BACKGROUND AND AIMS:Dyslipidemia is common in patients with MASLD, but the frequency and significance of inherited disorders of dyslipidemia are unclear. We investigated the prevalence and significance of pathogenic variants associated with selected monogenic disorders of dyslipidemia in 3358 patients with well-characterised MASLD. APPROACH:We identified clinically relevant variants in APOB, MTTP, PCSK9, ANGPTL3, LDLR and LDLRAP1 genes which can cause hypobetalipoproteinemia (HBL) and familial hypercholesterolemia (FH). Using ClinVar annotations as initial variant selection, we identified 2027 variants in those 6 genes which are reported as 'pathogenic' or 'likely pathogenic' (P/LP). We first assessed for the presence of P/LP variants in the study cohort and then investigated the effect of carrying P/LP variants on liver histology, by comparing ~4 matched controls for each APOB and LDLR carrier. As interpretative analyses, we also looked at the difference between liver enzymes, lipid measures and outcomes between the carriers and matched controls. RESULTS:Twenty-two variants among these 2027 P/LP variants were present in 24 out of 3358 patients (12 ApoB, 10 LDLR, 1 ANGPTL3 and 1 MTTP variant carriers). Compared to controls, APOB carriers had higher steatosis grade (2.4 vs. 1.7, p-value 0.0028), higher NAFLD activity score (NAS) (4.9 vs. 3.8, p-value 0.04), and numerically higher but statistically not significant fibrosis stage (1.2 vs. 1.1, p-value 0.75) and ALT (87.4 vs. 58.1 U/L, p-value 0.06). Their LDL-c (51 vs. 147.8 mg/dL, p-value 6.1E-09) and triglycerides (91.5 vs. 160.6 mg/dL, p-value 2.8E-03) were significantly lower. Compared to controls, LDLR carriers had numerically higher steatosis grade, NAS, fibrosis stage and LDL-c levels, but these were not statistically different. CONCLUSIONS:Monogenic disorders of dyslipidemia are rarely present in patients with MASLD and are sometimes associated with worse liver histology. Testing for these conditions may be considered on a case-by-case basis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) impacts more than a third of adults worldwide. The burgeoning epidemic of MASLD has led to increases in atherosclerotic cardiovascular disease (CVD), which is the leading cause of morbidity and mortality among affected individuals, particularly those with type 2 diabetes. Subsets of patients with MASLD will develop progressive liver disease, including metabolic dysfunction-associated steatohepatitis, advanced fibrosis, cirrhosis, and hepatocellular cancer. The association of progressive forms of MASLD with cardiometabolic disease has spurred interest in understanding how distinct pathways in hepatic lipid and lipoprotein metabolism may identify candidate therapeutic targets to mitigate CVD risk. A deeper understanding of hepatic lipid and lipoprotein homeostasis in MASLD is a pressing need for several reasons connected with some of the molecular and signaling adaptations occurring in obesity and type 2 diabetes. First, because these conditions individually and in combination accelerate the onset and progression of MASLD; second, because progressive MASLD may promote insulin resistance and type 2 diabetes; and third, because some CVD risk factors (dyslipidemia, obesity, type 2 diabetes) overlap with those for MASLD. Evidence suggests a multifactorial cause for MASLD, including genetic factors that either promote or mitigate liver disease progression and which, along with environmental modifiers (diet, obesity) and insulin resistance, contribute to CVD risk. Segregating these causes of MASLD by phenotype/metabotype reveals a systemic metabolic dysfunction (obesity, insulin resistance) dominant subtype, which is associated with increased CVD, and a second dominant subtype where familial and genetic factors predominate, which reflects impaired VLDL (very low-density lipoprotein) secretion and is associated with reduced CVD risk. Further study of these distinctive subtypes of MASLD may identify new, tailored approaches to prevent cardiometabolic disease.

Apolipoprotein B-containing (APOB-containing) lipoproteins contribute to atherosclerosis by entering the arterial wall through the endothelial cell (EC) surface receptors scavenger receptor-BI (SR-BI) and activin receptor-like kinase 1 (ALK1). We used N-terminal fragments of APOB, molecular modeling, and site-directed mutagenesis to identify and block the binding of chylomicrons and LDL to these receptors in cells and mice. We discovered that different APOB regions interact with SR-BI and ALK1 expressed on ECs. APOB48 lipoproteins were only internalized by SR-BI. A fragment of APOB comprising 18% of the N-terminal sequence, APOB18, reduced the uptake and transport of both chylomicrons and LDL by ECs, whereas a shorter fragment, APOB12, only blocked ALK1-mediated uptake of APOB100-containing lipoproteins. Importantly, overexpressing APOB18 decreased atherosclerosis in hypercholesterolemic mice. These findings identify the N-terminal region of APOB as the cause of atherosclerosis and illustrate an approach to treating or preventing vascular disease.

BACKGROUND & AIMS:Intestinal failure-associated liver disease results from massive small bowel resection (SBR). We assessed the impact of isocaloric enteral omega-3 (ω-3) and omega-6 (ω-6) polyunsaturated fatty acids (PUFAs) on intestinal function and liver injury following massive enterectomy. METHODS:Male C57BL/6J mice underwent SBR or sham operation. Mice received either ω-3-enriched, ω-6-enriched, or a balanced saturated/monounsaturated/PUFA (control) diet postoperatively. At 10 weeks, body composition, metabolic profiles, intestinal adaptation, and liver injury were assessed. Targeted lipidomic profiling and bulk RNA sequencing of liver and intestine was completed. The role of Pparα activity was assessed in vivo by genetic deletion and pharmacologic activation. RESULTS:ω-3-fed mice experienced improved weight recovery and more efficient energy metabolism following SBR. Compared with control mice, SBR mice fed either ω-3 or ω-6 diets showed reduced serum aspartate aminotransferase and alanine aminotransferase. Hepatic free fatty acid analysis reflected dietary intake. Hepatic steatosis and fibrosis was lowest in ω-3, intermediate in ω-6, and highest in control mice following SBR. Hepatic RNA sequencing found upregulation of fibrotic pathways in both the SBR control and SBR ω-6 with lipid metabolism upregulated in the SBR ω-3. Pparα was shown to be necessary for survival but not sufficient for hepatoprotection following SBR, which suggested that ω-3 fatty acids engage in Pparα-independent effects. RNA sequencing of the remnant ileum found ω-3 fatty acids promote upregulation of genes related to lipid metabolism and nutrient absorption consistent with a more proximal intestinal identity. CONCLUSIONS:These data suggest that enteral feeding with ω-3 PUFAs improves intestinal functional adaptation and protects the liver injury after SBR while improving weight recovery and energy expenditure.

Prediabetes associates with increased production of triglyceride-rich lipoproteins (TRLs), cardiovascular disease (CVD), and hepatic steatosis, which is linked to increased plasma levels of soluble TREM2 (sTREM2), the shed domain of TREM2 (triggering receptor expressed on myeloid cells 2). Whether and how TREM2 shedding contributes to elevated TRLs is unknown. By complementary analyses of individuals with prediabetes and hepatic steatosis and preclinical models, we show that plasma sTREM2 levels correlate positively with plasma apolipoprotein C3 (APOC3), an apolipoprotein that slows TRL catabolism and predicts CVD risk. Individuals with prediabetes and hepatic steatosis had higher plasma concentrations of APOC3-rich TRLs 35 to 60 nm in diameter than healthy controls. Mouse models of prediabetes with hepatic steatosis revealed that the increased plasma concentrations of sTREM2, APOC3, and TRLs were due to activation of macrophage ADAM17, a TREM2 sheddase. Preserving macrophage full-length TREM2 protected against the elevated plasma APOC3, sTREM2, dyslipidemia, and atherosclerosis, while TREM2-deficiency increased APOC3, TRLs, and atherosclerosis. Mechanistically, full-length TREM2 mediates macrophage TRL uptake, preventing excessive hepatic APOC3-rich TRL release and atherosclerosis. Our findings identify macrophage TREM2 shedding as an upstream contributor to the elevated TRLs in hepatic steatosis, providing a mechanistic link between hepatic steatosis and CVD risk in prediabetes.

Background Short gut syndrome (SGS) after extensive small bowel resection (SBR) is associated with intestinal failure associated liver disease (IFALD). Previously, we identified Annexin A2 (Anxa2) hepatocytes with fibrotic transcriptional alterations in a murine intestinal failure model. This study investigates Anxa2’s role in the pathogenesis of IFALD. Methods Wild type 8 to 12-week-old C57BL/6J (WT control) and B6J.129X1-Anxa2tm1Kah/Mmmh (A2KO) female mice underwent sham or 75% proximal SBR and sacrificed 10-weeks post-operatively. Liver injury was assessed with serum markers, histology and bulk RNA sequencing. Results A2KO SBR liver showed a reduction in liver injury marked by reduction in serum transaminases, liver steatosis, and liver fibrosis. Bulk RNA sequencing found A2KO SBR liver has upregulation of gene sets related to oxidative response and beta-oxidation with reduction in fibrotic and innate immune system responses. Conclusions Anxa2 appears to be a putative factor in fibrogenesis with multifactorial roles. Targeting Anxa2 may serve as a novel therapeutic approach in both IFALD and other liver pathologies.

Background & Aims Transmembrane protein 41B (TMEM41B) and vacuolar membrane protein 1 (VMP1) are endoplasmic reticulum (ER) scramblases whose roles in hepatic lipoprotein secretion and autophagy in metabolic-associated steatotic liver disease (MASLD) remain unclear. Methods We undertook lipidomic and functional studies in liver- and hepatocyte-specific Tmem41b knockout (KO) mice; Tmem41b knock-in (KI) mice, Tmem41b/Vmp1 double KO (DKO); Tmem41b KO/Vmp1 KI, and Vmp1 KO/Tmem41b KI mice. Results TMEM41B protein levels decreased in the livers of human subjects with MASLD. Loss of hepatic Tmem41b impaired very low-density lipoprotein (VLDL) secretion, with steatosis, inflammation, and fibrosis, whereas hepatic TMEM41B overexpression mitigated these effects. Tmem41b/Vmp1 DKO mice showed further impairment in VLDL secretion compared with single Tmem41b KO. Lipidomic analysis revealed decreased phosphatidylcholine and phosphatidylethanolamine, with increased neutral lipids in Tmem41b KO mice. VMP1 and TMEM41B localize at the mitochondrial-associated membrane with reduced mitochondria-ER contact in VMP1 and TMEM41B KO mice. Loss of hepatic VMP1 or TMEM41B increased levels of LC3B-II and p62/SQSTM1, which were not further changed in DKO mice. Restoring VMP1 in Tmem41b KO mice and TMEM41B in Vmp1 KO mice partially corrected defective VLDL secretion and hepatic steatosis in these single KO mice, respectively. Restoring VMP1 at a low but not a high dose corrected defective autophagy in Tmem41b KO mice, whereas overexpression of TMEM41B dose-dependently improved defective autophagy in Vmp1 KO mice. Conclusions Loss of hepatic VMP1 or TMEM41B reduces VLDL secretion and promotes MASLD via overlapping but distinct mechanisms that regulate lipoprotein secretion and autophagy.

Background and Aims:In inflammatory bowel disease, protein misfolding in the endoplasmic reticulum (ER) potentiates epithelial barrier dysfunction and impairs mucosal healing. Tauroursodeoxycholic acid (TUDCA), a naturally occurring bile acid, acts as a chemical chaperone to reduce protein aggregation and colitis severity in preclinical models. We conducted an open label trial evaluating oral TUDCA as therapy in patients with active ulcerative colitis (UC). Methods:Patients with moderate-to-severely active UC (Mayo score ≥6, endoscopic subscore ≥1) received oral TUDCA at 1.75 or 2 g/day for 6 weeks. Exclusion criteria included known hepatic disorders or change in UC therapy within 60 days. Clinical disease activity questionnaires, endoscopy with biopsy, blood, and stool were collected at enrollment and after 6 weeks. The primary outcome measure was change in ER stress markers while safety, tolerability and change in UC disease activity were secondary outcomes. Results:Thirteen participants completed the study with eleven evaluable for clinical response. TUDCA was well-tolerated with transient dyspepsia being the most common side effect. Mucosal biopsies revealed significant reductions in ER stress and inflammation as well as an increase in markers of epithelial restitution. Clinical, endoscopic, and histologic disease activity were significantly improved at week 6 (mean total Mayo Score: 9 to 4.5, p<0.001). Conclusions:Six weeks of oral TUDCA treatment was well-tolerated in patients with active ulcerative colitis and promoted mucosal healing, lessened ER stress, and reduced clinical disease activity. A randomized controlled trial of adjunctive TUDCA therapy in patients with UC is warranted. Trial registration:ClinicalTrials.gov (NCT04114292).

Intestinal epithelial cells (IECs) uniquely express two IRE1 paralogues, IRE1α and IRE1β, whose roles in intestinal physiology are incompletely understood. We examined the individual and cooperative functions of IRE1α and IRE1β in IECs using mice using intestine-specific deletion of Ire1α or germline Ire1β deletion, and subsequently with double deleted Ire1α, Ire1β mice. At baseline, intestine-specific Ire1α deleted mice and mice with germline Ire1β deletion exhibited no morphologic changes in small intestine or colon, but double deleted Ire1α -/- Ire1β -/- mice developed progressive intestinal and colonic injury and tumorigenesis. In contrast to single-deleted IECs, RNA-Seq from Ire1α -/- Ire1β -/- IECs revealed decreased expression of defense-associated mRNAs, together with increased expression of inflammatory and pathogenic mRNAs. Utilizing orthogonal models of intestinal tumorigenesis, reflecting either inflammatory-mutagenic injury (AOM-DSS) or spontaneous polyposis (APCmin), we observed that loss of either intestinal epithelial Ire1α or of Ire1β alone produced a growth advantage, increasing tumor burden. IRE1α mediated splicing of Xbp1 mRNA was maintained following Ire1β deletion but not in double deleted Ire1α -/- Ire1β -/- mice. Increased expression of either Ire1α or Ire1β mRNA was associated with improved survival in patients with colorectal cancer. Taken together our findings suggest IRE1 paralogues utilize essential but distinct mechanisms to safeguard intestinal homeostasis and suppress tumorigenesis.

Cardiovascular outcome trials are being considered for therapeutics that silence apolipoprotein C3 (APOC3) or angiopoietin-like 3 (ANGPTL3) because of their abilities to lower triglyceride-rich lipoproteins (TRLs) and their remnants in individuals with increased cardiovascular disease (CVD) risk1-4. Here we demonstrate that plasma APOC3 predicts CVD events in individuals with diabetes more strongly than in those without diabetes. Accordingly, plasma APOC3 levels are elevated, clearance of TRLs/remnants is slowed, and plasma TRL remnants are increased in two mouse models of diabetes-accelerated atherosclerosis. Silencing mouse APOC3 by a liver-targeted antisense oligonucleotide lowers both cholesterol and triglycerides carried by TRL/remnants and LDL and prevents aortic free cholesterol accumulation in diabetes, while ANGPTL3 silencing reduces triglycerides. Single-cell RNA-sequencing revealed that APOC3 silencing prevents a majority of diabetes-induced pathways in macrophages, endothelial cells, and smooth muscle cells, with inflammation as a major predicted upstream regulator, adding promise to APOC3 as a CVD target in diabetes.

Critical for cellular maintenance and repair, autophagy recycles damaged or redundant cellular components. Dysregulated autophagy is associated with progressive cellular injury in various liver pathologies. However, the role of autophagy in intestinal failure-associated liver disease has not previously been investigated. Using a preclinical murine model of short bowel syndrome, we explored the role of autophagy impairment at early and late time points. Regardless of sex, small bowel resected (SBR) mice demonstrated significant liver injury compared to sham controls, exhibiting elevated serum transaminases, altered liver histology, and increased mRNA expression of endoplasmic reticulum stress marker (Ddit3) and oxidative stress marker (Nqo1). Autophagy impairment was supported by increased p62 mRNA and protein expression in SBR liver and then confirmed with qualitative autophagy flux assay which suggested alteration of autophagy induction in SBR liver. Single nuclear RNA sequencing identified a cluster of hepatocytes in the SBR liver with increased p62 expression. These hepatocytes showed enrichment of genes related to the PI3K-Akt and ErbB pathways both which converge on mTORC1. Immunohistochemical distribution of pS6 confirmed increased mTORC1 activation in the SBR hepatocytes. Overall autophagy dysregulation is correlated with mTORC1 activation in SBR mice and is associated with greater liver injury.

Background:Transmembrane protein 41B (TMEM41B) and vacuolar membrane protein 1 (VMP1) are endoplasmic reticulum (ER) scramblases that shuttle phospholipids between the inner and outer leaflets of the ER membrane. Both TMEM41B and VMP1 also play critical roles in regulating hepatic lipoprotein secretion and autophagy. Despite these similarities, whether TMEM41B and VMP1 exhibit different roles in very low-density lipoprotein (VLDL) secretion and autophagy in the pathogenesis of metabolic-associated steatotic liver disease (MASLD) remains unclear. Methods:We created liver- and hepatocyte-specific single knockout (KO) and double knockout (DKO) mice for Tmem41b and Vmp1 , as well as overexpression knock-in (KI) mice with hepatic overexpression of TMEM41B, Tmem41b KO/ Vmp1 KI, and Vmp1 KO/ Tmem41b KI. We conducted lipidomic, metabolomic, biochemical, and functional studies in these mice, fed either a chow diet or a MASLD diet. Results:TMEM41B protein levels were decreased in the livers of human subjects with MASLD. The loss of hepatic Tmem41b impaired VLDL secretion, resulting in steatosis, inflammation, and fibrosis. Vmp1 KO mice exhibited similar phenotypes to DKO mice, displaying a more severe defect in VLDL secretion and greater liver injury than Tmem41b KO mice. Lipidomic analysis revealed decreased levels of phosphatidylcholine and phosphatidylethanolamine, along with increased neutral lipids in both Tmem41b KO and Vmp1 KO mice; however, these changes were generally more pronounced in Vmp1 KO mice. VMP1 and TMEM41B localized at the mitochondrial-associated membrane (MAM), and a reduction in mitochondria-ER contact was observed in hepatocytes deficient in either VMP1 or TMEM41B. Ultrastructural electron microscopy analysis showed increased accumulation of "lipid droplet" in the ER membrane bilayer and ER lumen in both Vmp1 KO and Tmem41b KO hepatocytes, with greater ER luminal "lipid droplet" accumulation in Tmem41b KO hepatocytes. The loss of hepatic Vmp1 or Tmem41b led to elevated levels of LC3-II and p62, with significantly higher levels of both markers in Vmp1 KO and DKO mouse livers compared to Tmem41b KO mouse livers. Restoring Vmp1 in Tmem41b KO mice partially improved defective VLDL secretion; however, high expression levels of VMP1 did not correct the hepatic autophagy defect. In contrast, restoring Tmem41b in Vmp1 KO mice dose-dependently enhanced both defective VLDL secretion and autophagy. Importantly, overexpression of hepatic TMEM41B mitigated diet-induced MASLD in mice. Conclusion:The loss of hepatic Vmp1 or Tmem41b decreases hepatic MAM and phospholipid content, leading to decreased VLDL secretion and promoting MASLD. While VMP1 and TMEM41B have overlapping functions, VMP1 appears to play a more critical role in regulating VLDL secretion and autophagy in mouse livers than TMEM41B.

Hypertriglyceridemia (HTG), particularly in combined hyperlipidemia, increases risk for atherosclerotic cardiovascular disease, but the underlying mechanisms remain incompletely understood. We sought to determine contributions of circulating monocytes to atherosclerosis associated with HTG in combined hyperlipidemia, created by transgenic expression of human apoCIII in Ldlr-/- mice (Ldlr-/-ApoCIIItg) fed Western high-fat diet (WD). Tissue culture with THP-1 and primary human monocytes was used to examine effects of triglyceride (TG)-rich lipoproteins on monocytes. Ldlr-/-ApoCIIItg mice were also treated with apoCIII antisense oligonucleotide (ASO) and examined for foamy monocytes and atherosclerosis. Compared to Ldlr-/- mice, Ldlr-/-ApoCIIItg mice fed WD had early and persistent increases in lipid accumulation within monocytes and enhanced atherosclerosis. Ldlr-/-ApoCIIItg mice versus Ldlr-/- mice had higher levels of CD11c, CD36, and cytokines in foamy monocytes, with increases in foamy monocyte adhesion to vascular cell adhesion molecule-1 and oxidized LDL uptake. Monocytes took up TG-rich lipoprotein in vivo and in vitro and changed phenotypes. Foamy monocytes infiltrated into atherosclerotic lesions, and specific and sustained depletion of CD11c+ (foamy) monocytes profoundly reduced atherosclerosis in Ldlr-/-ApoCIIItg mice on WD. Treatment with apoCIII ASO lowered plasma TG and cholesterol levels, improved foamy monocyte phenotypes, and reduced atherosclerosis in Ldlr-/-ApoCIIItg mice. In conclusion, HTG in combined hyperlipidemia accelerates atherosclerosis, in part, by increasing foamy monocyte formation and infiltration into atherosclerotic plaques. Treatment with apoCIII ASO is a potential new therapy for improving monocyte phenotypes and reducing atherosclerosis in combined hyperlipidemia.

Angiopoietin-like 3 (ANGPTL3) inhibits lipases that hydrolyze triglycerides (TGs) in TG-rich lipoproteins (TRLs). We evaluated TRL-TGs, TRL particle (apolipoprotein B), palmitate, and glucose kinetics during a mixed-meal test that included intravenous and oral tracer administrations in people with extremely rare compound heterozygous ANGTPL3 loss-of-function mutations (ANGPTL3-/- group, n = 3) and matched control participants (n = 7). Multi-organ (liver, muscle, and adipose tissue) insulin sensitivity was evaluated with a two-step hyperinsulinemic-euglycemic clamp procedure and glucose and palmitate tracer infusions. We find that plasma TG and TRL particle concentrations are more than 10-fold lower in the ANGPTL3-/- than in the control group due to both markedly reduced liver-derived TRL particle and TG secretion rates combined with increased plasma clearance of both liver- and gut-derived TRLs. Palmitate and glucose kinetics during the meal test are not different between the groups. We conclude that the biological function of ANGPTL3 reaches beyond inhibiting intravascular lipase activity.