Phosphatidylethanolamine Methyltransferase Deficiency Exacerbates Acute Alcohol-Induced Liver Injury

Alcohol-associated liver disease (ALD) is a global burden of healthcare and remains a major cause of morbidity and mortality worldwide. ALD includes a spectrum of injuries that progresses from hepatic steatosis, alcoholic hepatitis to alcohol-associated cirrhosis and even hepatocellular carcinoma with continued alcohol misuse. The development of ALD depends on several factors, including genetics. The liver enzyme phosphatidylethanolamine methyltransferase (PEMT) catalyzes three sequential methyl transfers to phosphatidylethanolamine, generating phosphatidylcholine (PC). The PC generated with PEMT-mediated catalysis is preferentially used in very-low-density-lipoprotein (VLDL) assembly and is required for its normal biogenesis and secretion (1-3). Alcohol affects the methylation potential and impairs PEMT activity, which by inhibiting VLDL synthesis contributes to the development of hepatic steatosis. Polymorphisms in the human PEMT gene causing loss of function confer susceptibility to metabolic-associated steatohepatitis. Based on these considerations, we hypothesized that PEMT deletion would exacerbate alcohol-induced liver injury. Animal Handling and Diet: Male and female wildtype (WT) and PEMT knock out (KO) mice (12 weeks of age) were subjected to ethanol binge feeding model. The animals were gavaged with maltose dextrin or ethanol (5g/Kg BW) twice, 12 hours apart. The mice were euthanized eight hours after the second dose, where the blood and liver were collected for the following analyses: AST and ALT levels: Serum AST and ALT were analyzed using a VITROS 5.1 FS Chemistry System. Hepatic histopathology: Neutral-buffered formalin fixed liver sections stained with hematoxylin & eosin (H & E) and picrosirius red were imaged using a Keyence BZ-810 microscope. HPLC Analysis: Liver tissues were homogenized in 0.5N perchloric acid and subjected to HPLC analysis to determine S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) levels (3,4) The SAM:SAH ratio, or methylation index, was calculated, as detailed (3,4). Triglyceride Quantification: Lipids were extracted by Folch method (6) and triglyceride levels were quantified using the Thermo DNA Kit (3,4). Enzymatic Activity: Lysosomal acid lipase and proteasome activities were determined in liver homogenates, as detailed (7) Statistical Analyses: Data are expressed as mean values ± standard error (SE). Values not sharing a common subscript letter are statistically different, p \u3c 0.05. We found deletion of PEMT exacerbates acute alcohol-induced liver injury in both males and females as evidenced by: •Increased AST and ALT levels •Increased fat accumulation by histopathological assessment •Decreased SAM levels causing a reduction in the methylation potential •Increased hepatic triglycerides •Decreased lysosomal acid lipase activity •Decreased proteasome activityhttps://digitalcommons.unmc.edu/surp2022/1036/thumbnail.jp

Phosphatidylethanolamine N-methyltransferase (PEMT) Knockout Mice Exhibit Worse Alcohol-Induced Liver Injury than Wildtype Mice

Phosphatidylethanolamine N-methyltransferase (PEMT) is an enzyme that catalyzes the successive transfer of 3 methyl groups from S-adenosylmethionine (SAM) to phosphatidylethanolamine (PE) to generate phosphatidylcholine (PC). PC is vital for exporting fat out of the liver, ultimately preventing hepatic steatosis. Alcohol also induces steatosis partly through damaging this pathway, so the purpose of this study was to investigate the relationship between alcohol and PEMT in the liver. PEMT -/- (KO) and wild-type (WT) mice were subjected to a chronic + binge alcohol treatment, and both serum and liver samples were analyzed. Triglyceride quantification, SAM and S-adenosylhomocysteine (SAH) levels, and histological analyses were performed on liver samples, while ALT levels were determined from serum samples. Our study showed that ethanol-fed PEMT KO mice exhibited worse liver injury compared to other treatment groups. Our results show increased triglyceride levels, increased ALT levels, decreased SAM:SAH ratio, and increased liver to body weight ratio. From these findings, we conclude that additional liver damage is observed with the combination of alcohol feeding and absence of the PEMT enzyme. The mechanism by which these two factors affect one another is a key area of future study.https://digitalcommons.unmc.edu/surp2021/1016/thumbnail.jp

Elevated S-Adenosylhomocysteine Induces Adipocyte Dysfunction to Promote Alcohol-Associated Liver Steatosis

It has been previously shown that chronic ethanol administration-induced increase in adipose tissue lipolysis and reduction in the secretion of protective adipokines collectively contribute to alcohol-associated liver disease (ALD) pathogenesis. Further studies have revealed that increased adipose S-adenosylhomocysteine (SAH) levels generate methylation defects that promote lipolysis. Here, we hypothesized that increased intracellular SAH alone causes additional related pathological changes in adipose tissue as seen with alcohol administration. To test this, we used 3-deazaadenosine (DZA), which selectively elevates intracellular SAH levels by blocking its hydrolysis. Fully differentiated 3T3-L1 adipocytes were treated in vitro for 48 h with DZA and analysed for lipolysis, adipokine release and differentiation status. DZA treatment enhanced adipocyte lipolysis, as judged by lower levels of intracellular triglycerides, reduced lipid droplet sizes and higher levels of glycerol and free fatty acids released into the culture medium. These findings coincided with activation of both adipose triglyceride lipase and hormone sensitive lipase. DZA treatment also significantly reduced adipocyte differentiation factors, impaired adiponectin and leptin secretion but increased release of pro-inflammatory cytokines, IL-6, TNF and MCP-1. Together, our results demonstrate that elevation of intracellular SAH alone by DZA treatment of 3T3-L1 adipocytes induces lipolysis and dysregulates adipokine secretion. Selective elevation of intracellular SAH by DZA treatment mimics ethanol\u27s effects and induces adipose dysfunction. We conclude that alcohol-induced elevations in adipose SAH levels contribute to the pathogenesis and progression of ALD

Acute Ethanol-Induced Liver Injury is Prevented by Betaine Administration

Binge drinking is the most common form of excessive alcohol use. Repeated episodes of binge drinking cause multiple organ injuries, including liver damage. We previously demonstrated that chronic ethanol administration causes a decline in the intrahepatic ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH). This decline causes impairments in essential methylation reactions that result in alcohol-induced fatty liver (steatosis) and other features of alcohol-associated liver disease (ALD). Co-treatment with betaine during chronic ethanol feeding, normalizes hepatocellular SAM:SAH ratio and alleviates many features of liver damage including steatosis. Here, we sought to examine whether betaine treatment similarly protects against liver injury in an alcohol binge-drinking model. We hypothesized that ethanol binge with prior or simultaneous betaine administration would prevent or attenuate acute alcohol-induced liver damage. Male C57Bl/6 mice were gavaged twice, 12 h apart, with either 6 g ethanol/kg BW or with an equal volume/kg BW of 0.9% NaCl. Two separate groups of mice (n = 5/group) were gavaged with 4 g betaine/kg BW, either 2 h before or simultaneously with the ethanol or saline gavages. All mice were sacrificed 8 h after the last gavage and serum and liver parameters were quantified. Ethanol binges caused a 50% decrease in hepatic SAM:SAH ratio and a \u3e3-fold rise in liver triglycerides (p ≤ 0.05). These latter changes were accompanied by elevated serum AST and ALT activities and blood alcohol concentrations (BAC) that were ∼three-times higher than the legal limit of intoxication in humans. Mice that were treated with betaine 2 h before or simultaneously with the ethanol binges exhibited similar BAC as in mice given ethanol-alone. Both betaine treatments significantly elevated hepatic SAM levels thereby normalizing the SAM:SAH ratio and attenuating hepatic steatosis and other injury parameters, compared with mice given ethanol alone. Simultaneous betaine co-administration with ethanol was more effective in preventing or attenuating liver injury than betaine given before ethanol gavage. Our findings confirm the potential therapeutic value of betaine administration in preventing liver injury after binge drinking in an animal model

Delays between the onset of symptoms and first rheumatology consultation in patients with rheumatoid arthritis in the UK: an observational study

Objective To investigate delays from symptom onset to rheumatology assessment for patients with a new onset of rheumatoid arthritis (RA) or unclassified arthritis. Methods Newly presenting adults with either RA or unclassified arthritis were recruited from rheumatology clinics. Data on the length of time between symptom onset and first seeing a GP (patient delay), between first seeing a general practitioner (GP) and being referred to a rheumatologist (general practitioner delay) and being seen by a rheumatologist following referral (hospital delay) were captured. Results 822 patients participated (563 female, mean age 55 years). The median time between symptom onset and seeing a rheumatologist was 27.2 weeks (IQR 14.1–66 weeks); only 20% of patients were seen within the first 3 months following symptom onset. The median patient delay was 5.4 weeks (IQR 1.4–26.3 weeks). Patients who purchased over-the-counter medications or used ice/heat packs took longer to seek help than those who did not. In addition, those with a palindromic or an insidious symptom onset delayed for longer than those with a non-palindromic or acute onset. The median general practitioner delay was 6.9 weeks (IQR 2.3–20.3 weeks). Patients made a mean of 4 GP visits before being referred. The median hospital delay was 4.7 weeks (IQR 2.9–7.5 weeks). Conclusion This study identified delays at all levels in the pathway towards assessment by a rheumatologist. However, delays in primary care were particularly long. Patient delay was driven by the nature of symptom onset. Complex multi-faceted interventions to promote rapid help seeking and to facilitate prompt onward referral from primary care should be developed

GWAS meta-analysis of intrahepatic cholestasis of pregnancy implicates multiple hepatic genes and regulatory elements

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder affecting 0.5–2% of pregnancies. The majority of cases present in the third trimester with pruritus, elevated serum bile acids and abnormal serum liver tests. ICP is associated with an increased risk of adverse outcomes, including spontaneous preterm birth and stillbirth. Whilst rare mutations affecting hepatobiliary transporters contribute to the aetiology of ICP, the role of common genetic variation in ICP has not been systematically characterised to date. Here, we perform genome-wide association studies (GWAS) and meta-analyses for ICP across three studies including 1138 cases and 153,642 controls. Eleven loci achieve genome-wide significance and have been further investigated and fine-mapped using functional genomics approaches. Our results pinpoint common sequence variation in liver-enriched genes and liver-specific cis-regulatory elements as contributing mechanisms to ICP susceptibility

Role of Elevated Intracellular S-Adenosylhomocysteine in the Pathogenesis of Alcohol-Related Liver Disease

Background: The earliest manifestation of alcohol-related liver disease (ALD) is steatosis, characterized by the accumulation of lipid droplets (LDs) in hepatocytes. Findings from our laboratory have indicated that many pathological changes, including steatosis, correlate with the alcohol-induced hepatocellular increases in S-adenosylhomocysteine (SAH). Based on these considerations, we hypothesized that an experimental increase in intracellular SAH alone will result in similar steatotic changes to those seen after alcohol exposure. Methods: Freshly isolated rat hepatocytes grown on collagen-coated plates were exposed to serum-free medium containing 50 µmol/L oleic acid and varying concentrations of 3-deazaadenosine (DZA) to experimentally elevate intracellular SAH levels. Results: Overnight exposure to DZA treatment dose-dependently increased hepatocellular triglyceride accumulation, which was also evident by morphological visualization of larger-sized LDs. The rise in triglycerides and LDs accompanied increases in mRNA and protein levels of several LD-associated proteins known to regulate LD number and size. Furthermore, DZA treatment caused a decline in the levels of lipases that prevent fat accumulation as well as increased the expression of factors involved in lipogenesis and fatty acid mobilization. Collectively, our results indicate that the elevation of intracellular SAH is sufficient to promote fat accumulation in hepatocytes, which is similar to that seen after alcohol exposure

Anticancer activity of crude acetone and water extracts of Tulbaghia violacea on human oral cancer cells

Objective: To evaluate the anticancer activity of crude acetone and water leaf extracts of Tulbaghia violacea on a human oral cancer cell line (KB). Methods: The antioxidant activity of the leaf extracts was evaluated by using the DPPH assay while the anti-proliferative activity was assessed by using the MTT assay. The morphological characteristics of apoptotic cells were examined by using the dual acridine orange/ethidium bromide staining. Flow cytometry was used to evaluate the induction of multi-caspase activity and changes in the cell cycle. Results: The acetone and water extracts exhibited antioxidant activity in a concentration dependent manner. The extracts inhibited the growth of the KB cell line with IC50 values of 0.2 mg/mL and 1 mg/mL, respectively for acetone and water. Morphological changes such as cell shrinkage, rounding and formation of membrane blebs were observed in the treated cells. In acridine orange/ethidium bromide staining, the number of apoptotic cells increased as the concentration of the extracts increased. The activation of multi-caspase activity in KB cells treated with Tulbaghia violacea extracts was concentration dependent, leading to cell death by apoptosis and cell cycle arrest at the G2/M phase. Conclusions: The acetone and water extracts of Tulbaghia violacea appear to have anti-cancer activity against human oral cancer cells and need to be investigated further

Role of ghrelin hormone in the development of alcohol-associated liver disease

Fatty liver is the earliest response of the liver to excessive alcohol consumption. Previously we identified that chronic alcohol administration increases levels of stomach-derived hormone, ghrelin, which by reducing circulating insulin levels, ultimately contributes to the development of alcohol-associated liver disease (ALD). In addition, ghrelin directly promotes fat accumulation in hepatocytes by enhancing de novo lipogenesis. Other than promoting ALD, ghrelin is known to increase alcohol craving and intake. In this study, we used a ghrelin receptor (GHSR) knockout (KO) rat model to characterize the specific contribution of ghrelin in the development of ALD with emphasis on energy homeostasis. Male Wistar wild type (WT) and GHSR-KO rats were pair-fed the Lieber-DeCarli control or ethanol diet for 6 weeks. At the end of the feeding period, glucose tolerance test was conducted, and tissue samples were collected. We observed reduced alcohol intake by GHSR-KOs compared to a previous study where WT rats were fed ethanol diet ad libitum. Further, when the WTs were pair-fed to GHSR-KOs, the KO rats exhibited resistance to develop ALD through improving insulin secretion/sensitivity to reduce adipose lipolysis and hepatic fatty acid uptake/synthesis and increase fatty acid oxidation. Furthermore, proteomic data revealed that ethanol-fed KO exhibit less alcohol-induced mitochondrial dysfunction and oxidative stress than WT rats. Proteomic data also confirmed that the ethanol-fed KOs are insulin sensitive and are resistant to hepatic steatosis development compared to WT rats. Together, these data confirm that inhibiting ghrelin action prevent alcohol-induced liver and adipose dysfunction independent of reducing alcohol intake

Beneficial Effects of Betaine: A Comprehensive Review

Medicinal herbs and many food ingredients possess favorable biological properties that contribute to their therapeutic activities. One such natural product is betaine, a stable, nontoxic natural substance that is present in animals, plants, and microorganisms. Betaine is also endogenously synthesized through the metabolism of choline or exogenously consumed through dietary intake. Betaine mainly functions as (i) an osmolyte and (ii) a methyl-group donor. This review describes the major physiological effects of betaine in whole-body health and its ability to protect against both liver- as well as non-liver-related diseases and conditions. Betaine’s role in preventing/attenuating both alcohol-induced and metabolic-associated liver diseases has been well studied and is extensively reviewed here. Several studies show that betaine protects against the development of alcohol-induced hepatic steatosis, apoptosis, and accumulation of damaged proteins. Additionally, it can significantly prevent/attenuate progressive liver injury by preserving gut integrity and adipose function. The protective effects are primarily associated with the regulation of methionine metabolism through removing homocysteine and maintaining cellular SAM:SAH ratios. Similarly, betaine prevents metabolic-associated fatty liver disease and its progression. In addition, betaine has a neuroprotective role, preserves myocardial function, and prevents pancreatic steatosis. Betaine also attenuates oxidant stress, endoplasmic reticulum stress, inflammation, and cancer development. To conclude, betaine exerts significant therapeutic and biological effects that are potentially beneficial for alleviating a diverse number of human diseases and conditions