Cardiac lipotoxicity and fibrosis underlie impaired contractility in a mouse model of metabolic dysfunction-associated steatotic liver disease.

The leading cause of death among patients with metabolic dysfunction-associated steatotic liver disease (MASLD) is cardiovascular disease. A significant percentage of MASLD patients develop heart failure driven by functional and structural alterations in the heart. Previously, we observed cardiac dysfunction in hepatocyte-specific peroxisome proliferator-activated receptor alpha knockout (Ppara HepKO), a mouse model that exhibits hepatic steatosis independent of obesity and insulin resistance. The goal of the present study was to determine mechanisms that underlie hepatic steatosis-induced cardiac dysfunction in Ppara HepKO mice. Experiments were performed in 30-week-old Ppara HepKO and littermate control mice fed regular chow. We observed decreased cardiomyocyte contractility (0.17 ± 0.02 vs. 0.24 ± 0.02 µm, p < 0.05), increased cardiac triglyceride content (0.96 ± 0.13 vs. 0.68 ± 0.06 mM, p < 0.05), collagen type 1 (4.65 ± 0.25 vs. 0.31 ± 0.01 AU, p < 0.001), and collagen type 3 deposition (1.32 ± 0.46 vs. 0.05 ± 0.03 AU, p < 0.05). These changes were associated with increased apoptosis as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling staining (30.9 ± 4.7 vs. 13.1 ± 0.8%, p < 0.006) and western blots showing increased cleaved caspase-3 (0.27 ± 0.006 vs. 0.08 ± 0.01 AU, p < 0.003) and pro-caspase-3 (5.4 ± 1.5 vs. 0.5 ± 0.3 AU, p < 0.02), B-cell lymphoma protein 2-associated X (0.68 ± 0.07 vs. 0.04 ± 0.04 AU, p < 0.001), and reduced B-cell lymphoma protein 2 (0.29 ± 0.01 vs. 1.47 ± 0.54 AU, p < 0.05). We further observed elevated circulating natriuretic peptides and exercise intolerance in Ppara HepKO mice when compared to controls. Our data demonstrated that lipotoxicity, and fibrosis underlie cardiac dysfunction in MASLD.

Mechanisms Linking Metabolic-Associated Fatty Liver Disease (MAFLD) to Cardiovascular Disease.

PURPOSE OF REVIEW: Metabolic-associated fatty liver disease (MAFLD) is a condition of fat accumulation in the liver that occurs in the majority of patients in combination with metabolic dysfunction in the form of overweight or obesity. In this review, we highlight the cardiovascular complications in MAFLD patients as well as some potential mechanisms linking MAFLD to the development of cardiovascular disease and highlight potential therapeutic approaches to treating cardiovascular diseases in patients with MAFLD. RECENT FINDINGS: MAFLD is associated with an increased risk of cardiovascular diseases (CVD), including hypertension, atherosclerosis, cardiomyopathies, and chronic kidney disease. While clinical data have demonstrated the link between MAFLD and the increased risk of CVD development, the mechanisms responsible for this increased risk remain unknown. MAFLD can contribute to CVD through several mechanisms including its association with obesity and diabetes, increased levels of inflammation, and oxidative stress, as well as alterations in hepatic metabolites and hepatokines. Therapies to potentially treat MAFLD-induced include statins and lipid-lowering drugs, glucose-lowering agents, antihypertensive drugs, and antioxidant therapy.

Loss of hepatic PPARα in mice causes hypertension and cardiovascular disease.

The leading cause of death in patients with nonalcoholic fatty liver disease (NAFLD) is cardiovascular disease (CVD). However, the mechanisms are unknown. Mice deficient in hepatocyte proliferator-activated receptor-α (PPARα) (PparaHepKO) exhibit hepatic steatosis on a regular chow diet, making them prone to manifesting NAFLD. We hypothesized that the PparaHepKO mice might be predisposed to poorer cardiovascular phenotypes due to increased liver fat content. Therefore, we used PparaHepKO and littermate control mice fed a regular chow diet to avoid complications with a high-fat diet, such as insulin resistance and increased adiposity. After 30 wk on a standard diet, male PparaHepKO mice exhibited elevated hepatic fat content compared with littermates as measured by Echo MRI (11.95 ± 1.4 vs. 3.74 ± 1.4%, P < 0.05), hepatic triglycerides (1.4 ± 0.10 vs. 0.3 ± 0.01 mM, P < 0.05), and Oil Red O staining, despite body weight, fasting blood glucose, and insulin levels being the same as controls. The PparaHepKO mice also displayed elevated mean arterial blood pressure (121 ± 4 vs. 108 ± 2 mmHg, P < 0.05), impaired diastolic function, cardiac remodeling, and enhanced vascular stiffness. To determine mechanisms controlling the increase in stiffness in the aorta, we used state-of-the-art PamGene technology to measure kinase activity in this tissue. Our data suggest that the loss of hepatic PPARα induces alterations in the aortas that reduce the kinase activity of tropomyosin receptor kinases and p70S6K kinase, which might contribute to the pathogenesis of NAFLD-induced CVD. These data indicate that hepatic PPARα protects the cardiovascular system through some as-of-yet undefined mechanism.

Bilirubin Nanoparticle Treatment in Obese Mice Inhibits Hepatic Ceramide Production and Remodels Liver Fat Content.

Studies have indicated that increasing plasma bilirubin levels might be useful for preventing and treating hepatic lipid accumulation that occurs with metabolic diseases such as obesity and diabetes. We have previously demonstrated that mice with hyperbilirubinemia had significantly less lipid accumulation in a diet-induced non-alcoholic fatty liver disease (NAFLD) model. However, bilirubin's effects on individual lipid species are currently unknown. Therefore, we used liquid chromatography-mass spectroscopy (LC-MS) to determine the hepatic lipid composition of obese mice with NAFLD treated with bilirubin nanoparticles or vehicle control. We placed the mice on a high-fat diet (HFD) for 24 weeks and then treated them with bilirubin nanoparticles or vehicle control for 4 weeks while maintaining the HFD. Bilirubin nanoparticles suppressed hepatic fat content overall. After analyzing the lipidomics data, we determined that bilirubin inhibited the accumulation of ceramides in the liver. The bilirubin nanoparticles significantly lowered the hepatic expression of two essential enzymes that regulate ceramide production, Sgpl1 and Degs1. Our results demonstrate that the bilirubin nanoparticles improve hepatic fat content by reducing ceramide production, remodeling the liver fat content, and improving overall metabolic health.

Suppressing Hepatic UGT1A1 Increases Plasma Bilirubin, Lowers Plasma Urobilin, Reorganizes Kinase Signaling Pathways and Lipid Species and Improves Fatty Liver Disease.

Several population studies have observed lower serum bilirubin levels in patients with non-alcoholic fatty liver disease (NAFLD). Yet, treatments to target this metabolic phenotype have not been explored. Therefore, we designed an N-Acetylgalactosamine (GalNAc) labeled RNAi to target the enzyme that clears bilirubin from the blood, the UGT1A1 glucuronyl enzyme (GNUR). In this study, male C57BL/6J mice were fed a high-fat diet (HFD, 60%) for 30 weeks to induce NAFLD and were treated subcutaneously with GNUR or sham (CTRL) once weekly for six weeks while continuing the HFD. The results show that GNUR treatments significantly raised plasma bilirubin levels and reduced plasma levels of the bilirubin catabolized product, urobilin. We show that GNUR decreased liver fat content and ceramide production via lipidomics and lowered fasting blood glucose and insulin levels. We performed extensive kinase activity analyses using our PamGene PamStation kinome technology and found a reorganization of the kinase pathways and a significant decrease in inflammatory mediators with GNUR versus CTRL treatments. These results demonstrate that GNUR increases plasma bilirubin and reduces plasma urobilin, reducing NAFLD and inflammation and improving overall liver health. These data indicate that UGT1A1 antagonism might serve as a treatment for NAFLD and may improve obesity-associated comorbidities.

The physiology of bilirubin: health and disease equilibrium.

Bilirubin has several physiological functions, both beneficial and harmful. In addition to reactive oxygen species-scavenging activities, bilirubin has potent immunosuppressive effects associated with long-term pathophysiological sequelae. It has been recently recognized as a hormone with endocrine actions and interconnected effects on various cellular signaling pathways. Current studies show that bilirubin also decreases adiposity and prevents metabolic and cardiovascular diseases. All in all, the physiological importance of bilirubin is only now coming to light, and strategies for increasing plasma bilirubin levels to combat chronic diseases are starting to be considered. This review discusses the beneficial effects of increasing plasma bilirubin, incorporates emerging areas of bilirubin biology, and provides key concepts to advance the field.

Sex and molecular differences in cardiovascular parameters at peak influenza disease in mice.

There is a growing interest in the detection of subtle changes in cardiovascular physiology in response to viral infection to develop better disease surveillance strategies. This is not only important for earlier diagnosis and better prognosis of symptomatic carriers but also useful to diagnose asymptomatic carriers of the virus. Previous studies provide strong evidence of an association between inflammatory biomarker levels and both blood pressure (BP) and heart rate (HR) during infection. The identification of novel biomarkers during an inflammatory event could significantly improve predictions for cardiovascular events. Thus, we evaluated changes in cardiovascular physiology induced in A/Puerto Rico/8/34 (PR8) influenza infections in female and male C57BL/6J mice and compared them with the traditional method of influenza disease detection using body weight (BW). Using radiotelemetry, changes in BP, HR, and activity were studied. Change in BW of infected females was significantly decreased from 5 to 13 days postinfection (dpi), yet alterations in normal physiology including loss of diurnal rhythm and reduced activity was observed starting at about 3 dpi for HR and 4 dpi for activity and BP; continuing until about 13 dpi. In contrast, males had significantly decreased BW 8 to 12 dpi and demonstrated altered physiological measurements for a shorter period compared with females with a reduction starting at 5 dpi for activity, 6 dpi for BP, and 7 dpi for HR until about 12 dpi, 10 dpi, and 9 dpi, respectively. Finally, females and males exhibited different patterns of inflammatory maker expression in lungs at peak disease by analyzing bulk RNA-sequencing data for lungs and Bio-plex cytokine assay for blood collected from influenza-infected and naïve C57BL/6J female and male mice at 7 dpi. In total, this study provides insight into cardiovascular changes and molecular markers to distinguish sex differences in peak disease caused by influenza virus infection.NEW & NOTEWORTHY This study performed longitudinal cardiovascular measurements of influenza viral infection and identified sex difference in both physiological and molecular markers at peak disease.

Novel Function for Bilirubin as a Metabolic Signaling Molecule: Implications for Kidney Diseases.

Bilirubin is the end product of the catabolism of heme via the heme oxygenase pathway. Heme oxygenase generates carbon monoxide (CO) and biliverdin from the breakdown of heme, and biliverdin is rapidly reduced to bilirubin by the enzyme biliverdin reductase (BVR). Bilirubin has long been thought of as a toxic product that is only relevant to health when blood levels are severely elevated, such as in clinical jaundice. The physiologic functions of bilirubin correlate with the growing body of evidence demonstrating the protective effects of serum bilirubin against cardiovascular and metabolic diseases. Although the correlative evidence suggests a protective effect of serum bilirubin against many diseases, the mechanism by which bilirubin offers protection against cardiovascular and metabolic diseases remains unanswered. We recently discovered a novel function for bilirubin as a signaling molecule capable of activating the peroxisome proliferator-activated receptor α (PPARα) transcription factor. This review summarizes the new finding of bilirubin as a signaling molecule and proposes several mechanisms by which this novel action of bilirubin may protect against cardiovascular and kidney diseases.

Molecular mechanisms of metabolic associated fatty liver disease (MAFLD): functional analysis of lipid metabolism pathways.

The metabolic-associated fatty liver disease (MAFLD) is a condition of fat accumulation in the liver in combination with metabolic dysfunction in the form of overweight or obesity and insulin resistance. It is also associated with an increased cardiovascular disease risk, including hypertension and atherosclerosis. Hepatic lipid metabolism is regulated by a combination of the uptake and export of fatty acids, de novo lipogenesis, and fat utilization by ß-oxidation. When the balance between these pathways is altered, hepatic lipid accumulation commences, and long-term activation of inflammatory and fibrotic pathways can progress to worsen the liver disease. This review discusses the details of the molecular mechanisms regulating hepatic lipids and the emerging therapies targeting these pathways as potential future treatments for MAFLD.

ßENaC and ASIC2 associate in VSMCs to mediate pressure-induced constriction in the renal afferent arteriole.

In independent studies, our laboratory has shown the importance of the degenerin proteins ß-epithelial Na+ channel (ßENaC) and acid-sensing ion channel 2 (ASIC2) in pressure-induced constriction (PIC) in renal interlobar arteries. Most, but not all, of the PIC response is abolished in mice lacking normal levels of ßENaC or in ASIC2-null mice, indicating that the functions of ßENaC and ASIC2 cannot fully compensate for the loss of the other. Degenerin proteins are known to associate and form heteromeric channels in expression systems, but whether they interact biochemically and functionally in vascular smooth muscle cells is unknown. We hypothesized that ßENaC and ASIC2 interact to mediate PIC responses in renal vessels. To address this possibility, we 1) used biochemical approaches to show that ßENaC associates into high-molecular-weight complexes and immunoprecipitants with ASIC2 in vascular smooth muscle cells and then 2) examined PIC in renal afferent arterioles in mice lacking normal levels of ßENaC (ßENaCm/m) or/and ASIC2 (ASIC2-/-) using the isolated afferent arteriole-attached glomerulus preparation. We found that the sensitivity of the PIC response (slope of the relationship between intraluminal pressure and percent myogenic tone) decreased to 26%, 27%, and -8% of wild-type controls in ASIC2-/-, ßENaCm/m, and ASIC2-/-/ßENaCm/m groups, respectively, suggesting that the PIC response was totally abolished in mice deficient in both ASIC2 and ßENaC. Surprisingly, we found that resting internal diameters were 20-30% lower (60 mmHg, Ca2+ free) in ASIC2-/-/ßENaCm/m (11.3 ± 0.5 µm) mice compared with control (14.4 ± 0.6 µm, P = 0.0007, independent two-tailed t test) or singly modified (15.7 ± 1.0 to 16.3 ± 1.1 µm) mice, suggesting compensatory vasoconstriction or remodeling. We then examined mean arterial blood pressure (MAP) using radiotelemetry and glomerular injury using histological examination of renal sections. We found that 24-h MAP was mildly elevated (+8 mmHg) in ASIC2-/-/ßENaCm/m mice versus wild-type controls and the glomerular injury score was modestly increased by 38%. These findings demonstrate that myogenic constriction in afferent arterioles is dependent on normal expression of ßENaC and ASIC2 and that mice lacking normal levels of ASIC2 and ßENaC have mild renal injury and increased MAP.NEW & NOTEWORTHY Transmission of systemic blood pressure to delicate renal microvessels is a primary determinant of vascular injury in chronic kidney disease progression to end-stage renal disease. Here, we identified two degenerin family members, with an evolutionary link to mechanosensing, that interact biochemically and functionally to regulate systemic blood pressure and renal injury. Thus, degenerin proteins may serve as a target for the development of therapies to prevent or delay renal disease progression.

Reactive Oxygen Species (ROS) and Antioxidants as Immunomodulators in Exercise: Implications for Heme Oxygenase and Bilirubin.

Exercise is commonly prescribed as a lifestyle treatment for chronic metabolic diseases as it functions as an insulin sensitizer, cardio-protectant, and essential lifestyle tool for effective weight maintenance. Exercise boosts the production of reactive oxygen species (ROS) and subsequent transient oxidative damage, which also upregulates counterbalancing endogenous antioxidants to protect from ROS-induced damage and inflammation. Exercise elevates heme oxygenase-1 (HO-1) and biliverdin reductase A (BVRA) expression as built-in protective mechanisms, which produce the most potent antioxidant, bilirubin. Together, these mitigate inflammation and adiposity. Moderately raising plasma bilirubin protects in two ways: (1) via its antioxidant capacity to reduce ROS and inflammation, and (2) its newly defined function as a hormone that activates the nuclear receptor transcription factor PPARα. It is now understood that increasing plasma bilirubin can also drive metabolic adaptions, which improve deleterious outcomes of weight gain and obesity, such as inflammation, type II diabetes, and cardiovascular diseases. The main objective of this review is to describe the function of bilirubin as an antioxidant and metabolic hormone and how the HO-1-BVRA-bilirubin-PPARα axis influences inflammation, metabolic function and interacts with exercise to improve outcomes of weight management.

Heme-oxygenase and lipid mediators in obesity and associated cardiometabolic diseases: Therapeutic implications.

Obesity-mediated metabolic syndrome remains the leading cause of death worldwide. Among many potential targets for pharmacological intervention, a promising strategy involves the heme oxygenase (HO) system, specifically its inducible form, HO-1. This review collects and updates much of the current knowledge relevant to pharmacology and clinical medicine concerning HO-1 in metabolic diseases and its effect on lipid metabolism. HO-1 has pleotropic effects that collectively reduce inflammation, while increasing vasodilation and insulin and leptin sensitivity. Recent reports indicate that HO-1 with its antioxidants via the effect of bilirubin increases formation of biologically active lipid metabolites such as epoxyeicosatrienoic acid (EET), omega-3 and other polyunsaturated fatty acids (PUFAs). Similarly, HO-1and bilirubin are potential therapeutic targets in the treatment of fat-induced liver diseases. HO-1-mediated upregulation of EET is capable not only of reversing endothelial dysfunction and hypertension, but also of reversing cardiac remodeling, a hallmark of the metabolic syndrome. This process involves browning of white fat tissue (i.e. formation of healthy adipocytes) and reduced lipotoxicity, which otherwise will be toxic to the heart. More importantly, this review examines the activity of EET in biological systems and a series of pathways that explain its mechanism of action and discusses how these might be exploited for potential therapeutic use. We also discuss the link between cardiac ectopic fat deposition and cardiac function in humans, which is similar to that described in obese mice and is regulated by HO-1-EET-PGC1α signaling, a potent negative regulator of the inflammatory adipokine NOV.

Cutting edge concepts: Does bilirubin enhance exercise performance?

Exercise performance is dependent on many factors, such as muscular strength and endurance, cardiovascular capacity, liver health, and metabolic flexibility. Recent studies show that plasma levels of bilirubin, which has classically been viewed as a liver dysfunction biomarker, are elevated by exercise training and that elite athletes may have significantly higher levels. Other studies have shown higher plasma bilirubin levels in athletes and active individuals compared to general, sedentary populations. The reason for these adaptions is unclear, but it could be related to bilirubin's antioxidant properties in response to a large number of reactive oxygen species (ROS) that originates from mitochondria during exercise. However, the mechanisms of these are unknown. Current research has re-defined bilirubin as a metabolic hormone that interacts with nuclear receptors to drive gene transcription, which reduces body weight. Bilirubin has been shown to reduce adiposity and improve the cardiovascular system, which might be related to the adaption of bilirubin increasing during exercise. No studies have directly tested if elevating bilirubin levels can influence athletic performance. However, based on the mechanisms proposed in the present review, this seems plausible and an area to consider for future studies. Here, we discuss the importance of bilirubin and exercise and how the combination might improve metabolic health outcomes and possibly athletic performance.

Pharmacological and Clinical Significance of Heme Oxygenase-1.

This Special Issue collates and updates the current knowledge of the pharmacology and clinical applications concerning the enzyme heme oxygenase (HO) [...].

Adipose-Specific PPARα Knockout Mice Have Increased Lipogenesis by PASK-SREBP1 Signaling and a Polarity Shift to Inflammatory Macrophages in White Adipose Tissue.

The nuclear receptor PPARα is associated with reducing adiposity, especially in the liver, where it transactivates genes for ß-oxidation. Contrarily, the function of PPARα in extrahepatic tissues is less known. Therefore, we established the first adipose-specific PPARα knockout (PparaFatKO) mice to determine the signaling position of PPARα in adipose tissue expansion that occurs during the development of obesity. To assess the function of PPARα in adiposity, female and male mice were placed on a high-fat diet (HFD) or normal chow for 30 weeks. Only the male PparaFatKO animals had significantly more adiposity in the inguinal white adipose tissue (iWAT) and brown adipose tissue (BAT) with HFD, compared to control littermates. No changes in adiposity were observed in female mice compared to control littermates. In the males, the loss of PPARα signaling in adipocytes caused significantly higher cholesterol esters, activation of the transcription factor sterol regulatory element-binding protein-1 (SREBP-1), and a shift in macrophage polarity from M2 to M1 macrophages. We found that the loss of adipocyte PPARα caused significantly higher expression of the Per-Arnt-Sim kinase (PASK), a kinase that activates SREBP-1. The hyperactivity of the PASK-SREBP-1 axis significantly increased the lipogenesis proteins fatty acid synthase (FAS) and stearoyl-Coenzyme A desaturase 1 (SCD1) and raised the expression of genes for cholesterol metabolism (Scarb1, Abcg1, and Abca1). The loss of adipocyte PPARα increased Nos2 in the males, an M1 macrophage marker indicating that the population of macrophages had changed to proinflammatory. Our results demonstrate the first adipose-specific actions for PPARα in protecting against lipogenesis, inflammation, and cholesterol ester accumulation that leads to adipocyte tissue expansion in obesity.

Bilirubin as a metabolic hormone: the physiological relevance of low levels.

Recent research on bilirubin, a historically well-known waste product of heme catabolism, suggests an entirely new function as a metabolic hormone that drives gene transcription by nuclear receptors. Studies are now revealing that low plasma bilirubin levels, defined as "hypobilirubinemia," are a possible new pathology analogous to the other end of the spectrum of extreme hyperbilirubinemia seen in patients with jaundice and liver dysfunction. Hypobilirubinemia is most commonly seen in patients with metabolic dysfunction, which may lead to cardiovascular complications and possibly stroke. We address the clinical significance of low bilirubin levels. A better understanding of bilirubin's hormonal function may explain why hypobilirubinemia might be deleterious. We present mechanisms by which bilirubin may be protective at mildly elevated levels and research directions that could generate treatment possibilities for patients with hypobilirubinemia, such as targeting of pathways that regulate its production or turnover or the newly designed bilirubin nanoparticles. Our review here calls for a shift in the perspective of an old molecule that could benefit millions of patients with hypobilirubinemia.

Increased Sirt1 secreted from visceral white adipose tissue is associated with improved glucose tolerance in obese Nrf2-deficient mice.

Obesity is associated with metabolic dysregulation characterized by insulin resistance and glucose intolerance. Nuclear factor E2-related factor (Nrf2) is a critical regulator of the stress response and Nrf2-deficient mice (Nrf2-/-) are protected against high fat diet (HFD)-induced metabolic derangement. We searched for factors that could underline this favorable phenotype and found that Nrf2-/- mice exhibit higher circulating levels of sirtuin 1 (Sirt1), a key player in cellular homeostasis and energy metabolism, compared to wild-type mice. Increased Sirt1 levels in Nrf2-/- mice were found not only in animals under standard diet but also following HFD. Interestingly, we report here that the visceral adipose tissue (eWAT) is the sole source of increased Sirt1 protein in plasma. eWAT and other fat depots displayed enhanced adipocytes lipolysis, increased fatty acid oxidation and glycolysis, suggesting autocrine and endocrine actions of Sirt1 in this model. We further demonstrate that removal of eWAT completely abolishes the increase in circulating Sirt1 and that this procedure suppresses the beneficial effect of Nrf2 deficiency on glucose tolerance, but not insulin sensitivity, following a HFD regime. Thus, in contrast to many other stressful conditions where Nrf2 deficiency exacerbates damage, our study indicates that up-regulation of Sirt1 levels specifically in the visceral adipose tissue of Nrf2-/- mice is a key adaptive mechanism that mitigates glucose intolerance induced by nutritional stress.