Hypolipidemic effect and modulation of hepatic enzymes by different edible oils in obese Wistar rats.

The current study assessed the hypolipidemic effect and modulation of hepatic enzymes by different edible oils in obese Wistar rats. In order to conduct this study, 36 Wistar rats that were collected at 5 weeks of age and weighed an average of 70 g were split into two groups: 28 of them were fed a high-fat diet (HFD) and 8 of them were fed a control diet. After 5 weeks of feeding, rats from the HFD (obese, n = 4) and the control diet group (n = 4) were sacrificed. Subsequently, the rest of obese rats (n = 24) were separated into six groups, including the continuing high-fat (CHF) diet group, rice bran oil (RBO) diet group, olive oil (OO) diet group, soybean oil (SO) diet group, cod liver oil (CLO) diet group, and sunflower oil (SFO) diet group, and the continuing control diet group (n = 4). Rats from each group were sacrificed following an additional 5 weeks, and all analytical tests were carried out. The results found that the interventions of RBO, CLO, and SFO in obese rats reduced their body weight non-significantly when compared with CHF. It was also observed that a non-significant reduction in weight of the heart, AAT, and EAT occurred by RBO, OO, SO, and CLO, while SFO reduced the AAT level significantly (p < 0.05). Besides, RBO, OO, SO, CLO, and SFO decreased IBAT and liver fat significantly compared to CHF. Similarly, the administration of RBO, OO, SO, and CLO reduced ALT significantly. RBO reduced GGT (p < 0.05) significantly, but other oils did not. The given oil has the efficiency to reduce TC, TAG, and LDL-C but increase HDL-C significantly. These findings suggest that different edible oils can ameliorate obesity, regulate lipid profiles, and modulate hepatic enzymes.

ENPP1 enzyme replacement therapy improves blood pressure and cardiovascular function in a mouse model of generalized arterial calcification of infancy.

Generalized arterial calcification of infancy (GACI) is a rare, life-threatening disorder caused by loss-of-function mutations in the gene encoding ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), which normally hydrolyzes extracellular ATP into AMP and pyrophosphate (PPi). The disease is characterized by extensive arterial calcification and stenosis of large- and medium-sized vessels, leading to vascular-related complications of hypertension and heart failure. There is currently no effective treatment available, but bisphosphonates - nonhydrolyzable PPi analogs - are being used off-label to reduce arterial calcification, although this has no reported impact on the hypertension and cardiac dysfunction features of GACI. In this study, the efficacy of a recombinant human ENPP1 protein therapeutic (rhENPP1) was tested in Enpp1asj-2J homozygous mice (Asj-2J or Asj-2J hom), a model previously described to show extensive mineralization in the arterial vasculature, similar to GACI patients. In a disease prevention study, Asj-2J mice treated with rhENPP1 for 3 weeks showed >95% reduction in aorta calcification. Terminal hemodynamics and echocardiography imaging of Asj-2J mice also revealed that a 6-week rhENPP1 treatment normalized elevated arterial and left ventricular pressure, which translated into significant improvements in myocardial compliance, contractility, heart workload and global cardiovascular efficiency. This study suggests that ENPP1 enzyme replacement therapy could be a more effective GACI therapeutic than bisphosphonates, treating not just the vascular calcification, but also the hypertension that eventually leads to cardiac failure in GACI patients.

G6PC mRNA Therapy Positively Regulates Fasting Blood Glucose and Decreases Liver Abnormalities in a Mouse Model of Glycogen Storage Disease 1a.

Glycogen storage disease type Ia (GSD1a) is an inherited metabolic disorder caused by the deficiency of glucose-6-phosphatase (G6Pase). GSD1a is associated with life-threatening hypoglycemia and long-term liver and renal complications. We examined the efficacy of mRNA-encoding human G6Pase in a liver-specific G6Pase-/- mouse model (L-G6PC-/-) that exhibits the same hepatic biomarkers associated with GSD1a patients, such as fasting hypoglycemia, and elevated levels of hepatic glucose-6-phosphate (G6P), glycogen, and triglycerides. We show that a single systemic injection of wild-type or native human G6PC mRNA results in significant improvements in fasting blood glucose levels for up to 7 days post-dose. These changes were associated with significant reductions in liver mass, hepatic G6P, glycogen, and triglycerides. In addition, an engineered protein variant of human G6Pase, designed for increased duration of expression, showed superior efficacy to the wild-type sequence by maintaining improved fasting blood glucose levels and reductions in liver mass for up to 12 days post-dose. Our results demonstrate for the first time the effectiveness of mRNA therapy as a potential treatment in reversing the hepatic abnormalities associated with GSD1a.

Silencing Myostatin Using Cholesterol-conjugated siRNAs Induces Muscle Growth.

Short interfering RNAs (siRNAs) are a valuable tool for gene silencing with applications in both target validation and therapeutics. Many advances have recently been made to improve potency and specificity, and reduce toxicity and immunostimulation. However, siRNA delivery to a variety of tissues remains an obstacle for this technology. To date, siRNA delivery to muscle has only been achieved by local administration or by methods with limited potential use in the clinic. We report systemic delivery of a highly chemically modified cholesterol-conjugated siRNA targeting muscle-specific gene myostatin (Mstn) to a full range of muscles in mice. Following a single intravenous injection, we observe 85-95% knockdown of Mstn mRNA in skeletal muscle and >65% reduction in circulating Mstn protein sustained for >21 days. This level of Mstn knockdown is also accompanied by a functional effect on skeletal muscle, with animals showing an increase in muscle mass, size, and strength. The cholesterol-conjugated siRNA platform described here could have major implications for treatment of a variety of muscle disorders, including muscular atrophic diseases, muscular dystrophy, and type II diabetes.

Impact of simvastatin on adipose tissue: pleiotropic effects in vivo.

Statins belong to a class of drugs well known for their ability to reduce circulating low-density lipoprotein cholesterol. In addition to cholesterol lowering, they also exhibit potential antiinflammatory and antioxidant properties, suggesting that tissues other than liver may be targeted by statins to exert their beneficial metabolic effects. Adipocytes have received very little attention as a potential target of these drugs, possibly because adipocytes are not a major source of biosynthetic cholesterol. Here, we examine the effects of simvastatin on the secretory pathway, inflammation, and cellular metabolism of adipocytes as well as on whole-body insulin sensitivity. We find that statins have a selective effect on the secretion of the insulin-sensitizing adipokine adiponectin by reducing circulating levels of the high-molecular-weight form of adiponectin specifically with a concomitant increase in intracellular adiponectin levels. However, these effects on adiponectin do not translate into changes in metabolism or whole-body insulin sensitivity, potentially due to additional antiinflammatory properties of statins. In addition, ob/ob mice treated with statins have reduced adiposity and an altered ultrastructure of the plasma membrane with respect to caveolar histology. Our data demonstrate that statins have major effects on the cellular physiology of the adipocyte on multiple levels.

Hypoxia-inducible factor 1alpha induces fibrosis and insulin resistance in white adipose tissue.

Adipose tissue can undergo rapid expansion during times of excess caloric intake. Like a rapidly expanding tumor mass, obese adipose tissue becomes hypoxic due to the inability of the vasculature to keep pace with tissue growth. Consequently, during the early stages of obesity, hypoxic conditions cause an increase in the level of hypoxia-inducible factor 1alpha (HIF1alpha) expression. Using a transgenic model of overexpression of a constitutively active form of HIF1alpha, we determined that HIF1alpha fails to induce the expected proangiogenic response. In contrast, we observed that HIF1alpha initiates adipose tissue fibrosis, with an associated increase in local inflammation. "Trichrome- and picrosirius red-positive streaks," enriched in fibrillar collagens, are a hallmark of adipose tissue suffering from the early stages of hypoxia-induced fibrosis. Lysyl oxidase (LOX) is a transcriptional target of HIF1alpha and acts by cross-linking collagen I and III to form the fibrillar collagen fibers. Inhibition of LOX activity by beta-aminoproprionitrile treatment results in a significant improvement in several metabolic parameters and further reduces local adipose tissue inflammation. Collectively, our observations are consistent with a model in which adipose tissue hypoxia serves as an early upstream initiator for adipose tissue dysfunction by inducing a local state of fibrosis.

Metabolic dysregulation and adipose tissue fibrosis: role of collagen VI.

Adipocytes are embedded in a unique extracellular matrix whose main function is to provide mechanical support, in addition to participating in a variety of signaling events. During adipose tissue expansion, the extracellular matrix requires remodeling to accommodate adipocyte growth. Here, we demonstrate a general upregulation of several extracellular matrix components in adipose tissue in the diabetic state, therefore implicating "adipose tissue fibrosis" as a hallmark of metabolically challenged adipocytes. Collagen VI is a highly enriched extracellular matrix component of adipose tissue. The absence of collagen VI results in the uninhibited expansion of individual adipocytes and is paradoxically associated with substantial improvements in whole-body energy homeostasis, both with high-fat diet exposure and in the ob/ob background. Collectively, our data suggest that weakening the extracellular scaffold of adipocytes enables their stress-free expansion during states of positive energy balance, which is consequently associated with an improved inflammatory profile. Therefore, the disproportionate accumulation of extracellular matrix components in adipose tissue may not be merely an epiphenomenon of metabolically challenging conditions but may also directly contribute to a failure to expand adipose tissue mass during states of excess caloric intake.

Secretion of the adipocyte-specific secretory protein adiponectin critically depends on thiol-mediated protein retention.

Adiponectin is a secretory protein abundantly secreted from adipocytes. It assembles into a number of different higher-order complexes. Adipocytes maintain tight control over circulating plasma levels, suggesting the existence of a complex, highly regulated biosynthetic pathway. However, the critical mediators of adiponectin maturation within the secretory pathway have not been elucidated. Previously, we found that a significant portion of de novo-synthesized adiponectin is not secreted and retained in adipocytes. Here, we show that there is an abundant pool of properly folded adiponectin in the secretory pathway that is retained through thiol-mediated retention, as judged by the release of adiponectin in response to treatment of adipocytes with reducing agents. Adiponectin is covalently bound to the ER chaperone ERp44. An adiponectin mutant lacking cysteine 39 fails to stably interact with ERp44, demonstrating that this residue is the primary site mediating the covalent interaction. Another ER chaperone, Ero1-Lalpha, plays a critical role in the release of adiponectin from ERp44. Levels of both of these proteins are highly regulated in adipocytes and are influenced by the metabolic state of the cell. While less critical for the secretion of trimers, these chaperones play a major role in the assembly of higher-order adiponectin complexes. Our data highlight the importance of posttranslational events controlling adiponectin levels and the release of adiponectin from adipocytes. One mechanism for increasing circulating levels of specific adiponectin complexes by peroxisome proliferator-activated receptor gamma agonists may be selective upregulation of rate-limiting chaperones.

beta-Amyloid promotes accumulation of lipid peroxides by inhibiting CD36-mediated clearance of oxidized lipoproteins.

BACKGROUND: Recent studies suggest that hypercholesterolemia, an established risk factor for atherosclerosis, is also a risk factor for Alzheimer's disease. The myeloid scavenger receptor CD36 binds oxidized lipoproteins that accumulate with hypercholesterolemia and mediates their clearance from the circulation and peripheral tissues. Recently, we demonstrated that CD36 also binds fibrillar beta-amyloid and initiates a signaling cascade that regulates microglial recruitment and activation. As increased lipoprotein oxidation and accumulation of lipid peroxidation products have been reported in Alzheimer's disease, we investigated whether beta-amyloid altered oxidized lipoprotein clearance via CD36. METHODS: The availability of mice genetically deficient in class A (SRAI & II) and class B (CD36) scavenger receptors has facilitated studies to discriminate their individual actions. Using primary microglia and macrophages, we assessed the impact of Abeta on: (a) cholesterol ester accumulation by GC-MS and neutral lipid staining, (b) binding, uptake and degradation of 125I-labeled oxidized lipoproteins via CD36, SR-A and CD36/SR-A-independent pathways, (c) expression of SR-A and CD36. In addition, using mice with targeted deletions in essential kinases in the CD36-signaling cascade, we investigated whether Abeta-CD36 signaling altered metabolism of oxidized lipoproteins. RESULTS: In primary microglia and macrophages, Abeta inhibited binding, uptake and degradation of oxidized low density lipoprotein (oxLDL) in a dose-dependent manner. While untreated cells accumulated abundant cholesterol ester in the presence of oxLDL, cells treated with Abeta were devoid of cholesterol ester. Pretreatment of cells with Abeta did not affect subsequent degradation of oxidized lipoproteins, indicating that lysosomal accumulation of Abeta did not disrupt this degradation pathway. Using mice with targeted deletions of the scavenger receptors, we demonstrated that Abeta inhibited oxidized lipoprotein binding and its subsequent degradation via CD36, but not SRA, and this was independent of Abeta-CD36-signaling. Furthermore, Abeta treatment decreased CD36, but not SRA, mRNA and protein, thereby reducing cell surface expression of this oxLDL receptor. CONCLUSIONS: Together, these data demonstrate that in the presence of beta-amyloid, CD36-mediated clearance of oxidized lipoproteins is abrogated, which would promote the extracellular accumulation of these pro-inflammatory lipids and perpetuate lipid peroxidation.

Fibrillar amyloid protein present in atheroma activates CD36 signal transduction.

The self-association of proteins to form amyloid fibrils has been implicated in the pathogenesis of a number of diseases including Alzheimer's, Parkinson's, and Creutzfeldt-Jakob diseases. We recently reported that the myeloid scavenger receptor CD36 initiates a signaling cascade upon binding to fibrillar beta-amyloid that stimulates recruitment of microglia in the brain and production of inflammatory mediators. This receptor plays a key role in the pathogenesis of atherosclerosis, prompting us to evaluate whether fibrillar proteins were present in atherosclerotic lesions that could initiate signaling via CD36. We show that apolipoprotein C-II, a component of very low and high density lipoproteins, readily forms amyloid fibrils that initiate macrophage inflammatory responses including reactive oxygen production and tumor necrosis factor alpha expression. Using macrophages derived from wild type and Cd36(-/-) mice to distinguish CD36-specific events, we show that fibrillar apolipoprotein C-II activates a signaling cascade downstream of this receptor that includes Lyn and p44/42 MAPKs. Interruption of this signaling pathway through targeted deletion of Cd36 or blocking of p44/42 MAPK activation inhibits macrophage tumor necrosis factor alpha gene expression. Finally, we demonstrate that apolipoprotein C-II in human atheroma co-localizes to regions positive for markers of amyloid and macrophage accumulation. Together, these data characterize a CD36-dependent signaling cascade initiated by fibrillar amyloid species that may promote atherogenesis.