3.1 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 deficiency 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, significantly increased and contributed to the elevated plasma glucose in older ETKO.
In addition, in vivo radiolabeling experiments showed that the incorporation of the undegradable [14C]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 [3H]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).
3.2 Enrichment gene analysis of early indicators of ETKO liver disease
To determine early changes in gene expression caused by Pcyt2 deficiency we assessed the existing microarray data for 2-mo old ETKO liver (GEO microarray data set:GSE55617) (Fig. 2A) and RTPCR-array data obtained for 6-mo old ETKO liver (Fig. 2B). At young age ETKO has no clinical symptoms of steatosis or insulin resistance (Fig. 1), yet the pathway analysis (Fig. 2A-a, Supplementary Table 2) of the 2-mo old ETKO downregulated genes (714 genes at p < 0.05) established that the most enriched pathways are for hepatic steatosis, abnormal liver physiology/morphology, increased triglyceride and ammonia, and abnormal amino-acid levels. The Gene Onthology (GO) analysis (Fig. 2A-b, Supplementary Table 2) further indicated that the most downregulated processes were synthesis and oxidation of fatty acids (gene cluster in Fig. 2A-b: ACOX1, ALDH3A2, PPAR-γ, ACAD11, ADIPOR2, ACADM, ACADL, CPT1A, ELOVL5, ACSL EHHADH, DECR2), CDP-Etn Kennedy pathway (PCYT2, CEPT) and nitrogen metabolism (nitric oxide and urea cycle genes: NOS3, ARG1, ASS1, ASL). As expected, because of Pcyt2 single-allele deletion, Pcyt2 was among those downregulated genes in 2-mo old ETKO (Fig. 2A-b). Insulin signaling and genes involved in glucose metabolism were not significantly changed in 2-mo old ETKO.
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 post-receptor 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 significantly 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 gene list and statistics for top 10 processes).
The upregulated pathways in 6-mo old ETKO (Fig. 3A-a, Supplementary Table 4, WIKI Pathway 2019 Mouse) included growth-promoting pathways (insulin, EGFR1, Jun), stem cell pluripotency, cell adhesion (focal/ integrin cell adhesion), as well as proinflammatory pathways (IL-6, Il-5, II-2). These pathways share a large set of genes participating in receptor activation, cell signaling and nuclear transcription: SHC1, GRB2, AKT1, PIK3R1, RAF1, MAPK, PIK3R, FOS, JUN, ARAF, KRAS) (Fig. 3A-a). In addition, GO analysis (Fig. 3A-b, Supplementary Table 4, Jansen Diseases) establish that the upregulated genes are involved in Hyperglycemia, Hyperinsulinemia, Fatty liver disease, Type-2 diabetes, Lipodystrophy, Arthritis, Neutropenia, Cancer, Lung disease and Noonan syndrome. The full list of the top 10 most significant genes is in Supplementary Table 4. The most shared upregulated genes in 6-mo ETKO liver were the lipogenic genes (SREBP1, PPARγ, RETN, LEP), the glucose metabolic genes (SLC2A1, G6PC, IRS2 AKT1) and the growth promoting genes (KRAS, JUN and VEGFA).
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 significantly modified, however, total Irs1 protein was reduced by 68% and pTyr-Irs1 was diminished by 60%. Pcyt2 deficiency caused a dramatic 88% reduction in p85PI3K and a 45–46% decrease in Akt1/2. Pdk mediated activation at pThr308-Akt is not affected however mTorc2 meditated pSer473-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.
3.3 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 modified 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.
3.4. Enrichment analysis of upregulated genes in 2-month-old ETKO liver predicts disease phenotype
We also assessed the upregulated gene sets in 2-mo old ETKO liver(GEO microarray data set:GSE55617) (Fig. 4, Supplementary Table 5). The enrichment analysis of 534 upregulated genes (Fig. 4, Supplementary Table 5) established multiple modified pathways. In agreement with immunoblotting, the angiopoietin signaling (ANGPT4;ANGPT2;FOXO1;LCK) (Fig. 4A-a) showed significant enrichment in 2mo-old ETKO. Also identified as significant were the endocannabinoid PE related pathway (NAPEPLD;DAGLB) and response to hypoxia (ARNT, EPGN). The pathway analysis with BioPlanet2019 and Reactome2016 both recognized as the most significant the transport processes of inorganic cations/anions and amino acids (SLC9A3;SLC6A19;SLC7A8;SLC34A1;SLC7A11;SLC12A6), G-protein activation (GNAZ;GNB4;GNG12) and Caspase 8 activation/apoptosis (CASP8;TNFSF10 MADD) (Fig. 4A-b,c). In agreement with this analysis, Gene Ontology (GO Biological processes 2018) analysis (Fig. 4. B-a, Supplementary Table 5) further indicated that angiopoietins as well as thromboxane signaling, (ANGPT4;ANGPT2;TBXA2R;MMRN2;MEOX2) (the gene cluster in Fig. 4B-a) are the most important for the negative regulation (disruption) of angiogenesis. GO Molecular function identified ligand binding membrane processes as most significant, including: T cell receptor binding, the binding of amino acids, aryl hydrocarbon receptor binding, phosphatidylinositol-3,4,5-trisphosphate binding, and amino acid- and peptide-transport activities (Fig. 4B-b, Supplementary Table 5). Interestingly, GO Human Phenotype analysis (Fig. 4B-c, Supplementary Table 5) of the upregulated genes in 2-mo old ETKO liver identified a disease network between Insulin-resistant Diabetes and Maternal Diabetes and Hypoalphalipoproteinemia (HDL lipoprotein deficiency), Hyperuricemia (high uric acid), Polydipsia (excessive thirst, abnormal drinking behavior), Rhabdomyosarcoma (skeletal muscle tumors) and immunity (Asthma and Complement deficiency). The muscle specific glycogen-associated regulatory subunit of Protein Phosphatase-1 (PPP1R3A) was the most common gene in this new network. PPP1R3A plays a critical role in glycogen synthesis that is independent of insulin 30.
3.5 PEtn modifies 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 deficiency. 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 specific 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 significant increase (15–17%) in the mRNA expression of the fatty acid and triglyceride metabolic regulators Ppar∝, Ppar𝛾 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).
3.6 PEtn attenuates ETKO liver inflammation.
Next, we probed whether PEtn could improve hepatic inflammation. Expression of proinflammatory cytokines Infγ, Tnfα and Il-6 were increased by 50%, 92%, and 49%, respectively, in ETKO liver (Fig. 7A). PEtn supplementation reduces elevated Infγ, Tnfα and Il-6 by 18%, 40% and 20%. Tnfr showed no significant differences across all groups nor did anti-inflammatory Tgfβ1, Tgfβ3 and Il-10. In addition, ETKO liver shows an increased collagen staining that was reduced with PEtn (Fig. 7B). We further investigated the involvement of PEtn in the liver Traf6/NfKB, Socs3/Stat3 (Fig. 7C), Keap1/Nrf2, Elf 2a (Fig. 7D) and Mapk and Jnk (Fig. 7E) signaling pathways.
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 modified 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 inflammation 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 inflammation that contribute to NASH development.