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...

Peroxisomal ascorbate peroxidase (APX) sorts indirectly via a subdomain of the ER (peroxisomal ER) to the boundary membrane of peroxisomes in tobacco Bright Yellow 2 cells. This novel subdomain characteristically appears as fluorescent reticular/circular compartments distributed variously in the cytoplasm. Further characterizations are presented herein. A peptide possessing the membrane targeting information for peroxisomal APX was fused to GFP (GFP-APX). Transiently expressed GFP-APX sorted to peroxisomes and to reticular/circular compartments; in both cases, the GFP moiety faced the cytosol. Of particular interest, both homotypic and heterotypic aggregates of peroxisomes, mitochondria, and/or plastids were formed. The latter two organelles comprised the circular portion of the reticular/circular compartments, apparently as a consequence of oligomerization (zippering) of the GFP moieties after insertion into the outer membranes of the affected organelles. These results, coupled with the accumulation of endogenous peroxisomal APX in cytoplasmic, noncircular compartment(s) following treatment with brefeldin A, indicate that authentic peroxisomal ER is composed only of a reticular compartment(s). Equally important, the data show that overexpressed, membrane-targeted GFP fusion proteins have a propensity to form organelle aggregates that may lead to misinterpretations of sorting pathways of trafficked proteins.

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