Palmitoleate Reverses High Fat-induced Proinflammatory Macrophage Polarization via AMP-activated Protein Kinase (AMPK)

A rise in tissue-embedded macrophages displaying “M1-like” proinflammatory polarization is a hallmark of metabolic inflammation during a high fat diet or obesity. Here we show that bone marrow-derived macrophages (BMDM) from high fat-fed mice retain a memory of their dietary environment in vivo (displaying the elevated proinflammatory genes Cxcl1, Il6, Tnf, Nos2) despite 7-day differentiation and proliferation ex vivo. Notably, 6-h incubation with palmitoleate (PO) reversed the proinflammatory gene expression and cytokine secretion seen in BMDM from high fat-fed mice. BMDM from low fat-fed mice exposed to palmitate (PA) for 18 h ex vivo also showed elevated expression of proinflammatory genes (Cxcl1, Il6, Tnf, Nos2, and Il12b) associated with M1 polarization. Conversely, PO treatment increased anti-inflammatory genes (Mrc1, Tgfb1, Il10, Mgl2) and oxidative metabolism, characteristic of M2 macrophages. Therefore, saturated and unsaturated fatty acids bring about opposite macrophage polarization states. Coincubation of BMDM with both fatty acids counteracted the PA-induced Nos2 expression in a PO dose-dependent fashion. PO also prevented PA-induced IκBα degradation, RelA nuclear translocation, NO production, and cytokine secretion. Mechanistically, PO exerted its anti-inflammatory function through AMP-activated protein kinase as AMP kinase knockout or inhibition by Compound C offset the PO-dependent prevention of PA-induced inflammation. These results demonstrate a nutritional memory of BMDM ex vivo, highlight the plasticity of BMDM polarization in response to saturated and unsaturated fatty acids, and identify the potential to reverse diet- and saturated fat-induced M1-like polarization by administering palmitoleate. These findings could have applicability to reverse obesity-linked inflammation in metabolically relevant tissues.     

Augmentation of myelopoiesis in a murine host bearing a T cell lymphoma following in vivo administration of proton pump inhibitor pantoprazole

Proton pump inhibitors (PPI) are being proposed as potent antitumor agents, owing to their ability to specifically induce tumor cell death by reversing H⁺ ion homeostasis. As tumor growth induces myelosuppression in tumor-bearing hosts, it remains unclear if PPI can also modulate tumor-induced myelosuppression. Thus, we studied the effect of in vivo administration of pantoprazole (PPZ), a PPI, on myelopoiesis in a murine model of a transplantable T cell lymphoma, designated as Dalton’s lymphoma (DL). Intraperitoneal administration of PPZ to tumor-bearing mice resulted in an enhanced bone marrow cellularity, inhibited induction of apoptosis and augmented bone marrow cell (BMC) survival. BMC of PPZ-administered tumor-bearing mice showed elevated number of F4/80 positive cells, augmented colony forming ability and differentiation in bone marrow-derived macrophages (BMDM) with higher expression of F4/80 and CD11c markers. This study also presents evidences to indicate that PPZ-dependent augmentation of myelopoiesis in the tumor-bearing host is dependent on an enhanced expression of M-CSF and receptors for M-CSF & GM-CSF in BMC, along with a modulation in the expression of cell survival regulatory molecules PUMA, Bcl2, p53 and caspase-activated DNase (CAD). BMDM obtained from PPZ-administered tumor-bearing mice also showed an augmented expression of TLR-2, tumoricidal activity, production of NO and monokines: IL-1, IL-6 & TNF-α. The study discusses the possible mechanisms underlying PPZ-dependent augmentation of myelopoiesis. Taken together, the present study proposes that a PPZ-dependent alleviation of tumor-induced myelosuppression could contribute to an augmented myelopoiesis.     

Myelopoietic Efficacy of Orlistat in Murine Hosts Bearing T Cell Lymphoma: Implication in Macrophage Differentiation and Activation

Orlistat, an inhibitor of fatty acid synthase (FASN), acts as an antitumor agent by blocking de novo fatty acid synthesis of tumor cells. Although, myelopoiesis also depends on de novo fatty acid synthesis, the effect of orlistat on differentiation of macrophages, which play a central role in host’s antitumor defence, remains unexplored in a tumor-bearing host. Therefore, the present investigation was undertaken to examine the effect of orlistat administration on macrophage differentiation in a T cell lymphoma bearing host. Administration of orlistat (240 mg/kg/day/mice) to tumor-bearing mice resulted in a decline of tumor load accompanied by an augmentation of bone marrow cellularity and survival of bone marrow cells (BMC). The expression of apoptosis regulatory caspase-3, Bax and Bcl2 was modulated in the BMC of orlistat-administered tumor-bearing mice. Orlistat administration also resulted in an increase in serum level of IFN-γ along with decreased TGF-β and IL-10. BMC of orlistat-administered tumor-bearing mice showed augmented differentiation into macrophages accompanied by enhanced expression of macrophage colony stimulating factor (M-CSF) and its receptor (M-CSFR). The macrophages differentiated from BMC of orlistat-administered mice showed characteristic features of M₁ macrophage phenotype confirmed by expression of CD11c, TLR-2, generation of reactive oxygen species, phagocytosis, tumor cell cytotoxicity, production of IL-1,TNF-α and nitric oxide. These novel findings indicate that orlistat could be useful to support myelopoesis in a tumor-bearing host.     

Astaxanthin exerts anti-inflammatory and antioxidant effects in macrophages in NRF2-dependent and independent manners

Although anti-inflammatory effects of astaxanthin (ASTX) have been suggested, the underlying mechanisms have not been fully understood. Particularly, the modulatory action of ASTX in the interplay between nuclear factor E2-related factor 2 (NRF2) and nuclear factor κB (NFκB) to exert its anti-inflammatory effect in macrophages is unknown. The effect of ASTX on mRNA and protein expression of pro-inflammatory and antioxidant genes and/or cellular reactive oxygen species (ROS) accumulation were determined in RAW 264.7 macrophages, bone marrow-derived macrophages (BMDM) from wild-type (WT) and Nrf2-deficient mice, and/or splenocytes and peritoneal macrophages of obese mice fed ASTX. The effect of ASTX on M1 and M2 macrophage polarization was evaluated in BMDM. ASTX significantly decreased LPS-induced mRNA expression of interleukin 6 (Il-6) and Il-1β by inhibiting nuclear translocation of NFκB p65; and attenuated LPS-induced ROS with an increase in NRF2 nuclear translocation, concomitantly decreasing NADPH oxidase 2 expression in RAW 264.7 macrophages. In BMDM of WT and Nrf2-deficient mice, ASTX decreased basal and LPS-induced ROS accumulation. The induction of Il-6 mRNA by LPS was repressed by ASTX in both types of BMDM while Il-1β mRNA was decreased only in WT BMDM. Furthermore, ASTX consumption lowered LPS sensitivity of splenocytes in obese mice. ASTX decreased M1 polarization of BMDM while increasing M2 polarization. ASTX exerts its anti-inflammatory effect by inhibiting nuclear translocation of NFκB p65 and by preventing ROS accumulation in NRF2-dependent and -independent mechanisms. Thus, ASTX is an agent with anti-inflammatory and antioxidant properties that may be used for the prevention of inflammatory conditions.     

Depletion of Cutaneous Macrophages and Dendritic Cells Promotes Growth of Basal Cell Carcinoma in Mice

Basal cell carcinoma (BCC) belongs to the group of non-melanoma skin tumors and is the most common tumor in the western world. BCC arises due to mutations in the tumor suppressor gene Patched1 (Ptch). Analysis of the conditional Ptch knockout mouse model for BCC reveals that macrophages and dendritic cells (DC) of the skin play an important role in BCC growth restraining processes. This is based on the observation that a clodronate-liposome mediated depletion of these cells in the tumor-bearing skin results in significant BCC enlargement. The depletion of these cells does not modulate Ki67 or K10 expression, but is accompanied by a decrease in collagen-producing cells in the tumor stroma. Together, the data suggest that cutaneous macrophages and DC in the tumor microenvironment exert an antitumor effect on BCC.     

The Alteration of MiR-222 and Its Target Genes in Nickel-Induced Tumor

Nickel is an important kind of metal and a necessary trace element in people’s production and livelihood; it is also a well-confirmed human carcinogen. In the past few years, researchers did a large number of studies about the molecular mechanisms of nickel carcinogenesis, and they focused on activation of proto-oncogenes and inactivation of anti-oncogenes caused by gene point mutation, gene deletion, gene amplification, DNA methylation, chromosome condensation, and so on that were induced by nickel. However, the researches on tumorigenic molecular mechanisms regulated by microRNAs (miRNAs) are rare. In this study, we established nickel-induced tumor by injecting Ni₃S₂ compounds to Wistar Rattus. By establishing a cDNA library of miRNA from rat muscle tumor tissue induced by Ni₃S₂, we found that the expression of miR-222 was significantly upregulated in tumor tissue compared with the normal tissue. As we expected, the expression levels of target genes of miR-222, CDKN1B and CDKN1C, were downregulated in the nickel-induced tumor. The same alteration of miR-222 and its target genes was also found in malignant 16HBE cells induced with Ni₃S₂ compounds. We conclude that miR-222 may promote cell proliferation infinitely during nickel-induced tumorigenesis in part by regulating the expression of its target genes CDKN1B and CDKN1C. Our study elucidated a novel molecular mechanism of nickel-induced tumorigenesis.     

Stimulation and inhibition of neoplastic cell growth by tumor promoter-treated macrophages

Macrophages treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent inflammatory and tumor-promoting agent, can have the diametrically opposed functions of contact-mediated tumor cytotoxicity and release of soluble clonal proliferation factor(s) for tumor cells. In vitro TPA treatment of macrophages at 1.0 ng/ml induced prostaglandin E₂ release and morphological changes analogous to cell activation. In addition, conditioned medium from macrophages pulsed with TPA enhanced M109 carcinoma colony formation in vitro. Although macrophages were not rendered tumoricidal by TPA in vitro, cytotoxic macrophages were recovered from mice following ip treatment with TPA at 1–100 μg/kg. This indicated an indirect pathway for the activation of macrophages by TPA. The very weak tumor promoting 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate lacked effects on macrophages at all doses tested. The possibility that macrophage secretions (e.g., prostaglandin E₂, angiogenesis-stimulating factor(s), and clonal proliferation factor(s) for carcinogen-triggered cells) may be involved in the tumor promotion process is discussed.     

Loss of Nrf2 in bone marrow-derived macrophages impairs antigen-driven CD8+ T cell function by limiting GSH and Cys availability

NF-E2-related factor 2 (Nrf2), known to protect against reactive oxygen species, has recently been reported to resolve acute inflammatory responses in activated macrophages. Consequently, disruption of Nrf2 promotes a proinflammatory macrophage phenotype. In the current study, we addressed the impact of this macrophage phenotype on CD8⁺ T cell activation by using an antigen-driven coculture model consisting of Nrf2^−/− and Nrf2^+/+ bone marrow-derived macrophages (BMDMΦ) and transgenic OT-1 CD8⁺ T cells. OT-1 CD8⁺ T cells encode a T cell receptor that specifically recognizes MHC class I-presented ovalbumin OVA(257–264) peptide, thereby causing a downstream T cell activation. Interestingly, coculture of OVA(257–264)-pulsed Nrf2^−/− BMDMΦ with transgenic OT-1 CD8⁺ T cells attenuated CD8⁺ T cell activation, proliferation, and cytotoxic function. Since the provision of low-molecular-weight thiols such as glutathione (GSH) or cysteine (Cys) by macrophages limits antigen-driven CD8⁺ T cell activation, we quantified the amounts of intracellular and extracellular GSH and Cys in both cocultures. Indeed, GSH levels were strongly decreased in Nrf2^−/− cocultures compared to wild-type counterparts. Supplementation of thiols in Nrf2^−/− cocultures via addition of glutathione ester, N-acetylcysteine, β-mercaptoethanol, or cysteine itself restored T cell proliferation as well as cytotoxicity by increasing intracellular GSH. Mechanistically, we identified two potential Nrf2-regulated genes involved in thiol synthesis in BMDMΦ: the cystine transporter subunit xCT and the modulatory subunit of the GSH-synthesizing enzyme γ-GCS (GCLM). Pharmacological inhibition of γ-GCS-dependent GSH synthesis as well as knockdown of the cystine antiporter xCT in Nrf2^+/+ BMDMΦ mimicked the effect of Nrf2^−/− BMDMΦ on CD8⁺ T cell function. Our findings demonstrate that reduced levels of GCLM as well as xCT in Nrf2^−/− BMDMΦ limit GSH availability, thereby inhibiting antigen-induced CD8⁺ T cell function.     

Pten Null Prostate Epithelium Promotes Localized Myeloid-Derived Suppressor Cell Expansion and Immune Suppression during Tumor Initiation and Progression

Chronic inflammation is known to be associated with prostate cancer development, but how epithelium-associated cancer-initiating events cross talk to inflammatory cells during prostate cancer initiation and progression is largely unknown. Using the Pten null murine prostate cancer model, we show an expansion of Gr-1⁺ CD11b⁺ myeloid-derived suppressor cells (MDSCs) occurring intraprostatically immediately following epithelium-specific Pten deletion without expansion in hematopoietic tissues. This MDSC expansion is accompanied by sustained immune suppression. Prostatic Gr-1⁺ CD11b⁺ cells, but not those isolated from the spleen of the same tumor-bearing mice, suppress T cell proliferation and express high levels of Arginase 1 and iNOS. Mechanistically, the loss of PTEN in the epithelium leads to a significant upregulation of genes within the inflammatory response and cytokine-cytokine receptor interaction pathways, including Csf1 and Il1b, two genes known to induce MDSC expansion and immunosuppressive activities. Treatment of Pten null mice with the selective CSF-1 receptor inhibitor GW2580 decreases MDSC infiltration and relieves the associated immunosuppressive phenotype. Our study indicates that epithelium-associated tumor-initiating events trigger the secretion of inflammatory cytokines and promote localized MDSC expansion and immune suppression, thereby promoting tumor progression.     

Cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber Holothuria moebii

BackgroundWhether sulfated saponins from Holothuria moebii inhibit the proliferation of colorectal cancer cells and have anti-colorectal tumor effects in animal model has not been investigated.PurposeTo evaluate the cytotoxic and anti-colorectal tumor effects of sulfated saponins from sea cucumber Holothuria moebii.Method(1) Column chromatography was used to prepare the total and individual saponins and HPLC was applied to define the components of the total saponins; (2) the activity of the total and individual saponins inhibiting the proliferation of human colorectal cancer cells was determined by SRB assay and the apoptosis induced by the saponins was qualified using cytometric analysis with Annexin V-FITC/PI double staining; and (3) the antitumor effects of the sulfated saponins on colorectal CT-26 tumor-bearing Balb/c mice were tested.ResultsThe total and individual sulfated saponins significantly inhibited the proliferation of four different human colorectal cancer cells with IC₅₀ values ranging from 1.04 to 4.08 μM (or 1.46 to 3.24 μg/ml for total saponins) and induced late apoptosis at an early treatment time in cancer cells. The total saponins (120 mg/kg) had antitumor activity in colorectal CT-26 tumor-bearing Balb/c mice.ConclusionThe sulfated saponins from H. moebii remarkably inhibited the proliferation of different human colorectal cancer cells and had significant anti-colorectal tumor activity in animal model.     

Lipocalin (LCN) 2 Mediates Pro-Atherosclerotic Processes and Is Elevated in Patients with Coronary Artery Disease

Background

Lipocalin (LCN) 2 is associated with multiple acute and chronic inflammatory diseases but the underlying molecular and cellular mechanisms remain unclear. Here, we investigated whether LCN2 is released from macrophages and contributes to pro-atherosclerotic processes and whether LCN2 plasma levels are associated with the severity of coronary artery disease progression in humans.

Methods and Results

In an autocrine-paracrine loop, tumor necrosis factor (TNF)-α promoted the release of LCN2 from murine bone-marrow derived macrophages (BMDM) and vice versa. Moreover, LCN2 stimulation of BMDM led to up-regulation of M1 macrophage markers. In addition, enhanced migration of monocytic J774A.1 cells towards LCN2 was observed. Furthermore, LCN2 increased the expression of the scavenger receptors Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) as well as scavenger receptor class A-1 (SRA-1) and induced the conversion of macrophages to foam cells. In atherosclerotic lesions of low density lipoprotein receptor-deficient (ldlr ^−/−) mice fed a high fat, high cholesterol diet, LCN2 was found to be co-localized with macrophages in the shoulder region of the atherosclerotic plaque. In addition, LCN2 plasma levels were significantly increased in plasma samples of these mice. Finally, LCN2 plasma levels correlated with the severity of coronary artery disease (CAD) in patients as determined by coronary angiography.

Conclusions

Here we demonstrated that LCN2 plays a pivotal role in processes involved in atherogenesis by promoting polarization and migration of monocytic cells and development of macrophages towards foam cells. Moreover, LCN2 may be used as a prognostic marker to determine the status of CAD progression.     

Impact of microbial tolerance in persistent secondary Klebsiella pneumoniae peritonitis

Purpose and methodsMicrobial tolerance represents a diminished pro-inflammatory response following repeated stimulation by a host of pathogen-associated molecular patterns (PAMPs) of varying origins. Toll-like receptors (TLRs) have been centrally implicated in the development of tolerance. The purpose of this study was to investigate the impact of tolerance in a previously described murine model.C57BL/6 mice were pretreated intraperitoneally with phosphate buffered saline (PBS), heat-killed Klebsiella 2 × 10⁸ CFU (hkKlebsiella), LPS 10 mg/kg (LPS 10), or BLP 10 mg/kg (BLP 10). Following pretreatment, peritonitis was induced 24 h later using 10³ intraperitoneal Klebsiella CFU. Peritoneal concentrations of TNF-α, IL-10 and nitric oxide (NO), as well as characteristic cell patterns, were determined. Long-term consequences of microbial tolerance were assessed by measuring survival and weight-loss.ResultsFollowing in vitro stimulation with Klebsiella 10⁵ and 10³ CFU, TNF-α and IL-10 secretion were diminished in macrophages harvested from mice pretreated with hkKlebsiella, LPS 10 and BLP 10. Pretreated animals had significantly lower bacterial counts. Conversely, local NO levels were elevated. Survival was not different between the groups.ConclusionPretreatment with TLR ligands induced microbial tolerance, with reduced peritoneal cytokine concentrations and enhanced early bacterial clearance. However, this did not translate into improved survival.     

Analysis of inducible nitric oxide synthase gene polymorphisms in vitiligo in Han Chinese people

Background

Vitiligo is a chronic depigmented skin disorder with regional melanocytes depletion. The pathogenesis was not completely clarified. Recently, more and more evidence suggested that polymorphisms of some genes are associated with vitiligo risk. Here, we want to examine the association between the inducible nitric oxide synthase (iNOS) gene polymorphisms and the risk of vitiligo in Chinese populations.

Methods and Principal Findings

In a hospital-based case-control study of 749 patients with vitiligo and 763 age- and sex-matched healthy controls, three polymorphisms of iNOS gene were genotyped by using the PCR-restriction fragment length polymorphism (PCR-RFLP) and mutagenically separated PCR (MS-PCR) methods, respectively. We found the iNOS-954 polymorphism was associated with a significantly higher risk of vitiligo (adjusted OR = 1.36, 95% CI = 1.02–1.81). Furthermore, this association is more pronounced in vulgaris vitiligo, active vitiligo and vitiligo without other autoimmune diseases in the stratification study. Analysis of haplotypes showed increased risk for the C _-1173 C _-954 C_{Ex 16+14} (OR = 1.44, 95% CI = 1.01–1.74). In addition, the serum iNOS activity is significantly associated with iNOS-954 combined genotype (GC+CC) and is much higher in vitiligo patients than in the controls (P<0.01). Logistic regression analysis of iNOS activity showed increased risk between higher activity and iNOS-954 G→C variant genotype carriers (P_trend<0.001).

Conclusions and Significance

INOS gene polymorphisms may play an important role in the genetic susceptibility to the development of vitiligo.     

Antitumor and immunomodulating activity of polysaccharides from Enteromorpha intestinalis

Two polysaccharides (WEA and WEB) were isolated from Enteromorpha intestinalis by hot water extraction, anion-exchange, and gel-permeation chromatography. The average molecular weights (Mw) of the two fractions were 72.03 kDa (WEA) and 60.12 kDa (WEB). WEA was composed of Rha, Xyl, Man, and Glc in a molar ratio of 1.39:1.00:0.13:3.23. WEB consisted of Rha, Xyl, Gal, and GlcA (glucuronic acid) in a molar ratio of 7.32:1.00:0.51:1.28. Both polysaccharides could inhibit tumor growth in S180 tumor-bearing mice, and increased the relative spleen and thymus weight. They also increased the expression of tumor necrosis factor-alpha (TNF-α) in serum. WEA and WEB induced lymphocyte proliferation, increased the production of TNF-α in macrophages, and stimulated macrophages to produce nitric oxide dose-dependently through the up-regulation of inducible NO synthase activity. However, no direct cytotoxicity against Sarcoma 180 was investigated in vitro. These results indicate that the antitumor effects of these polysaccharides are associated with immunostimulation.     

Chlamydia psittaci plasmid-encoded CPSIT_P7 induces macrophage polarization to enhance the antibacterial response through TLR4-mediated MAPK and NF-κB pathways

Although the protective effects of Chlamydia psittaci plasmid-encoded protein CPSIT_P7 as vaccine antigens to against chlamydial infection have been confirmed in our previous study, the function and mechanism of CPSIT_P7 inducing innate immunity in the antibacterial response remain unknown. Here, we found that plasmid protein CPSIT_P7 could induce M1 macrophage polarization upregulating the genes of the surface molecule CD86, proinflammatory cytokines (TNF-α, IL-6, and IL-1β), and antibacterial effector NO synthase 2 (iNOS). During M1 macrophage polarization, macrophages acquire phagocytic and microbicidal competence, which promotes the host antibacterial response. As we observed that CPSIT_P7-induced M1 macrophages could partially reduce the infected mice pulmonary Chlamydia psittaci load. Furthermore, CPSIT_P7 induced M1 macrophage polarization through the TLR4-mediated MAPK and NF-κB pathways. Collectively, our results highlight the effect of CPSIT_P7 on macrophage polarization and provide new insights into new prevention and treatment strategies for chlamydial infection.     

IFN-γ upregulates survivin and Ifi202 expression to induce survival and proliferation of tumor-specific T cells

Background

A common procedure in human cytotoxic T lymphocyte (CTL) adoptive transfer immunotherapy is to expand tumor-specific CTLs ex vivo using CD3 mAb prior to transfer. One of the major obstacles of CTL adoptive immunotherapy is a lack of CTL persistence in the tumor-bearing host after transfer. The aim of this study is to elucidate the molecular mechanisms underlying the effects of stimulation conditions on proliferation and survival of tumor-specific CTLs.

Methodology/Principal Findings

Tumor-specific CTLs were stimulated with either CD3 mAb or cognate Ag and analyzed for their proliferation and survival ex vivo and persistence in tumor-bearing mice. Although both Ag and CD3 mAb effectively induced the cytotoxic effecter molecules of the CTLs, we observed that Ag stimulation is essential for sustained CTL proliferation and survival. Further analysis revealed that Ag stimulation leads to greater proliferation rates and less apoptosis than CD3 mAb stimulation. Re-stimulation of the CD3 mAb-stimulated CTLs with Ag resulted in restored CTL proliferative potential, suggesting that CD3 mAb-induced loss of proliferative potential is reversible. Using DNA microarray technology, we identified that survivin and ifi202, two genes with known functions in T cell apoptosis and proliferation, are differentially induced between Ag- and CD3 mAb-stimulated CTLs. Analysis of the IFN-γ signaling pathway activation revealed that Ag stimulation resulted in rapid phosphorylation of STAT1 (pSTAT1), whereas CD3 mAb stimulation failed to activate STAT1. Chromatin immunoprecipitation revealed that pSTAT1 is associated with the promoters of both survivin and ifi202 in T cells and electrophoresis mobility shift assay indicated that pSTAT1 directly binds to the gamma activation sequence element in the survivin and ifi202 promoters. Finally, silencing ifi202 expression significantly decreased T cell proliferation.

Conclusions/Significance

Our findings delineate a new role of the IFN-γ signaling pathway in regulating T cell proliferation and apoptosis through upregulating survivin and ifi202 expression.     

Involvement of apoptosis and autophagy in the death of RPMI 8226 multiple myeloma cells by two enantiomeric sigma receptor ligands

Over-expression of σ receptors by many tumor cell lines makes ligands for these receptors attractive as potential chemotherapeutic drugs. Enantiomeric piperazines (S)-4 and (R)-4 were prepared as potential σ-receptor ligands in a chiral pool synthesis starting from (S)- and (R)-aspartate. Both compounds showed high affinities for the σ₁ and σ₂ receptors. In the human multiple myeloma cell line RPMI 8226, a line expressing high levels of σ receptors, both compounds inhibited cell proliferation with IC₅₀ values in the low μM range. No chiral differentiation between either the σ receptor binding affinity or the cytotoxicity of the two enantiomers was observed. Both compounds induced apoptosis, which was evidenced by nuclear condensation, binding of annexin-V to phosphatidylserine in the outer leaf of the cell membrane, cleavage products of poly(ADP-ribose) polymerase-1 (PARP-1) and caspase-8 as well as the expression of bcl₂ family members bax, bad and bid. However, apoptosis appeared to be caspase independent. Increased levels of the phosphorylated form of the microtubule associated protein light chain 3-II (LC3-II), an autophagosome marker, gave evidence that both compounds induced autophagy. However, further data (e.g., treatment with wortmannin) indicate that autophagy is incomplete and not cytoprotective. Lipid peroxidation (LPO) was observed in RPMI 8226 cells treated with the two compounds, and the lipid antioxidant α-tocopherol attenuated LPO. Interestingly, α-tocopherol reduced significantly both apoptosis and autophagy induced by the compounds. These results provide evidence that, by initiating LPO and changes in mitochondrial membrane potential, both compounds induce apoptosis and autophagy in RPMI 8226 cells.     

PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese,Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

Synthetic peroxisome proliferator-activated receptor (PPAR) agonists are used to treat dyslipidemia and insulin resistance. In this study, we examined molecular mechanisms that explain differential effects of a PPARα agonist (fenofibrate) and a PPARγ agonist (rosiglitazone) on macrophages during obesity-induced atherogenesis. Twelve-week-old mice with combined leptin and LDL-receptor deficiency (DKO) were treated with fenofibrate, rosiglitazone or placebo for 12 weeks. Only rosiglitazone improved adipocyte function, restored insulin sensitivity, and inhibited atherosclerosis by decreasing lipid-loaded macrophages. In addition, it increased interleukin-1 receptor-associated kinase-3 (Irak3) and decreased monocyte chemoattractant protein-1 (Mcp1) expressions, indicative of a switch from M1 to M2 macrophages. The differences between fenofibrate and rosiglitazone were independent of Pparγ expression. In bone marrow-derived macrophages (BMDM), we identified the rosiglitazone-associated increase in adiponectin as cause of the increase in Irak3. Interestingly, the deletion of Irak3 in BMDM (IRAK3^−/− BMDM) resulted in activation of the canonical NFκB signaling pathway and increased Mcp1 protein secretion. Rosiglitazone could not decrease the elevated Mcp1 secretion in IRAK3^−/− BMDM directly and fenofibrate even increased the secretion, possibly due to increased mitochondrial reactive oxygen species production. Furthermore, aortic extracts of high-fat insulin-resistant LDL-receptor deficient mice, with lower adiponectin and Irak3 and higher Mcp1, showed accelerated atherosclerosis. In aggregate, our results emphasize an interaction between PPAR agonist-mediated increase in adiponectin and macrophage-associated Irak3 in the protection against atherosclerosis by PPAR agonists.     

L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS-Dependent Mechanism

Dietary L-citrulline is thought to modulate muscle protein turnover by increasing L-arginine availability. To date, the direct effects of increased L-citrulline concentrations in muscle have been completely neglected. Therefore, we determined the role of L-citrulline in regulating cell size during catabolic conditions by depriving mature C2C12 myotubes of growth factors (serum free; SF) or growth factors and nutrients (HEPES buffered saline; HBS). Cells were treated with L-citrulline or equimolar concentrations of L-arginine (positive control) or L-alanine (negative control) and changes in cell size and protein turnover were assessed. In myotubes incubated in HBS or SF media, L-citrulline improved rates of protein synthesis (HBS: +63%, SF: +37%) and myotube diameter (HBS: +18%, SF: +29%). L-citrulline treatment substantially increased iNOS mRNA expression (SF: 350%, HBS: 750%). The general NOS inhibitor L-NAME and the iNOS specific inhibitor aminoguanidine prevented these effects in both models. Depriving myotubes in SF media of L-arginine or L-leucine, exacerbated wasting which was not attenuated by L-citrulline. The increased iNOS mRNA expression was temporally associated with increases in mRNA of the endogenous antioxidants SOD1, SOD3 and catalase. Furthermore, L-citrulline prevented inflammation (LPS) and oxidative stress (H₂O₂) induced muscle cell wasting. In conclusion, we demonstrate a novel direct protective effect of L-citrulline on skeletal muscle cell size independent of L-arginine that is mediated through induction of the inducible NOS (iNOS) isoform. This discovery of a nutritional modulator of iNOS mRNA expression in skeletal muscle cells could have substantial implications for the treatment of muscle wasting conditions.