Objective-Monocyte/macrophage inflammation is an important contributor to diabetes and cardiovascular disease.Studies have suggested saturated fatty acids (SFA) induce monocyte inflammation in a Toll-like receptor-4 -dependent manner, but recent data suggest SFA do not directly interact with Toll-like receptor-4. The present study tests the novel hypothesis that metabolism of SFA cooperatively amplifies Toll-like receptor-4 -mediated inflammation. Methods and Results-THP-1 monocytes exposed to 100 mol/L SFA in vitro for 16 hours followed by 1 ng/mL lipopolysaccharide demonstrated enhanced IL-6 and IL-8 mRNA and protein expression (Ϸ3-fold higher than the sum of individual responses to SFA and lipopolysaccharide). SFA had similar effects on THP-1 macrophages and primary human monocytes. This amplified lipopolysaccharide response could be blocked by inhibition of SFA metabolism to ceramide and restored by cell-permeable ceramide. Both SFA and ceramide activated PKC-and the mitogen-activated protein kinases Erk, JNK, and p38. Inhibition of these pathways prevented the SFA-induced increase in cytokine expression. Conclusion-These results provide evidence for potent amplification of monocyte/macrophage innate immune responses by a novel pathway requiring metabolism of SFA to ceramide and activation of PKC-/mitogen-activated protein kinases. These findings demonstrate how nutrient excess may modulate innate immune system activation and possibly contribute to development of diabetes and cardiovascular disease. Key Words: ceramide Ⅲ mitogen-activated protein kinase Ⅲ monocytes Ⅲ protein kinase C Ⅲ Toll-like receptor I n vitro and animal studies have implicated chronic inflammation in the development and progression of insulin resistance and type 2 diabetes, and in the excess cardiovascular disease risk associated with diabetes. 1 These findings closely parallel results from epidemiological studies that demonstrate plasma inflammatory markers (such as the acute phase proteins, C-reactive protein and serum amyloid A, and the proinflammatory cytokines, IL-6, IL-1␤, and possibly tumor necrosis factor-␣) as predictors, and potential mediators, of these conditions. [1][2][3] See accompanying article on page 692Monocytes and macrophages are critical cells in the development of insulin resistance and cardiovascular disease. They accumulate early in atherosclerotic lesion development and remain throughout the transition to advanced atheroma. 4 They are closely linked to the chronic inflammatory processes that underlie plaque formation. Similarly, macrophages and macrophage-derived proinflammatory factors have been detected in adipose and other tissues 5 and appear to contribute to development of insulin resistance and hyperglycemia. 6,7 Determining what factors play important roles in activating monocytes/macrophages and contribute to systemic inflammation is essential to understanding the pathophysiology of insulin resistance and cardiovascular disease.Bacterial lipopolysaccharide (LPS) is a potent inducer of inflammation in m...

BackgroundThe objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions.MethodologyBuffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot.Principal FindingsThe established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence.ConclusionsWe have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions.

Previous studies have reported that elevated myocardial lipids in a model of mild-to-moderate heart failure increased mitochondrial function, but did not alter left ventricular function. Whether more prolonged exposure to high dietary lipids would promote a lipotoxic phenotype in mitochondrial and myocardial contractile function has not been determined. We tested the hypothesis that prolonged exposure to high dietary lipids, following coronary artery ligation, would preserve myocardial and mitochondrial function in heart failure. Rats underwent ligation or sham surgery and were fed normal (10% kcal fat) (SHAM, HF) or high fat diet (60% kcal saturated fat) (SHAM+FAT, HF+FAT) for sixteen weeks. Although high dietary fat was accompanied by myocardial tissue triglyceride accumulation (SHAM 1.47±0.14; SHAM+FAT 2.32±0.14; HF 1.34±0.14; HF+FAT 2.21±0.20 μmol/gww), fractional shortening was increased 16% in SHAM+FAT and 28% in HF+FAT compared to SHAM and HF, respectively. Despite increased medium-chain acyl-CoA dehydrogenase (MCAD) activity in interfibrillar mitochondria (IFM) of both SHAM+FAT and HF +FAT, dietary lipids also were associated with decreased state 3 respiration using palmitoylcarnitine (SHAM 369±14; SHAM+FAT 307±23; HF 354±13; HF+FAT 366±18 nAO·min −1 ·mg −1 ) in SHAM +FAT compared to SHAM and HF+FAT. State 3 respiration in IFM also was decreased in SHAM +FAT relative to SHAM using succinate and DHQ. In conclusion, high dietary lipids promoted myocardial lipid accumulation, but were not accompanied by alterations in myocardial contractile function typically associated with lipotoxicity. In normal animals, high dietary fat decreased mitochondrial respiration, but also increased MCAD activity. These studies support the concept that high fat feeding can modify multiple cellular pathways that differentially affect mitochondrial function under normal and pathological conditions.