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Elevated skeletal muscle diacylglycerols (DAGs) and ceramides can impair insulin signaling, and acylcarnitines (acylCNs) reflect impaired mitochondrial fatty acid oxidation, thus, the intramuscular lipid profile is indicative of insulin resistance. Acute (i.e., postprandial) hyperinsulinemia has been shown to elevate lipid concentrations in healthy muscle and is an independent risk factor for type 2 diabetes (T2D). However, it is unclear how the relationship between acute hyperinsulinemia and the muscle lipidome interacts across metabolic phenotypes, thus contributing to or exacerbating insulin resistance. We therefore investigated the impact of acute hyperinsulinemia on the skeletal muscle lipid profile to help characterize the physiological basis in which hyperinsulinemia elevates T2D risk. In a cross-sectional comparison, endurance athletes (n = 12), sedentary lean adults (n = 12), and individuals with obesity (n = 13) and T2D (n = 7) underwent a hyperinsulinemic-euglycemic clamp with muscle biopsies. Although there were no significant differences in total 1,2-DAG fluctuations, there was a 2% decrease in athletes versus a 53% increase in T2D during acute hyperinsulinemia (P = 0.087). Moreover, C18 1,2-DAG species increased during the clamp with T2D only, which negatively correlated with insulin sensitivity (P < 0.050). Basal muscle C18:0 total ceramides were elevated with T2D (P = 0.029), but not altered by clamp. Acylcarnitines were universally lowered during hyperinsulinemia, with more robust reductions of 80% in athletes compared with only 46% with T2D (albeit not statistically significant, main effect of group, P = 0.624). Similar fluctuations with acute hyperinsulinemia increasing 1,2 DAGs in insulin-resistant phenotypes and universally lowering acylcarnitines were observed in male mice. In conclusion, acute hyperinsulinemia elevates muscle 1,2-DAG levels with insulin-resistant phenotypes. This suggests a possible dysregulation of intramuscular lipid metabolism in the fed state in individuals with low insulin sensitivity, which may exacerbate insulin resistance. NEW & NOTEWORTHY: Postprandial hyperinsulinemia is a risk factor for type 2 diabetes and may increase muscle lipids. However, it is unclear how the relationship between acute hyperinsulinemia and the muscle lipidome interacts across metabolic phenotypes, thus contributing to insulin resistance. We observed that acute hyperinsulinemia elevates muscle 1,2-DAGs in insulin-resistant phenotypes, whereas ceramides were unaltered. Insulin-mediated acylcarnitine reductions are also hindered with high-fat feeding. The postprandial period may exacerbate insulin resistance in metabolically unhealthy phenotypes. [ABSTRACT FROM AUTHOR]

PurposeThe cell surface receptor CD40 is required for the development of retinopathies induced by diabetes and ischemia/reperfusion. The purpose of this study was to identify signaling pathways by which CD40 triggers proinflammatory responses in retinal cells, since this may lead to pharmacologic targeting of these pathways as novel therapy against retinopathies.MethodsRetinal endothelial and Müller cells were transduced with vectors that encode wild-type CD40 or CD40 with mutations in sites that recruit TNF receptor associated factors (TRAF): TRAF2,3 (ΔT2,3), TRAF6 (ΔT6), or TRAF2,3 plus TRAF6 (ΔT2,3,6). Cells also were incubated with CD40-TRAF2,3 or CD40-TRAF6 blocking peptides. We assessed intercellular adhesion molecule-1 (ICAM-1), CD40, monocyte chemoattractant protein-1 (MCP-1), VEGF, and prostaglandin E₂ (PGE₂) by fluorescence-activated cell sorting (FACS), ELISA, or mass spectrometry. Mice (B6 and CD40(-/-)) were made diabetic using streptozotocin. The MCP-1 mRNA was assessed by real-time PCR.ResultsThe CD40-mediated ICAM-1 upregulation in endothelial and Müller cells was markedly inhibited by expression of CD40 ΔT2,3 or CD40 ΔT6. The CD40 was required for MCP-1 mRNA upregulation in the retina of diabetic mice. The CD40 stimulation of endothelial and Müller cells enhanced MCP-1 production that was markedly diminished by CD40 ΔT2,3 or CD40 ΔT6. Similar results were obtained in cells incubated with CD40-TRAF2,3 or CD40-TRAF6 blocking peptides. The CD40 ligation upregulated PGE₂ and VEGF production by Müller cells, that was inhibited by CD40 ΔT2,3 or CD40 ΔT6. All cellular responses tested were obliterated by expression of CD40 ΔT2,3,6.ConclusionsBlockade of a single CD40-TRAF pathway was sufficient to impair ICAM-1, MCP-1, PGE₂, and VEGF upregulation in retinal endothelial and/or Müller cells. Blockade of CD40-TRAF signaling may control retinopathies.

Profita, Mirella, Angelo Sala, Loredana Riccobono, Elisabetta Pace, Alessandra Paterno, Simona Zarini, Liboria Siena, Angela Mirabella, Giovanni Bonsignore, and Antonio M. Vignola. 15(S)-HETE modulates [LTB.sub.4] production and neutrophil chemotaxis in chronic bronchitis. Am J Physiol Cell Physiol 279: C1249-C1258, 2000.--We evaluated the levels of 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] and the expression of 15-lipoxygeanse (15-LO) mRNA in induced sputum obtained from 10 control and 15 chronic bronchitis subjects. 15(S)-HETE was evaluated by reverse phase high-performance liquid chromatography separation followed by specific RIA. 15-LO mRNA expression was determined by primed in situ labeling. The levels of both soluble and cell-associated 15(S)-HETE resulted significantly higher in chronic bronchitis than in control subjects. The percentage of cells expressing 15-LO mRNA was significantly higher in chronic bronchitis than in control subjects (P [is less than] 0.01). Double staining for specific cell type markers and 15-LO mRNA showed macrophages and neutrophils positive for 15-LO, whereas similar staining of peripheral blood neutrophils did not show evidence for 15-LO expression, suggesting that expression of 15-LO in neutrophils takes place on migration into the airways. Because 15(S)-HETE inversely correlated with the percentage of neutrophils in sputum of chronic bronchitis subjects, we studied the effect of 15(S)-HETE on leukotriene [B.sub.4] ([LTB.sub.4]) production in vitro and evaluated the concentration of [LTB.sub.4] in induced sputum and the contribution of [LTB.sub.4] to the chemotactic activity of induced sputum samples ex vivo. The results obtained indicate that macrophages and neutrophils present within the airways of chronic bronchitis subjects express 15-LO mRNA; increased basal levels of 15(S)-HETE may contribute to modulate, through the inhibition of 5-lipoxygenase metabolites production, neutrophil infiltration and airway inflammation associated with chronic bronchitis. 5-lipoxygenase; 15-lipoxygenase

The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine mediate an array of pro- and anti-inflammatory effects. These effects are related, in part, to their metabolism by eicosanoid biosynthetic enzymes. For example, N-arachidonoyl-ethanolamine and 2-arachidonoyl-glycerol can be metabolized by cyclooxygenase-2 into PG-ethanolamide (PG-EA) and PG-glycerol (PG-G), respectively. Although PGE2 is a recognized suppressor of neutrophil functions, the impact of cyclooxygenase-derived endocannabinoids such as PGE2-EA or PGE2-G on neutrophils is unknown. This study's aim was to define the effects of these mediators on neutrophil functions and the underlying cellular mechanisms involved. We show that PGE2-G, but not PGE2-EA, inhibits leukotriene B4 biosynthesis, superoxide production, migration, and antimicrobial peptide release. The effects of PGE2-G were prevented by EP1/EP2 receptor antagonist AH-6809 but not the EP4 antagonist ONO-AE2-227. The effects of PGE2-G required its hydrolysis into PGE2, were not observed with the non-hydrolyzable PGE2-serinol amide, and were completely prevented by methyl-arachidonoyl-fluorophosphate and palmostatin B, and partially prevented by JZL184 and WWL113. Although we could detect six of the documented PG-G hydrolases in neutrophils by quantitative PCR, only ABHD12 and ABHD16A were detected by immunoblot. Our pharmacological data, combined with our protein expression data, did not allow us to pinpoint one PGE2-G lipase, and rather support the involvement of an uncharacterized lipase and/or of multiple hydrolases. In conclusion, we show that PGE2-G inhibits human neutrophil functions through its hydrolysis into PGE2, and by activating the EP2 receptor. This also indicates that neutrophils could regulate inflammation by altering the balance between PG-G and PG levels in vivo. [ABSTRACT FROM AUTHOR]