Enpp1 ameliorates NAFLD by regulating hepatocyte lipid metabolism through the AMPK/PPARα signaling pathway

doi:10.21203/rs.3.rs-4877420/v1

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Enpp1 ameliorates NAFLD by regulating hepatocyte lipid metabolism through the AMPK/PPARα signaling pathway

https://doi.org/10.21203/rs.3.rs-4877420/v1

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Background Non-alcoholic fatty liver disease (NAFLD) has become the leading chronic liver disease worldwide, and there are no approved pharmacotherapies to treat this disease. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) has been found to be related to insulin resistance and lipid accumulation. However, the role and mechanism of Enpp1 in the development of NAFLD remain unknown.

Results Here we discovered that Enpp1 is lowly expressed in the liver of NAFLD patients by clinical investigation. Knocking out Enpp1 in the liver of mice aggravated obesity, insulin resistance and hepatic steatosis, and these effects were reversed by liver-specific Enpp1 over-expression. Then, through transcriptomic data mining and experimental validation, we demonstrated that Enpp1 deficiency inhibited the expression of AMPK (energy receptor) and PPARα (nuclear transcription factor for lipid metabolism), thereby promoting the transcription of lipid synthesis factors and mediating the progression of NAFLD. Mechanistically, Enpp1 enhanced the activity of AMPK by increasing the AMP-to-ATP ratio, which in turn raised the level of PPARα and promotes the transcription of its downstream lipid metabolism factors. Pharmacological inhibition of AMPK activity abolished the promoting effect of Enpp1 on PPARα protein expression.

Conclusion This study indicate that Enpp1 can effectively ameliorate NAFLD through AMPK/PPARα signaling pathway-mediated lipid metabolism, and reveal the significance of Enpp1 as a promising therapeutic target against NAFLD.

Non-alcoholic fatty liver disease

Enpp1

lipid metabolism

AMPK

PPARα

Non-alcoholic fatty liver disease (NAFLD) is characterized by the abnormal buildup of fats in the liver in the absence of excessive alcohol intake and other causes of liver fat accumulation. This condition can develop into non-alcoholic steatohepatitis (NASH), which is a leading cause of liver fibrosis, cirrhosis and hepatocellular carcinoma [1, 2]. NAFLD has become the leading chronic liver disease worldwide, which prevalence will reach 25% of the global population, with a notable increase among adolescents and children [3]. There is still no effective treatment for this chronic liver disease, highlighting the urgent need for the identification of new therapeutic targets. Insulin resistance, an early indicator of various metabolic disorders, has been identified as a key factor associated with liver fat accumulation [4]. This largely attributed to the reduced ability of insulin to suppress fat breakdown in fat tissue, resulting in an increased flow of free fatty acids to the liver and synthesis of triglycerides, which eventually aggravates hepatic steatosis [5, 6]. Therefore, identifying potential therapeutic targets that can increase insulin sensitivity and control lipid metabolism is considered crucial for the treatment of NAFLD.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1), a type II transmembrane metalloenzyme, hydrolyzes extracellular ATP and GTP to form AMP and GMP, playing a role in the regulation of mineralization in skeletal and soft tissues [7]. High Enpp1 expression levels are also detected in many solid tumors including glioblastoma, ovarian, and breast among others [8–10]. It is reported that Enpp1 promotes chemotactic infiltration of polymorphonuclear marrow-derived inhibitory cells and inhibits tumor infiltrating cytotoxic T cells hindering anti-tumor immune attack in breast cancer model [10]. Several previous studies have suggested that Enpp1 also contributes to a range of metabolic diseases including diabetes, cardiovascular disease and NAFLD [7, 11, 12]. They found the Enpp1 121Gln polymorphisms tended to aggravate hyperlipidemia and liver injury in patients with NAFLD. There was research reported that Enpp1 can aggravate insulin resistance [13]. In addition, research has shown that Enpp1 can inhibit adipocyte maturation by reducing the expression of adipogenic genes, suggesting that Enpp1 may participate in lipid metabolism [14]. This permits us to speculate the value of Enpp1 in defending against NAFLD through a global modulation of metabolic disorders.

In this study, we conducted a systematic evaluation of Enpp1 expression in NAFLD. Subsequently, we investigated the effects of Enpp1 on the obesity and insulin resistance in NAFLD mice, which are key mechanisms of liver lipid accumulation. The results showed that liver Enpp1 expression were decreased in NAFLD patients and NAFLD mouse models, while NAFLD mice lacking the Enpp1 gene exhibited significant obesity, insulin resistance and hepatic steatosis. Furthermore, to evaluate the molecular mechanisms by which Enpp1 regulates lipid metabolism in NALFD liver, we initially analyzed transcriptomic data of NAFLD mouse liver and identified AMPK/PPARα as a target of Enpp1. Since AMPK/PPARα plays a critical role in regulating liver lipid metabolism, we performed in vivo and vitro experiments to validate that Enpp1 can induce AMPK phosphorylation and promote PPARα expression, thereby inhibiting the transcription of liver lipid synthesis genes and improving lipid accumulation in NAFLD liver. Additionally, the use of compound C, a small-molecule inhibitor of AMPK enzymatic activity, eliminated the promoting effect of Enpp1 on PPARα protein expression. These findings emphasize the role of Enpp1 in alleviating NAFLD progression by enhancing the expression of AMPK/PPARα. Therefore, this study elucidates a novel mechanism by which Enpp1 regulates liver lipid metabolism and provides a new therapeutic strategy for treating NAFLD.

Human liver samples

Human liver specimens were obtained from 11 patients diagnosed with gallstones at the Ningxia Medical University Second Affiliated Hospital (Yinchuan, China). And then they were divided into NAFLD group (n = 5) and NAFLD control group (Non-NALFD, n = 6) by ultrasound and pathological analysis of liver biopsy. This study was approved by the Institutional Ethics Committee of Ningxia Medical University and Ningxia Medical University Second Affiliated Hospital (approval number: 2019 − 228). All research was conducted in accordance with both the Declarations of Helsinki and Istanbul. Written consent was given in writing by all subjects. The steatosis indices of these liver specimens were accessed using H&E and Oil Red O staining.

Animal experiments

All institutional and national guidelines for the care and use of laboratory animals were followed. All the animal experiments were approved by the Animal Care and Use Committee of the Institute of Experimental Animals, Chinese Academy of Medical Sciences and Beijing Union Medical College (yzw22002). All the experiments were conducted with male mice. Liver-specific Enpp1 knockout (Enpp1^fl/fl-Alb-Cre, CKO) mice were generated by crossing a male Alb-Cre^+/− mouse to a female Enpp1^fl/fl mouse, which were developed by Beijing Viewsolid Biotech. The db/db mice (genetic models of NAFLD) were also purchased from Beijing Viewsolid Biotech. CKO mice and controls were fed with a normal chow diet (NCD) or a high-fat diet (HFD: 60% calories from fat, 0.28% from cholesterol, and 20% calories from carbohydrate; Research Diets catalog MD12033, Medi-science, China) for 16 weeks.

For the over-expression of hepatic Enpp1, C57BL/6J male mice (8 weeks of age) were maintained on a HFD for 8 weeks and then injected with an Enpp1 liver-specific over-expression adenovirus (AAV8·Enpp1, 2×10¹² genome copies) purchased from Vigene Bioscience, Shandong, China; and the mice were maintained on HFD diet for another 8 weeks. Mice were fasted for 6 h and anesthetized, after which the serum, liver tissue, and adipose tissue from the epididymis were collected.

Serum enzyme and lipid assays

All groups of mice were fasted for 6 h and then anesthetized for enucleation to collect serum samples. A biochemical analyzer ((Mindray, China) was used to measure the serum levels of the liver enzymes alanine transaminase (ALT) and aspartate transaminase (AST), and the serum lipid contents of the triglyceride (TG), total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C) according to the manufacturer’s instructions. In liver tissues and cells, the TG content was measured using a triglyceride assay kit (A110-1-11, Nanjing Jiancheng) and the liver total cholesterol (T-CHO) was measured by a total cholesterol assay kit (A111-1-11, Nanjing Jiancheng), then normalize the obtained result to the protein concentration.

Intravenous glucose tolerance test (IGTT) and insulin tolerance test (ITT)

Glucose tolerance tests and insulin tolerance tests were conducted in mice that had been fasted for 6 h, using intraperitoneal injections of glucose (2 mg/g) or insulin (0.75 IU/10 g). Blood glucose levels were determined through tail bleeding (Sanocare Stable Non-adjustable Blood Glucose Test Strips, China) at predetermined intervals (0, 30, 60, 90, 120 min). The area under the curve (AUC) was calculated by summation of trapezoids.

Western blotting

Cells and liver tissues were lysed with RIPA lysis buffer containing a protease inhibitor and phosphatase inhibitor. Subsequently, the protein lysis solution was centrifuged to obtain a supernatant, after which the protein concentration was measured. The protein samples were separated via 10% SDS-PAGE and transferred onto an NC membrane. All the antibodies were used in this study including anti-Enpp1 (1:2000, #2061S, Cell Signaling Technology), anti-phospho-AMPK (Thr172) (1:2000, #50081S, Cell Signaling Technology), anti-AMPK (1:2000, #2532S, Cell Signaling Technology), anti-PPARα (1:2000, #15540-1-AP, Proteintech), HRP-conjugated anti-β-actin antibodies (1:5000, #KC-5A08, KANGCHEN) and HRP-conjugated anti-rabbit IgG antibodies (1:10000, #b31402, Thermo Fisher Scientific).

Quantitative PCR (qPCR)

Total RNA was extracted from liver tissue using a Total RNA Isolation Kit (#RC112-01, Vazyme) and was subsequently reverse-transcribed into cDNA via an RT reagent kit. Real-time qPCR was performed with SYBR Green PCR Master Mix (#Q311-02, Vazyme) according to the manufacturer’s protocol. Relative changes in mRNA expression were calculated by the 2^−ΔΔCt method. Information about the sequences of primers used is listed in Table S1.

Oil red O staining

Fresh liver tissues were submerged in a 30% sucrose solution at 4°C for dehydration. Subsequently, the tissues were excised into 8 µm thick frozen section, and Oil red O staining was carried out using an Oil red O stain kit (#G1261, Solarbio) as the instructions provided by the manufacturer. In the case of hepatocyte Oil red O staining, AML-12 cells were fixed in 4% paraformaldehyde for 30 min at room temperature, then stained with the oil red O staining solution for 15 min and rinsed with PBS.

Periodic Acid-Schiff staining

Mouse liver tissues were fixed in a 10% neutral formalin solution. Following fixation, the tissues were processed for paraffin embedding and sectioned into thin slices using standard histological techniques. The paraffin sections were then subjected to Periodic Acid-Schiff (PAS) staining according to the manufacturer's protocol provided with the staining kit (#S1281, Solairbio). The positive areas of PAS staining were quantified using Image J software.

Quantitation of adipocyte size

Tissue sections from epididymal adipose tissue were stained with hematoxylin and eosin (H&E). Image J software was used to measure adipocyte area, which is represented as the average adipocyte area (in µm²). Adipocyte size was measured from five mice per group (> 300 cells/group).

RNA sequencing

Total RNA was extracted, and cDNA libraries were constructed for profiling of gene expression differences. DESeq2 was used to calculate differential gene expression. Genes with a |log2FC|>1 and a P-value < 0.05 were defined as differential genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations of all genes in the reference genome were downloaded from the KEGG database. Pathways with P values less than 0.05 were defined as significantly enriched pathways.

GEO database mining

Raw data were deposited in the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) under the accession number of GSE126848. Genes showing |log2 fold change | > 0.5 and adjusted P values < 0.05 were considered to present differential expression.

Cell treatment and transfection

AML-12 cells (normal mouse hepatocytes) were cultured in DMEM/F12 medium (#ZQ606, Shanghai Zhong Qiao Xin Zhou Biotechnology Co.,Ltd.), supplemented with 10% FBS, 100 U/ml penicillin and streptomycin. The cells were plated at a density of 3 x 10⁵ cells on 6-well collagen (Sigma) coated plates and transfected with 100 nM Enpp1-siRNA (F:5'-GUCUCAGUGUCCAAUCAAATT-3'; R:5'-UUUGAU-

UGGACACUGAGACTT-3') or 4 µg/µl Enpp1 over-expression plasmid according to the manufacturer’s instructions, and then treated with 200 µM Palmitic acid (PA) to constructe the NAFLD cell model.

Measurement of the AMP-to-ATP Ratio in Cells

The extraction of intracellular AMP and ATP from according to previously reported methods [15]. Then the concentration of AMP and ATP was measured with corresponding assay kits (#V5011, Promega and #S0026, Solarbio) according to the protocol supplied by the manufacturer.

Statistical analysis

The statistical data were analyzed by using GraphPad Prism 9. Comparisons between two groups were determined by an unpaired two-tailed t test. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was used. The data were expressed as the mean ± SEM. P < 0.05 was considered statistically significant.

Hepatic Enpp1 expression is decreased in both NAFLD patients and mouse models

To investigated the genetic basis of NAFLD progression, a human RNA-seq dataset for liver simples across the spectrum of normal, non-alcoholic fatty liver (NAFL), and non-alcoholic steatohepatitis (NASH) were obtained from the Gene Expression Omnibus (GEO) database. To identify the specific genes involved in NAFLD progression, we focused on the differentially expressed genes (DEGs) (|log2 fold change | > 0.5 and adjusted P value < 0.05) that showed significant changes between the NAFL/NASH group and control group. After overlapping, 1892 up-regulated genes and 1612 down-regulated genes were differentially expressed in both NAFL and NASH (Fig. 1A). During these DEGs, we noted that Enpp1, a novel lipid metabolism biomarker [16], mRNA expression levels were degraded in liver tissues of both NAFL and NASH compared with normal liver tissues (Fig. 1B, C). To verify these findings, we examined Enpp1 expression in our cohort of NAFLD samples. Western blotting analysis demonstrated lower Enpp1 protein expression in NAFLD liver tissues compared with paired normal liver tissues (Fig. 1D). Furthermore, we also examined the protein expression of Enpp1 in liver tissues of high-fat diet (HFD) induced NAFLD mouse models and NAFLD gene models (db/db mice). The results demonstrated a significant decrease in Enpp1 expression in NAFLD models mice fed a HFD (Fig. 1E, F) and in db/db obese mice models (Fig. 1G, H). These data suggest the essential function of Enpp1 in NAFLD progression.

Hepatic Enpp1 deficiency aggravates obesity and insulin resistance in mice fed a high-fat diet

To further investigate the effects of Enpp1 on the progression of NAFLD, we established a mouse that specifically knocking out the Enpp1 gene in the liver of mice. And simulate NAFLD model by feeding liver-specific Enpp1 knockout (CKO) mice and the control (Flox) mice with high-fat diet (HFD) or normal chow diet (NCD) for 16 weeks, and CKO mice and Flox mice show no significant difference in food intake (Fig. S1). Compared with HFD-fed Flox mice, HFD-fed CKO mice displayed more obvious obesity (Fig. 2A), greater body weight (Fig. 2B) and larger adipocytes size in the epididymal fat by Hematoxylin-eosin (H&E) staining (Fig. 2C). Periodic acid-Schiff (PAS) staining of mouse livers showed that glycogen content was decreased significantly in HFD-fed CKO mice (Fig. 2D), and the expression of gluconeogenesis genes in the livers of HFD-fed CKO mice were increased (Fig. S2A). Additionally, compared with HFD-Flox mice, HFD-fed CKO mice had higher fasting blood glucose levels (Fig. S2B). We also found that HFD-fed CKO mice showed more areas under the curve (AUC) for intravenous glucose tolerance test (IGTT) (Fig. 2E) and insulin tolerance test (ITT) (Fig. 2F) than HFD-fed Flox mice. These findings suggest that liver-specific Enpp1 knockout mice with HFD-fed exhibited exacerbate obesity and insulin resistance.

Hepatic Enpp1 deletion exacerbates hepatic steatosis in NAFLD mice

We next investigated the effect of Enpp1 on hepatic steatosis by using liver-conditional knockout of Enpp1 (CKO) mice. HFD feeding for a duration of 16 weeks, but not NCD, increased the liver wet weight and steatosis in CKO mice (Fig. 3A-C) and resulted in an increase in liver triglycerides (TG) and liver total cholesterol (T-CHO) compared with those in controls (Fig. 3D, E). Additionally, Serum TG, T-CHO and low-density lipoprotein cholesterol (LDL-C) levels were also increased in HFD-fed CKO group compared with those in HFD-fed Flox group, suggesting the enhanced HFD-induced hepatocellular lipid accumulation in CKO mice (Fig. 3F-H). And the serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels were significantly elevated in the HFD-fed CKO group, revealing that HFD-induced hepatocyte damage was aggravated in the CKO mice (Fig. 3I, J). To elucidate the effect of Enpp1 on lipid metabolism, we measured the mRNA levels of hepatic regulators of fatty acid synthesis (Fasn, Scd1, Elov5 and Srebf1), fatty acid oxidation (Cpt1α, Acadm and Acox1), TG synthesis (Dgat1 and Dgat2) and fatty acid transportation (Ldlr and Cd36). Under HFD conditions, the livers of CKO mice exhibited significant upregulation of the expression of all genes involved in fatty acid synthesis, while the expression of genes involved in fatty acid oxidation were decreased (Fig. 3K). These findings above clarify that hepatic Enpp1 deficiency can exacerbate the progression of hepatic steatosis.

Enpp1 over-expression alleviates hepatic steatosis and metabolic deterioration

Therefore, we then analyzed the influence of over-expression of hepatic Enpp1 on hepatic steatosis. Wild-type C57BL/6J mice (8 weeks of age) were fed with either NCD or HFD for 8 weeks. HFD-fed mice were then injected via the tail vein with empty vector adenovirus (AAV8·EV, 2 × 10¹² genome copies per mouse) or the Enpp1 liver-specific over-expression adenovirus (AAV8·Enpp1, 2 × 10¹² genome copies per mouse) in the eighth week and continued to be fed for another 8 weeks (Fig. 4A). Compared with vehicle-treated HFD-fed mice, Enpp1 over-expressed HFD-fed mice had a significant reduction in body weight and liver wet weight (Fig. 4B, C). We found that Enpp1 over-expression mice showed improvement in glucose tolerance (Fig. 4D) and insulin resistance (Fig. 4E). H&E and Oil red O staining revealed that Enpp1 over-expression reduced hepatic steatosis (Fig. 4F). Consistent with these results, a notable reduction in liver TG levels, liver T-CHO levels and serum TG levels were observed in Enpp1-overexpressed HFD-fed mice (Fig. 4G-I). Importantly, a significant decrease in serum AST levels was also observed following Enpp1 over-expression (Fig. 4J), indicating liver damage was reduced. In addition, Enpp1 over-expression under HFD conditions decreased the hepatic key regulators of fatty acid synthesis (Srebf1 and Elov5), TG synthesis (Dgat2) and fatty acid transportation (Cd36), while increased the expression of Cpt1α and Acadl, which are rate-limiting enzymes for fatty acid oxidation (Fig. 4K). These findings collectively suggest that Enpp1 exerts its protective effects in vivo by down-regulating lipid synthesis and enhancing fatty acid oxidation, to mitigate the progression of NAFLD.

Enpp1 regulates hepatic lipid metabolism through activating the AMPK/PPARα signaling pathway

To explore the molecular mechanisms underlying the effects of Enpp1 on lipid metabolism, RNAseq data from liver samples of HFD-fed CKO mice and HFD-fed Flox mice were analyzed via Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. We found that the genes whose expression was differentially expressed were mainly associated with the lipid metabolism pathway, including peroxisome the proliferator-activated receptor α (PPARα) and AMP-activated protein kinase (AMPK) (Fig. 5A, B). Then we verified the correlation between Enpp1 and the AMPK/PPARα pathway. The results showed that the HFD-fed CKO group had significantly inhibited phosphorylation of AMPK, as well as PPARα expression in the liver (Fig. 5C). And, the expression of downstream target genes of PPARα (Fabl1 and Acsl1) in the liver of HFD-fed CKO mice was significantly lower than HFD-fed Flox mice, which indicated that fatty acid oxidation in the liver was inhibited (Fig. 5D). However, Enpp1 over-expression induced AMPK phosphorylation and PPARα expression (Fig. 5E), as well as the expression levels of PPARα downstream target genes (Fig. 5F). These findings suggest that Enpp1 down-regulates lipid synthesis via AMPK/PPARα activation, thus exerting its protective effect in vivo.

Enpp1 activates the AMPK/PPARα axis via increasing AMP-to-ATP ratio to inhibits TG accumulation in hepatocytes

To further determine Enpp1’s function in regulating the expression of AMPK/PPARα, we conducted in vitro experiments using siRNA and plasmid transfection to knockdown or overexpress Enpp1 in AML-12 cells (normal mouse hepatocytes) induced by PA. Considering the role of Enpp1 in hydrolyzing ATP into AMP and PPi, and the AMP-to-ATP ratio is the core factor affecting AMPK phosphorylation [17, 18], we speculate that Enpp1 may activate the AMPK/PPARα signaling pathway by regulating the AMP-to-ATP ratio. The resluts indicated that knocking down Enpp1 significantly reduced AMP-to-ATP ratio (Fig. 6A), the levels of phosphorylated AMPK and PPARα (Fig. 6B), and the expression of target genes downstream of PPARα in AML-12 cells treated with PA (Fig. S3A). More importantly, after knocking down Enpp1, the lipid droplets and TG content in AML-12 cells significantly increased (Fig. 6C, D). In contrast, Enpp1 over-expression increased the AMP-to-ATP ratio (Fig. 6E) and stimulated AMPK phosphorylation and PPARα protein expression accompanied by an increase in the expression of PPARα downstream target genes in AML-12 cells treated with PA (Fig. 6F and Fig. S3B). And Enpp1 over-expression suppressed lipid accumulation and TG levels in AML-12 cells treated with PA (Fig. 6G, H). Furthermore, to validate the crosstalk between Enpp1 and PPARα relies on AMPK modification, we introduced a small-molecule inhibitor of AMPK, namely, compound C (CC). AML-12 transfected with Enpp1 over-expression plasmid were divided into the control group (DMSO for 24 h) and the CC group (20 µM for 24 h). By western blot analysis, we found that the protein levels of AMPK phosphorylation and PPARα were significantly downregulated after CC intervention compared with the control group, which suggested that CC can block the protein synthesis of PPARα by inhibiting Enpp1-mediated AMPK (Fig. 6I). These findings demonstrated that Enpp1 can activate the AMPK/PPARα signaling pathway by increasing AMP-to-ATP ratio, alleviating lipid accumulation in hepatocytes.

NAFLD is a condition of fat accumulation in the liver in combination with metabolic dysfunction in the form of obesity and insulin resistance [19]. Enpp1, a key protein closely associated with NAFLD progression, requires further exploration regarding its functional role in the hepatic steatosis and metabolic dysregulation of NAFLD. A previous study showed that Enpp1 gene polymorphisms increase the risk of maternal and neonatal obesity [16]. Interestingly, another study revealed that high expression of Enpp1 in subcutaneous adipose tissue was the pathogenic factor of obesity [20]. Recent study further confirmed that Enpp1 is either over-expressed or overactive in muscles, adipose tissues, fibroblasts and other tissues of non-diabetic and diabetic insulin-resistant individuals [19]. While, another research suggested mice with impaired glucose tolerance were inclined to have decreased liver Enpp1 protein levels, under the condition of high circulating insulin level [20]. In this study, we analyzed the expression of Enpp1 in the public databases and validated it through western blotting analysis of clinical specimens from NAFLD patients. The result indicated that there is low expression level of Enpp1 in NAFLD.

To elucidate the role of Enpp1 on hepatic steatosis and metabolic dysregulation in NAFLD, we introduced liver-specific knockout of Enpp1 (CKO) mice Enpp1 and liver-specific over-expression of Enpp1 mice. In vivo experiments, we found the deletion of Enpp1 obviously decreased the insulin sensitivity under the high-fat diet (HFD) condition, while, slightly but not significantly improved insulin sensitivity under normal chow diet (NCD) condition. Consistent with previous studies showing that the impaired insulin sensitivity induced a pathological increase in hepatic gluconeogenesis [21], Enpp1 deletion increased the expression of gluconeogenic genes and declined liver glycogen content. These results indicate that Enpp1 can mitigate NAFLD associated with obesity and insulin resistance, however the causal relationship among obesity, insulin resistance and NAFLD regulated by Enpp1 remains to be investigated. Additionally, we observed HFD fed liver-specific Enpp1 knockout (CKO) mice showed significantly worse hepatic steatosis, on the contrary, specific over-expression of Enpp1 in the liver markedly mitigated liver lipid accumulation.

Through deep mining of RNAseq data from liver samples of HFD-fed CKO mice and HFD-fed Flox mice, we identified AMP-activated protein kinase (AMPK) and proliferator-activated receptor α (PPARα ) as downstream targets of Enpp1 in the lipid metabolism pathway. Subsequently, we confirmed HFD-fed CKO group had significantly inhibited phosphorylation of AMPK, as well as PPARα expression in the liver. After observing the Enpp1-mediated signaling regulation of AMPK/PPARα, we attempted to elucidate the specific mechanism. Intracellular AMP is the main activator of AMPK [22, 23], which originates from the hydrolysis of intracellular ATP and other purine nucleotides in various physiological processes [24]. Referring to studies of the function of Enpp1 that involving in the hydrolysis of different purine nucleotides across a range of physiological processes, notably hydrolyzing ATP to AMP [24, 25], we modulate Enpp1 expression in AML-12 cells to evaluate AMP-to-ATP ratio. In cell experiments, by knocking down Enpp1 expression in AML-12 cells induced by PA, we observed a significant decrease in the AMP-to-ATP ratio and a corresponding reduction in AMPK phosphorylation and PPARα protein levels, and an increase in intracellular lipid accumulation and triglyceride content. Conversely, in Enpp1 over-expressing cells, the AMP-to-ATP ratio increased, AMPK phosphorylation and PPARα protein levels increased, while intracellular triglyceride content decreased. Aligning with our data plasma AMP-to-ATP ratio was lower in patients deficiency of Enpp1 [26, 27]. These experiments support the mechanism by which Enpp1 increase activated AMPK/PPARα pathway by increasing AMP-to-ATP ratio, thereby facilitating lipid metabolism.

However, this study has some limitations. For example, we did not extensively investigate the specific effect of Enpp1 on lipid metabolism through regulating AMPK/PPARα pathway. However, relevant literature has reported activation of parenchymal cell PPARα improves hepatic lipid metabolism by increasing ‌ω-oxidation as well as peroxisomal and mitochondrial β-oxidation [28]. Additionally, although the effects of Enpp1 on lipid metabolism of NAFLD have been demonstrated already, further exploration is warranted regarding its effects on immune phenotypes. NAFLD is accompanied by the increase of inflammatory mediators (including cytokines and chemokines), which leads to the intrahepatic infiltration of various types of immune cells [27, 29]. Enpp1, as an extracellular enzyme, can hydrolyze extracellular cGAMP, causing the inhibition of STING pathway, and ultimately inhibit the production of Interferon type I (IFN-1) and other pro-inflammatory cytokines [30]. Enpp1 also participates in the occurrence and development of hepatocellular carcinoma by regulating the infiltration of NK cells, dendritic cells and Th17 immune cells [16]. Consequently, Enpp1 may also exert important roles in inflammatory response in the process of NAFLD/NASH disease, which requires in-depth research.

In summary, this research provides both clinical and experimental evidence demonstrating that Enpp1 plays an important role in NAFLD. Deficiency of Enpp1 in liver exhibits abnormal lipid accumulation so as to accelerate the development of NAFLD. Over-expression of Enpp1 curtails lipid accumulation in hepatocytes, thereby ameliorating NAFLD. Mechanistically, Enpp1 activates AMPK/PPARα signaling by increasing AMP-to-ATP ratio to reduce lipid content in hepatocytes. Therefore, this study not only uncovered a novel molecular mechanism of NAFLD progression but also identified the Enpp1 protein as a potential new molecular target for the clinical management of NAFLD.

NAFLD Non-alcoholic Fatty Liver Disease

HFD High fat diet

NCD Normal Chow Diet

TG Triglycerides

T-CHO Total Cholesterol

AMPK AMP-activated protein kinase

p-AMPK Phospho-AMP-activated protein kinase

PPAR Peroxisome Proliferator-Activated Receptor

AMP Adenosine Monophosphate

ATP Adenosine Triphosphate

H&E Hematoxylin and Eosin

PAS Periodic Acid-Schiff

CKO Conditional Knockout

OE Over Expression

EV Empty Vector

KEGG Kyoto Encyclopedia of Genes and Genomes

PPi Pyrophosphate

DEGs Different Expressed Genes

Acknowledgements

We appreciate Professor Weibo Xia from Peking Union Medical College Hospital for providing the Enpp1^fl/fl mice.

Author contributions

Drafting of the manuscript: Xiaohui Liu. Research conception and design: Zhiwei Yang, Yuhan Li and Xiaohui Liu. Data analysis and interpretation: Xiaohui Liu, Yuhan Li. Statistical analysis: Xiaohui Liu. Literature retrieval: Yuhan Li, Xianxian Wu, Xiaoliang Jiang. Experiment performing: Xiaohui Liu, Shuai Chen and Xing Liu. Critical revision of the manuscript: Zhiwei Yang. Approval of the final manuscript: all authors.

Funding

The work was supported by National key research and development program (2022YFF0710600) and National Natural Science Foundation of China (82300653).

Data availability

The data that support the findings in this study are available in the manuscript and Supplementary Materials of this article. The RNA-Seq datasets generated during the current study were submitted to Genome Sequence Archive (GSA). All other relevant data are available from the corresponding author on request.

Ethics approval and consent to participate

All the animal experiments were approved by the Animal Care and Use Committee of the Institute of Experimental Animals, Chinese Academy of Medical Sciences and Beijing Union Medical College (yzw22002). This clinical study was approved by the Institutional Ethics Committee of Ningxia Medical University and Ningxia Medical University Second Affiliated Hospital (approval number: 2019-228). All research was conducted in accordance with both the Declarations of Helsinki and Istanbul. Written consent was given in writing by all subjects.

Consent for publication

Not applicable.

Competing interests

The authors have declared no competing interests.

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Editorial decision: Major revision
02 Nov, 2024
Reviewers agreed at journal
30 Aug, 2024
Reviewers invited by journal
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Editor assigned by journal
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Enpp1 ameliorates NAFLD by regulating hepatocyte lipid metabolism through the AMPK/PPARα signaling pathway

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Version 1

Abstract

Figures

INTRODUCTION

METHODS

Human liver samples

Animal experiments

Serum enzyme and lipid assays

Intravenous glucose tolerance test (IGTT) and insulin tolerance test (ITT)

Western blotting

Quantitative PCR (qPCR)

Oil red O staining

Periodic Acid-Schiff staining

Quantitation of adipocyte size

RNA sequencing

GEO database mining

Cell treatment and transfection

Measurement of the AMP-to-ATP Ratio in Cells

Statistical analysis

RESULTS

Hepatic Enpp1 expression is decreased in both NAFLD patients and mouse models

Hepatic Enpp1 deficiency aggravates obesity and insulin resistance in mice fed a high-fat diet

Hepatic Enpp1 deletion exacerbates hepatic steatosis in NAFLD mice

Enpp1 over-expression alleviates hepatic steatosis and metabolic deterioration

Enpp1 regulates hepatic lipid metabolism through activating the AMPK/PPARα signaling pathway

Enpp1 activates the AMPK/PPARα axis via increasing AMP-to-ATP ratio to inhibits TG accumulation in hepatocytes

DISSCUSSION

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1