Choline is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC), and can enter one-carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses and can impact phospholipid metabolism. In the current study we sought to interrogate if HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing if the inhibition of choline uptake and metabolism upon concurrent HCV infection alters viral replication and infectivity. Additionally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS- and HS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter, choline transporter-like family (CTL). HCV infection in FBS, but not HS-cultured cells diminished CTL1 transcript and protein expression at 24 h post-infection, which was associated with lower choline uptake and lower incorporation of choline into PC. No changes in other transporters were observed and at 96 h post-infection, all differences were normalized. Reciprocally, limiting the availability of choline for PC synthesis by use of a choline uptake inhibitor resulted in increased HCV replication at this early stage (24 h post-infection) in both FBS- and HS-cultured cells. Finally, in chronic infection (96 h post-infection), inhibiting choline uptake and metabolism significantly impaired the production of infectious virions. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV infection.

Elevated circulating dipeptidyl peptidase-4 (DPP4) is a biomarker for liver disease, but its involvement in gluconeogenesis and metabolic associated fatty liver disease progression remains unclear. Here, we identified that DPP4 in hepatocytes but not TEK receptor tyrosine kinase–positive endothelial cells regulates the local bioactivity of incretin hormones and gluconeogenesis. However, the complete absence of DPP4 ( Dpp4 ^–/– ) in aged mice with metabolic syndrome accelerates liver fibrosis without altering dyslipidemia and steatosis. Analysis of transcripts from the livers of Dpp4 ^–/– mice displayed enrichment for inflammasome, p53, and senescence programs compared with littermate controls. High-fat, high-cholesterol feeding decreased Dpp4 expression in F4/80 ⁺ cells, with only minor changes in immune signaling. Moreover, in a lean mouse model of severe nonalcoholic fatty liver disease, phosphatidylethanolamine N -methyltransferase mice, we observed a 4-fold increase in circulating DPP4, in contrast with previous findings connecting DPP4 release and obesity. Last, we evaluated DPP4 levels in patients with hepatitis C infection with dysglycemia (Homeostatic Model Assessment of Insulin Resistance > 2) who underwent direct antiviral treatment (with/without ribavirin). DPP4 protein levels decreased with viral clearance; DPP4 activity levels were reduced at long-term follow-up in ribavirin-treated patients; but metabolic factors did not improve. These data suggest elevations in DPP4 during hepatitis C infection are not primarily regulated by metabolic disturbances.

The dysregulation of macrophage lipid metabolism drives atherosclerosis. AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics and plays essential roles regulating macrophage lipid dynamics. Here, we investigated the consequences of atherogenic lipoprotein-induced foam cell formation on downstream immunometabolic signaling in primary mouse macrophages. A variety of atherogenic low-density lipoproteins (acetylated, oxidized, and aggregated forms) activated AMPK signaling in a manner that was in part due to CD36 and calcium-related signaling. In quiescent macrophages, basal AMPK signaling was crucial for maintaining markers of lysosomal homeostasis as well as levels of key components in the lysosomal expression and regulation network. Moreover, AMPK activation resulted in targeted upregulation of members of this network via transcription factor EB. However, in lipid-induced macrophage foam cells, neither basal AMPK signaling nor its activation affected lysosomal-associated programs. These results suggest that while the sum of AMPK signaling in cultured macrophages may be anti-atherogenic, atherosclerotic input dampens the regulatory capacity of AMPK signaling.

Choline is an essential nutrient that is critical component of the membrane phospholipid phosphatidylcholine (PC), the neurotransmitter acetylcholine and the methylation pathway. In the liver specifically, PC is the major membrane constituent and can be synthesized by the CDP-choline or the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway. With the continuing global rise in the rates of obesity and non-alcoholic fatty liver disease, we sought to explore how excess fatty acids (FA), typical of an obesity and hepatic steatosis, affect choline uptake and metabolism in primary hepatocytes. Our results demonstrate that hepatocytes chronically treated with palmitate, but not oleate or a mixture, had decreased choline uptake, which was associated with lower choline incorporation into PC and lower expression of choline transport proteins. Interestingly, a reduction in the rate of degradation spared PC levels in response to palmitate when compared to control. PE synthesis was slightly diminished; however, no compensatory changes in the PEMT pathway were observed. We next hypothesized that ER stress may be a potential mechanism by which palmitate treatment diminished choline. However, when we exposed primary hepatocytes to the common ER stress inducing compound tunicamycin, choline uptake, contrary to our expectation was augmented, concomitant with the transcript expression of choline transporters. Moreover, tunicamycin-induced ER stress divorced the observed increase in choline uptake from CDP-choline pathway flux since ER stress significantly diminished the incorporation and total PC content, similar to PE. Conclusion : Therefore, our results suggest that the altered FA milieu seen in obesity and fatty liver disease progression may adversely affect choline metabolism, but that compensatory mechanisms work to maintain phospholipid homeostasis. Abstract Figure: