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The liver is a central organ in regulating the whole body metabolic homeostasis, and, among many other processes, it plays a crucial role in lipoprotein metabolism. The liver controls the secretion of very-low-density lipoproteins (VLDLs), particles specialized in the transport of liver lipids, mainly triglycerides (TGs), to the adipose tissue, heart, and muscle, among other tissues, providing fatty acids to be stored or to be used as an energy source. The analysis of this metabolic process provides relevant information about the crosstalk between the liver and other organs. It also helps to identify how the liver is able to secrete lipids to reduce its accumulation. This protocol shows how to analyze the liver TG secretion rate blocking the VLDL clearance from the blood by the administration of poloxamer 407. In addition, it shows how to isolate the VLDL produced by the liver at the end of the experiment, so that the apolipoprotein and lipid content and size can be measured. Using antisense oligonucleotides (ASOs) for silencing target proteins involved in metabolic diseases has emerged as a new promising therapeutic approach. Thus, the usage of ASOs has also been included in this protocol. As a conclusion, evaluation of TG secretion rate in mice provides key information to understand the organ crosstalk in metabolic diseases and the capacity of the liver to secrete lipids to blood., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Unlabelled: Very low-density lipoprotein (VLDL) secretion provides a mechanism to export triglycerides (TG) from the liver to peripheral tissues, maintaining lipid homeostasis. In nonalcoholic fatty liver disease (NAFLD), VLDL secretion disturbances are unclear. Methionine adenosyltransferase (MAT) is responsible for S-adenosylmethionine (SAMe) synthesis and MAT I and III are the products of the MAT1A gene. Deficient MAT I and III activities and SAMe content in the liver have been associated with NAFLD, but whether MAT1A is required for normal VLDL assembly remains unknown. We investigated the role of MAT1A on VLDL assembly in two metabolic contexts: in 3-month-old MAT1A-knockout mice (3-KO), with no signs of liver injury, and in 8-month-old MAT1A-knockout mice (8-KO), harboring nonalcoholic steatohepatitis. In 3-KO mouse liver, there is a potent effect of MAT1A deletion on lipid handling, decreasing mobilization of TG stores, TG secretion in VLDL and phosphatidylcholine synthesis via phosphatidylethanolamine N-methyltransferase. MAT1A deletion also increased VLDL-apolipoprotein B secretion, leading to small, lipid-poor VLDL particles. Administration of SAMe to 3-KO mice for 7 days recovered crucial altered processes in VLDL assembly and features of the secreted lipoproteins. The unfolded protein response was activated in 8-KO mouse liver, in which TG accumulated and the phosphatidylcholine-to-phosphatidylethanolamine ratio was reduced in the endoplasmic reticulum, whereas secretion of TG and apolipoprotein B in VLDL was increased and the VLDL physical characteristics resembled that in 3-KO mice. MAT1A deletion also altered plasma lipid homeostasis, with an increase in lipid transport in low-density lipoprotein subclasses and decrease in high-density lipoprotein subclasses., Conclusion: MAT1A is required for normal VLDL assembly and plasma lipid homeostasis in mice. Impaired VLDL synthesis, mainly due to SAMe deficiency, contributes to NAFLD development in MAT1A-KO mice., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Osteopontin (OPN), a senescence‐associated secretory phenotype factor, is increased in patients with nonalcoholic fatty liver disease (NAFLD). Cellular senescence has been associated with age‐dependent hepatosteatosis. Thus, we investigated the role of OPN in the age‐related hepatosteatosis. For this, human serum samples, animal models of aging, and cell lines in which senescence was induced were used. Metabolic fluxes, lipid, and protein concentration were determined. Among individuals with a normal liver, we observed a positive correlation between serum OPN levels and increasing age. This correlation with age, however, was absent in patients with NAFLD. In wild‐type (WT) mice, serum and liver OPN were increased at 10 months old (m) along with liver p53 levels and remained elevated at 20m. Markers of liver senescence increased in association with synthesis and concentration of triglycerides (TG) in 10m OPN‐deficient (KO) hepatocytes when compared to WT hepatocytes. These changes in senescence and lipid metabolism in 10m OPN‐KO mice liver were associated with the decrease of 78 kDa glucose‐regulated protein (GRP78), induction of ER stress, and the increase in fatty acid synthase and CD36 levels. OPN deficiency in senescent cells also diminished GRP78, the accumulation of intracellular TG, and the increase in CD36 levels. In 20m mice, OPN loss led to increased liver fibrosis. Finally, we showed that OPN expression in vitro and in vivo was regulated by p53. In conclusion, OPN deficiency leads to earlier cellular senescence, ER stress, and TG accumulation during aging. The p53‐OPN axis is required to inhibit the onset of age‐related hepatosteatosis. [ABSTRACT FROM AUTHOR]

Background & Aims Very-low-density lipoproteins (VLDLs) export lipids from the liver to peripheral tissues and are the precursors of low-density-lipoproteins. Low levels of hepatic S-adenosylmethionine (SAMe) decrease triglyceride (TG) secretion in VLDLs, contributing to hepatosteatosis in methionine adenosyltransferase 1A knockout mice but nothing is known about the effect of SAMe on the circulating VLDL metabolism. We wanted to investigate whether excess SAMe could disrupt VLDL plasma metabolism and unravel the mechanisms involved. Methods Glycine N -methyltransferase ( GNMT ) knockout (KO) mice, GNMT and perilipin-2 ( PLIN2 ) double KO ( GNMT - PLIN2 -KO) and their respective wild type (WT) controls were used. A high fat diet (HFD) or a methionine deficient diet (MDD) was administrated to exacerbate or recover VLDL metabolism, respectively. Finally, 33 patients with non-alcoholic fatty-liver disease (NAFLD); 11 with hypertriglyceridemia and 22 with normal lipidemia were used in this study. Results We found that excess SAMe increases the turnover of hepatic TG stores for secretion in VLDL in GNMT -KO mice, a model of NAFLD with high SAMe levels. The disrupted VLDL assembly resulted in the secretion of enlarged, phosphatidylethanolamine-poor, TG- and apoE-enriched VLDL-particles; special features that lead to increased VLDL clearance and decreased serum TG levels. Re-establishing normal SAMe levels restored VLDL secretion, features and metabolism. In NAFLD patients, serum TG levels were lower when hepatic GNMT-protein expression was decreased. Conclusions Excess hepatic SAMe levels disrupt VLDL assembly and features and increase circulating VLDL clearance, which will cause increased VLDL-lipid supply to tissues and might contribute to the extrahepatic complications of NAFLD. [ABSTRACT FROM AUTHOR]