (Sigma).

Glucokinase and Glucose-6 Phosphatase Activities
Hepatic glucokinase (Gk) activity was measured as previously described 27 with some modi cation. Liver samples (100 mg) were homogenized in 50 mM HEPES, 100 mM KCl, 1 mM EDTA, 5 mM MgCl 2 , and 2.5 mM dithioerythritol. Homogenates were brie y centrifuged and incubated for 10 min on ice with 25% PEG. The microsomal fraction/glucose 6-phosphatase activity was then removed by ultracentrifugation (100,000xg; 30 min; 4 0 C). Glucose phosphorylating activities were measured by the production of NADPH from NADP + in the presence of glucose 6-phosphate dehydrogenase (G6PDH) and either 100 mM glucose or 0.5 mM glucose, to distinguish hexokinase (Hk) from glucokinase (Gk) activity. The activity obtained at 0.5 mM glucose is considered the HK activity. The subtraction of the activity measured at 100 mM glucose from the activity measured at 0.5 mM glucose is considered the Gk activity.
The glucose-6 phosphatase (G6Pase) assay was based on the hydrolysis of glucose-6-phosphate to Pi by the microsomal fractions isolated above. The microsomal fractions were incubated with 10 mM glucose-6-phosphate at 37 0 C and the reaction was stopped after 20 min with acid molybdate containing 2/9 volume of 10% SDS and 1/9 volume of 10% ascorbic acid. The mixture was then incubated for 20 min at 45 0 C and the Pi-molybdate complex absorbance read at 820 nm. Protein concentration was measured using BCA assay (Thermo Fisher Sci).

Glycogen Content
The glycogen content was determined as previously described 28 . In brief, livers (50mg; n = 12 per group) were immersed in 500 µl 30% potassium hydroxide saturated with sodium sulfate and boiled for 20-30 mins until homogenous solution was obtained. Glycogen was precipitated with cold 95% ethanol, separated by centrifugation, and dissolved in distilled water. Glycogen content was determined at 490 nm after the addition of 5% phenol and 95% sulfuric acid. Standard curve was generated using pure glycogen (Roche).

Liver histology and immunohistochemistry
Livers (n = 4 per group) were xed in 10% formalin in PBS at room temperature for 12-16 h and embedded in para n until histopathologic examination. Sections were de-waxed in xylene and rehydrated in a series of ethanol washes. Sections of 10 µm were stained with hematoxylin and eosin (H&E) to examine lipid droplets, periodic acid-Schiff reagent (PAS) for glycogen and 0.1% Picrosirius red (Sigma) for collagen content and were visualized with light microscopy using standard techniques. All staining was performed at the Ontario Veterinary College, Department of Pathobiology, University of Guelph.

Hepatic triglyceride content
Livers (200 mg; n = 9 per group) were homogenized in 500 µL of PBS and 5% Tween 20. Samples were heated for 5 mins at 95ºC and cooled to room temperature. Heating/cooling process was repeated, and insoluble material was removed through centrifugation. A TAG assay kit (Wako Diagnostics 994-02891 and 998-02992) was used to quantify TAG content.
2.11 2-Bromopalmitate and 2-deoxyglucose uptake by liver Mice were fasted for 12 h (n = 6 per group) and retro-orbitally injected with 5 µCi 2-deoxy[ 14 C]glucose and 1.5 µCi BSA-complexed 2-bromo-[ 3 H]palmitate. After 5 mins post injection, blood was drawn and radioactivity in serum determined by liquid scintillation counting (LSC). After 1 h post injection, livers were harvested and homogenized in PBS. Radioactivity was determined by LCS and calculated as a portion of the initial radioactivity present in 5 µl of serum 5 mins after injection, to adjust for the injected dose.

RT-PCR array
Liver RNA from WT ant ETKO (n = 3 for each group) was extracted using the RNeasy Mini Kit (QIAGEN). The samples were pooled together and complementary DNA synthesis, 1 µg total RNA was reverse transcribed using the High Capacity RNA-to DNA Master Mix (Applied Biosystems). Complementary DNA, equivalent to 40 ng RNA, was used as a template for real-time reverse-transcription PCR (RT-PCR) using an Applied Biosystems 7900HT Fast Real-time PCR system (Applied Biosystems). Insulin Signaling Pathway PCR Array Mouse Gene Expression Array -PAMM-030Z (SA-Biosciences) was used for expression of 84 genes. PCR array data were calculated by the comparative cycle threshold method, normalized against multiple housekeeping genes, and expressed as mean fold change.
The enrichment analysis tool Enrichr (https://maayanlab.cloud/Enrichr/# ) was used for the analysis of the microarray data (GEO microarrays GSE55617) and RTPCR array data. The bar charts of the top 10 enriched terms from the selected libraries and clustergrams of the input genes vs. the enriched terms were produced separately for upregulated and down-regulated genes. The Manhattan and Volcano plots that establish the signi cance of each gene set vs. its odds ratio were visualized with Appyter (https://appyters.maayanlab.cloud/#/Enrichment_Analysis_Visualizer).

Statistical analysis
Data was analyzed using two-tailed unpaired t-test, and for differences between more than 2 groups oneway ANOVA with Tukey's post hoc test was performed. Signi cance was rejected at p ≥ 0.05. Results are represented as mean ± SD. All statistical tests were performed with Graphpad Prism 6 software.

Liver glucose contributes to increased glucose tolerance in older ETKO
To help elucidate the mechanism by which heterozygous ablation of Pcyt2 affects glucose metabolism by age, we performed glucose (GTT) and pyruvate (PTT) and tolerance tests on 2-mo and 8-mo old mice.
ETKO fasting glucose levels are unaltered at 2-mo of age, but by 8-mo are 20% higher than age-matched WT littermates (Fig. 1A). Two-month ETKO maintain normal glucose levels in response to the GTT while 8-mo ETKO are hyperglycemic compared to WT littermates ( Fig. 1B and C). GTT area under the curve (AUC) was elevated by 38% in 8-mo old ETKO, showing age-dependent and reduced glucose clearance from plasma (Fig. 1C).
We determined the ability of the ETKO liver to utilize pyruvate for glucose production through an intraperitoneal injection of sodium pyruvate and measurement of the subsequent rise in plasma glucose levels. Glucose production is unchanged in 2-mo old ETKO but increased in 8-mo old ETKO, with a 50% elevation in PTT AUC, relative to WT littermates ( Fig. 1D and E). To reinforce the concept that Pcyt2 de ciency augments liver glucose production we determined glucose release from primary hepatocytes isolated from 8-mo old mice. Indeed, primary ETKO hepatocytes exhibit a 64% increased glucose output compared to WT hepatocytes (Fig. 1F).
Further indicators of elevated glucose production are shown in the increased expression of the key liver enzymes in the gluconeogenic pathway. In fasted 8-mo old ETKO mice, mRNA levels of Pepck and G6Pase are increased by 2.36-and 2.21-fold, respectively, along with a 2.37-fold increase in G6Pase enzyme activity (Fig. 1G and H). The expression of glycolytic L-Pk was modestly reduced by 31% but Gk expression was reduced 3.58-fold and Gk activity by 46% showing reduced glucose utilization by glycolysis in old ETKO liver ( Fig. 1I and J). Together these data show that liver glucose production by gluconeogenesis was normal at younger age, however, signi cantly increased and contributed to the elevated plasma glucose in older ETKO.
In addition, in vivo radiolabeling experiments showed that the incorporation of the undegradable [ 14 C]deoxyglucose is increased 2.36-fold in fasted 8-mo old ETKO liver relative to WT littermates. The FA uptake measured by the metabolically stable [ 3 H]bromopalmitate is increased by 49% demonstrating that both glucose and FA are more readily available in fasted ETKO than in fasted WT mice ( Fig. 1J and K).
Liver staining displayed altered glycogen storage and quantitative analysis revealed a 75% increase in glycogen content in older ETKO liver ( Fig. 1L and M).
The enrichment analysis of RTPCR-arrays ( Fig. 2B-a, Supplementary Table 3: KEGG Pathway 2019 Mouse) indicated that insulin signaling, FoxO, mTOR, AMPK and several growth factors that share postreceptor regulation with insulin (cluster: MAPK2K1, SOS1, RAF1. PI3KCA, PI3KR2, BRAF in Fig. 2B-a) were downregulated in 6-mo old ETKO liver. Gene ontology (GO: Biological processes) analysis established that the most signi cantly downregulated were responses to insulin/peptide hormones/insulin receptor/tyrosine kinase signaling and pyruvate/glycolytic process/glucose homeostasis (Fig. 2B-b and Supplementary Table 3  Older ETKO liver develops impaired Pi3k/Akt signalling We next examined the activity of the insulin signaling pathway in 6-mo old ETKO liver. As shown in Fig. 3B, older ETKO shows severe impairment in the Irs1/Pi3K/Akt pathway in fasted state. Insulin receptor IR was not signi cantly modi ed, however, total Irs1 protein was reduced by 68% and pTyr-Irs1 was diminished by 60%. Pcyt2 de ciency caused a dramatic 88% reduction in p85PI3K and a 45-46% decrease in Akt1/2. Pdk mediated activation at pThr 308 -Akt is not affected however mTorc2 meditated pSer 473 -Akt activation was diminished by 56%. Together, the results indicated that the insulin signaling pathway in older ETKO liver was profoundly diminished at the level of protein content and activation of Irs1, Akt1/2 and Pi3k in fasted state. ETKO liver did not show impairments in the insulin signaling in fed state (Fig. 3B) when phosphorylated and total content of Irs1, p85PI3K and Akt1/2 were unchanged.

Early defects in ETKO fatty acid metabolism remain impaired with ageing
Since young ETKO had normal GTT and PTT tests (Fig. 1B and D) and the microarray analysis indicated an early impairment in fatty acid metabolism ( Fig. 2A) we next checked the activity of mitochondrial and fatty acid metabolic pathways (Fig. 3C). Indeed, fasted 2-mo old ETKO showed reduced levels and activity of the mitochondrial activators Sirt1 and Ampkα, 34% and 49% respectively, along with an expected 87% increase in pAcc-the rate limiting enzyme in fatty acid synthesis. The key nuclear regulator of fatty acid synthesis by lipogenesis, Srebp1 both in precursor and active forms were raised by 46% and 84%, respectively. Pkcα and Pkcβ1, well known DAG regulated kinases, increased by 52% and 72% while Pkcβ2 underwent a modest increase of 11%. Additionally, the important angiogenic factor and inhibitor of lipolysis Angptl4 exhibited a 43% elevation in 2-mo old ETKO (Fig. 3C). Importantly Angpt4, and Angpt2 gene expression and signaling via STAT and FOXO1 pathways were also upregulated in 2-mo old ETKO (Fig. 4A,B). As shown in Supplementary Fig. 1 and as it will be shown later as part of the PEtn study some of those activities continued to be modi ed in 6-mo old ETKO. Fasted 6-mo old ETKO ( Supplementary  Fig. 1) exhibit reduced Sirt1 by 31%, total Ampkα and p-Ampkα:Ampkα ratio decreased 56% and 43% and highly increased Srebp1c in both the precursor form (2.95-fold) and the active form (2.40-fold), along with a substantial (6.40-fold) increase in Acc. DAG regulated Pkcα and Pkcβ1/2, are also drastically elevated by 3.25-fold, 4.87-fold and 7.45-fold.
Together these data show that an early reduction in mitochondria energy production and increased fatty acid synthesis by lipogenesis 29 preceded development of adult ETKO liver steatosis and insulin resistance, and became even more impaired with ageing.

PEtn modi es phospholipid metabolic genes and cell signaling proteins
As shown in (Fig. 2C) in 8-mo old ETKO hepatic TAG content was elevated by 76% relative to WT littermates and attenuated 26% by PEtn supplementation (ETKO + PEtn). Accordingly, PEtn reduced lipid droplet accumulation (Fig. 2C). Since PEtn was constantly supplemented in drinking water, we decided to monitor the molecular effect of PEtn in the fed state. As expected for single-allele deletion, in addition to the reduced mRNA ( Fig. 2A-b), Pcyt2 protein was also reduced in ETKO and unaltered with its substrate-PEtn supplementation (Fig. 5A). The Kennedy pathway transporter Ctl1 31 was increased by PEtn at the mRNA level but protein content was not changed. PEtn increased Pss1 by 32% in ETKO, suggesting that PS synthesis from PC readily occurs in Pcyt2 de ciency. PEtn also increased PS decarboxylase (Psd) and Pss2 expression by 52% and 41%, respectively, indicating that PEtn stimulated an increase in the conversions of PE to PS by Pss2 and Psd decarboxylation of PS to PE. Since Pss2 utilizes PE at the level of the ER, this indicates that PEtn stimulation of the CDP-Etn Kennedy pathway was balanced by an increase in both Pss2 and Psd pathways, i.e., increased PE degradation to PS in the ER mitochondria associated membranes (MAM) by Pss2 occurs simultaneously with increased PS degradation to PE by Psd in the mitochondria. Taken together, such speci c stimulatory effect of PEtn on Pss1, Pss2 and Psd genes that control the PC-PS-PE cycle showed that PEtn was readily metabolized to PE by the CDP-Etn Kennedy pathway. In addition, PEtn caused small but signi cant increase (15-17%) in the mRNA expression of the fatty acid and triglyceride metabolic regulators Ppar∝, Ppar and Atgl (Fig. 5B).
The most prominent effect of PEtn on old ETKO liver was the increased phosphorylation/inactivation of Foxo1. pFoxo1 was almost absent (reduced 86%) in ETKO (Fig. 6A) and probably is the most responsible for the increased gluconeogenesis in ETKO liver (Fig. 1). PEtn also reduced the elevated (43%) Pkcα by 3.29-fold and did not affect Pkcβ (Fig. 6A). On the other hand, mTorc1 and p-p70S6K increased 2.28-and 5.76-fold in ETKO and an additional 77% and 2.3-fold with PEtn. Sirt1 and Ampkα activity were unchanged in fed ETKO and unaltered by PEtn. Pgc1α reduced 50-57% in ETKO was not altered with PEtn supplementation (Fig. 6B). Taken together, supplementation had a prominent effect on amino acid metabolism, by a strong inhibitory effect on gluconeogenesis (Foxo1) and stimulatory effect on protein synthesis (mTorc1).
ETKO mice exhibit Traf6 increase by 2.55-fold and NFkB increase by 77%. Furthermore, nuclear form of NFκb was elevated 56% indicating its increased activation in ETKO and not modi ed by PEtn. Socs3 protein was elevated by 8.32-fold and Stat3 by 3.22-fold in ETKO. Nuclear Stat3 was minimally raised in ETKO relative to WT, and PEtn induced a dramatic (7.25-fold) increase in nuclear Stat3 activation. Keap1, the negative regulator of cytoprotective nuclear factor Nrf2, was increased by 2.43-fold and accordingly nuclear Nrf2 was decreased by 72% in ETKO and a further 40% with PEtn. Phosphorylation of translation initiation factor Eif2α was increased by 72% in ETKO and decreased by 64% by PEtn. Phosphorylation of Erk1/2 and Jnk1/2 were unaffected across all groups however, p-p38 Mapk that was decreased by 54% in ETKO was improved 73% by PEtn supplementation. Taken together, these data established that old ETKO liver in ammation was characterized with upregulated Nfkb and pEif2a and reduced Keap1/Nrf2, Stat3, and p-p38 activity that all except Nfkb were regressed with 2 months of PEtn supplementation at physiologically relevant levels.
The working model for the signaling perturbations in ETKO NASH is illustrated in Fig. 8. It shows how reduced de novo synthesis of the membrane PE phospholipid results in metabolic and genetic adaptations to maintain membrane bilayers and accommodate unused metabolic intermediates, resulting in changes in glucose and FA metabolism and in ammation that contribute to NASH development.

Discussion
In this study we focus on the mechanisms of age-related development of NASH in Pcyt2 heterozygous mice ETKO. We show that at young age (2-mo) ETKO mice exhibit normal liver lipids, insulin signaling and glucose handling, but at adult stage (8-mo) they develop steatosis, in ammation, and augmented liver glucose production, predicted at young age by gene expression analysis. We further demonstrated that adult ETKO exhibit a de cit in Pi3k/Akt signalling with a shift to increased glucose production by gluconeogenesis, leading to an increase in liver lipid and glycogen content. In fasted state adult ETKO also exhibited reduced lipolysis and FA oxidation 20 , leading to lipid accumulation and development of steatosis.
A major paradox of type 2 diabetes is the selective impairment in the insulin mediated liver processes. The adult ETKO model recapitulates this by exemplifying the insulin resistant condition at older age with concomitantly increased gluconeogenesis and lipogenesis. In fasting, the contribution of glucose to the liver energy production is low, as shown by reduced glucose uptake and expression/activity of the glycolytic genes in older ETKO. Previous metabolic pro ling demonstrated increased plasma glucose, reduced plasma glycerol (reduced lipolysis) and increased plasma acyl carnitines (reduced FA oxidation), and an altered amino acid metabolism in older ETKO, with the notable increase in major anaplerotic precursor, glutamine 20,32 . A suppression of glucose and FA utilization limits the available pathways for energy production and thus, forces anaplerosis to replenish TCA cycle intermediates and permit its continued function. Subsequent obligate cataplerosis is linked to glucose and lipid synthesis in the liver 33 and thus, an active contribution of glutamine/amino acids to TCA reconciles the contradiction of simultaneously increased hepatic glucose and FA production in older ETKO. This mechanism is corroborated in mice 34 and human subjects with NAFLD 35 , showing the connection between increased anaplerosis and intrahepatic TAG accumulation, gluconeogenesis and insulin resistance.
We established that the mitochondria regulators Sirt1/p-Ampkα are inhibited and the main regulator of FA synthesis Srebp1c is upregulated early in asymptomatic young ETKO, as direct consequence of Pcyt2 gene deletion, not because of liver steatosis or insulin resistance, which develops at adult age. Pcyt2 heterozygosity leads to reduced ux by the Kennedy pathway and an accumulation of unused intermediates (DAG, ATP) that need to be accommodated 21 . An early increase in Srebp1 and FA synthesis is necessary to form TAG from DAG to reduce DAG levels and thus, even at young age, glucose and FA usage for energy are suppressed 21 . The function of conventional, DAG dependant PKCα/β1/2 is tightly linked to phospholipid homeostasis and stimulates ET/Pcyt2 activity 3,8 and not surprisingly they were also constitutively upregulated in young ETKO. PKCα and PKC β1/2 among many other functions, are well-known inhibitors of insulin signaling 36 . As DAG/TAG accumulate, constitutively increased PKC, which occurs prior to NASH development, could progressively diminish insulin signaling in older ETKO. These ndings are in line with previously established reduced FA oxidation and increased DAG in asymptomatic young ETKO 21,37 , indicating that these are the main drivers of impaired insulin signaling and adult-onset NASH developemnt even on a regular chow diet. Together the inherent transcriptional and metabolic adaptations to reduced Pcyt2 expression and activity cause a progressive metabolic dysfunction, culminating in general obesity, insulin resistance, hypertriglyceridemia 21,29 .
ETKO liver shows heavily diminished Irs1, p-Irs1, PI3K and Akt1/2 and pSer 473 -Akt activation by Torc2 speci cally in the fasted state, suggesting an increased protein degradation which agree with higher anaplerotic demands for amino acids, described above. There is also an increased gluconeogenesis in fasted state, showing a failed blockade of glucose production by insulin and increased Pepck and G6Pase expression and activity, controlled by gluconeogenic transcription factor Foxo1 38 . Indeed, ETKO phosphorylation of Foxo1 is decreased, leading to increased transcriptional activity which is in congruance with observed increase in gluconeogenesis. Supplementation of substrate PEtn is able to ameliorate ETKO NASH. PEtn supplementation increases total mTorc1 and mTorc1 substrate p70S6K levels, indicting a stimulation protein synthesis, which may help rectify the abnormal amino acid metabolism shown in 6-mo ETKO microarray. PEtn increases the activation of Stat3 and restores the diminished phosphorylation of Foxo1, allowing for the improvement in NASH 39,40 . Notably, DAG-regulated Pkcα is reduced by PEtn supplementation, suggesting an attenuation of DAG accumulation and improvement in lipid metabolism. PEtn supplementation marginally increased Atgl and PParα and parallels the effects of over expression of ATGL, an important lipase that governs hepatic TAG turnover and PPARα. Overexpression of ATGL increases FA oxidation, preventing hepatic lipid accumulation and increases PPARα activity, whereas knockdown causes steatosis 41,42 .Thus, it is conceivable that PEtn is able to improve ETKO NASH through stimulating metabolic ux by the Kennedy pathway, restoring membrane phospholipid homeostasis and increasing the utilization of DAG to reestablish energy balance and improve lipid and glucose metabolism.
The bene cial effect of PEtn was also evident on several characteristics of the in ammatory response associated with NASH. ETKO livers exhibit an elevated content in ammatory factors Il-6, Tnfα, Socs3, Traf6, and Nfκb, demonstrating the chronic in ammation characteristic of NASH. PEtn signi cantly attenuated the proin ammatory cytokines but was not able to reduce the nuclear content of Nfκb. Traf6 activates Nfκb through PI3k signaling 43 and ETKO Pi3k was not modi ed by PEtn, leading to the consistent activation of NFκb 44 . However, PEtn appears to alleviate oxidative stress as p38 Mapk undergoes dephosphorylation in ETKO that is partially restored with PEtn supplementation. While p38 activity is typically associated with in ammation, dephosphorylation of p38 Mapk occurs in response to increased Pi3k activity and oxidative stress and is shown to be regulated independently from Erk/Jnk 45,46 . Moreover, p-Eif2α was reduced by PEtn, indicating an improvement of ETKO oxidative stress. In mice with NAFLD induced with high fructose diet, phosphorylation of Eif2α is increased to protect hepatocytes from oxidative stress, brosis and death 47 . Nuclear content of Stat3 is dramatically increased with PEtn. Stat3 plays a complex role in liver in ammation, having both pro-and antiin ammatory functions, but importantly STAT3 has been shown protect against hepatocellular damage and attenuate the in ammatory response in models of liver injury 48,49 .
An important mechanism in brogenesis is the generation of mesenchymal cells through epithelial-tomesenchymal transition, which deposit extracellular matrix once activated 50 . PEtn most likely attenuated this process through stimulation of the Kennedy pathway, known to be a regulator of the reverse process, i.e., the mesenchymal-to-epithelial transition 51 and therefore inhibition of extracellular matrix deposition. The reduction in brosis is in line with an improvement in hepatic oxidative stress and the reduction of Tnfα and Il-6 52 .
In summary, this work emphasizes the importance of membrane phospholipid homeostasis in metabolic disease development and progression. Our work established for the rst time that a negative metabolic energy balance, due to reduced membrane lipid synthesis, that results in excessive production of FA in order to accommodate unused intermediates could lead to all known features of NASH, including steatosis and in ammation. PEtn supplementation was able to reverse ETKO hepatic steatosis, oxidative stress and in ammation. These effects indicate the CDP-Etn Kennedy pathway as a target in fatty liver disease and the therapeutic potential of PEtn. Given the increasing global presence of obesity and type 2 diabetes and lack of drug therapy for NASH, identifying new treatment options is critical.  Immunoblot analysis of PI3K/AKT pathway in fasted and fed 6-mo old ETKO (n=4) ( C) Immunoblot analysis of lipid pathways in fasted and fed 2-mo old WT and ETKO (n=4). Data are presented as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.  PEtn stimulates phospholipid and triglyceride metabolic genes. (A) Western blot analysis of liver Pcyt2 and Ctl1 in 8-mo WT, ETKO and ETKO + PEtn mice treated for 12 weeks (n=3-4). Relative mRNA analysis of phospholipid metabolism genes (B) and lipolytic and lipogenic (C) genes in WT, ETKO and ETKO + PEtn mice. GAPDH used as loading control. Band intensities were measured using ImageJ. Data are presented as mean ± SD. *p<0.05, **p<0.01 relative to WT; #p<0.05, ##p<0.01 relative to ETKO. PEtn inactivates FOXO1 and activates mTORC1 signaling pathway. (A) Western blot analysis of liver PKCα, PKCβ1, p-Foxo and (B) Sirt1, Pgc1α, p-Ampkα, Ampkα, p70S6K, and mTorc1 in 8-mo fed WT, ETKO and ETKO + PEtn mice (n=3-5). β-Actin was used as loading controls. Band intensities were measured using ImageJ. Data are presented as mean ± SD. *p<0.05, **p<0.01 relative to WT; #p<0.05, ##p<0.01 relative to ETKO.