Ouliana Ziouzenkova

Lipolysis of triglyceride-rich lipoproteins generates PPAR ligands: Evidence for an antiinflammatory role for lipoprotein lipase

Increased levels of triglyceride-rich lipoproteins provoke lipid accumulation in the artery wall, triggering early inflammatory responses central to atherosclerosis like endothelial adhesion molecule expression. The endogenous mechanisms limiting such reactions remain poorly defined. Lipoprotein lipase (LPL) plays a central role in lipid metabolism by hydrolyzing triglyceride rich lipoproteins and releasing fatty acids. We found that LPL treatment reversed tumor necrosis factor α and very low-density lipoprotein (VLDL)-stimulated endothelial vascular cell adhesion molecule 1 (VCAM1) induction and VCAM1 promoter responses, thus recapitulating effects reported with synthetic peroxisome proliferator-activated receptor (PPAR) agonists. In fact, these LPL effects on VCAM1 were absent in endothelial cells isolated from PPARα-deficient mice. This finding suggests a novel antiinflammatory role for LPL. Further studies reveal specificity for PPAR activation through lipolysis in regards to lipoprotein substrate (VLDL ≫ LDL > HDL), PPAR isoform (PPARα ≫ PPARδ > PPARγ), and among fatty acid-releasing lipases. These PPAR responses required intact LPL catalytic activity. In vivo, transgenic mice overexpressing LPL had increased peroxisome proliferation, but not in the genetic absence of PPARα. Although human plasma possesses minimal PPARα activation despite containing abundant free fatty acids, marked PPARα activation is seen with human plasma after LPL is added in vitro or systemically released in vivo. These data suggest a previously uncharacterized pathway in which the key lipolytic enzyme LPL can act on circulating lipoproteins to generate PPARα ligands, providing a potentially important link between lipoprotein metabolism and distal PPARα transcriptional effects.

High-Density Lipoprotein Hydrolysis by Endothelial Lipase Activates PPARα. A Candidate Mechanism for High-Density Lipoprotein-Mediated Repression of Leukocyte Adhesion

Although high-density lipoprotein (HDL) is known to inhibit endothelial adhesion molecule expression, the mechanism for this anti-inflammatory effect remains obscure. Surprisingly, we observed that HDL no longer decreased adhesion of U937 monocytoid cells to tumor necrosis factor (TNF)&agr;-stimulated human endothelial cells (EC) in the presence of the general lipase inhibitor tetrahydrolipstatin. In considering endothelial mechanisms responsible for this effect, we found that endothelial lipase (EL) overexpression in both EC and non-EL–expressing NIH/3T3 mouse embryonic fibroblasts cells significantly decreased TNF&agr;-induced VCAM1 expression and promoter activity in a manner dependent on HDL concentration and intact EL activity. Given recent evidence for lipolytic activation of peroxisome proliferator-activated receptors (PPARs)—nuclear receptors implicated in metabolism, atherosclerosis, and inflammation—we hypothesized HDL hydrolysis by EL is an endogenous endothelial mechanism for PPAR activation. In both EL-transfected NIH cells and bovine EC, HDL significantly increased PPAR ligand binding domain activation in the order PPAR-&agr;≫-&ggr;>-&dgr;. Moreover, HDL stimulation induced expression of the canonical PPAR&agr;-target gene acyl-CoA-oxidase (ACO) in a PPAR&agr;-dependent manner in ECs. Conditioned media from EL-adenovirus transfected cells but not control media exposed to HDL also activated PPAR&agr;. PPAR&agr; activation by EL was most potent with HDL as a substrate, with lesser effects on LDL and VLDL. Finally, HDL inhibited leukocyte adhesion to TNF&agr;-stimulated ECs isolated from wild-type but not PPAR&agr;-deficient mice. This data establishes HDL hydrolysis by EL as a novel, distinct natural pathway for PPAR&agr; activation and identifies a potential mechanism for HDL-mediated repression of VCAM1 expression, with significant implications for both EL and PPARs in inflammation and vascular biology.

Autocrine Function of Aldehyde Dehydrogenase 1 as a Determinant of Diet- and Sex-Specific Differences in Visceral Adiposity

Mechanisms for sex- and depot-specific fat formation are unclear. We investigated the role of retinoic acid (RA) production by aldehyde dehydrogenase 1 (Aldh1a1, -a2, and -a3), the major RA-producing enzymes, on sex-specific fat depot formation. Female Aldh1a1−/− mice, but not males, were resistant to high-fat (HF) diet–induced visceral adipose formation, whereas subcutaneous fat was reduced similarly in both groups. Sexual dimorphism in visceral fat (VF) was attributable to elevated adipose triglyceride lipase (Atgl) protein expression localized in clusters of multilocular uncoupling protein 1 (Ucp1)-positive cells in female Aldh1a1−/− mice compared with males. Estrogen decreased Aldh1a3 expression, limiting conversion of retinaldehyde (Rald) to RA. Rald effectively induced Atgl levels via nongenomic mechanisms, demonstrating indirect regulation by estrogen. Experiments in transgenic mice expressing an RA receptor response element (RARE-lacZ) revealed HF diet–induced RARE activation in VF of females but not males. In humans, stromal cells isolated from VF of obese subjects also expressed higher levels of Aldh1 enzymes compared with lean subjects. Our data suggest that an HF diet mediates VF formation through a sex-specific autocrine Aldh1 switch, in which Rald-mediated lipolysis in Ucp1-positive visceral adipocytes is replaced by RA-mediated lipid accumulation. Our data suggest that Aldh1 is a potential target for sex-specific antiobesity therapy.

Zinc Deficiency Augments Leptin Production and Exacerbates Macrophage Infiltration into Adipose Tissue in Mice Fed a High-Fat Diet

Zinc (Zn) deficiency and obesity are global public health problems. Zn deficiency is associated with obesity and comorbid conditions that include insulin resistance and type 2 diabetes. However, the function of Zn in obesity remains unclear. Using a mouse model of combined high-fat and low-Zn intake (0.5-1.5 mg/kg), we investigated whether Zn deficiency exacerbates the extent of adiposity as well as perturbations in metabolic and immune function. C57BL/6 mice were randomly assigned to receive either a high-fat diet (HFD) or a control (C) diet for 6 wk, followed by further subdivision into 2 additional groups fed Zn-deficient diets (C-Zn, HFD-Zn), along with a C diet and an HFD, for 3 wk (n = 8-9 mice/group). The extent of visceral fat, insulin resistance, or systemic inflammation was unaffected by Zn deficiency. Strikingly, Zn deficiency significantly augmented circulating leptin concentrations (HFD-Zn vs. HFD: 3.15 ± 0.16 vs. 2.59 ± 0.12 μg/L, respectively) and leptin signaling in the liver of obese mice. Furthermore, gene expression of macrophage-specific markers ADAM8 (A disintegrin and metalloproteinase domain-containing protein 8) and CD68 (cluster of differentiation 68) was significantly greater in adipose tissue in the HFD-Zn group than in the HFD group, as confirmed by CD68 protein analysis, indicative of increased macrophage infiltration. Inspection of Zn content and mRNA profiles of all Zn transporters in the adipose tissue revealed alterations of Zn metabolism to obesity and Zn deficiency. Our results demonstrate that Zn deficiency increases leptin production and exacerbates macrophage infiltration into adipose tissue in obese mice, indicating the importance of Zn in metabolic and immune dysregulation in obesity.

Lipolytic PPAR activation: new insights into the intersection of triglycerides and inflammation?

Purpose of reviewTo examine connections between triglyceride metabolism and inflammation, especially as they relate to transcriptional regulation through peroxisomal proliferator activated receptors activation. Recent findingsPeroxisomal proliferator activated receptors, members of the steroid hormone nuclear receptor family, have been of particular interest as a mechanism through which different dietary components might control gene expression. Extensive prior work has defined the central role peroxisomal proliferator activated receptors play in many key metabolic responses, including glucose control and lipid metabolism. Emerging evidence suggests peroxisomal proliferator activated receptor activation may limit inflammation and atherosclerosis. The demonstration that certain fatty acids can activate peroxisomal proliferator activated receptors belies the potential link between nutritional components and peroxisomal proliferator activated receptor responses. Interest in this connection had been heightened by recent evidence that lipolysis in certain situations can both generate peroxisomal proliferator activated receptor ligands and limit some known inflammatory responses. SummaryLipolytic peroxisomal proliferator activated receptor activation suggests new ways in which to reconsider triglycerides and the distal consequences of their metabolism, including the possible effects on inflammation and atherosclerosis.

Aldehyde dehydrogenase 1A1: friend or foe to female metabolism?

In this review, we summarize recent advances in understanding vitamin A-dependent regulation of sex-specific differences in metabolic diseases, inflammation, and certain cancers. We focus on the characterization of the aldehyde dehydrogenase-1 family of enzymes (ALDH1A1, ALDH1A2, ALDH1A3) that catalyze conversion of retinaldehyde to retinoic acid. Additionally, we propose a “horizontal transfer of signaling” from estrogen to retinoids through the action of ALDH1A1. Although estrogen does not directly influence expression of Aldh1a1, it has the ability to suppress Aldh1a2 and Aldh1a3, thereby establishing a female-specific mechanism for retinoic acid generation in target tissues. ALDH1A1 regulates adipogenesis, abdominal fat formation, glucose tolerance, and suppression of thermogenesis in adipocytes; in B cells, ALDH1A1 plays a protective role by inducing oncogene suppressors Rara and Pparg. Considering the conflicting responses of Aldh1a1 in a multitude of physiological processes, only tissue-specific regulation of Aldh1a1 can result in therapeutic effects. We have shown through successful implantation of tissue-specific Aldh1a1−/− preadipocytes that thermogenesis can be induced in wild-type adipose tissues to resolve diet-induced visceral obesity in females. We will briefly discuss the emerging role of ALDH1A1 in multiple myeloma, the regulation of reproduction, and immune responses, and conclude by discussing the role of ALDH1A1 in future therapeutic applications.

Graded Maximal Exercise Testing to Assess Mouse Cardio-Metabolic Phenotypes

Functional assessments of cardiovascular fitness (CVF) are needed to establish animal models of dysfunction, test the effects of novel therapeutics, and establish the cardio-metabolic phenotype of mice. In humans, the graded maximal exercise test (GXT) is a standardized diagnostic for assessing CVF and mortality risk. These tests, which consist of concurrent staged increases in running speed and inclination, provide diagnostic cardio-metabolic parameters, such as, VO2max, anaerobic threshold, and metabolic crossover. Unlike the human-GXT, published mouse treadmill tests have set, not staged, increases in inclination as speed progress until exhaustion (PXT). Additionally, they often lack multiple cardio-metabolic parameters. Here, we developed a mouse-GXT with the intent of improving mouse-exercise testing sensitivity and developing translatable parameters to assess CVF in healthy and dysfunctional mice. The mouse-GXT, like the human-GXT, incorporated staged increases in inclination, speed, and intensity; and, was designed by considering imitations of the PXT and differences between human and mouse physiology. The mouse-GXT and PXTs were both tested in healthy mice (C57BL/6J, FVBN/J) to determine their ability to identify cardio-metabolic parameters (anaerobic threshold, VO2max, metabolic crossover) observed in human-GXTs. Next, theses assays were tested on established diet-induced (obese-C57BL/6J) and genetic (cardiac isoform Casq2-/-) models of cardiovascular dysfunction. Results showed that both tests reported VO2max and provided reproducible data about performance. Only the mouse-GXT reproducibly identified anaerobic threshold, metabolic crossover, and detected impaired CVF in dysfunctional models. Our findings demonstrated that the mouse-GXT is a sensitive, non-invasive, and cost-effective method for assessing CVF in mice. This new test can be used as a functional assessment to determine the cardio-metabolic phenotype of various animal models or the effects of novel therapeutics.

Moderate vitamin A supplementation in obese mice regulates tissue factor and cytokine production in a sex-specific manner

Vitamin A (vitA) regulates obesity, insulin resistance, inflammation, dyslipidemia and hemostasis through its metabolites retinaldehyde (Rald) and retinoic acid (RA) produced in endogenous enzymatic reactions. Combination of at least 3 of these conditions leads to development of metabolic syndrome (Msyn) and, consequently, type 2 diabetes and/or cardiovascular disease. Although many foods are fortified with vitA, it remains unknown what conditions of Msyn are influenced by moderate dietary vitA supplementation. A family of aldehyde dehydrogenase 1 (Aldh1) enzymes is a key contributor to obesity via sex- and fat depot-specific production of RA in adipose tissue. Therefore, we studied effects of moderate vitamin A supplementation of an obesogenic high-fat (HF) diet (4 IU vitA/g and 20 IU vitA/g HF diet) on multiple conditions and mediators of Msyn in wild-type (WT, C57Bl/6) and Aldh1a1(-/-) mice. We found that mild vitamin A supplementation did not influence obesity, fat distribution, and glucose tolerance in males and females of the same genotype. In contrast, multiplex analysis of bioactive proteins in blood showed moderately increased concentrations (10-15%) of inflammatory IL-18 and MIP-1γ in vitA supplemented vs. control WT males. Marked decrease (28-31%) in concentrations of lymphotactin and tissue factor, a key protein contributing to thrombogenesis during injury, was achieved by vitA supplementation in WT females compared to control WT females. Aldh1a1 deficiency reduced obesity, insulin resistance, suppressed many pro-inflammatory cytokines, and abolished the effects of vitA supplementation seen in WT mice. Our study revealed specific inflammatory and pro-thrombotic proteins in plasma regulated by dietary vitamin A and the critical role of endogenous vitA metabolism in these processes. The sex-specific decrease of plasma tissue factor concentrations by moderate dietary vitA supplementation could potentially reduce pro-thrombotic states in obese females.

Nitrones reverse hyperglycemia-induced endothelial dysfunction in bovine aortic endothelial cells.

Hyperglycemia has been implicated in the development of endothelial dysfunction through heightened ROS production. Since nitrones reverse endothelial nitric oxide synthase (eNOS) dysfunction, increase antioxidant enzyme activity, and suppress pro-apoptotic signaling pathway and mitochondrial dysfunction from ROS-induced toxicity, the objective of this study was to determine whether nitrone spin traps DMPO, PBN and PBN-LA were effective at duplicating these effects and improving glucose uptake in an in vitro model of hyperglycemia-induced dysfunction using bovine aortic endothelial cells (BAEC). BAEC were cultured in DMEM medium with low (5.5mM glucose, LG) or high glucose (50mM, HG) for 14 days to model in vivo hyperglycemia as experienced in humans with metabolic disease. Improvements in cell viability, intracellular oxidative stress, NO and tetrahydrobiopterin (BH4)​ levels, mitochondrial membrane potential, glucose transport, and activity of antioxidant enzymes were measured from single treatment of BAEC with nitrones for 24h after hyperglycemia. Chronic hyperglycemia significantly increased intracellular ROS by 50%, decreased cell viability by 25%, reduced NO bioavailability by 50%, and decreased (BH4) levels by 15% thereby decreasing NO production. Intracellular glucose transport and superoxide dismutase (SOD) activity were also decreased by 50% and 25% respectively. Nitrone (PBN and DMPO, 50 μM) treatment of BAEC grown in hyperglycemic conditions resulted in the normalization of outcome measures except for SOD and catalase activities. Our findings demonstrate that the nitrones reverse the deleterious effects of hyperglycemia in BAEC. We believe that in vivo testing of these nitrone compounds in models of cardiometabolic disease is warranted.

Aldehyde dehydrogenase-1a1 induces oncogene suppressor genes in B cell populations

The deregulation of B cell differentiation has been shown to contribute to autoimmune disorders, hematological cancers, and aging. We provide evidence that the retinoic acid-producing enzyme aldehyde dehydrogenase 1a1 (Aldh1a1) is an oncogene suppressor in specific splenic IgG1(+)/CD19(-) and IgG1(+)/CD19(+) B cell populations. Aldh1a1 regulated transcription factors during B cell differentiation in a sequential manner: 1) retinoic acid receptor alpha (Rara) in IgG1(+)/CD19(-) and 2) zinc finger protein Zfp423 and peroxisome proliferator-activated receptor gamma (Pparg) in IgG1(+)/CD19(+) splenocytes. In Aldh1a1(-/-) mice, splenic IgG1(+)/CD19(-) and IgG1(+)/CD19(+) B cells acquired expression of proto-oncogenic genes c-Fos, c-Jun, and Hoxa10 that resulted in splenomegaly. Human multiple myeloma B cell lines also lack Aldh1a1 expression; however, ectopic Aldh1a1 expression rescued Rara and Znf423 expressions in these cells. Our data highlight a mechanism by which an enzyme involved in vitamin A metabolism can improve B cell resistance to oncogenesis.