The Expression of Peroxisome Proliferator‐Activated Receptor γ in Pig Fetal Tissue and Primary Stromal‐Vascular Cultures

Objective: This study was designed to determine when peroxisome proliferator‐activated receptor γ (PPARγ) is expressed in developing fetal adipose tissue and stromal‐vascular adipose precursor cells derived from adipose tissue. In addition we examined developing tissue for CCAAT/enhancer‐binding protein β (C/EBPβ) expression to see if it was correlated with PPARγ expression. Pituitary function and hormones involved with differentiation (dexamethasone and retinoic acid) were also tested for their effects on PPARγ expression to determine if hormones known to affect differentiation also effect PPARγ expression in vivo and in cell culture. Research Methods and Procedures: Developing subcutaneous adipose tissues from the dorsal region of the fetal pig were collected at different gestation times and assayed using Western blot analysis to determine levels of PPARγ and C/EBPβ. Hypophysectomy was performed on 75‐day pig fetuses and tissue samples were then taken at 105 days for Western blot analysis. Adipose tissue was also taken from postnatal pigs to isolate stromal‐vascular (S‐V) cells. These adipose precursor cells were grown in culture and samples were taken for Western blot analysis to determine expression levels of PPARγ. Results: Our results indicate that PPARγ is expressed as early as 50 days of fetal development in adipose tissue and continues through 105 days. Expression of PPARγ was found to be significantly enhanced in adipose tissue from hypophysectomized fetuses at 105 days of fetal development (p < 0.05). C/EBPβ was not found in 50‐ or 75‐day fetal tissues and was found only at low levels in 105‐day tissues. C/EBPβ was not found in hypophysectomized (hypoxed) 105‐day tissue where PPARγ was elevated. S‐V cells freshly isolated from adipose tissue of 5‐ to 7‐day postnatal pigs showed the expression of PPARγ1. When S‐V cells were cultured, both PPARγ1 and 2 were expressed after the first day and continued as cells differentiated. High concentrations of retinoic acid decreased PPARγ expression in early S‐V cultures (p < 0.05). Discussion: Our data indicate that PPARγ is expressed in fetal adipose tissue very early before distinct fat cells are observed and can be expressed without the expression of C/EBPβ. The increase in PPARγ expression after hypophysectomy may explain the increase in fat cell size under these conditions. Adipose precursor cells (S‐V cells) from 5‐ to 7‐day postnatal pigs also express PPARγ in the tissue before being induced to differentiate in culture. Thus S‐V cells from newborn pig adipose tissue are probably more advanced in development than the 3T3‐L1 cell model. S‐V cells may be in a state where PPARγ and C/EBPα are expressed but new signals or vascularization are needed before cells are fully committed and lipid filling begins.     

Inflammation inhibits the expression of phosphoenolpyruvate carboxykinase in liver and adipose tissue

Inhibition of adipocyte triglyceride biosynthesis is required for fatty acid mobilization during inflammation. Triglyceride biosynthesis requires glycerol 3-phosphate and phosphoenolpyruvate carboxykinase (PEPCK) plays a key role. We demonstrate that LPS, zymosan, and TNF-α decrease PEPCK in liver and fat. Turpentine decreases PEPCK in liver, but not in fat. The LPS-induced decrease in PEPCK does not occur in TLR4 deficient animals, indicating that this receptor is required. The LPS-induced decrease in hepatic PEPCK does not occur in TNF receptor/IL-1 receptor knockout mice, but occurs in fat, indicating that TNF-α/IL-1 is essential for the decrease in liver but not fat. In 3T3-L1 adipocytes TNF-α, IL-1, IL-6, and IFNγ inhibit PEPCK indicating that there are multiple pathways by which PEPCK is decreased in adipocytes. The binding of PPARγ and RXRα to the PPARγ response element in the PEPCK promoter is markedly decreased in adipose tissue nuclear extracts from LPS treated animals. Lipopolysaccharide and zymosan reduce PPARγ and RXRα expression in fat, suggesting that a decrease in PPARγ and RXRα accounts for the decrease in PEPCK. Thus, there are multiple cytokine pathways by which inflammation inhibits PEPCK expression in adipose tissue which could contribute to the increased mobilization of fatty acids during inflammation.     

Overexpressing STAMP2 attenuates adipose tissue angiogenesis and insulin resistance in diabetic ApoE−/−/LDLR−/− mouse via a PPARγ/CD36 pathway

The aim of this study was to investigate whether overexpression of STAMP2 improves insulin resistance by regulating angiogenesis in adipose tissues. The characteristics of diabetic mice were measured by serial metabolite and pathology tests. Samples were obtained from epididymal, subcutaneous and brown adipose tissues. Histological and morphological analysis demonstrated that STAMP2 gene overexpression reduced adipocyte size, angiogenesis in epididymal and brown adipose tissues. On aortic ring assay, microvessels sprouting from aortas were significantly inhibited after STAMP2 gene overexpression. The cellular effect of STAMP2 on angiogenesis was explored in human umbilical vein endothelial cells (HUVECs) model. Correlation of STAMP2 and angiogenesis was validated by Ad‐STAMP2 transfection and STAMP2 siRNA inhibition. In vitro, overexpression of STAMP2 significantly inhibited endothelial cell migration, tube formation. The effects of Ad‐STAMP2 transfection on HUVECs were abolished by treatment with PPARγ antagonist GW9662 (2.5 μM), and the roles of STAMP2 siRNA on HUVECs were also reversed by treatment with PPARγ agonist rosiglitazone (RSG) (0.1 mM). RT‐PCR indicated that STAMP2 could regulate levels of adhesion molecules, vascular endothelial growth factor A and CD36. The expression of PPARγ and CD36 was decreased when STAMP2 was inhibited by siRNA, while PPARγ and CD36 were highly expressed after overexpression of STAMP2. Our results suggested that STAMP2 gene overexpression may improve insulin resistance via attenuating angiogenesis in epididymal and brown adipose tissues through the PPARγ/CD36 signalling pathway.     

Green tea catechins prevent obesity through modulation of peroxisome proliferator-activated receptors

Epidemiological evidence and experimental studies suggest that drinking green tea is associated with a lower risk of obesity and related diseases. However, the mechanisms of these effects are not clear. In the present study, we investigated the anti-obesity mechanisms of green tea catechins (GTCs) through modulation of peroxisome proliferator activated-receptor (PPAR) pathways in high-fat diet-induced obesity in rats. GTC supplementation significantly attenuated the increased body and liver weights and the elevated serum and liver triglyceride levels. Meanwhile, GTCs increased the PPARγ levels in subcutaneous white adipose tissue (SWAT) and decreased the PPAR levels in visceral white adipose tissue (VWAT). In addition, GTC treatment up-regulated the levels of PPARδ in SWAT, VWAT, and brown adipose tissue and increased the expression of genes involved in fatty acid oxidation in brown adipose tissue. Our results suggest that GTCs exert their anti-obesity mechanism in part by modulating PPAR signaling pathways.     

Troglitazone activates TRPV1 and causes deacetylation of PPARγ in 3T3-L1 cells

Published research suggests that activation of transient receptor potential vanilloid subfamily 1 (TRPV1) enhances the expression and deacetylation of peroxisome proliferator-activated receptor gamma (PPARγ) to cause browning of white adipose tissue. Here, we show that TRPV1 activation by capsaicin significantly prevents high fat diet-induced obesity in mice. This is associated with an increase in the expression and deacetylation of PPARγ in the epididymal fat of these mice. Consistent with the TRPV1 activation in vivo, overexpression of TRPV1 enhanced the PPARγ and other thermogenic genes in cultured 3T3-L1 preadipocytes. To determine the interaction between TRPV1 and PPARγ signaling, we analyzed the effect of Troglitazone (Trog; a thiazolidinedione derivative and an agonist of PAARγ) treatment on cultured 3T3-L1 cells. Trog enhanced the expression of TRPV1, PPARγ and thermogenic proteins in undifferentiated 3T3-L1 cells but not in differentiated cells. Acute application of Trog stimulated a robust Ca²⁺ influx into 3T3-L1 cells and TRPV1 inhibition by capsazepine prevented this. More interestingly, Trog or capsaicin treatment caused the deacetylation of PPARγ in 3T3-L1 cells and inhibition of TRPV1 or Sirtuin 1 - prevented this. Our data suggest a novel effect of Trog to induce PPARγ deacetylation by activating TRPV1. This research has a significant implication on the role of TRPV1 and PPARγ signaling in the browning of white adipose tissue.     

Activation of peroxisome proliferator-activated receptor-α enhances fatty acid oxidation in human adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPARα in adipocytes have been unclarified. We examined the functions of PPARα using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPARα by GW7647, a potent PPARα agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPARγ, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPARα activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPARγ is activated. On the other hand, PPARα activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPARα-dependent manner. Moreover, PPARα activation increased the production of CO₂ and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPARα stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPARα agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPARα activation are very valuable for managing diabetic conditions accompanied by obesity, because PPARγ agonists, usually used as antidiabetic drugs, induce excessive lipid accumulation in adipocytes in addition to improvement of insulin resistance.     

Mitochondrial long chain fatty acid β-oxidation in man and mouse

Several mouse models for mitochondrial fatty acid β-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD^−/−), very long chain acyl-CoA dehydrogenase knockout (VLCAD^−/−) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.     

Osthol inhibits fatty acid synthesis and release via PPARα/γ-mediated pathways in 3T3-L1 adipocytes

Osthol is an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson (Apiaceae), and has obvious therapeutic effect on fatty liver, but its mechanisms are not yet understood completely. One potential link between adipose tissue and fatty liver may be circulating fatty acids. In the present study, the effect of osthol on fatty acid synthesis and release in cultured 3T3-L1 adipocytes was observed. Following treatment of adipocytes with osthol, the intracellular levels of free fatty acids (FFA) and triacylglycerols as well as cultured supernatant level of FFA were decreased, and some lipogenic gene and protein expressions were also decreased, while the peroxisome proliferator-activated receptor (PPAR) α/γ mRNA expressions were increased. Osthol-reduced lipogenic gene expressions were decreased or abrogated after pretreatment with specific inhibitor(s) of PPARα and/or PPARγ.     

Activation of peroxisome proliferator-activated receptor-γ by rosiglitazone improves lipid homeostasis at the adipose tissue-liver axis in ethanol-fed mice

The development of alcohol-induced fatty liver is associated with a reduction of white adipose tissue (WAT). Peroxisome proliferator-activated receptor (PPAR)-γ prominently distributes in the WAT and plays a crucial role in maintaining adiposity. The present study investigated the effects of PPAR-γ activation by rosiglitazone on lipid homeostasis at the adipose tissue-liver axis. Adult C57BL/6 male mice were pair fed liquid diet containing ethanol or isocaloric maltose dextrin for 8 wk with or without rosiglitazone supplementation to ethanol-fed mice for the last 3 wk. Ethanol exposure downregulated adipose PPAR-γ gene and reduced the WAT mass in association with induction of inflammation, which was attenuated by rosiglitazone. Ethanol exposure stimulated lipolysis but reduced fatty acid uptake capacity in association with dysregulation of lipid metabolism genes. Rosiglitazone normalized adipose gene expression and corrected ethanol-induced lipid dyshomeostasis. Ethanol exposure induced steatosis and upregulated inflammatory genes in the liver, which were attenuated by rosiglitazone. Hepatic peroxisomal fatty acid β-oxidation was suppressed by ethanol in associated with inhibition of acyl-coenzyme A oxidase 1. Rosiglitazone elevated plasma adiponectin level and normalized peroxisomal fatty acid β-oxidation rate. However, rosiglitazone did not affect ethanol-reduced very low-density lipoprotein secretion from the liver. These results demonstrated that activation of PPAR-γ by rosiglitazone reverses ethanol-induced adipose dysfunction and lipid dyshomeostasis at the WAT-liver axis, thereby abrogating alcoholic fatty liver.     

Bezafibrate activation of PPAR drives disturbances in mitochondrial redox bioenergetics and decreases the viability of cells from patients with VLCAD deficiency

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common inborn long-chain fatty acid oxidation (FAO) disorder. VLCAD deficiency is characterized by distinct phenotypes. The severe phenotypes are potentially life-threatening and affect the heart or liver, with a comparatively milder phenotype characterized by myopathic symptoms. There is an unmet clinical need for effective treatment options for the myopathic phenotype. The molecular mechanisms driving the gradual decrease in mitochondrial function and associated alterations of muscle fibers are unclear.The peroxisome proliferator-activated receptor (PPAR) pan-agonist bezafibrate is a potent modulator of FAO and multiple other mitochondrial functions and has been proposed as a potential medication for myopathic cases of long-chain FAO disorders. In vitro experiments have demonstrated the ability of bezafibrate to increase VLCAD expression and activity. However, the outcome of small-scale clinical trials has been controversial.We found VLCAD deficient patient fibroblasts to have an increased oxidative stress burden and deranged mitochondrial bioenergetic capacity, compared to controls. Applying heat stress under fasting conditions to bezafibrate pretreated patient cells, caused a marked further increase of mitochondrial superoxide levels. Patient cells failed to maintain levels of the essential thiol peptide antioxidant glutathione and experienced a decrease in cellular viability. Our findings indicate that chronic PPAR activation is a plausible initiator of long-term pathogenesis in VLCAD deficiency. Our findings further implicate disruption of redox homeostasis as a key pathogenic mechanism in VLCAD deficiency and support the notion that a deranged thiol metabolism might be an important pathogenic factor in VLCAD deficiency.     

Structural basis for substrate fatty acyl chain specificity: crystal structure of human very-long-chain acyl-CoA dehydrogenase

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a member of the family of acyl-CoA dehydrogenases (ACADs). Unlike the other ACADs, which are soluble homotetramers, VLCAD is a homodimer associated with the mitochondrial membrane. VLCAD also possesses an additional 180 residues in the C terminus that are not present in the other ACADs. We have determined the crystal structure of VLCAD complexed with myristoyl-CoA, obtained by co-crystallization, to 1.91-A resolution. The overall fold of the N-terminal approximately 400 residues of VLCAD is similar to that of the soluble ACADs including medium-chain acyl-CoA dehydrogenase (MCAD). The novel C-terminal domain forms an alpha-helical bundle that is positioned perpendicular to the two N-terminal helical domains. The fatty acyl moiety of the bound substrate/product is deeply imbedded inside the protein; however, the adenosine pyrophosphate portion of the C14-CoA ligand is disordered because of partial hydrolysis of the thioester bond and high mobility of the CoA moiety. The location of Glu-422 with respect to the C2-C3 of the bound ligand and FAD confirms Glu-422 to be the catalytic base. In MCAD, Gln-95 and Glu-99 form the base of the substrate binding cavity. In VLCAD, these residues are glycines (Gly-175 and Gly-178), allowing the binding channel to extend for an additional 12A and permitting substrate acyl chain lengths as long as 24 carbons to bind. VLCAD deficiency is among the more common defects of mitochondrial beta-oxidation and, if left undiagnosed, can be fatal. This structure allows us to gain insight into how a variant VLCAD genotype results in a clinical phenotype.     

Loss of mitogen-activated protein kinase phosphatase-1 protects from hepatic steatosis by repression of cell death-inducing DNA fragmentation factor A (DFFA)-like effector C (CIDEC)/fat-specific protein 27

The integration of metabolic signals required for the regulation of hepatic lipid homeostasis is complex. Previously, we showed that mice lacking expression of the mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) have increased fatty acid oxidation and are protected from the development of hepatic steatosis. Here, we show that leptin receptor-deficient (db/db) mice lacking MKP-1 are also resistant to the development of hepatic steatosis. Microarray analyses of livers from db/db mice lacking MKP-1 showed suppression of peroxisome proliferator-activated receptor γ (PPARγ) target genes. We identified the fat-specific protein 27 (Fsp27), which promotes PPARγ-mediated hepatic steatosis, as repressed in livers of both db/db and high fat diet-fed mice lacking MKP-1. Hepatocytes from MKP-1-deficient mice exhibited reduced PPARγ-induced lipid droplet formation. Mechanistically, loss of MKP-1 inhibited PPARγ function by increasing MAPK-dependent phosphorylation on PPARγ at its inhibitory residue of serine 112. These results demonstrate that in addition to inhibiting hepatic fatty acid oxidation, MKP-1 promotes hepatic lipogenic gene expression through PPARγ. Hence, MKP-1 plays an important role in MAPK-mediated control of hepatic lipid homeostasis.     

Cloning of human very-long-chain acyl-coenzyme A dehydrogenase and molecular characterization of its deficiency in two patients.

Two overlapping cDNA clones (1,991 bp and 736 bp, respectively) encoding the precursor of human mitochondrial very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) were cloned and sequenced. The cDNA inserts of these clones together encompass a region of 2,177 bases, encoding the entire protein of 655 amino acids, including a 40-amino acid leader peptide and a 615-amino acid mature polypeptide. PCR-amplified VLCAD cDNAs were sequenced in cultured fibroblasts from two VLCAD-deficient patients. In both patients, a 105-bp deletion encompassing bases 1078-1182 in VLCAD cDNA was identified. The deletion seems to occur due to exon skipping during processing of VLCAD pre-mRNA. This is the first demonstration of a mutation causing VLCAD deficiency. Quantitative cDNA expression of normal human VLCAD was performed in the patients' fibroblasts, using vaccinia viral system, which demonstrated that the deficiency of the normal VLCAD protein causes impaired long-chain fatty acid beta-oxidation activity in the patients' fibroblasts. In patient fibroblasts, raising VLCAD activity to approximately 20% of normal control fibroblast activity raised palmitic acid beta-oxidation flux to the level found in control fibroblasts, which may offer important information for the rational design of future somatic gene therapy for VLCAD deficiency.