Hepatic fibrosis is the first stage of liver disease, and can progress to a chronic status, such as cirrhosis or hepatocellular carcinoma. Excessive production of extracellular matrix (ECM) components plays an important role in the development of fibrosis. Mechanistically, transforming growth factor beta (TGFβ)-induced phosphorylation of Smad is thought to be a key signaling pathway in the development of liver fibrosis. Although the natural isoquinoline alkaloid oxoglaucine (1,2,9,10-tetramethoxy-7H-dibenzo(de,g)quinolin-7-one) exerts numerous beneficial effects, including anti-cancer, anti-inflammatory, and anti-osteoarthritic effects in diverse cell types, the effects of oxoglaucine on liver fibrosis and fibrogenic gene expression have not been fully elucidated. The aim of this study is to evaluate the signaling pathway and antifibrotic activity of isoquinoline alkaloid oxoglaucine in TFGβ-induced hepatic fibrosis in vitro. Using Hepa1c1c7 cells and primary hepatocytes, we demonstrated that oxoglaucine treatment resulted in inhibition of the expression of fibrosis markers such as collagen, fibronectin, and alpha-SMA. Subsequent experiments showed that oxoglaucine suppressed TGFβ-induced phosphorylation of Smad2 and reactive oxygen species (ROS) generation, without altering cell proliferation. We further determined that the increase in Smad7 by oxoglaucine treatment is responsible for the inhibition of Smad2 phosphorylation and the anti-fibrogenic effects. These findings indicate that oxoglaucine plays a crucial role in suppression of fibrosis in hepatocytes, thereby making it a potential drug candidate for treatment of liver fibrosis.

27-Hydroxycholesterol (27OHC), an endogenous oxysterol, has been implicated in various physiological processes, including regulation of estrogen receptor activity and lipid metabolism. However, studies on how 27OHC affects the metabolic changes associated with lipogenesis inhibition in the liver remain limited. This study aimed to investigate the systemic effects of 27OHC on hepatocytes through a comparative proteomic analysis of the proteomes in the 27OHC-treated AML12 cells. Ingenuity Pathway Analysis revealed significant downregulation of certain metabolic pathways, such as cholesterol biosynthesis and glycolysis, which are highly associated with lipid metabolism, following 27OHC treatment. Furthermore, in vitro biochemical analysis revealed significant inhibition of the expression of genes associated with the mevalonate pathway and a decrease in the total cellular cholesterol levels in AML12 cells and primary hepatocytes following 27OHC treatment. In addition, it was observed that 27OHC significantly reduced the transcripts levels of critical glycolytic enzymes such as aldolase, phosphofructokinase, and pyruvate kinase. This inhibition resulted in decreased lactate production and extracellular acidification (ECAR), indicating suppression of glycolytic flux. Concurrently, we proved that downregulation of reactive oxygen species generation and HIF-1α expression following 27OHC treatment partially contributed to glycolysis inhibition. Overall, we demonstrated the inhibitory effects of 27OHC on the hepatic mevalonate pathway and glycolysis, revealing a novel mechanism by which 27OHC regulates lipid metabolism. As the accumulation of cholesterol and lipids promotes hepatic fatty liver disease and increased glycolysis contributes to triacylglycerol maturation, the suppressive effects of 27OHC on hepatic lipid and glucose metabolism may contribute to protect against fatty liver development.

Fatty acids (FA) are a common supplement for lactating cows to increase energy supply, potentially improving feed efficiency and milk and milk fat yields. However, different FA can cause varying responses probably because of different FA digestibility and interactions with other nutrients (e.g., fibers). Lysophospholipids (LPL) are natural emulsifiers and LPL as a feed additive have shown increases in FA digestibility and nutrient absorption in non-ruminant animals. This study aimed to investigate the effects of FA sources with LPL supplementation on nutrient digestibility in lactating cows. Mid-lactation cows (n = 48) were allocated into 12 blocks, and cows in each block were randomly assigned to the following dietary treatments:1) a diet with a FA supplement [1.8% of dietary dry matter (DM)] mainly containing palmitic acid (PA); 2) the PA diet with LPL (0.003% of dietary DM); 3) a diet with a FA supplement (1.8% of dietary DM) mainly containing stearic acid (SA); and 4) the SA diet with LPL (0.003% of DM). The experiment was conducted for 6 weeks, and spot samples of feces and urine were collected over 3 days (every 3 hours within a 24-hour cycle) during the last week of the experiment to determine nutrient digestibility and utilization. Data were analyzed using the mixed model with FA source, LPL, and their interaction as fixed effects and block as a random effect. The digestibility of DM, organic matter, and crude protein were not affected by the FA source and no interaction between FA and LPL was observed. However, PA had greater neutral detergent fiber (NDF) digestibility (41.4 vs. 38.8%; P = 0.04) than SA with no interaction between the FA source and LPL. In addition, as expected, PA decreased (P < 0.01) the digestibility of 16-carbon FA compared with SA due to the greater C16:0 supply, and SA decreased (P < 0.01) that of 18-carbon FA compared with PA due to the greater C18:0 supply. Supplementation of LPL did not affect digestibility of DM, OM, CP, and NDF, but increased total FA digestibility (64.7 vs. 60.5%; P = 0.02) compared with no LPL. In addition, LPL increased 18-carbon FA digestibility (68.2 vs. 74.1%; P < 0.01) compared with no LPL. An interaction between LPL and FA was observed for 16-carbon digestibility because LPL increased 16-carbon digestibility with SA but not with PA. In conclusion, PA supplementation improved fiber digestibility compared with SA, which could be a factor for increased productivity (e.g., milk fat). Supplementation of LPL was effective in increasing FA digestibility and may have potential to increase dietary energy supply or provide a similar energy supply with less dietary fat content in practice. However, more studies are needed to understand different interactions of LPL with FA sources.

The modern concept of diet formulation for protein is to balance for amino acids (AA). The expectation of feeding a diet with balanced AA is better dietary N utilization, leading to decreased protein content in a diet and less N excretion. Feeding rumen protected (RP) AA can be a good option to increase some of AA that are short in supply. RP-Met and -Lys have been largely studied with early- and mid-lactation cows, and meta-analyses are available. Although studies that showed no responses to RP-AA exist, results of meta-analyses were consistent that RP-Met is effective to increase milk protein and fat yield. When RP-Met and -Lys were fed together, positive effects on milk yield and composition were also shown. If a diet adequate or slightly deficient in protein is fed especially at commercial farms, RP-His has large potential to increase production of high producing cows. Although cows have the ability to utilize digestible His efficiently for milk protein when the supply is short, studies consistently showed that additional supply of His increased milk yield and milk protein yield when His supply is insufficient. However, RP-His is not commercially available yet. Studies about transition cows with RP-AA are relatively limited, and results are variable. The inclusion of RP-Met and Lys in prepartum and postpartum diets were determined assuming that the requirements of AA are the same as early- or mid-lactating cows. In the transition cow studies, if positive production effects (e.g., milk and milk protein yield) were observed, DMI was often increased with RP-AA. If DMI was not affected, then production was not affected by RP-AA or milk fat yield was sometimes increased. The inconsistent results likely indicate potential differences in AA requirements between transition cows and early lactation cows. Because of the negative energy and protein balance postpartum, studies showed positive production responses consistently to increased dietary protein concentration. One study compared production responses between a postpartum diet with increased protein without considering AA profile and diet with increased protein with considering AA profile (casein AA). No difference in milk yield and composition was found, suggesting that the quantity of protein supply is more important than quality, or digestible AA mimicking casein AA is not ideal for transition cows. There are key components to consider when RP-AA are fed to cows to potentially increase responses. Because utilization efficiency of AA for protein synthesis decreases as the supply increases, excessive supply of AA should be avoided. To adequately supply certain AA via RP-AA, knowing accurate bioavailability of RP-AA is essential. However, recent studies questioned in vitro and in situ methods about its accuracy. Development of a gold standard in vivo method to calibrate in vitro or in situ methods is necessary.