Kate J. Claycombe

(n-3) Fatty Acids Alleviate Adipose Tissue Inflammation and Insulin Resistance: Mechanistic Insights

Obesity is associated with the metabolic syndrome, a significant risk factor for developing type 2 diabetes and cardiovascular diseases. Chronic low-grade inflammation occurring in the adipose tissue of obese individuals is causally linked to the pathogenesis of insulin resistance and the metabolic syndrome. Although the exact trigger of this inflammatory process is unknown, adipose tissue hypoxia, endoplasmic reticular stress, and saturated fatty acid-mediated activation of innate immune processes have been identified as important processes in these disorders. Furthermore, macrophages and T lymphocytes have important roles in orchestrating this immune process. Although energy restriction leading to weight loss is the primary dietary intervention to reverse these obesity-associated metabolic disorders, other interventions targeted at alleviating adipose tissue inflammation have not been explored in detail. In this regard, (n-3) PUFA of marine origin both prevent and reverse high-fat-diet-induced adipose tissue inflammation and insulin resistance in rodents. We provide an update on the pathogenesis of adipose tissue inflammation and insulin resistance in obesity and discuss potential mechanisms by which (n-3) PUFA prevent and reverse these changes and the implications in human health.

Eicosapentaenoic Acid Prevents and Reverses Insulin Resistance in High-Fat Diet-Induced Obese Mice via Modulation of Adipose Tissue Inflammation

We investigated the effects of eicosapentaenoic acid (EPA) on prevention (P) and reversal (R) of high saturated-fat (HF) diet-induced obesity and glucose-insulin homeostasis. Male C57BL/6J mice were fed low-fat (LF; 10% energy from fat), HF (45% energy from fat), or a HF-EPA-P (45% energy from fat; 36 g/kg EPA) diet for 11 wk. A 4th group was initially fed HF for 6 wk followed by the HF-EPA-R diet for 5 wk. As expected, mice fed the HF diet developed obesity and glucose intolerance. In contrast, mice fed the HF-EPA-P diet maintained normal glucose tolerance despite weight gain compared with the LF group. Whereas the HF group developed hyperglycemia and hyperinsulinemia, both HF-EPA groups (P and R) exhibited normal glycemia and insulinemia. Further, plasma adiponectin concentration was lower in the HF group but was comparable in the LF and HF-EPA groups, suggesting a role of EPA in preventing and improving insulin resistance induced by HF feeding. Further analysis of adipose tissue adipokine levels and proteomic studies in cultured adipocytes indicated that dietary EPA supplementation of HF diets was associated with reduced adipose inflammation and lipogenesis and elevated markers of fatty acid oxidation. In C57BL/6J mice, EPA minimized saturated fat-induced insulin resistance and this is in part mediated by its effects on fatty acid oxidation and inflammation.

Immunity as a link between obesity and insulin resistance.

Obesity is a major public health problem in the United States and worldwide. Further, obesity is causally linked to the pathogenesis of insulin resistance, metabolic syndrome and type-2 diabetes (T2D). A chronic low-grade inflammation occurring in adipose tissue is at least in part responsible for the obesity-induced insulin resistance. This adipose tissue inflammation is characterized by changes in immune cell populations giving rise to altered adipo/cytokine profiles, which in turn induces skeletal muscle and hepatic insulin resistance. Detailed molecular mechanisms of insulin resistance, adipose tissue inflammation and the implications of these findings on therapeutic strategies are discussed in this review.

Ceramide-induced and Age-associated Increase in Macrophage COX-2 Expression Is Mediated through Up-regulation of NF-κB Activity

We have shown that the age-associated increase in lipopolysaccharide (LPS)-stimulated macrophages (Mφ) prostaglandin E2 (PGE2) production is because of ceramide-induced up-regulation of cyclooxygenase (COX)-2 transcription that leads to increased COX-2 expression and enzyme activity. To determine the mechanism of the age-related and ceramide-dependent increase in COX-2 transcription, we investigated the role of various transcription factors involved in COX-2 gene expression. The results showed that LPS-initiated activations of both consensus and COX-2-specific NF-κB, but not AP-1 and CREB, were significantly higher in Mφ from old mice than those from young mice. We further showed that the higher NF-κB activation in old Mφ was because of greater IκB degradation in the cytoplasm and p65 translocation to the nucleus. An IκB phosphorylation inhibitor, Bay 11-7082, inhibited NF-κB activation, as well as PGE2 production, COX activity, COX-2 protein, and mRNA expression in both young and old Mφ. Similar results were obtained by blocking NF-κB binding activity using a NF-κB decoy. Furthermore, NF-κB inhibition resulted in significantly greater reduction in PGE2 production and COX activity in old compared with young Mφ. Addition of ceramide to the young Mφ, in the presence or absence of LPS, increased NF-κB activation in parallel with PGE2 production. Bay 11-7082 or NF-κB decoy prevented this ceramide-induced increase in NF-κB binding activity and PGE2 production. These findings strongly suggest that the age-associated and ceramide-induced increase in COX-2 transcription is mediated through higher NF-κB activation, which is, in turn, because of a greater IκB degradation in old Mφ.

Effects of eicosapentaenoic and γ-linolenic acid on lung permeability and alveolar macrophage eicosanoid synthesis in endotoxic rats

OBJECTIVES Proinflammatory eicosanoids (cyclooxgenase and lipoxygenase metabolites of arachidonic acid) released by alveolar macrophages play an important role in endotoxin-induced acute lung injury. We investigated the effect of prefeeding rats for 21 days with enteral diets that provided the anti-inflammatory fatty acids, eicosapentaenoic acid and gamma-linolenic acid (derived from fish oil and borage oil, respectively), as compared with an n-6 fatty acid-enriched diet (corn oil) on the following: a) lung microvascular protein permeability, arterial blood pressure, and platelet and white blood cells in a model of endotoxin-induced acute lung injury; b) alveolar macrophage prostaglandin and leukotriene synthesis; and c) liver and alveolar macrophage phospholipid fatty acid composition. DESIGN Prospective, randomized, controlled, double-blind study. SETTING Research laboratory at a university medical center. SUBJECTS Male Long-Evans rats, weighing 250 g. INTERVENTIONS Rats were randomized into four dietary treatment groups and fed nutritionally complete diets (300 kcal/kg/day), containing 55.2% of the total calories from fat with either 97% corn oil, 20% fish oil, 20% fish and 5% borage oil, or 20% fish and 20% borage oil for 21 days. On day 22, lung microvascular protein permeability, mean arterial pressure, and platelet and white blood cell counts were determined for 2 hrs after an intravenous injection of Salmonella enteritidis endotoxin (10 mg/kg). In a second group of prefed rats, the phospholipid fatty acid composition was determined in liver and alveolar macrophages. Alveolar macrophages were harvested by bronchoalveolar lavage and stimulated in vitro with a calcium ionophore (A23187), and the concentrations of leukotrienes B4 and B5, thromboxane A2, prostaglandin E2, and 6-keto-prostaglandin F1 alpha were measured in a third group of prefed rats. MEASUREMENT AND MAIN RESULTS Lung permeability was greatest with corn oil and was significantly attenuated with 20% fish oil and 20% fish and 5% borage oil, and this effect approached significance with 20% fish and 20% borage oil (p = .06). The early and late hypotensive effects of endotoxin were attenuated with 20% fish oil, 20% fish and 5% borage oil, and 20% fish and 20% borage oil, as compared with corn oil. Concentrations of leukotriene B4, prostaglandin E2, and thromboxane B2 released from A23187-stimulated alveolar macrophages were significantly lower with 20% fish oil and 20% fish and 20% borage oil, as compared with corn oil. The increase in lung microvascular protein permeability with 20% fish and 20% borage oil was not significantly different than the lung microvascular protein permeability that was found in animals receiving 20% fish oil (p = .20) and 20% fish and 5% borage oil (p = .31). Alveolar macrophage and liver phospholipid concentrations of arachidonic acid were lower, and the concentrations of eicosapentaenoic acid and docosahexaenic acid were higher, with 20% fish oil, and 5% borage oil, and 20% fish and 20% borage oil, as compared with corn oil. Dihomo-gamma-linolenic acid, the desaturated and elongated intermediate of gamma-linolenic acid, was increased with 20% fish and 20% borage oil, as compared with 20% fish oil and 20% fish and 5% borage oil. CONCLUSIONS The severity of pulmonary microvascular protein permeability and the degree of hypotension were reduced with fish or fish and borage oil diets, as compared with corn oil, in endotoxic rats. The reduced synthesis of the proinflammatory arachidonic acid-derived mediators, leukotriene B4, thromboxane B2, and prostaglandin E2 from stimulated alveolar macrophages was indicative of a decrease in arachidonic acid and an increase in eicosapentaenoic acid and docosahexaenoic acid in cell membrane phospholipids.

Homocysteine increases monocyte and T-cell adhesion to human aortic endothelial cells

Although hyperhomocysteinemia has been recognized as an independent risk factor for atherosclerosis, its mechanism(s) are not well understood. Because chemotaxis and accumulation of leukocytes such as monocytes and T cells have been demonstrated to be critical events in the initiation and development of atherosclerosis, we investigated the effect of homocysteine (HCY) on U937 monocytic cells- and Jurkat T-cell-human aortic endothelial cell (HAEC) interactions under inflammatory cytokine-stimulated conditions. When HAEC were pretreated with HCY followed by stimulation with IL-1 beta, U937 and Jurkat T-cell adhesion to HAEC increased in a dose-dependent manner. The significant increase in U937 cell adhesion to HAEC was also observed when U937 cells were treated with HCY or when both cell types were treated with HCY. We also demonstrated that HCY increases endothelial surface expression and mRNA level of adhesion molecules, VCAM-1 and E-selectin. Attenuation of Jurkat T-cell and U937 cell adhesion to HAEC by monoclonal antibodies directed to specific adhesion molecules demonstrated that both VCAM-1 and E-selectin are involved in Jurkat T-cell adhesion, and VCAM-1 in U937 cell adhesion. Supplementation of HAEC with vitamin E was effective in preventing HCY-stimulated Jurkat T-cell adhesion and VCAM-1 and E-selectin expression in HAEC. These results indicate that HCY-mediated leukocyte-endothelial cell interaction is one potential mechanism by which homocysteinemia may lead to the development of atherosclerosis under inflammatory conditions. Dietary antioxidants such as vitamin E may attenuate HCY-stimulated activation of the endothelium and may help reduce the risk of vascular disease associated with hyperhomocysteinemia.

Epigenetics in Adipose Tissue, Obesity, Weight Loss, and Diabetes

Given the role that diet and other environmental factors play in the development of obesity and type 2 diabetes, the implication of different epigenetic processes is being investigated. Although it is well known that external factors can cause cell type-dependent epigenetic changes, including DNA methylation, histone tail modifications, and chromatin remodeling, the regulation of these processes, the magnitude of the changes and the cell types in which they occur, the individuals more predisposed, and the more crucial stages of life remain to be elucidated. There is evidence that obese and diabetic people have a pattern of epigenetic marks different from nonobese and nondiabetic individuals. The main long-term goals in this field are the identification and understanding of the role of epigenetic marks that could be used as early predictors of metabolic risk and the development of drugs or diet-related treatments able to delay these epigenetic changes and even reverse them. But weight gain and insulin resistance/diabetes are influenced not only by epigenetic factors; different epigenetic biomarkers have also been identified as early predictors of weight loss and the maintenance of body weight after weight loss. The characterization of all the factors that are able to modify the epigenetic signatures and the determination of their real importance are hindered by the following factors: the magnitude of change produced by dietary and environmental factors is small and cumulative; there are great differences among cell types; and there are many factors involved, including age, with multiple interactions between them.

Vitamin E and genome stability.

Free radicals and reactive oxygen species (ROS) which are generated continuously cause mutagenic alterations resulting in cancer, aging and abnormalities in the nervous system. Accumulating evidence indicates that Vitamin E, the most potent lipid peroxyl radical scavenger, may reduce free radical induced chromosomal damages through inhibition of free radical formation, and activation of endonuclease that can be triggered by intracellular oxidative stress, and by increasing the rate of removal of damaged DNA. Although some studies suggest a potential usefulness of Vitamin E in the prevention of mutagenic effects caused by genotoxic free radicals, other studies report no effects. Thus the data are not conclusive enough to be used as a basis to change the current recommended dietary allowances (RDA). Future research should address molecular mechanisms underlying the protective effects of Vitamin E and develop appropriate biologically relevant biomarkers of DNA damage to further help in determining the dietary levels of Vitamin E needed to protect the genetic pool from internally and externally induced DNA damages.

Noninvasive Detection and Localization of Vulnerable Plaque and Arterial Thrombosis With Computed Tomography Angiography/Positron Emission Tomography

Background— It has been shown that plaque uptake of fluorodeoxyglucose is proportional to macrophage density. We tested the hypothesis that arterial thrombosis occurs in areas with high fluorodeoxyglucose uptake and that computed tomography angiography (CTA) can detect thrombi in vessels. Methods and Results— Twenty New Zealand White rabbits were studied before and after atherosclerosis induction through de-endothelialization and a high-cholesterol diet; 14 were then thrombus triggered. CTA/positron emission tomography scans were performed before cholesterol diet, at the middle diet feeding, at the end of diet feeding, and after triggering. Serum inflammatory markers were measured. Maximal standardized uptake value was measured over the thoracic and upper and lower abdominal aortas and correlated with thrombosis and macrophage density on sections from the same sites. Aortic diameters averaged 2.84±1.16 mm. The sensitivity, specificity, and accuracy of CTA for detecting thrombi were 92%, 89%, and 90%, respectively. Plasminogen activator inhibitor-1 and C-reactive protein levels increased with atherosclerosis and thrombosis triggering. Maximal standardized uptake value at baseline was 0.62±0.13, 0.96±0.33 at the middle of feeding, and 1.06±0.38 at the end of feeding. Segments that developed thrombosis had the highest maximal standardized uptake value of 1.32±0.69 (113% increase; P=0.002) and had a 129% increase in macrophage density compared with segments without thrombi (P=0.01). Conclusions— Fluorodeoxyglucose uptake was proportional to the duration of cholesterol feeding and peaked with plaque disruption and thrombosis. CTA was highly accurate in detecting thrombi. Our findings in this animal model of atherosclerotic plaques with high macrophage density showed that CTA/positron emission tomography can be used to identify and localize inflamed plaques and thrombosis. With the currently available technology and nuclear tracers, however, many challenges remain before clinical applications are possible.

Insulin increases fatty acid synthase gene transcription in human adipocytes

The purpose of this study was to investigate the molecular mechanism whereby insulin increases expression of a key de novo lipogenic gene, fatty acid synthase ( FAS), in cultured human adipocytes and hepatoma cells. RNA isolated from cultured adipocytes or from Hep G2 cells treated with or without insulin (20 nM) was analyzed. In addition, run-on transcription assays and measurements of RNA half-life were performed to determine the controlled step in FAS gene regulation by insulin. We demonstrated that FAS mRNA was expressed in both Hep G2 cells and human adipocytes. Insulin induced an approximately five- and threefold increase in FAS mRNA content in adipocytes and hepatoma cells, respectively. Similar regulation of FAS was observed in adipocytes from lean and obese human subjects. Furthermore, we demonstrated that the induction of human FAS expression by insulin was due to increased transcription rate of the FAS gene in human adipocytes, whereas mRNA stabilization accounted for increased FAS mRNA content in hepatoma cells. In conclusion, we report here for the first time expression of human FAS mRNA and its specific transcriptional induction by insulin in cultured human adipocytes.