Apolipoprotein B production reduces lipotoxic cardiomyopathy: studies in heart-specific lipoprotein lipase transgenic mouse

Lipid accumulation is associated with cardiac dysfunction in diabetes and obesity. Transgenic mice expressing non-transferable lipoprotein lipase (LpL) with a glycosylated phosphatidyl-inositol (GPI) anchor in cardiomyocytes have dilated cardiomyopathy. However, the mechanisms responsible for lipid accumulation and cardiomyopathy are not clear. Hearts from 3-month-old mice expressing GPI-anchored human LpL (hLpLGPI) mice had increased fatty acid oxidation and heart failure genes and decreased glucose transporter genes. 6-month-old mice had increased mRNA expression and activation of the apoptosis marker caspase-3. Moreover, hLpLGPI hearts had significant cytochrome c release from mitochondria to cytosol. Low density lipoprotein uptake was greater in hLpLGPI hearts, and this was associated with more intracellular apolipoprotein B (apoB). To test whether lipid accumulation in the hLpLGPI heart is reduced by cardiac expression of apoB, hLpLGPI mice were bred with transgenic human apoB (HuB)-expressing mice. Hearts of HuB/hLpLGPI mice had less triglyceride (38%) and free fatty acids (19%), secreted more apoB, and expressed less atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) and more glucose transporter 4 (GLUT4). The increased mortality of the mice was abrogated by the transgenic expression of apoB. Therefore, we hypothesize that cardiac apoB expression improves cardiomyopathy by increasing lipid resecretion from the heart.     

Overexpression of apolipoprotein B in the heart impedes cardiac triglyceride accumulation and development of cardiac dysfunction in diabetic mice

The heart secretes apolipoprotein B (apoB) containing lipoproteins. Herein, we examined whether the overexpression of a human apoB transgene in the heart affects triglyceride accumulation and development of cardiac dysfunction in streptozotocin-treated diabetic mice. Blood glucose, plasma free fatty acids, and plasma triglycerides were similarly affected in diabetic wild type mice and diabetic apoB transgenic mice as compared with non-diabetic mice of the same genotype. After 12 weeks, heart triglycerides were increased by 48% in diabetic wild type mice. These mice displayed an increased expression of brain natriuretic peptide and deterioration of heart function on echocardiography. In diabetic apoB transgenic mice, heart triglyceride levels were identical to those in non-diabetic wild type and apoB transgenic mice, and brain natriuretic peptide expression as well as echocardiographic indexes of heart function were only marginally affected or unaffected. The findings suggest that triglyceride accumulation in the heart is important for development of diabetic cardiomyopathy in mice, and that lipoprotein formation by cardiomyocytes plays an integrated role in cardiac lipid metabolism.     

The function of porcine PPARγ and dietary fish oil effect on the expression of lipid and glucose metabolism related genes

Peroxisome-proliferator-activated receptor γ (PPARγ) plays a critical role in regulation of adipocyte differentiation and insulin sensitivity. To become functional, PPARγ must be activated by binding an appropriate ligand. Polyunsaturated fatty acids (PUFA) are potential ligands for PPARγ. The current experiment was designed to determine the potential for PUFA, particularly eicosapentaenoic acid and docosahexaenoic acid, to activate the function of porcine PPARγ in vivo. Transgenic mice, expressing porcine PPARγ in skeletal muscle were generated and fed with a high-saturated fat (beef tallow) or high-unsaturated fat (fish oil) diet for 4 months. When transgenic mice were fed a fish oil supplemented diet, the expression of adipogenic and glucose uptake genes was increased, leading to reduced plasma glucose concentration. The PPARγ transgene increased the expression of Glut4 in the muscle. This result suggests that there was increased glucose utilization and, therefore, a reduced blood glucose concentration in the transgenic mice. Also, the plasma adiponectin was elevated by fish oil treatment, suggesting a role of adiponectin in mediating the PUFA effect. These results suggest that PUFA may serve as a natural regulator of glucose uptake in vivo and these effects are mainly through PPARγ function.     

Ghrelin is a signal to facilitate the utilization of fatty acids and save glucose by the liver,skeletal muscle,and adipose tissues in chicks

Ghrelin, classically known as a central appetite-stimulating hormone, has recently been recognized to play an important role in peripheral tissue energy metabolism. In chicken, contrary to mammal, ghrelin acts as an anorexia signal, increased by fasting and further elevated after refed. In the present study, the effect of ghrelin on glucose/lipid utilization by peripheral tissues was investigated. Injection of exogenous acyl ghrelin reduced plasma triglyceride and glucose levels of chickens at both fasting and fed status. In the in vitro cultured chicken primary hepatocytes, adipocytes, and myoblasts, ghrelin suppressed glucose uptake, stimulated fatty acids uptake and oxidation, and decreased TG content. In hepatocyte, ghrelin increased the activities of LPL and HL, and upregulated the expression levels of gene ACC, CPT1, and PPARα. Ghrelin treatment markedly increased the protein level of p-ACC, PPARγ, PGC1α, and CPT1 in hepatocytes, adipocytes and myoblasts. Inhibition of AMPK activity by Compound C had no influence on glucose uptake by hepatocyte, adipocyte, and myoblast, but further amplified the stimulated fatty acid uptake of adipocyte by ghrelin. The present result demonstrates that ghrelin facilitates the uptake and oxidation of fatty acid and cut down the utilization of glucose by the liver, muscle, and adipose tissues. The result suggests that ghrelin functions as a signal of fatty acid oxidation. The study provides a vital framework for understanding the intrinsic role of ghrelin as a crucial factor in the concerted regulation of metabolic substrate of hepatocytes, adipocytes, and myoblasts.     

Beta-arrestin-1 protein represses adipogenesis and inflammatory responses through its interaction with peroxisome proliferator-activated receptor-gamma (PPARgamma)

One of the master regulators of adipogenesis and macrophage function is peroxisome proliferator-activated receptor-γ (PPARγ). Here, we report that a deficiency of β-arrestin-1 expression affects PPARγ-mediated expression of lipid metabolic genes and inflammatory genes. Further mechanistic studies revealed that β-arrestin-1 interacts with PPARγ. β-Arrestin-1 suppressed the formation of a complex between PPARγ and 9-cis-retinoic acid receptor-α through its direct interaction with PPARγ. The interaction of β-arrestin-1 with PPARγ repressed PPARγ/9-cis-retinoic acid receptor-α function but promoted PPARγ/nuclear receptor corepressor function in PPARγ-mediated adipogenesis and inflammatory gene expression. Consistent with these results, a deficiency of β-arrestin-1 binding to PPARγ abolished its suppression of PPARγ-dependent adipogenesis and inflammatory responses. These results indicate that the regulation of PPARγ by β-arrestin-1 is critical. Furthermore, in vivo expression of β-arrestin-1 (but not the binding-deficient mutant) significantly repressed adipogenesis, macrophage infiltration, and diet-induced obesity and improved glucose tolerance and systemic insulin sensitivity. Therefore, our findings not only reveal a molecular mechanism for the modulation of obesity by β-arrestin-1 but also suggest a potential tactical approach against obesity and its associated metabolic disorders.     

The Expression of GHS-R in Primary Neurons Is Dependent upon Maturation Stage and Regional Localization

Ghrelin is a hormone with a crucial role in the regulation of appetite, regulation of inflammation, glucose metabolism and cell proliferation. In the brain ghrelin neurons are located in the cortex (sensorimotor area, cingular gyrus), and the fibres of ghrelin neurons in hypothalamus project directly to the dorsal vagal complex (DVC). Ghrelin binds the growth hormone secretagogue receptor (GHS-R) a G-protein-coupled receptor with a widespread tissue distribution, indeed these receptors are localized both in nonnervous, organs/tissues (i.e. adipose tissue, myocardium, adrenals, gonads, lung, liver, arteries, stomach, pancreas, thyroid, and kidney) as well as in central nervous system (CNS) and higher levels of expression in the pituitary gland and the hypothalamus and lower levels of expression in other organs, including brain. A GHS-R specific monoclonal antibody has been developed and characterized and through it we demonstrate that GHS-R is expressed in primary neurons and that its expression is dependent upon their developmental stage and shows differences according to the brain region involved, with a more pronounced expression in hippocampal rather than cortical neurons. A characterization of GHS-R within the central nervous system is of extreme importance in order to gain insights on its role in the modulation of neurodegenerative events such as Alzheimer’s disease.     

Transgenic overexpression of intraislet ghrelin does not affect insulin secretion or glucose metabolism in vivo

Whereas ghrelin is produced primarily in the stomach, a small amount of it is produced in pancreatic islets. Although exogenous administration of ghrelin suppresses insulin secretion in vitro or in vivo, the role of intraislet ghrelin in the regulation of insulin secretion in vivo remains unclear. To understand the physiological role of intraislet ghrelin in insulin secretion and glucose metabolism, we developed a transgenic (Tg) mouse model, rat insulin II promoter ghrelin-internal ribosomal entry site-ghrelin O-acyl transferase (RIP-GG) Tg mice, in which mouse ghrelin cDNA and ghrelin O-acyltransferase are overexpressed under the control of the rat insulin II promoter. Although pancreatic desacyl ghrelin levels were elevated in RIP-GG Tg mice, pancreatic ghrelin levels were not altered in animals on a standard diet. However, when Tg mice were fed a medium-chain triglyceride-rich diet (MCTD), pancreatic ghrelin levels were elevated to ～16 times that seen in control animals. It seems likely that the gastric ghrelin cells possess specific machinery to provide the octanoyl acid necessary for ghrelin acylation but that this machinery is absent from pancreatic β-cells. Despite the overexpression of ghrelin, plasma ghrelin levels in the portal veins of RIP-GG Tg mice were unchanged from control levels. Glucose tolerance, insulin secretion, and islet architecture in RIP-GG Tg mice were not significantly different even when the mice were fed a MCTD. These results indicate that intraislet ghrelin does not play a major role in the regulation of insulin secretion in vivo.     

Early life overfeeding decreases acylated ghrelin circulating levels and upregulates GHSR1a signaling pathway in white adipose tissue of obese young mice

Ghrelin is a hormone synthesized by the stomach that acts in different tissues via a specific receptor (GHS-R1a), including hypothalamus and adipose tissue. For instance, recent reports have shown that ghrelin has a direct action on hypothalamic regulation of food intake mainly inducing an orexigenic effect. On the other hand, ghrelin also modulates energy stores and expenditure in the adipocytes. This dual action has suggested that this hormone may act as a link between the central nervous system and peripheral mechanisms. Furthermore, concerning nutritional disorders, it has been suggested that obesity may be considered an impairment of the above cited link. Therefore, considering that neonatal overfeeding induces obesity in adulthood by unknown mechanisms, in this study we examined the effects of early life overnutrition on the development of obesity and in particular on adipose tissue ghrelin signaling in young mice. Our data demonstrated that overnutrition during early life induces a significant increase in body weight of young mice, starting at 10 days, and this increase in weight persisted until adulthood (90 days of age). In these animals, blood glucose, liver weight and visceral fat weight were found higher at 21 days when compared to the control group. Acylated ghrelin circulating levels were found lower in the young obese pups. In addition, in white adipose tissue ghrelin receptor (GHS-R1a) expression increased and was associated to positive modulation of content and phosphorylation of proteins involved in cell energy store and use as AKT, PI3K, AMPK, GLUT-4, and CPT1. However, PPARγ content decreased in obese group. Basically, we showed that adipose tissue metabolism is altered in early life acquired obesity and probably due to such modification a new pattern of ghrelin signaling pathway takes place.     

Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. Identifying novel regulators of mitochondrial bioenergetics will broaden our understanding of regulatory checkpoints that coordinate complex metabolic pathways. We previously showed that Nur77, an orphan nuclear receptor of the NR4A family, regulates the expression of genes linked to glucose utilization. Here we demonstrate that expression of Nur77 in skeletal muscle also enhances mitochondrial function. We generated MCK-Nur77 transgenic mice that express wild-type Nur77 specifically in skeletal muscle. Nur77-overexpressing muscle had increased abundance of oxidative muscle fibers and mitochondrial DNA content. Transgenic muscle also exhibited enhanced oxidative metabolism, suggestive of increased mitochondrial activity. Metabolomic analysis confirmed that Nur77 transgenic muscle favored fatty acid oxidation over glucose oxidation, mimicking the metabolic profile of fasting. Nur77 expression also improved the intrinsic respiratory capacity of isolated mitochondria, likely due to the increased abundance of complex I of the electron transport chain. These changes in mitochondrial metabolism translated to improved muscle contractile function ex vivo and improved cold tolerance in vivo. Our studies outline a novel role for Nur77 in the regulation of oxidative metabolism and mitochondrial activity in skeletal muscle.     

Effects of dietary sea cucumber saponin on the gene expression rhythm involved in circadian clock and lipid metabolism in mice during nighttime-feeding

In mammals, clock rhythms exist not only in the suprachiasmatic nucleus, which is entrained by light/dark (LD) cycles, but also in most peripheral tissues. Recent studies have revealed that most physiology and behavior are subject to well-controlled daily oscillations; similarly, metabolic state influences the diurnal rhythm too. Previous studies have indicated that dietary sea cucumber saponin (SCS) could improve glucose and lipid metabolism of rodent. However, whether SCS could affect the expression of clock genes, which is involved in lipid metabolism, is unknown at present. The aim of this study is to investigate the effects of SCS on the clock and clock-controlled genes involved in lipid metabolism. ICR male mice were divided into a control and SCS group mice (add 0.03 % sea cucumber saponin to regular chow) and were fed at night (2030–0830 hours). After 2 weeks, clock genes expression in brain and liver, blood glucose, hormones, and lipid metabolic markers were analyzed. The results showed that dietary SCS caused alteration in rhythms and/or amplitudes of clock genes was more significant in brain than in liver. In addition, peroxisome proliferator-activated receptor (PPARα), sterol regulatory element binding protein-1c (SREBP-1c), together with their target genes carnitine palmitoyl transferase (CPT), and fatty acid synthase (FAS) showed marked changes in rhythm and/or amplitude in SCS group mice. These results suggested that SCS could affect the daily expression patterns of clock genes in brain and liver tissues, and alter the clock-controlled genes involved in lipid metabolism.     

Ghrelin inhibits early osteogenic differentiation of C3H10T1/2 cells by suppressing Runx2 expression and enhancing PPARγ and C/EBPα expression

Ghrelin is a 28‐residue peptide identified in the stomach as an endogenous ligand of the growth hormone secretagogue receptor that is expressed in a variety of peripheral tissues, as well as in the brain. In previous studies, ghrelin has been shown to stimulate both adipogenic differentiation from preadipocytes and osteogenic differentiation from preosteoblasts or primary osteoblasts. This study was undertaken to investigate the direct effect of ghrelin on the lineage allocation of mesenchymal stem cells (MSCs). We identified ghrelin receptor mRNA in C3H10T1/2 cells, and we found the levels of this mRNA to be attenuated during osteogenic differentiation. Treatment of cells with ghrelin resulted in both proliferation and inhibition of caspase‐3 activity. In addition, ghrelin decreased serum deprivation‐induced bax protein expression and release of cytochrome c from the mitochondria, whereas it increased bcl‐2 protein expression. Moreover, ghrelin inhibited early osteogenic differentiation, as shown by alkaline phosphatase activity and staining, and inhibited osteoblast‐specific genes expression by altering Runx2, PPARγ, and C/EBPα protein expression. J. Cell. Biochem. 106: 626–632, 2009. © 2009 Wiley‐Liss, Inc.     

PPARγ的翻译后修饰

过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)是一种配体依赖性核转录因子,它具有调控细胞分化、脂肪代谢、糖代谢及炎症等多种生物学功能.机体对PPARγ转录活性的调控方式是多种多样的,包括蛋白表达水平、配体以及转录辅助因子等不同层次上的调控.近年来众多证据揭示,蛋白翻译后修饰(posttranslational modifications,PTMs)是机体调节PPARγ转录活性的另一重要方式.目前,已报道的PPARγ翻译后修饰包括磷酸化、泛素化、SUMO化和亚硝基化等,它们能够改变蛋白构象、调控蛋白相互作用、改变受体与配体间的亲和力,从而调控PPARγ下游基因的转录.重要的是,PPARγ的翻译后修饰与一些疾病如糖尿病、动脉粥样硬化、肿瘤等密切相关.本文将主要围绕PPARγ的各种翻译后修饰及其在疾病的发生、发展和治疗中的意义作一综述.