Abstract 13164: Tm6sf2 is a Regulator of Liver Fat Metabolism in Smooth Er Influencing Vldl Lipidation

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Fei Luo ◽  
Helen H Hobbs ◽  
Jonathan C Cohen
Keyword(s):  
Secretory Pathway ◽  
Fat Metabolism ◽  
Liver Fat ◽  
Cell Fractionation ◽  
Loss Of Function ◽  
Nonalcoholic Fatty Liver ◽  
Golgi Membranes ◽  
Exon 1 ◽  
Rough Er ◽  
Reduced Rate

Backgrounds: A variant in Transmembrane 6 Superfamily Member 2 [ TM6SF2 (E167K)] that is associated with a loss of function is strongly associated with both alcoholic and nonalcoholic fatty liver disease. TM6SF2 is a polytopic protein that is expressed predominantly in the liver and small intestines. Immunolocalization studies are consistent with the protein being present in the endoplasmic reticulum (ER) and the Golgi complex. Tm6sf2 -/- mice replicate the phenotype of individuals with TM6SF2 -167K variant: hepatic steatosis accompanied by hypocholesterolemia, and transaminitis. These mice have a reduced rate of secretion of VLDL-TG without any change in the rate of secretion of ApoB. Thus, TM6SF2 is required for normal lipidation of VLDL. To determine where in the secretory pathway TM6SF2 promotes lipid addition to nascent VLDL, we have generated Tm6sf2 -/- rats. Methods: Two lines of Tm6sf2 -/- rats with different frameshift mutations in exon 1 were generated using CRISPR/Cas9 technology. Rats were fasted 4 hours and tissues were collected and frozen at -80°C. Cell fractionation studies were performed to isolate the smooth and rough ER along with trans - and cis -Golgi membranes. Result: Both lines of rats were confirmed by immunoblotting to produce no TM6SF2 in the liver or intestines. The phenotype of the Tm6sf2 -/- rats resembles that of mice and humans. Cell fractionation studies revealed that TM6SF2 mainly localized to the smooth ER. Unlike what was observed previously by immunocytochemistry, no TM6SF2 was found in the Golgi fraction. Analysis of the lipid profile of the Golgi apparatus in the KO rat revealed that the majority triglyceride (TG) and fatty acids (FA) subclasses were decreased by one to two folds compared to WT in terms of TG or FA to ApoB48 ratios. Conclusions: TM6SF2 is localized predominantly in the smooth ER and regulates the lipidation of VLDL.

scholarly journals Hypolipidemia associated with inactivation of TM6SF2 is due to decreased VLDL-lipids secretion

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
F Luo ◽  
E Smagris ◽  
J A Fletcher ◽  
J C Cohen ◽  
H H Hobbs
Keyword(s):  
Plasma Lipid ◽  
Microscopic Analysis ◽  
Missense Variant ◽  
Cell Fractionation ◽  
Loss Of Function ◽  
Lipid Levels ◽  
Funding Sources ◽  
Golgi Compartment ◽  
Exon 1 ◽  
Biochemical Analyses

Abstract Background A missense variant in Transmembrane 6 Superfamily Member 2 [TM6SF2 (E167K)] is associated with reduced plasma lipid levels and protection from coronary atherosclerosis. The substitution of lysine for glutamate at residue 167 is associated with a marked decrease in TM6SF2 protein expression, consistent with a loss-of-function mutation. However the biological role of TM6SF2 is not known, and the mechanism(s) responsible for the hypolipidemia associated with mutation gene has not been fully defined. To elucidate the pathological mechanism for the hypolipidemia associated with TM6SF2 deficiency, we inactivated Tm6sf2 in mice and rats. Methods Tm6sf2−/− mice were generated as described previously. Two lines of Tm6sf2−/− rats with different frameshift mutations in exon 1 were generated using CRISPR/Cas9 technology. Primary hepatocytes were isolated from WT and Tm6sf2−/− mice for microscopy. Rats were fasted 16 or 4 hours and tissues were collected on ice for cell fractionation, and in liquid nitrogen for biochemical analyses. Frozen samples were stored at −80°C for subsequent analyses. Result In both mice and rats, inactivation of Tm6sf2 recapitulated the phenotype of humans with the E167K substitution: steatosis, reduced plasma lipid levels, and transaminitis. The phenotype was readily apparent in animals fed chow diets. Both species had reduced secretion of VLDL-TG, as determined by TRITON WR1399 injection, with no decrease in secretion of ApoB. Experiments in isolated perfused livers from WT and Tm6sf2−/− mice confirmed that the decreased TG secretion observed in intact animals reflected reduced TG secretion from the liver. Lipidomic analysis of the liver perfusates by by LC-MS indicated that secretion of cholesteryl esters, and phospholipids was also decreased in the KO animals. Taken together, these findings are consistent with a role for TM6SF2 in lipidation of ApoB-containing lipoproteins. To further elucidate the function of TM6SF2, we used fluorescence microscopy and cell fractionation to determine the subcellular localization of the protein. Microscopic analysis showed that TM6SF2 co-localized with ER and Golgi markers, but cell fractionation studies indicated that the protein is located primarily in the smooth ER. The ratio of TG to ApoB was lower in Golgi fractions from TM6sf2−/− rats than in corresponding fractions from WT animals. Conclusions Since the sequela of TM6SF2 inactivation are already apparent in the Golgi, we speculate that TM6SF2 promotes lipidation of VLDL in a pre-Golgi compartment. We are currently performing additional studies to further define the specific mechanism whereby TM6SF2 promotes lipidation of ApoB-containing lipoproteins. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): National Institutes of Health

scholarly journals Cofilin-mediated sorting and export of specific cargo from the Golgi apparatus in yeast

2012 ◽  
Vol 23 (12) ◽  
pp. 2327-2338 ◽  
Author(s):  
Amy J. Curwin ◽  
Julia von Blume ◽  
Vivek Malhotra
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Calcium Atpase ◽  
Carboxypeptidase Y ◽  
Secretory Proteins ◽  
Golgi Membranes ◽  
Golgi Compartment ◽  
Reduced Rate ◽  
Golgi Network

The mechanism of cargo sorting at the trans-Golgi network (TGN) for secretion is poorly understood. We previously reported the involvement of the actin-severing protein cofilin and the Ca2+ ATPase secretory pathway calcium ATPase 1 (SPCA1) in the sorting of soluble secretory cargo at the TGN in mammalian cells. Now we report that cofilin in yeast is required for export of selective secretory cargo at the late Golgi membranes. In cofilin mutant (cof1-8) cells, the cell wall protein Bgl2 was secreted at a reduced rate and retained in a late Golgi compartment, whereas the plasma membrane H+ ATPase Pma1, which is transported in the same class of carriers, reached the cell surface. In addition, sorting of carboxypeptidase Y (CPY) to the vacuole was delayed, and CPY was secreted from cof1-8 cells. Loss of the yeast orthologue of SPCA1 (Pmr1) exhibited similar sorting defects and displayed synthetic sickness with cof1-8. In addition, overexpression of PMR1 restored Bgl2 secretion in cof1-8 cells. These findings highlight the conserved role of cofilin and SPCA1/Pmr1 in sorting of the soluble secretory proteins at the TGN/late Golgi membranes in eukaryotes.

scholarly journals Lrrk regulates the dynamic profile of dendritic Golgi outposts through the golgin Lava lamp

2015 ◽  
Vol 210 (3) ◽  
pp. 471-483 ◽  
Author(s):  
Chin-Hsien Lin ◽  
Hsun Li ◽  
Yi-Nan Lee ◽  
Ying-Ju Cheng ◽  
Ruey-Meei Wu ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Kinase Activity ◽  
Dominant Negative ◽  
Dendritic Arbor ◽  
Leucine Rich Repeat ◽  
Loss Of Function ◽  
Negative Effects ◽  
Golgi Membranes ◽  
Prominent Component ◽  
Dynamic Profile

Constructing the dendritic arbor of neurons requires dynamic movements of Golgi outposts (GOPs), the prominent component in the dendritic secretory pathway. GOPs move toward dendritic ends (anterograde) or cell bodies (retrograde), whereas most of them remain stationary. Here, we show that Leucine-rich repeat kinase (Lrrk), the Drosophila melanogaster homologue of Parkinson’s disease–associated Lrrk2, regulates GOP dynamics in dendrites. Lrrk localized at stationary GOPs in dendrites and suppressed GOP movement. In Lrrk loss-of-function mutants, anterograde movement of GOPs was enhanced, whereas Lrrk overexpression increased the pool size of stationary GOPs. Lrrk interacted with the golgin Lava lamp and inhibited the interaction between Lva and dynein heavy chain, thus disrupting the recruitment of dynein to Golgi membranes. Whereas overexpression of kinase-dead Lrrk caused dominant-negative effects on GOP dynamics, overexpression of the human LRRK2 mutant G2019S with augmented kinase activity promoted retrograde movement. Our study reveals a pathogenic pathway for LRRK2 mutations causing dendrite degeneration.

scholarly journals Involvement of intracellular calcium in protein secretion in Dictyostelium discoideum

1992 ◽  
Vol 103 (2) ◽  
pp. 371-380 ◽  
Author(s):  
M.B. Coukell ◽  
A.M. Cameron ◽  
N.R. Adames
Keyword(s):  
Dictyostelium Discoideum ◽  
Translational Control ◽  
Secretory Pathway ◽  
Normal Function ◽  
Cell Fractionation ◽  
Inhibitory Process ◽  
Cellular Energy ◽  
Selective Degradation ◽  
Preferential Loss ◽  
Rough Er

We reported previously that Ca2+ depletion of Dictyostelium discoideum cells severely inhibits extracellular cyclic nucleotide phosphodiesterase (PD) synthesis at a post-transcriptional step. In this study, further experiments were performed to learn more about the nature of this phenomenon. Examination of the polysomal distribution of PD transcripts in control cells and in cells depleted of Ca2+ by incubation with EGTA and A23187 (EA) suggested that inhibition of PD production does not involve translational control. Kinetic analysis of this inhibitory process revealed that soluble, intracellular PD activity, synthesized from either the 2.4 or 1.9 kb PD mRNA, decreased very rapidly upon addition of EA. Furthermore, this decrease in activity was accompanied by the preferential loss of PD-related polypeptides, indicating a proteolytic event. EA-induced PD degradation required cellular energy and concomitant protein synthesis but was unaffected by most of the lysosomotropic agents tested. Therefore, PD proteolysis might not occur in the lysosome. In cell fractionation experiments, the EA-sensitive, intracellular PD activity comigrated with a rough ER marker in Percoll/KCl gradients. In addition to its effect on the PD, EA were also observed to inhibit production and rapidly lower the intracellular levels of another secreted glycoprotein, the PD inhibitor. Together, these results suggest that depletion of some intracellular Ca2+ store(s) in Dictyostelium, possibly the ER, disrupts the normal function of the secretory pathway, resulting in selective degradation of certain proteins.

scholarly journals Identification of a novel homozygous loss-of-function mutation in FUCA1 gene causing severe fucosidosis: A case report

2021 ◽  
Vol 49 (4) ◽  
pp. 030006052110059
Author(s):  
Xinwen Zhang ◽  
Shaozhi Zhao ◽  
Hongwei Liu ◽  
Xiaoyan Wang ◽  
Xiaolei Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Lysosomal Storage Disorder ◽  
Autosomal Recessive ◽  
Signs And Symptoms ◽  
Lysosomal Storage ◽  
Loss Of Function ◽  
Wild Type ◽  
Single Nucleotide ◽  
Whole Exome ◽  
Exon 1

Fucosidosis is a rare lysosomal storage disorder characterized by deficiency of α-L-fucosidase with an autosomal recessive mode of inheritance. Here, we describe a 4-year-old Chinese boy with signs and symptoms of fucosidosis but his parents were phenotypically normal. Whole exome sequencing (WES) identified a novel homozygous single nucleotide deletion (c.82delG) in the exon 1 of the FUCA1 gene. This mutation will lead to a frameshift which will result in the formation of a truncated FUCA1 protein (p.Val28Cysfs*105) of 132 amino acids approximately one-third the size of the wild type FUCA1 protein (466 amino acids). Both parents were carrying the mutation in a heterozygous state. This study expands the mutational spectrum of the FUCA1 gene associated with fucosidosis and emphasises the benefits of WES for accurate and timely clinical diagnosis of this rare disease.

scholarly journals Sodium-Glucose Cotransporter-2 Inhibitors for Treatment of Nonalcoholic Fatty Liver Disease: A Meta-Analysis of Randomized Controlled Trials

Metabolites ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 22
Author(s):  
Alessandro Mantovani ◽  
Graziana Petracca ◽  
Alessandro Csermely ◽  
Giorgia Beatrice ◽  
Giovanni Targher
Keyword(s):  
Liver Disease ◽  
Fatty Liver ◽  
Fatty Liver Disease ◽  
Meta Analysis ◽  
Liver Fat ◽  
Controlled Trials ◽  
Liver Fat Content ◽  
Nonalcoholic Fatty Liver ◽  
Randomized Controlled ◽  
Sodium Glucose Cotransporter

Recent randomized controlled trials (RCTs) tested the efficacy of sodium-glucose cotransporter-2 (SGLT-2) inhibitors to specifically treat nonalcoholic fatty liver disease (NAFLD). We systematically searched three electronic databases (up to 31 October 2020) for identifying placebo-controlled or head-to-head RCTs that used SGLT-2 inhibitors for treatment of NAFLD. No published RCTs with paired liver biopsy data were available for the meta-analysis. Primary outcome measures were changes in serum liver enzyme levels and liver fat content on imaging techniques. Overall, we included a total of twelve RCTs testing the efficacy of dapagliflozin (n = six RCTs), empagliflozin (n = three RCTs), ipragliflozin (n = two RCTs) or canagliflozin (n = one RCT) to specifically treat NAFLD for a median period of 24 weeks with aggregate data on 850 middle-aged overweight or obese individuals with NAFLD (90% with type 2 diabetes). Compared to placebo/reference therapy, treatment with SGLT-2 inhibitors significantly decreased serum alanine aminotransferase (weighted mean differences (WMD): −10.0 IU/L, 95%CI −12.2 to −7.79 IU/L; I2 = 10.5%) and gamma-glutamyltransferase levels (WMD: −14.49 IU/L, 95%CI −19.35 to −9.63 IU/L, I2 = 38.7%), as well as the absolute percentage of liver fat content on magnetic resonance-based techniques (WMD: −2.05%, 95%CI −2.61 to −1.48%; I2 = 0%). In conclusion, SGLT-2 inhibitors seem to be a promising treatment option for NAFLD.

scholarly journals Prevalence of NAFLD in Healthy and Young Male Individuals

2011 ◽  
Vol 2011 ◽  
pp. 1-4 ◽  
Author(s):  
Asif Niaz ◽  
Zafar Ali ◽  
Shaista Nayyar ◽  
Naureen Fatima
Keyword(s):  
Liver Disease ◽  
Fatty Liver ◽  
Alcohol Intake ◽  
Young Male ◽  
Difficult Problem ◽  
Liver Fat ◽  
Renal Diseases ◽  
Nonalcoholic Fatty Liver ◽  
History Of ◽  
Ultrasound Evidence

Introduction. Nonalcoholic fatty liver disease (NAFLD) is an important cause of liver disease in adults and the most common cause of liver disease in children (Lavine and Schwimmer 2004). The abnormalities include increased liver fat without inflammation (steatosis) and nonalcoholic steatohepatitis (NASH). NASH may lead to fibrosis, cirrhosis, and ultimately liver failure if it is not treated (Matteoni et al. 1999). The objective of the study is to estimate the magnitude of the problem which will help us to formulate strategies in managing the potentially difficult problem. Materials and Methods. We included 1000 individuals between the ages of 30 and 50 years who came for annual checkup. The patients with other comorbidities like diabetes, ischemic heart disease, chronic liver disease, or renal diseases were excluded from the study. History of alcohol ingestion was also taken; any individual with history of alcohol intake was also excluded. All of them underwent investigations including CBC, LFTs, height and weight. The individuals who were found to have increased ALT (50 to 150 u/L) further underwent investigations including ultrasound of abdomen hepatitis b and c serology RA and ANA antibodies. All the individuals who were found to have viral or autoimmune illness were excluded from the study. The individuals having raised ALT levels and ultrasound evidence of fatty liver were taken. Results. 13.5% of the individuals were found to have NAFLD among those selected for the study. Conclusion. Mass campaign regarding physical and dietary measures needs to be undertaken in general masses regarding the gravity and potential prevention of the disease.

Abstract 007: Nonalcoholic Fatty Liver Disease and Subclinical Atherosclerosis: Analyses from the Coronary Artery Risk Development in Young Adults (CARDIA) Study

Circulation ◽  
2013 ◽  
Vol 127 (suppl_12) ◽  
Author(s):  
Lisa B VanWagner ◽  
Christina M Shay ◽  
Hongyan Ning ◽  
John Wilkins ◽  
Cora E Lewis ◽  
...  
Keyword(s):  
Risk Factors ◽  
Young Adults ◽  
Liver Disease ◽  
Coronary Artery ◽  
Fatty Liver ◽  
Fatty Liver Disease ◽  
Liver Fat ◽  
Cvd Risk Factors ◽  
Cvd Risk ◽  
Nonalcoholic Fatty Liver

Background: Nonalcoholic Fatty Liver Disease (NAFLD) and excess visceral adipose tissue (VAT) are associated with cardiovascular disease (CVD). Recent studies suggest that NAFLD and coronary artery calcification (CAC) are related independent of VAT. In a population-based cross-sectional sample of black and white adults free from prevalent liver or heart disease, we tested the hypothesis that NAFLD is associated with the presence of CAC and abdominal aortoiliac calcification (AAC) independent of VAT and other CVD risk factors. Methods: Participants from the Coronary Artery Risk Development in Young Adults study (Y25 exam) with concurrent computed tomography quantification of liver fat, CAC and AAC were included (n=2,163). NAFLD was defined as liver attenuation ≤ 40 Hounsfield Units after exclusion of other causes of liver fat (medication/alcohol use). Using the Agatston method, CAC/AAC presence was defined as a score > 0. Logistic regression models were used to calculate odds ratios and 95% confidence intervals. Results: Participant age was 49.9 (3.7) years and the sample was equally distributed by sex (55.6% female) and race (50.1% black). Mean BMI was 30.6 (7.1). The CAC and AAC prevalence was 26.5% and 49.6%. NAFLD prevalence was 9.6%. NAFLD participants were 50.1 (3.7) years old and more likely to be male (59.8% vs. 51.7%, p<0.0001), white (56.5% vs. 49.3%, p<0.05) and have the metabolic syndrome (70.1% vs. 22.6%, p<0.0001) than those with no NAFLD. They were also more likely to have CAC (37.2%) and AAC (60.9%) than those with no NAFLD (25.4% and 49.4%, respectively). In multivariable analyses adjusted for demographics and health behaviors, NAFLD was associated with the presence of CAC and AAC (Table 1). This association was attenuated after adjustment for CVD risk factors and VAT. Effect modification by race and sex was not statistically significant. Conclusion: In contrast to prior studies, our results suggest that the relationship between NAFLD and subclinical CVD is mediated by the presence of other CVD risk factors.

Abstract 05: Association of 25-Year Body Mass Index Trajectories Throughout Early Adulthood With Nonalcoholic Fatty Liver Disease in Middle Age: The Coronary Artery Risk Development in Young Adults (CARDIA) Study

Circulation ◽  
2017 ◽  
Vol 135 (suppl_1) ◽  
Author(s):  
Lisa B VanWagner ◽  
Sadiya Khan ◽  
Hongyan NIng ◽  
Juned Siddique ◽  
Cora E Lewis ◽  
...  
Keyword(s):  
Body Mass Index ◽  
Liver Disease ◽  
Fatty Liver ◽  
Nonalcoholic Fatty Liver Disease ◽  
Body Mass ◽  
Fatty Liver Disease ◽  
Early Adulthood ◽  
Liver Fat ◽  
Nonalcoholic Fatty Liver ◽  
Bmi Trajectory

Background: Nonalcoholic Fatty Liver Disease (NAFLD) has increased in parallel with obesity, is a risk factor for cirrhosis and liver cancer, and has few effective treatments. Identifying modifiable risk factors for NAFLD development is essential to effectively design prevention programs. We tested whether trajectories of body mass index (BMI) change throughout early adulthood were associated with risk of prevalent NAFLD in midlife independent of current BMI. Methods: Participants from the CARDIA study, a prospective multicenter population-based biracial cohort of adults (baseline age 18-30 years), underwent BMI measurement at exam years 0, 2, 5, 7, 10, 15, 20, and 25. At Year 25 (Y25, 2010-2011), liver fat was assessed by computed tomography. NAFLD was identified after exclusion of other causes of liver fat (alcohol/hepatitis). Latent mixture modeling was used to identify 25-year trajectories in BMI percent (%) change relative to baseline BMI over time. Multivariable logistic regression models were used to assess associations between BMI trajectory group and prevalent NAFLD with adjustment for baseline or current Y25 BMI. Results: Among 4,423 participants, we identified 4 distinct trajectories of BMI %change: stable BMI (26.2% of the cohort, 25-year mean BMI Δ=0.7 kg/m 2 ), mild increase (46.0%, BMI Δ=5.2 kg/m 2 ), moderate increase (20.9%, BMI Δ=10.0 kg/m 2 ), and extreme increase (6.9%, BMI Δ=15.1 kg/m 2 ) (Figure). NAFLD prevalence at Y25 was higher with increasing BMI trajectory: 4.1%, 9.3%, 13.0%, and 17.6% (p-trend <0.0001). At baseline, 34.6% of participants had overweight or obesity. After adjustment for confounders, trajectories of greater BMI increase were associated with greater NAFLD prevalence independent of baseline or current Y25 BMI (Figure). Conclusion: Weight gain throughout adulthood is associated with greater prevalence of NAFLD in midlife independent of baseline or current BMI. These findings highlight weight maintenance throughout adulthood as a potential target for primary prevention of NAFLD.

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