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Long-term ad libitum dietary restriction such as low-protein diet (LPD) improves metabolic health and extend the life spans of mice and possibly human. However, most studies conducted thus far have mainly focused on preventive rather than the treatment potential of LP diets. To determine whether dietary protein or caloric restriction could be used to treat preexisting metabolic symptoms, particularly in a more severely affected mouse model, we subjected hyperglycemic lipodystrophic IRFKO (adipose-specific insulin receptor knockout) mice to LPD (5.1% kcal from protein). Interestingly, postprandial blood glucose (BG) values of the IRFKO mice began to improve 10 days after the introduction of LPD. The IRFKO mice became normoglycemic 2 weeks after the onset of the LPD treatment. Activation of adaptive thermogenesis in brown (BAT) and beige adipose tissues promotes energy expenditure (EE) and utilization of BG and triglyceride (TG). A LPD has been previously shown to promote EE via the sympathetic nervous system (SNS). Consistent with previous study, we observed that a LPD acutely and sustainably upregulates EE in both the WT and IRFKO mice. This is likely due to LPD dramatically improves BAT functions by transforming the whitened unilocular lipid droplet BAT in the IRFKO mice into normal looking brown multi-locular lipid droplet BAT within 1 week of LPD treatment. This is further supported by significant increase in thermogenic markers expression in the BAT after LPD. Besides thermogenic markers, we also observed a time-dependent regulation of gene markers for fatty acid transport, oxidation and glucose metabolism in BAT after LPD. Together with changes in blood chemistry in the IRFKO mice, we think that glucose and lipid-lowering effects of LPD is the consequence of continuous activation of EE leading to utilization and depletion of systemic thermogenic fuels. Taken together, our data suggest that short-term LPD could be a potential strategy for the treatment of metabolic syndrome. Disclosure M. D. Munoz: None. M. A. Mccann: None. P. Kim: None. C. Liew: None.

The body contains two kinds of fat: white fat, which acts as an energy storage organ, and brown fat, which utilizes energy to generate heat. White fat consists of the subcutaneous and the visceral fat. Accumulation of visceral fat contributes more to metabolic dysfunction due to its increased rate of lipolysis, reduced ability to form brown-like adipocytes known as “beige” cells, and its more inflammatory nature. In this study, we investigated the role that cyclic-AMP Responsive Element Binding Protein 3-like-3 (CREB3L3) plays in adipose tissue. CREB3L3 is an ER-bound transcription factor that has previously been described as liver specific, and an important regulator of lipid metabolism. Upon discovering that CREB3L3 is not only expressed in adipose tissue, but selectively downregulated in the more “metabolically healthy” subcutaneous fat in both obese mice and humans, we investigated how ablating this transcription factor would affect adipose biology. To study this, we created a CREB3L3 fat-specific knockout (fKO) mouse. When challenged with high-fat diet, the fKO mice became 18% heavier than floxed controls. They had significant expansion of their epididymal and inguinal adipose depots, due to a reduction in whole-body energy expenditure and oxygen consumption as measured by indirect calorimetry. The fKO mice are also more resistant to browning during cold exposure and treatment with the β3-adrenergic receptor agonist CL316,243, suggesting impairment of adaptive thermogenesis in the subcutaneous fat. Expansion of the visceral epididymal fat caused the tissue to become more inflammatory as evidenced by an increase in the number of crown-like structures and expression of the macrophage marker F/480 in the fKO tissue. This led to the fKO mice becoming more insulin resistant following high-fat feeding. Together, ablation of CREB3L3 enhances adiposity and insulin resistance during obesity due to a reduction in browning potential and amplified visceral inflammation. Disclosure M.A. McCann: None. M.D. Munoz: None. V. Gil: None. K. Zhang: None. C. LIew: None. Funding National Institutes of Health (R01DK109015-01); American Heart Association (16PRE30190011)

The metabolic syndrome (MS) is a deadly metabolic abnormality-associated to obesity. The pulmonary microvasculature is a sink of circulating neutrophils; as well as this is highly sensitive to small changes in the systemic oxidative/inflammatory profile, as occur in chronic inflammatory diseases, such as obesity. Previously, we have characterized a MS-mouse model in which animals were fed for 16 weeks a 22% p/p chicken-fat rich diet and 10 % fructose in the drinking water. These animals show several features of MS including obesity, central adiposity, insulin resistance (IR), hypertension, dyslipidemia and streatohepatitis. Using this model we envisioned to test whether MS predisposes to pulmonary retention/activation of neutrophils and how it affects whole-body IR. To accomplish this goal we studied MS and control mice (B6 mice fed for 16 weeks with low-fat diet/tap water). MS mice had more IR than control mice. MS mice had also higher concentration of circulating inflammatory mediators than control mice. The lung tissue of MS mice was lighter and expressed more inflammation mediators (TNF-α; IL-6 and inducible nitric oxide) than the control lung. The lung of MS mice had more neutrophils (NIMP-14 + cells), myeloperoxidase (MPO) activity and chlorotyrosine than control mice. ICAM-1 expression in MS mice’s lung tissue was higher than control mice. In relation to control mice, intratracheal instillation (ITI) 2.5 ug lipopolysaccharide (LPS)/mouse to MS mice caused more retention/activation of neutrophils, ICAM-1 expression, MPO activity, chlorotyrosine, circulating inflammatory mediators and also worsened IR. These effects where damped by ITI of 5 nmol of 5,5-dimetil-1-pirrolina N-oxido/mouse. Our data suggest that retention/activation of neutrophils in the lung may be a potential therapeutic target to reduce IR and other complications of obesity. Supported by PROICO 2-3214 & PICT-2014-3369 (toDCR), PROICO 10-0414 (ToSEGM) and PIP2015-2017-112215-0100603CO (To DCR, SEA & SEGM).

The nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was originally synthesized for the study of free radicals. However we found that DMPO can also prevent inflammatory activation in RAW264.7 cells primed with lipopolysaccharide (LPS). Additionally, DMPO prevents lung damage, adipose tissue inflammation, systemic inflammation and insulin resistance in a mouse model of acute distress respiratory syndrome induced by intratracheal instillation with LPS. These effects of DMPO are more likely caused by changes in gene expression in innate immune cells. Thus herein we tested whether DMPO by itself can change the transcriptome and interactome in RAW 264.7 cells, as a model of macrophage. To accomplish our goal we incubated RAW 264.7 cells with (DMPO) or without (control) 50 mM DMPO for 6h. The transcriptome was analyzed using micro-array (Illumina) and corroborated by Nanostring (nCounter) technology. Bioinformatics analysis showed 79 differentially expressed genes (DEGs) in DMPO vs control comparison (ONE-way ANOVA; FDR = 0.05). DAVID databases for identifying enriched Gene Onthology terms and Ingenuity Pathway Analysis (IPA) for functional analysis showed that DMPO DEGs were consistent with a negative regulation of innate immune response. Functional analysis indicated that IRF7 and TLRs were related (predicted inhibitions) to the observed transcriptomic effects of DMPO. Functional data analyses are consistent with DMPO dampening LPS-induced inflammatory activation of RAW 264.7 cells by down regulating several genes related to the innate immune response. These effects were also supported by Nanostring data showing that DMPO downregulates the expression of inflammation markers (CCL2, IFNβ, COX-2 and iNOS). Remarkably, our data suggest that DMPO by itself shifts the transcriptomic profile of RAW264.7 cells towards a negative modulation of innate immune response.

5,5-dimethyl-1-pirroline N-oxide (DMPO) is a nitrone spin trap originally synthesized as a nitrone spin trap to study free radicals by electron spin resonance spectroscopy and recently by immuno-spin trapping. Herein we envisioned at studying what are the mechanisms involved in these anti-inflammatory effects of this old drug with new properties. To accomplish this goal we used a well known model of macrophage-like cells (RAW264.6) primed with LPS; which induce a well known MAPK signaling cascade that ends in activation of NF-kB—the master regulator of inflammation, inducible nitric oxide (iNOS) expression and nitric oxide synthesis. DMPO blocked NO synthesis, iNOS induction and MAPK signaling; but it did not affect LPS binding to LPS to membrane receptors. Thus we hypothesized that DMPO, and likewise other nitrones, may somehow affect very early LPS triggered signaling downstream of LPS-receptor binding. In silico data showed that DMPO binds to a very narrow sequence of aminoacids inside the TIR domain of TLR-2. TIR domains are conserved throughout TLRs (TLR-4; 6; 10) and species, particularly in a region called BB-loop which is responsible for downstream signal transduction. Molecular dynamics data shows that DMPO binds almost exclusively to these residues located at the BB-loop. Taking together, our data indicate that DMPO anti-inflammatory effect, is at least in part due to its binding to specific residues in the cytoplasmic portion of TLRs, thus further signaling is damped. Supported by PROICO 2-3214 & PICT-2014-3369 (to DCR), PROICO 10-0414 (To SEGM) and PIP2015-2017-112215-0100603CO (To DCR, SEA & SEGM).

Macrophages are tissue cells from the innate-immune system where they play a number of homeostatic and defense functions. Inside the tissues and under tissue-specific microenvironmental pressures monocytes are recruited and differentiated to specific phenotypes. This phenotype is a consequence of the expression of specific genes that are under the control of one or more transcription factors. In this context, inflammatory phenotype of adipose tissue (AT) macrophages (ATM-M1) is responsible for adipose tissue oxidative stress and inflammation mediators that reduce whole-body insulin sensitivity and cause a number of metabolic abnormalities-associated to obesity. Intratracheal instillation of the nitrone spin trap 5,5-dimethyl-1-pirroline N -oxide (DMPO) to diet-induced obese-mice reduced markers of AT oxidative stress and inflammation, reduced serum concentration of inflammatory cytokines and improved insulin sensitivity. Thus we hypothesized that DMPO may produce transcripcional effects in macrophages at the AT and maybe other tissues. To approach this hypothesis we determined the transcripcional effects of DMPO in RAW264.7 cells after 6h incubation and with or without lipopolysaccharide (LPS) to model transcriptional profile of ATM. Microarray data showed that LPS caused an M1-transcriptional pattern, whereas DMPO reduced these changes. Remarkable effects were observed in the expression of IRF-7 and PPAR-d, master regulators of genes that determine M1 and M2 macrophage phenotype. LPS induced IRF-7, but reduced PPARd expression; whereas DMPO reduced IRF-7, but induced PPAR-d expression. Taking together our data suggest that DMPO may serve as a structural platform for the design of novel compounds to reduce AT inflammation and, thus other inflammatory abnormalities-associated to obesity, such as insulin resistance and metabolic syndrome. Supported by PROICO 2-3214 & PICT-2014-3369 (to DCR), PROICO 10-0414 (To SEGM) and PIP2015-2017-112215-0100603CO (To DCR, SEGM & SEGM).