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Nonalcoholic fatty liver disease (NAFLD) has emerged as a leading cause of chronic liver disease worldwide, primarily because of the massive global increase in obesity. Despite intense research efforts in this field, the factors that govern the initiation and subsequent progression of NAFLD are poorly understood, which hampers the development of diagnostic tools and effective therapies in this area of high unmet medical need. Here we describe a regulator in molecular pathogenesis of NAFLD: STE20‐type protein kinase MST4. We found that MST4 expression in human liver biopsies was positively correlated with the key features of NAFLD (i.e., hepatic steatosis, lobular inflammation, and hepatocellular ballooning). Furthermore, the silencing of MST4 attenuated lipid accumulation in human hepatocytes by stimulating β‐oxidation and triacylglycerol secretion, while inhibiting fatty acid influx and lipid synthesis. Conversely, overexpression of MST4 in human hepatocytes exacerbated fat deposition by suppressing mitochondrial fatty acid oxidation and triacylglycerol efflux, while enhancing lipogenesis. In parallel to these reciprocal alterations in lipid storage, we detected substantially decreased or aggravated oxidative/endoplasmic reticulum stress in human hepatocytes with reduced or increased MST4 levels, respectively. Interestingly, MST4 protein was predominantly associated with intracellular lipid droplets in both human and rodent hepatocytes. Conclusion: Together, our results suggest that hepatic lipid droplet–decorating protein MST4 is a critical regulatory node governing susceptibility to NAFLD and warrant future investigations to address the therapeutic potential of MST4 antagonism as a strategy to prevent or mitigate the development and aggravation of this disease. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) contributes to the pathogenesis of type 2 diabetes and cardiovascular disease, and patients with nonalcoholic steatohepatitis (NASH) are also at risk of developing cirrhosis, liver failure, and hepatocellular carcinoma. To date, no specific therapy exists for NAFLD/NASH, which has been recognized as one of the major unmet medical needs of the twenty‐first century. We recently identified serine/threonine protein kinase (STK)25 as a critical regulator of energy homeostasis and NAFLD progression. Here, we investigated the effect of antisense oligonucleotides (ASOs) targeting Stk25 on the metabolic and molecular phenotype of mice after chronic exposure to dietary lipids. We found that Stk25 ASOs efficiently reversed high‐fat diet‐induced systemic hyperglycemia and hyperinsulinemia, improved whole‐body glucose tolerance and insulin sensitivity, and ameliorated liver steatosis, inflammatory infiltration, apoptosis, hepatic stellate cell activation, and nutritional fibrosis in obese mice. Moreover, Stk25 ASOs suppressed the abundance of liver acetyl‐coenzyme A carboxylase (ACC) protein, a key regulator of both lipid oxidation and synthesis, revealing the likely mechanism underlying repression of hepatic fat accumulation by ASO treatment. We also found that STK25 protein levels correlate significantly and positively with NASH development in human liver biopsies, and several common nonlinked single‐nucleotide polymorphisms in the human STK25 gene are associated with altered liver fat, supporting a critical role of STK25 in the pathogenesis of NAFLD in humans. Conclusion: Preclinical validation for the metabolic benefit of pharmacologically inhibiting STK25 in the context of obesity is provided. Therapeutic intervention aimed at reducing STK25 function may provide a new strategy for the treatment of patients with NAFLD, type 2 diabetes, and related complex metabolic diseases. (Hepatology Communications 2018;2:69–83) [ABSTRACT FROM AUTHOR]

The article examines the roles of perlecan heparan sulfate (HS) chains through animal experimentation. The roles of lipid retention, lipid permeability, and smooth muscle cell proliferation are discussed. The study shows that the perlecan (HS) chains are proatherogenic in mice, which is most likely due to increased lipoproteins retention.

Purpose of Review: Abdominal obesity is associated with a number of important metabolic abnormalities including liver steatosis, insulin resistance and an atherogenic lipoprotein profile (termed dyslipidemia). The purpose of this review is to highlight recent progress in understanding the pathogenesis of this dyslipidemia., Recent Findings: Recent results from kinetic studies using stable isotopes indicate that the hypertriglyceridemia associated with abdominal obesity stems from dual mechanisms: (1) enhanced secretion of triglyceride-rich lipoproteins and (2) impaired clearance of these lipoproteins. The over-secretion of large triglyceride-rich VLDLs from the liver is linked to hepatic steatosis and increased visceral adiposity. The impaired clearance of triglyceride-rich lipoproteins is linked to increased levels of apolipoprotein C-III, a key regulator of triglyceride metabolism., Summary: Elucidation of the pathogenesis of the atherogenic dyslipidemia in abdominal obesity combined with the development of novel treatments based on apolipoprotein C-III may in the future lead to better prevention, diagnosis and treatment of the atherogenic dyslipidemia in abdominal obesity.

Purpose of Review: Today, it is no longer a hypothesis, but an established fact, that increased plasma concentrations of cholesterol-rich apolipoprotein-B (apoB)-containing lipoproteins are causatively linked to atherosclerotic cardiovascular disease (ASCVD) and that lowering plasma LDL concentrations reduces cardiovascular events in humans. Here, we review evidence behind this assertion, with an emphasis on recent studies supporting the 'response-to-retention' model - namely, that the key initiating event in atherogenesis is the retention, or trapping, of cholesterol-rich apoB-containing lipoproteins within the arterial wall., Recent Findings: New clinical trials have shown that ezetimibe and anti-PCSK9 antibodies - both nonstatins - lower ASCVD events, and they do so to the same extent as would be expected from comparable plasma LDL lowering by a statin. These studies demonstrate beyond any doubt the causal role of apoB-containing lipoproteins in atherogenesis. In addition, recent laboratory experimentation and human Mendelian randomization studies have revealed novel information about the critical role of apoB-containing lipoproteins in atherogenesis. New information has also emerged on mechanisms for the accumulation in plasma of harmful cholesterol-rich and triglyceride-rich apoB-containing remnant lipoproteins in states of overnutrition. Like LDL, these harmful cholesterol-rich and triglyceride-rich apoB-containing remnant lipoprotein remnants become retained and modified within the arterial wall, causing atherosclerosis., Summary: LDL and other cholesterol-rich, apoB-containing lipoproteins, once they become retained and modified within the arterial wall, cause atherosclerosis. This simple, robust pathophysiologic understanding may finally allow us to eradicate ASCVD, the leading killer in the world.

In this short review, we discuss apolipoprotein B100 and the assembly of very low-density lipoproteins. In particular, we address the nature and importance of co- and posttranslational degradation of apolipoprotein B100 during the assembly process. We also provide a short historical background to the development of the current model for the degradation of apolipoprotein B100.

Objective: Obesity increases the risk of cardiovascular disease and premature death. However, not all obese subjects develop the metabolic abnormalities associated with obesity. The aim of this study was to clarify the mechanisms that induce dyslipidemia in obese subjects., Methods and Results: Stable isotope tracers were used to elucidate the pathophysiology of the dyslipidemia in hypertriglyceridemic (n=14) and normotriglyceridemic (n=14) obese men (with comparable body mass index and visceral fat volume) and in normotriglyceridemic nonobese men (n=10). Liver fat was determined using proton magnetic resonance spectroscopy, and subcutaneous abdominal and visceral fat were measured by magnetic resonance imaging. Serum triglycerides in obese subjects were increased by the combination of increased secretion and severely impaired clearance of triglyceride-rich very-low-density lipoprotein(1) particles. Furthermore, increased liver and subcutaneous abdominal fat were linked to increased secretion of very-low-density lipoprotein 1 particles, whereas increased plasma levels of apolipoprotein C-III were associated with impaired clearance in obese hypertriglyceridemic subjects., Conclusions: Dual metabolic defects are required to produce hypertriglyceridemia in obese subjects with similar levels of visceral adiposity. The results emphasize the clinical importance of assessing hypertriglyceridemic waist in obese subjects to identify subjects at high cardiometabolic risk.

Objective: Enhanced endothelial permeability leading to intimal accumulation of low-density lipoproteins (LDL) stimulates the formation of atherosclerotic lesions. Histamine is known to increase vascular permeability. Whether this affects the formation of atherosclerotic lesions, however, remains elusive., Methods and Results: Apolipoprotein E-null (ApoE(-/-)) mice treated with a histamine H1 receptor but not an H2 receptor antagonist developed 40% fewer atherosclerotic lesions in the aorta than placebo-treated controls. Similarly, genetic deletion of the H1 but not the H2 receptor resulted in a 60% reduction of lesions compared with ApoE(-/-) controls. The H1 receptor enhanced LDL permeability and lipid accumulation in the aorta, whereas plasma lipoprotein levels remained unaltered. In contrast, the H1 receptor did not affect proliferation and migration of vascular smooth muscle cells. Bone marrow transplantation confirmed that the formation of atherosclerotic lesions depended on the H1 receptor in vascular cells, whereas its presence in bone marrow-derived cells was irrelevant for plaque development. Mice expressing the H1 receptor exhibited higher levels of the chemokine (C-C motif) ligand 5 and higher numbers of macrophages and T-helper lymphocytes in plaques, higher numbers of circulating lymphocytes, and larger spleens., Conclusion: These data indicate that H1 but not H2 receptor activation drives the formation of atherosclerotic lesions through an increased vascular permeability for LDL, which is associated with an enhanced secondary aortic and systemic inflammation. These data open novel perspectives for the prevention and treatment of atherosclerotic vascular disease.

Insulin resistance is a key feature of the metabolic syndrome and often progresses to type 2 diabetes. Both insulin resistance and type 2 diabetes are characterized by dyslipidemia, which is an important and common risk factor for cardiovascular disease. Diabetic dyslipidemia is a cluster of potentially atherogenic lipid and lipoprotein abnormalities that are metabolically interrelated. Recent evidence suggests that a fundamental defect is an overproduction of large very low-density lipoprotein (VLDL) particles, which initiates a sequence of lipoprotein changes, resulting in higher levels of remnant particles, smaller LDL, and lower levels of high-density liporotein (HDL) cholesterol. These atherogenic lipid abnormalities precede the diagnosis of type 2 diabetes by several years, and it is thus important to elucidate the mechanisms involved in the overproduction of large VLDL particles. Here, we review the pathophysiology of VLDL biosynthesis and metabolism in the metabolic syndrome. We also review recent research investigating the relation between hepatic accumulation of lipids and insulin resistance, and sources of fatty acids for liver fat and VLDL biosynthesis. Finally, we briefly discuss current treatments for lipid management of dyslipidemia and potential future therapeutic targets.

The key initiating process in atherogenesis is the subendothelial retention of apolipoprotein B-containing lipoproteins. Local biological responses to these retained lipoproteins, including a chronic and maladaptive macrophage- and T-cell-dominated inflammatory response, promote subsequent lesion development. The most effective therapy against atherothrombotic cardiovascular disease to date--low density lipoprotein-lowering drugs--is based on the principle that decreasing circulating apolipoprotein B lipoproteins decreases the probability that they will enter and be retained in the subendothelium. Ongoing improvements in this area include more aggressive lowering of low-density lipoprotein and other atherogenic lipoproteins in the plasma and initiation of low-density lipoprotein-lowering therapy at an earlier age in at-risk individuals. Potential future therapeutic approaches include attempts to block the interaction of apolipoprotein B lipoproteins with the specific subendothelial matrix molecules that mediate retention and to interfere with accessory molecules within the arterial wall that promote retention such as lipoprotein lipase, secretory sphingomyelinase, and secretory phospholipase A2. Although not the primary focus of this review, therapeutic strategies that target the proatherogenic responses to retained lipoproteins and that promote the removal of atherogenic components of retained lipoproteins also hold promise. The finding that certain human populations of individuals who maintain lifelong low plasma levels of apolipoprotein B lipoproteins have an approximately 90% decreased risk of coronary artery disease gives hope that our further understanding of the pathogenesis of this leading killer could lead to its eradication.

Objective: Atherosclerotic lesions have regions that are hypoxic. Because the lesion contains macrophages that are loaded with lipid, we investigated whether hypoxia can influence the accumulation of lipids in these cells., Methods and Results: Exposure of human macrophages to hypoxia for 24 hours resulted in an increased formation of cytosolic lipid droplets and an increased accumulation of triglycerides. Exposure of the macrophages to oxidized low-density lipoprotein (oxLDL) increased the accumulation of cytosolic lipid droplets because of an increase in cellular cholesterol esters. The accumulation of lipid droplets in oxLDL-treated cells was further increased after hypoxia, caused by an increased level of triglycerides. Expression analyses combined with immunoblot or RT-PCR demonstrated that hypoxia increased the expression of several genes that could promote the accumulation of lipid droplets. Hypoxia increased the mRNA and protein levels of adipocyte differentiation-related protein (ADRP). It is well known that an increased expression of ADRP increases the formation of lipid droplets. Hypoxia decreased the expression of enzymes involved in beta-oxidation (acyl-coenzyme A synthetase and acyl-coenzyme A dehydrogenase) and increased the expression of stearoyl-coenzyme A desaturase, an important enzyme in the fatty acid biosynthesis. Moreover, exposure to hypoxia decreased the rate of beta-oxidation, whereas the accumulation of triglycerides increased., Conclusions: The results demonstrate that exposure of human macrophages to hypoxia causes an accumulation of triglyceride-containing cytosolic lipid droplets. This indicates that the hypoxia present in atherosclerotic lesions can contribute to the formation of the lipid-loaded macrophages that characterize the lesion and to the accumulation of triglycerides in such lesions.

Objective: We investigated the role of adipocyte differentiation-related protein (ADRP) in triglyceride turnover and in the secretion of very low-density lipoprotein (VLDL) from McA-RH7777 cells and primary rat hepatocytes., Methods and Results: An increase in the expression of ADRP increased triglyceride accumulation in cytosolic lipid droplets and prevented the incorporation of fatty acids into secretable triglycerides, thereby reducing the secretion of triglycerides as well as of apolipoprotein B-100 (apoB-100) and apoB-48 VLDL. The ability of ADRP to block the secretion of apoB-100 VLDL1 decreased with increasing quantities of fatty acids in the medium, indicating a saturable process and emphasizing the importance of sequestering of fatty acids for the effect of ADRP on VLDL secretion. Knockdown (small interfering RNA) of ADRP decreased the pool of cytosolic lipid droplets but increased only the secretion of apoB-48 VLDL1. Additionally, there was an increased flow of fatty acids into beta-oxidation., Conclusions: ADRP is essential for the accumulation of triglycerides in cytosolic lipid droplets. An increase in ADRP prevents the formation of VLDL by diverting fatty acids from the VLDL assembly pathway into cytosolic triglycerides, whereas a decrease of the protein increases the sorting of fatty acids to beta-oxidation and promotes the secretion of apoB-48 VLDL1.

Purpose of Review: Diabetic dyslipidaemia is a cluster of plasma lipid and lipoprotein abnormalities that are metabolically interrelated. The increase of large type 1 very low density lipoprotein particles in type 2 diabetes initiates a sequence of events that generates atherogenic remnants, small dense low-density lipoprotein and small dense high-density lipoprotein particles. Thus, it is of great importance to elucidate the mechanisms behind the overproduction of large very low density lipoprotein particles in diabetic dyslipidaemia. This review discusses the pathophysiology of very low density lipoprotein metabolism in type 2 diabetes and recent concepts of lipid management of diabetic dyslipidaemia., Recent Findings: Results indicate that triglyceride and apolipoprotein B production in types 1 and 2 very low density lipoprotein are significantly correlated, suggesting a coupling of the two processes governing the metabolism of these lipoprotein subpopulations. Insulin resistance, hyperglycaemia, and liver fat were associated with excess hepatic production of type 1 but not type 2 very low density lipoprotein particles. These data provide support for the independent regulation of types 1 and 2 very low density lipoprotein apolipoprotein B production., Summary: Recent data suggest that the assembly of very low density lipoprotein is fundamentally altered in type 2 diabetes, explaining the overproduction of large type 1 very low density lipoprotein as well as the inability of insulin to suppress production of type 1 very low density lipoprotein in type 2 diabetes. Future discoveries hopefully will delineate the regulatory steps to allow more targeted treatment of diabetic dyslipidaemia.

Objective: Adipocyte differentiation-related protein (ADRP)-containing lipid droplets have an essential role in the development of insulin resistance and atherosclerosis. Such droplets form in a cell-free system with a diameter of 0.1 to 0.4 microm, while the droplets present in cells vary in size, from small to very large, suggesting that the droplets can increase in size after being assembled. We have addressed this possibility., Methods and Results: Experiments in NIH 3T3 cells demonstrated that the lipid droplets could increase in size independently of triglyceride biosynthesis. NIH 3T3 cells were either microinjected with ADRP-GFP (green fluorescent protein) or stained with Nile Red and followed by confocal microscopy and time-lapse recordings. The results showed that lipid droplets formed complexes with each other, with a volume equal to the sum of the merging particles. The formation of complexes could be inhibited by the nocodazole-induced depolymerization of the microtubules; thus, the process is dependent on microtubules. The presence of dynein on ADRP-containing droplets supports a role for this motor protein., Conclusions: Lipid droplets can grow after they have been assembled. This increase in size is independent of triglyceride biosynthesis and involves formation of complexes, which requires intact microtubules.

Objective: We sought to compare the synthesis and metabolism of VLDL1 and VLDL2 in patients with type 2 diabetes mellitus (DM2) and nondiabetic subjects., Methods and Results: We used a novel multicompartmental model to simultaneously determine the kinetics of apolipoprotein (apo) B and triglyceride (TG) in VLDL1 and VLDL2 after a bolus injection of [2H3]leucine and [2H5]glycerol and to follow the catabolism and transfer of the lipoprotein particles. Our results show that the overproduction of VLDL particles in DM2 is explained by enhanced secretion of VLDL1 apoB and TG. Direct production of VLDL2 apoB and TG was not influenced by diabetes per se. The production rates of VLDL1 apoB and TG were closely related, as were the corresponding pool sizes. VLDL1 and VLDL2 compositions did not differ in subjects with DM2 and controls, and the TG to apoB ratio of newly synthesized particles was very similar in the 2 groups. Plasma glucose, insulin, and free fatty acids together explained 55% of the variation in VLDL1 TG production rate., Conclusions: Insulin resistance and DM2 are associated with excess hepatic production of VLDL1 particles similar in size and composition to those in nondiabetic subjects. We propose that hyperglycemia is the driving force that aggravates overproduction of VLDL1 in DM2.

Objective: We investigated the role of ADP ribosylation factor 1 (ARF1) in the assembly of very-low-density lipoproteins (VLDLs)., Methods and Results: The dominant-negative ARF1 mutant, T31N, decreased the assembly of apoB-100 VLDL 1 (Svedberg floatation units [Sf] 60 to 400) by 80%. The decrease coincided with loss of coatamer I (COPI) from the Golgi apparatus and inhibition of anterograde transport, as demonstrated by time-lapse studies of the vesicular stomatitis virus G protein. The VLDL 1 assembly was also completely inhibited at 15 degrees C. Thus, the antegrade transport is essential for the assembly of VLDL 1. Intracellular localization of N-acetylgalactosaminyl transferase 2 indicated that the Golgi apparatus was at least partly intact when the VLDL assembly was inhibited. Transient transfection with phospholipase D 1 increased the assembly of VLDL 1 and VLDL 2 (Sf 20 to 60). Overexpression of ARF1 in stably transfected McA-RH7777 cells increased the secretion of VLDL 2 but not of VLDL 1, which was dependent on the availability of oleic acid. Secretion of VLDL 1 increased with increasing amounts of oleic acid, and VLDL 2 secretion decreased simultaneously., Conclusions: Overexpression of ARF1 increased the assembly of VLDL 2 but not of VLDL 1, whose production was dependent on both anterograde transport and the availability of fatty acids.

Purpose of Review: Considerable evidence suggests that the subendothelial retention of atherogenic lipoproteins is a key early step in atherogenesis. In humans and experimental animals, elevated levels of plasma lipoproteins are associated with increased atherosclerosis, and lipoproteins with higher affinity for arterial proteoglycans are more atherogenic. Here we discuss the molecular mechanisms underlying lipoprotein retention in the arterial wall and how this interaction can be modulated., Recent Findings: Functional proteoglycan binding sites in lipoproteins containing apolipoprotein B have been identified and shown to have atherogenic potential in vivo. In addition to apolipoprotein B, novel bridging molecules, those that can interact with both proteoglycans and lipoproteins, have been identified that mediate the retention of atherogenic particles in the vessel wall. The interaction between lipoproteins and proteoglycans can be enhanced by the modification of lipoproteins in the circulation and in the arterial wall, by alterations in the subendothelium, and by changes in proteoglycan synthesis that result in a more atherogenic profile. The retention of atherogenic lipoproteins is a potential target for therapies to reverse atherosclerosis, and in-vitro studies have identified compounds that decrease the affinity of proteoglycans for lipoproteins., Summary: Considerable progress has been made in understanding the association between lipoproteins and cardiovascular disease. This review highlights the importance of the interaction between lipoproteins and the arterial matrix.

Objective: To investigate the effect of complement deficiency on atherogenesis and lipidemia, we used mice deficient in the third complement component (C3-/-) or factor B (FB-/-)., Methods and Results: Complement-deficient mice were crossed with mice deficient in both apolipoprotein E and the low-density lipoprotein receptor (Apoe-/- LDLR-/-). The percent lesion area in the aorta at 16 weeks, determined by en face analysis, was 84% higher in C3-/- mice than in controls (11.8%+/-0.4% versus 6.4%+/-0.8%, mean+/-SEM, P<0.00005). The C3-/- mice also had 58% higher serum triglyceride levels (P<0.05) and a more proatherogenic lipoprotein profile, with significantly more low-density lipoprotein cholesterol and very-low-density lipoprotein triglycerides than control mice. The C3-/- mice weighed 13% less (P<0.01) and had a lower body fat content (3.5%+/-1.0% versus 13.1%+/-3.0%, P<0.01). There were no differences between FB-/- mice and controls., Conclusions: Complement activation by the classical or lectin pathway exerts atheroprotective effects, possibly through the regulation of lipid metabolism.

Objective: The aim of this study was to investigate the molecular mechanism for changes in proteoglycan binding and LDL receptor affinity on two compositional changes in LDL that have been associated with atherosclerosis: cholesterol enrichment of the core and modification by secretory group IIA phospholipase A2 (sPLA2) of the surface., Methods and Results: Transgenic mice expressing recombinant apolipoprotein (apo) B and sPLA2 were generated. Recombinant LDL were isolated and tested for their proteoglycan and LDL receptor-binding activity. The results show site A (residues 3148-3158) in apoB100 becomes functional in sPLA2-modified LDL and that site A acts cooperatively with site B (residues 3359-3369), the primary proteoglycan-binding site in native LDL, in the binding of sPLA2-modified LDL to proteoglycans. Our results also show that cholesterol enrichment of LDL is associated with increased affinity for proteoglycans and for the LDL receptor. This mechanism is likely mediated by a conformational change of site B and is independent of site A in apoB100., Conclusions: Site A in apoB100 becomes functional in sPLA2-modified LDL and acts cooperatively with site B resulting in increased proteoglycan-binding activity. The increased binding for proteoglycans of cholesterol-enriched LDL is solely dependent on site B.