In established obesity, inflammation and macrophage recruitment likely contribute to the development of insulin resistance. In the current study, we set out to explore whether adipose tissue infiltration by neutrophils that occurs early (3 days) after initiating a high-fat diet (HFD) could contribute to the early occurrence of hepatic insulin resistance and to determine the role of cytosolic phospholipase A2α (cPLA2α) in this process. The 3-day HFD caused a significant upregulation of cPLA2α in periepididymal fat and in the liver. A specific antisense oligonucleotide (AS) effectively prevented cPLA2α induction, neutrophil infiltration into adipose tissue (likely involving MIP-2), and protected against 3-day HFD–induced impairment in hepatic insulin signaling and glucose over-production from pyruvate. To sort out the role of adipose neutrophil infiltration independent of cPLA2α induction in the liver, mice were injected intraperitoneally with anti–intracellular adhesion molecule-1 (ICAM-1) antibodies. This effectively prevented neutrophil infiltration without affecting cPLA2α or MIP-2, but like AS, prevented impairment in hepatic insulin signaling, the enhanced pyruvate-to-glucose flux, and the impaired insulin-mediated suppression of hepatic glucose production (assessed by clamp), which were induced by the 3-day HFD. Adipose tissue secretion of tumor necrosis factor-α (TNF-α) was increased by the 3-day HFD, but not if mice were treated with AS or ICAM-1 antibodies. Moreover, systemic TNF-α neutralization prevented 3-day HFD–induced hepatic insulin resistance, suggesting its mediatory role. We propose that an acute, cPLA2α-dependent, neutrophil-dominated inflammatory response of adipose tissue contributes to hepatic insulin resistance and glucose overproduction in the early adaptation to high-fat feeding.

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal multifactorial neurodegenerative disease characterized by selective death of motor neurons in the cortex, brainstem, and spinal cord. Cytosolic phospholipase A2 alpha (cPLA2α) upregulation and activation in the spinal cord of patients with sporadic ALS and in the spinal cord of human mutant SOD1G93A (hmSOD1) transgenic mice were recently reported.MethodscPLA2α upregulation in the brainstem and spinal cord was reduced by brain infusion of a specific antisense oligonucleotide against cPLA2α (AS), and the effect was evaluated on disease progression and brain cell activation.ResultsWe found that the elevation of cPLA2α protein expression in the spinal cord was first detected at 6-week-old hmSOD1 mice and remained elevated during their whole life span. Reduction of the elevated expression of cPLA2α in the spinal cord of hmSOD1 mice by brain infusion of an AS at week 15 (shortly before the appearance of the disease symptoms), for a duration of 6 weeks, delayed the loss of motor neuron function in comparison with hmSOD1 mice and with sense brain-infused hmSOD1 mice. To characterize the effect of cPLA2α upregulation on different processes taking place at the appearance of the disease symptoms, mice were brain infused with AS or with sense at week 15 for 3–4 weeks. The AS treatment that reduced cPLA2α upregulation in the spinal cord of AS-treated hmSOD1 mice (as analyzed at week 18–19) prevented the reduction in the number of the neurons (detected by NeuN) and inhibited astrocyte activation (detected by GFAP) and microglia activation (detected by Iba-1 and by CD40). In addition, AS treatment blunted the upregulation of the proinflammatory enzyme-inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) detected in hmSOD1 mice.ConclusionsSince specific reduction of cPLA2α in the brainstem and spinal cord significantly attenuated the development of the disease, cPLA2α may offer an efficient target for treatment of ALS.

Amyloid-b peptides generated by proteolysis of the b-amyloid precursor protein (APP) play an important role in the pathogenesis of Alzheimer's disease. The present study aimed to determine whether cytosolic phospholipase A 2 a (cPLA 2 a) plays a role in elevated APP protein expression induced by aggregated amyloid-b 1-42 (Ab) in cortical neurons and to elucidate its specific role in signal events leading to APP induction. Elevated cPLA 2 a and its activity determined by phosphorylation on serine 505 as well as elevated APP protein expression, were detected in primary rat cortical neuronal cultures exposed to Ab for 24 h and in cortical neuron of human amyloid-b 1-42 brain infused mice. Prevention of cPLA 2 a up-regulation and its activity by oligonucleotide antisense against cPLA 2 a (AS) prevented the elevation of APP protein in cortical neuronal cultures and in mouse neuronal cortex. To determine the role of cPLA 2 a in the signals leading to APP induction, increased cPLA 2 a expression and activity induced by Ab was prevented by means of AS in neuronal cortical cultures. Under these conditions, the elevated cyclooxygenase-2 and the production of prostaglandin E 2 (PGE 2 ) were prevented. Addition of PGE 2 or cyclic AMP analogue (dbcAMP) to neuronal cultures significantly increased the expression of APP protein, while the presence protein kinase A inhibitor (H-89) attenuated the elevation of APP induced by Ab. Inhibition of elevated cPLA 2 a by AS prevented the activation of cAMP response element binding protein (CREB) as detected by its phosphorylated form, its translocation to the nucleus and its DNA binding induced by Ab which coincided with cPLA 2 a dependent activation of CREB in the cortex of Ab brain infused mice. Our results show that accumulation of Ab induced elevation of APP protein expression mediated by cPLA 2 a, PGE 2 release, and CREB activation via protein kinase A pathway.

Collagen is the major structural protein in the extracellular matrix of skin produced by fibroblasts. UV exposure results in infiltration of neutrophils within the epidermis and dermis, inducing collagen damage and contributing to the process of photo-aging. Collagen-3 is an integral structural component with collagen-1, and is an important regulator of collagen-1 fibrillogenesis. Addition of neutrophils activated with TNFα to normal human dermal fibroblast cultures, but not their supernatant, caused significant collagen-3 damage. To study whether Lumenato can protect from collagen-3 damage, it was added to co-cultures of Normal human dermal fibroblasts and neutrophils activated with TNFα. Lumenato prevented collagen-3 damage induced by activated neutrophils in a dose-dependent manner in the co-cultures. Lumenato also induced a low rate of collagen-3 synthesis in a dose-dependent manner detected by pro-collagen-3 secretion, but did not affect fibroblast cell number. Although Lumenato inhibited MMP-8, MMP-9, and elastase secreted from neutrophils, its main effect was in inhibiting both NADPH oxidase-producing superoxides and MPO activity-producing halides in a dose-dependent manner that correlated with protection from collagen-3 damage. In conclusion, the results suggest that Lumenato induces low levels of collagen-3 that may contribute for skin health and is very effective in defending the co-cultures from collagen-3 damage by inhibiting free radicals secreted from neutrophils, thus, indicating Lumenato's possible potential for skin protection.

Colitis, an inflammation of the colon, is a well-characterized massive tissue injury. Cytosolic phospholipase A 2 α (cPLA 2 α) upregulation plays an important role in the development of several inflammatory diseases. The aim of the present study was to define the role of cPLA 2 α upregulation in the development of colitis. We used a mouse model of dextran sulfate sodium induced colitis. Immunoblotting analysis showed that cPLA 2 α and NF-κB were upregulated and activated in the colon from day 2 of colitis induction. This molecular event preceded the development of the disease, as determined by Disease Activity Index score, body weight, colon length, and the expression of colonic inflammatory markers, including neutrophil infiltration detected by myeloperoxidase and by NIMP-R14, ICAM-1, COX-2, iNOS upregulation and LTB 4 and TNF-α secretion. Prevention of cPLA 2 α upregulation and activity in the colon by i.v. administration of specific antisense oligonucleotides against cPLA 2 α 1 day prior and every day of exposure to dextran sulfate sodium significantly impeded the development of the disease and prevented NF-κB activation, neutrophils infiltration into the colonic mucosa, and expression of proinflammatory proteins in the colon. Our results demonstrate a critical role of cPLA 2 α upregulation in inflammation and development of murine colitis.Keywords: COX-2 r Cytosolic phospholipase A 2 α (cPLA 2 α) r DAI: Disease Activity Index r DSS-induced colitis r Inflammatory bowel disease (IBD) r Neutrophil NF-κB r TNF-α