Heterozygous midnolin knockout attenuates severity of nonalcoholic fatty liver disease in mice fed a Western-style diet high in fat, cholesterol, and fructose.

Although midnolin has been studied for over 20 years, its biological roles in vivo remain largely unknown, especially due to the lack of a functional animal model. Indeed, given our recent discovery that the knockdown of midnolin suppresses liver cancer cell tumorigenicity and that this antitumorigenic effect is associated with modulation of lipid metabolism, we hypothesized that knockout of midnolin in vivo could potentially protect from nonalcoholic fatty liver disease (NAFLD) which has become the most common cause of chronic liver disease in the Western world. Accordingly, in the present study, we have developed and now report on the first functional global midnolin knockout mouse model. Although the overwhelming majority of global homozygous midnolin knockout mice demonstrated embryonic lethality, heterozygous knockout mice were observed to be similar to wild-type mice in their viability and were used to determine the effect of reduced midnolin expression on NAFLD. We found that global heterozygous midnolin knockout attenuated the severity of NAFLD in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism. Collectively, our results support a role for midnolin in regulating cholesterol/lipid metabolism in the liver. Thus, midnolin may represent a novel therapeutic target for NAFLD. Finally, our observation that midnolin was essential for survival underscores the broad importance of this gene beyond its role in liver biology.NEW & NOTEWORTHY We have developed and now report on the first functional global midnolin knockout mouse model. We found that global heterozygous midnolin knockout attenuated the severity of nonalcoholic fatty liver disease (NAFLD) in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism.

Lecithin:cholesterol acyltransferase: symposium on 50 years of biomedical research from its discovery to latest findings.

LCAT converts free cholesterol to cholesteryl esters in the process of reverse cholesterol transport. Familial LCAT deficiency (FLD) is a genetic disease that was first described by Kaare R. Norum and Egil Gjone in 1967. This report is a summary from a 2017 symposium where Dr. Norum recounted the history of FLD and leading experts on LCAT shared their results. The Tesmer laboratory shared structural findings on LCAT and the close homolog, lysosomal phospholipase A2. Results from studies of FLD patients in Finland, Brazil, Norway, and Italy were presented, as well as the status of a patient registry. Drs. Kuivenhoven and Calabresi presented data from carriers of genetic mutations suggesting that FLD does not necessarily accelerate atherosclerosis. Dr. Ng shared that LCAT-null mice were protected from diet-induced obesity, insulin resistance, and nonalcoholic fatty liver disease. Dr. Zhou presented multiple innovations for increasing LCAT activity for therapeutic purposes, whereas Dr. Remaley showed results from treatment of an FLD patient with recombinant human LCAT (rhLCAT). Dr. Karathanasis showed that rhLCAT infusion in mice stimulates cholesterol efflux and suggested that it could also enhance cholesterol efflux from macrophages. While the role of LCAT in atherosclerosis remains elusive, the consensus is that a continued study of both the enzyme and disease will lead toward better treatments for patients with heart disease and FLD.

Novel metabolic phenotypes in lecithin cholesterol acyltyransferase-deficient mice.

PURPOSE OF REVIEW: Lecithin cholesterol acyltyransferase (LCAT) deficiency is a rare monogenic disorder causing lipoprotein dysregulation and multiple organ dysfunctions, including renal impairment. LCAT knockout mice have been shown informative in elucidating mechanisms of many major clinical morbid phenotypes. Extended characterization of the LDL receptor/LCAT double knockout (Ldlr/Lcat-DKO or DKO) mice had led to the discovery of a number of novel protective metabolic phenotypes, including resistance to obesity, nonalcoholic steatohepatitis (NASH) and insulin resistance. We seek to integrate the findings to explore novel pathogenic pathways. RECENT FINDINGS: The chow fed DKO mice were found more insulin sensitive than their Ldlr-KO controls. Joint analyses of the three strains (DKO, Ldlr-KO and wild-type) revealed differential metabolic responses to a high cholesterol diet (HCD) vs. high-fat diet (HFD). DKO mice are protected from HFD-induced obesity, hepatic endoplasmic reticulum (ER) stress, insulin resistance, ER cholesterol and NASH markers (steatosis and inflammasomes). Joint analysis revealed the HFD-induced NASH is dependent on de-novo hepatic cholesterol biosynthesis. DKO mice are protected from HCD-induced hepatic ER stress, ER cholesterol, but not NASH, the latter likely due to cholesterol crystal accumulation. DKO mice were found to develop ectopic brown adipose tissue (BAT) in skeletal muscle. Ectopic BAT derived in part from myoblast in utero and from adult satellite cells. Primed expression of PRDM16 and UCP in quiescent satellite cell caused by LCAT deficiency synergizes with cell cholesterol depletion to induce satellite cell-to-BAT transdifferentiation. SUMMARY: Metabolic phenotyping of selective LCAT null mice led to the discovery of novel metabolically protective pathways.

Pak1 mediates the stimulatory effect of insulin and curcumin on hepatic ChREBP expression.

Insulin can stimulate hepatic expression of carbohydrate-responsive element-binding protein (ChREBP). As recent studies revealed potential metabolic beneficial effects of ChREBP, we asked whether its expression can also be regulated by the dietary polyphenol curcumin. We also aimed to determine mechanisms underlying ChREBP stimulation by insulin and curcumin. The effect of insulin on ChREBP expression was assessed in mouse hepatocytes, while the effect of curcumin was assessed in mouse hepatocytes and with curcumin gavage in mice. Chemical inhibitors for insulin signaling molecules were utilized to identify involved signaling molecules, and the involvement of p21-activated protein kinase 1 (Pak1) was determined with its chemical inhibitor and Pak1-/- hepatocytes. We found that both insulin and curcumin-stimulated ChREBP expression in Akt-independent but MEK/ERK-dependent manner, involving the inactivation of the transcriptional repressor Oct-1. Aged Pak1-/- mice showed reduced body fat volume. Pak1 inhibition or its genetic deletion attenuated the stimulatory effect of insulin or curcumin on ChREBP expression. Our study hence suggests the existence of a novel signaling cascade Pak1/MEK/ERK/Oct-1 for both insulin and curcumin in exerting their glucose-lowering effect via promoting hepatic ChREBP production, supports the recognition of beneficial functions of ChREBP, and brings us a new overview on dietary polyphenols.

Cellular cholesterol accumulation modulates high fat high sucrose (HFHS) diet-induced ER stress and hepatic inflammasome activation in the development of non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH), is the form of non-alcoholic fatty liver disease posing risk to progress into serious long term complications. Human and pre-clinical models implicate cellular cholesterol dysregulation playing important role in its development. Mouse model studies suggest synergism between dietary cholesterol and fat in contributing to NASH but the mechanisms remain poorly understood. Our laboratory previously reported the primary importance of hepatic endoplasmic reticulum cholesterol (ER-Chol) in regulating hepatic ER stress by comparing the responses of wild type, Ldlr-/-xLcat+/+ and Ldlr-/-xLcat-/- mice, to a 2% high cholesterol diet (HCD). Here we further investigated the roles of ER-Chol and ER stress in HFHS diet-induced NASH using the same strains. With HFHS diet feeding, both WT and Ldlr-/-xLcat+/+ accumulate ER-Chol in association with ER stress and inflammasome activation but the Ldlr-/-xLcat-/- mice are protected. By contrast, all three strains accumulate cholesterol crystal, in correlation with ER-Chol, albeit less so in Ldlr-/-xLcat-/- mice. By comparison, HCD feeding per se (i) is sufficient to promote steatosis and activate inflammasomes, and (ii) results in dramatic accumulation of cholesterol crystal which is linked to inflammasome activation in Ldlr-/-xLcat-/- mice, independent of ER-Chol. Our data suggest that both dietary fat and cholesterol each independently promote steatosis, cholesterol crystal accumulation and inflammasome activation through distinct but complementary pathways. In vitro studies using palmitate-induced hepatic steatosis in HepG2 cells confirm the key roles by cellular cholesterol in the induction of steatosis and inflammasome activations. These novel findings provide opportunities for exploring a cellular cholesterol-focused strategy for treatment of NASH.

Lecithin:Cholesterol Acyltransferase (LCAT) Deficiency Promotes Differentiation of Satellite Cells to Brown Adipocytes in a Cholesterol-dependent Manner.

Our laboratory previously reported that lecithin:cholesterol acyltransferase (LCAT) and LDL receptor double knock-out mice (Ldlr(-/-)xLcat(-/-) or DKO) spontaneously develop functioning ectopic brown adipose tissue (BAT) in skeletal muscle, putatively contributing to protection from the diet-induced obesity phenotype. Here we further investigated their developmental origin and the mechanistic role of LCAT deficiency. Gene profiling of skeletal muscle in DKO newborns and adults revealed a classical lineage. Primary quiescent satellite cells (SC) from chow-fed DKO mice, not in Ldlr(-/-)xLcat(+/+) single-knock-out (SKO) or C57BL/6 wild type, were found to (i) express exclusively classical BAT-selective genes, (ii) be primed to express key functional BAT genes, and (iii) exhibit markedly increased ex vivo adipogenic differentiation into brown adipocytes. This gene priming effect was abrogated upon feeding the mice a 2% high cholesterol diet in association with accumulation of excess intracellular cholesterol. Ex vivo cholesterol loading of chow-fed DKO SC recapitulated the effect, indicating that cellular cholesterol is a key regulator of SC-to-BAT differentiation. Comparing adipogenicity of Ldlr(+/+)xLcat(-/-) (LCAT-KO) SC with DKO SC identified a role for LCAT deficiency in priming SC to express BAT genes. Additionally, we found that reduced cellular cholesterol is important for adipogenic differentiation, evidenced by increased induction of adipogenesis in cholesterol-depleted SC from both LCAT-KO and SKO mice. Taken together, we conclude that ectopic BAT in DKO mice is classical in origin, and its development begins in utero. We further showed complementary roles of LCAT deficiency and cellular cholesterol reduction in the SC-to-BAT adipogenesis.

Revising the high-density lipoprotein targeting strategies - insights from human and preclinical studies.

In recent years, the high-density lipoprotein (HDL) hypothesis has been challenged. Several completed randomized clinical trials continue to fall short in demonstrating HDL, or at least HDL-cholesterol (HDL-C) levels, as being a consistent target in the prevention of cardiovascular diseases. However, population studies and findings in lipid modifying trials continue to strongly support HDL-C as a superb risk predictor. It is increasingly evident that the complexity of HDL metabolism confounds the use of HDL-C concentration as a unified target. However, important insights continue to emerge from the post hoc analyses of recently completed (i) fibrate-based FIELD and ACCORD trials, including the unexpected beneficial effect of fibrates in microvascular diseases, (ii) the niacin-based AIM-HIGH and HPS2-THRIVE studies, (iii) recombinant HDL-based as well as (iv) the completed CETP inhibitor-based trials. These together with on-going mechanistic studies on novel pathways, which include the unique roles of microRNAs, post-translational remodeling of HDL and novel pathways related to HDL modulators will provide valuable insights to guide how best to refocus and redesign the conceptual framework for selecting HDL-based targets.

Diagnosis, prevention, and management of statin adverse effects and intolerance: Canadian Working Group Consensus update.

The Proceedings of a Canadian Working Group Consensus Conference, first published in 2011, provided a summary of statin-associated adverse effects and intolerance and management suggestions. In this update, new clinical studies identified since then that provide further insight into effects on muscle, cognition, cataracts, diabetes, kidney disease, and cancer are discussed. Of these, the arenas of greatest controversy pertain to purported effects on cognition and the emergence of diabetes during long-term therapy. Regarding cognition, the available evidence is not strongly supportive of a major adverse effect of statins. In contrast, the linkage between statin therapy and incident diabetes is more firm. However, this risk is more strongly associated with traditional risk factors for new-onset diabetes than with statin itself and any possible negative effect of new-onset diabetes during statin treatment is far outweighed by the cardiovascular risk reduction benefits. Additional studies are also discussed, which support the principle that systematic statin rechallenge, and lower or intermittent statin dosing strategies are the main methods for dealing with suspected statin intolerance at this time.

Lecithin cholesterol acyltransferase deficiency protects from diet-induced insulin resistance and obesity--novel insights from mouse models.

Reduced plasma level of high-density lipoprotein cholesterol is an independent risk factor for atherosclerotic heart disease and is also a major diagnostic feature for the metabolic syndrome. Lecithin cholesterol acyltransferase (LCAT), an enzyme mediating the esterification of cholesterol in circulating lipoproteins, is one of the major modulators of high-density lipoprotein levels and composition. Loss-of-function mutations of LCAT invariably results in profound HDL deficiency and also modest hypertriglyceridemia (HTG). While intense effort has been devoted to investigate the role of LCAT in atherogenesis, which remains controversial, much less is known about whether LCAT also modulates glucose and energy homeostasis. In recent years, findings from studying the LCAT knockout mice began to suggest that LCAT deficiency, in spite of its unfavorable high triglyceride/low HDL lipid phenotypes, may confer protection from the development of insulin resistance and obesity. To date, alterations in specific metabolic pathways in liver, white adipose tissue, and skeletal muscle have been implicated. A better mechanistic understanding in the metabolic linkage between the primary biochemical action of LCAT and the downstream protective phenotypes will greatly facilitate the identification of potential novel pathways and targets in the treatment of obesity and diabetes.

2012 update of the Canadian Cardiovascular Society guidelines for the diagnosis and treatment of dyslipidemia for the prevention of cardiovascular disease in the adult.

Many developments have occurred since the publication of the widely-used 2009 Canadian Cardiovascular Society (CCS) Dyslipidemia guidelines. Here, we present an updated version of the guidelines, incorporating new recommendations based on recent findings and harmonizing CCS guidelines with those from other Societies. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) system was used, per present standards of the CCS. The total cardiovascular disease Framingham Risk Score (FRS), modified for a family history of premature coronary disease, is recommended for risk assessment. Low-density lipoprotein cholesterol remains the primary target of therapy. However, non-high density lipoprotein cholesterol has been added to apolipoprotein B as an alternate target. There is an increased emphasis on treatment of higher risk patients, including those with chronic kidney disease and high risk hypertension. The primary panel has recommended a judicious use of secondary testing for subjects in whom the need for statin therapy is unclear. Expanded information on health behaviours is presented and is the backbone of risk reduction in all subjects. Finally, a systematic approach to statin intolerance is advocated to maximize appropriate use of lipid-lowering therapy. This document presents the recommendations and principal conclusions of this process. Along with associated Supplementary Material that can be accessed online, this document will be part of a program of knowledge translation. The goal is to increase the appropriate use of evidence-based cardiovascular disease event risk assessment in the management of dyslipidemia as a fundamental means of reducing global risk in the Canadian population.

HDL--is it too big to fail?

The HDL hypothesis has suffered damage in the past few years. Clinical trials have shown that raising HDL cholesterol levels does not improve cardiovascular disease (CVD) outcomes. In addition, Mendelian randomization studies have shown that DNA variants that alter HDL cholesterol levels in populations are unrelated to incident CVD events. Balancing this deluge of negative data are substantial basic science data supporting the concept that raising HDL cholesterol levels reduces CVD risk. Also, functionally relevant HDL subfractions might be more important determinants of risk than overall HDL cholesterol levels. But, while wobbly, the HDL hypothesis is still standing, seemingly too big to fail owing to past intellectual, economic and psychological investments in the idea.

Diabetic dyslipidemia: from evolving pathophysiological insight to emerging therapeutic targets.

Diabetic dyslipidemia is characterized by hepatic very low density lipoprotein (VLDL) and intestinal chylomicron overproduction, reduced high density lipoprotein cholesterol (HDL-C) level, increased propensity of small dense LDL (sdLDL) and increased postprandial lipemia. This dyslipidemic profile is also strongly linked to other features of the metabolic syndrome. Diabetic dyslipidemia is a well-recognized risk factor for atherosclerotic cardiovascular diseases. Currently, statins remain the first line therapy primarily through reducing the atherogenic LDL. Clinical trials on other lipid modifying agents were met with variable success in selective patient populations. Emerging new insights into the pathophysiology of lipid metabolism, in general, and diabetic dyslipidemia, in particular, have opened up potentially novel therapeutic strategies to further reduce the risk associated with diabetic dyslipidemia and insulin resistant state.

Lecithin:cholesterol acyltransferase deficiency protects against cholesterol-induced hepatic endoplasmic reticulum stress in mice.

We recently reported that lecithin:cholesterol acyltransferase (LCAT) knock-out mice, particularly in the LDL receptor knock-out background, are hypersensitive to insulin and resistant to high fat diet-induced insulin resistance (IR) and obesity. We demonstrated that chow-fed Ldlr-/-xLcat+/+ mice have elevated hepatic endoplasmic reticulum (ER) stress, which promotes IR, compared with wild-type controls, and this effect is normalized in Ldlr-/-xLcat-/- mice. In the present study, we tested the hypothesis that hepatic ER cholesterol metabolism differentially regulates ER stress using these models. We observed that the Ldlr-/-xLcat+/+ mice accumulate excess hepatic total and ER cholesterol primarily attributed to increased reuptake of biliary cholesterol as we observed reduced biliary cholesterol in conjunction with decreased hepatic Abcg5/g8 mRNA, increased Npc1l1 mRNA, and decreased Hmgr mRNA and nuclear SREBP2 protein. Intestinal NPC1L1 protein was induced. Expression of these genes was reversed in the Ldlr-/-xLcat-/- mice, accounting for the normalization of total and ER cholesterol and ER stress. Upon feeding a 2% high cholesterol diet (HCD), Ldlr-/-xLcat-/- mice accumulated a similar amount of total hepatic cholesterol compared with the Ldlr-/-xLcat+/+ mice, but the hepatic ER cholesterol levels remained low in conjunction with being protected from HCD-induced ER stress and IR. Hepatic ER stress correlates strongly with hepatic ER free cholesterol but poorly with hepatic tissue free cholesterol. The unexpectedly low ER cholesterol seen in HCD-fed Ldlr-/-xLcat-/- mice was attributable to a coordinated marked up-regulation of ACAT2 and suppressed SREBP2 processing. Thus, factors influencing the accumulation of ER cholesterol may be important for the development of hepatic insulin resistance.