Secretory Factors from Calcium-Sensing Receptor-Activated SW872 Pre-Adipocytes Induce Cellular Senescence and A Mitochondrial Fragmentation-Mediated Inflammatory Response in HepG2 Cells.

Adipose tissue inflammation in obesity has a deleterious impact on organs such as the liver, ultimately leading to their dysfunction. We have previously shown that activation of the calcium-sensing receptor (CaSR) in pre-adipocytes induces TNF-α and IL-1ß expression and secretion; however, it is unknown whether these factors promote hepatocyte alterations, particularly promoting cell senescence and/or mitochondrial dysfunction. We generated conditioned medium (CM) from the pre-adipocyte cell line SW872 treated with either vehicle (CMveh) or the CaSR activator cinacalcet 2 µM (CMcin), in the absence or presence of the CaSR inhibitor calhex 231 10 µM (CMcin+cal). HepG2 cells were cultured with these CM for 120 h and then assessed for cell senescence and mitochondrial dysfunction. CMcin-treated cells showed increased SA-ß-GAL staining, which was absent in TNF-α- and IL-1ß-depleted CM. Compared to CMveh, CMcin arrested cell cycle, increased IL-1ß and CCL2 mRNA, and induced p16 and p53 senescence markers, which was prevented by CMcin+cal. Crucial proteins for mitochondrial function, PGC-1α and OPA1, were decreased with CMcin treatment, concomitant with fragmentation of the mitochondrial network and decreased mitochondrial transmembrane potential. We conclude that pro-inflammatory cytokines TNF-α and IL-1ß secreted by SW872 cells after CaSR activation promote cell senescence and mitochondrial dysfunction, which is mediated by mitochondrial fragmentation in HepG2 cells and whose effects were reversed with Mdivi-1. This investigation provides new evidence about the deleterious CaSR-induced communication between pre-adipocytes and liver cells, incorporating the mechanisms involved in cellular senescence.

Lampaya Medicinalis Phil. decreases lipid-induced triglyceride accumulation and proinflammatory markers in human hepatocytes and fat body of Drosophila melanogaster.

BACKGROUND: Excess hepatic triglyceride (TG) accumulation (steatosis) commonly observed in obesity, may lead to non-alcoholic fatty liver disease (NAFLD). Altered regulation of intracellular lipid droplets (LD) and TG metabolism, as well as activation of JNK-mediated proinflammatory pathways may trigger liver steatosis-related disorders. Drosophila melanogaster is an animal model used for studying obesity and its associated disorders. In Drosophila, lipids and glycogen are stored in the fat body (FB), which resembles mammalian adipose tissue and liver. Dietary oversupply leads to obesity-related disorders, which are characterized by FB dysfunction. Infusions of Lampaya medicinalis Phil. (Verbenaceae) are used in folk medicine of Chile to counteract inflammatory diseases. Hydroethanolic extract of lampaya (HEL) contains considerable amounts of flavonoids that may explain its anti-inflammatory effect. METHODS: We studied whether HEL affects palmitic acid (PA, C16:0) and oleic acid (OA; C18:1)-induced TG accumulation and proinflammatory marker content in HepG2 hepatocytes as well as impaired lipid storage and proinflammatory molecule expression in Drosophila melanogaster fed a high-fat diet (HFD). RESULTS: In HepG2 hepatocytes, exposure to OA/PA elevated TG content, FABP4, ATGL and DGAT2 expression, and the JNK proinflammatory pathway, as well as TNF-α and IL-6 production, while diminished FAS expression. These effects were prevented by HEL co-treatment. In Drosophila larvae fed a HFD, HEL prevented TG accumulation and downregulated proinflammatory JNK pathway activation. CONCLUSION: HEL effect counteracting OA/PA- and HFD-induced lipid accumulation and proinflammatory marker expression in HepG2 hepatocytes and Drosophila larvae may represent a preventive approach against hepatic steatosis and inflammation, associated to obesity and NAFLD.

Calcium-Sensing Receptor in Adipose Tissue: Possible Association with Obesity-Related Elevated Autophagy.

Autophagy is upregulated in adipose tissue (AT) from people with obesity. We showed that activation of the calcium-sensing receptor (CaSR) elevates proinflammatory cytokines through autophagy in preadipocytes. Our aim is to understand the role of CaSR on autophagy in AT from humans with obesity. We determined mRNA and protein levels of CaSR and markers of autophagy by qPCR and western blot in human visceral AT explants or isolated primary preadipocytes (60 donors: 72% female, 23-56% body fat). We also investigated their association with donors' anthropometric variables. Donors' % body fat and CaSR mRNA expression in AT were correlated (r = 0.44, p < 0.01). CaSR expression was associated with mRNA levels of the autophagy markers atg5 (r = 0.37, p < 0.01), atg7 (r = 0.29, p < 0.05) and lc3b (r = 0.40, p < 0.01). CaSR activation increased becn and atg7 mRNA expression in AT. CaSR activation also upregulated LC3II by ~50%, an effect abolished by the CaSR inhibitor. Spermine (CaSR agonist) regulates LC3II through the ERK1/2 pathway. Structural equation model analysis suggests a link between donors' AT CaSR expression, AT autophagy and expression of Tumor Necrosis Factor alpha TNF-α. CaSR expression in visceral AT is directly associated with % body fat, and CaSR activation may contribute to obesity-related disruption in AT autophagy.

GDF-11 prevents cardiomyocyte hypertrophy by maintaining the sarcoplasmic reticulum-mitochondria communication.

Growth differentiation factor 11 (GDF11) is a novel factor with controversial effects on cardiac hypertrophy both in vivo and in vitro. Although recent evidence has corroborated that GDF11 prevents the development of cardiac hypertrophy, its molecular mechanism remains unclear. In our previous work, we showed that norepinephrine (NE), a physiological pro-hypertrophic agent, increases cytoplasmic Ca2+ levels accompanied by a loss of physical and functional communication between sarcoplasmic reticulum (SR) and mitochondria, with a subsequent reduction in the mitochondrial Ca2+ uptake and mitochondrial metabolism. In order to study the anti-hypertrophic mechanism of GDF11, our aim was to investigate whether GDF11 prevents the loss of SR-mitochondria communication triggered by NE. Our results show that: a) GDF11 prevents hypertrophy in cultured neonatal rat ventricular myocytes treated with NE. b) GDF11 attenuates the NE-induced loss of contact sites between both organelles. c) GDF11 increases oxidative mitochondrial metabolism by stimulating mitochondrial Ca2+ uptake. In conclusion, the GDF11-dependent maintenance of physical and functional communication between SR and mitochondria is critical to allow Ca2+ transfer between both organelles and energy metabolism in the cardiomyocyte and to avoid the activation of Ca2+-dependent pro-hypertrophic signaling pathways.

Calcium-sensing receptor activates the NLRP3 inflammasome in LS14 preadipocytes mediated by ERK1/2 signaling.

The study of the mechanisms that trigger inflammation in adipose tissue is key to understanding and preventing the cardiometabolic consequences of obesity. We have proposed a model where activation of the G protein-coupled calcium sensing receptor (CaSR) leads to inflammation and dysfunction in adipose cells. Upon activation, CaSR can mediate the expression and secretion of proinflammatory factors in human preadipocytes, adipocytes, and adipose tissue explants. One possible pathway involved in CaSR-induced inflammation is the activation of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome, that promotes maturation and secretion of interleukin (IL)-1ß. The present work aimed to study whether CaSR mediates the activation of NLRP3 inflammasome in the human adipose cell model LS14. We assessed NLRP3 inflammasome priming and assembly after cinacalcet-induced CaSR activation and evaluated if this activation is mediated by downstream ERK1/2 signaling in LS14 preadipocytes. Exposure to 2 µM cinacalcet elevated mRNA expression of NLRP3, CASP-1, and IL-1ß, as well as an increase in pro-IL-1ß protein. In addition, CaSR activation triggered NLRP3 inflammasome assembly, as evidenced by a 25% increase in caspase-1 activity and 63% IL-1ß secretion. CaSR silencing (siRNA) abolished the effect. Upstream ERK pathway inhibition decreased cinacalcet-dependent activation of NLRP3 inflammasome. We propose CaSR-dependent NLRP3 inflammasome activation in preadipocytes through ERK signaling as a novel mechanism for the development of adipose dysfunction, that may favor the cardiovascular and metabolic consequences of obesity. To the best of our knowledge, this is the first report linking the inflammatory effect of CaSR to NLRP3 inflammasome induction in adipose cells.

ER-to-mitochondria miscommunication and metabolic diseases.

Eukaryotic cells contain a variety of subcellular organelles, each of which performs unique tasks. Thus follows that in order to coordinate these different intracellular functions, a highly dynamic system of communication must exist between the various compartments. Direct endoplasmic reticulum (ER)-mitochondria communication is facilitated by the physical interaction of their membranes in dedicated structural domains known as mitochondria-associated membranes (MAMs), which facilitate calcium (Ca(2+)) and lipid transfer between organelles and also act as platforms for signaling. Numerous studies have demonstrated the importance of MAM in ensuring correct function of both organelles, and recently MAMs have been implicated in the genesis of various human diseases. Here, we review the salient structural features of interorganellar communication via MAM and discuss the most common experimental techniques employed to assess functionality of these domains. Finally, we will highlight the contribution of MAM to a variety of cellular functions and consider the potential role of MAM in the genesis of metabolic diseases. In doing so, the importance for cell functions of maintaining appropriate communication between ER and mitochondria will be emphasized.

Regulation of cardiomyocyte autophagy by calcium.

Calcium signaling plays a crucial role in a multitude of events within the cardiomyocyte, including cell cycle control, growth, apoptosis, and autophagy. With respect to calcium-dependent regulation of autophagy, ion channels and exchangers, receptors, and intracellular mediators play fundamental roles. In this review, we discuss calcium-dependent regulation of cardiomyocyte autophagy, a lysosomal mechanism that is often cytoprotective, serving to defend against disease-related stress and nutrient insufficiency. We also highlight the importance of the subcellular distribution of calcium and related proteins, interorganelle communication, and other key signaling events that govern cardiomyocyte autophagy.

Calcium sensing receptor effects in adipocytes and liver cells: Implications for an adipose-hepatic crosstalk.

The calcium sensing receptor (CaSR) is expressed in human adipose cells, and its activation may associate with adipose tissue (AT) dysfunction. We evaluated whether CaSR stimulation influences adipocyte triglyceride (TG) and fatty acid binding protein 4 (aP2) content, and hepatocyte TGs and proinflammatory cytokine expression. The effect of the calcimimetic cinacalcet on TGs (fluorimetry), lipogenic genes (qPCR) and aP2 (immunoblot) was evaluated in LS14 adipocytes or AT. In the human HepG2 hepatic cell line, we assessed CaSR expression and cinacalcet effect on TGs and lipogenic and proinflammatory genes. CaSR activation decreased adipocyte TG content by 20% and the expression of GPD and LPL by 34% and 20%, respectively. Cinacalcet increased aP2 protein expression by 60%. CaSR expression was shown in HepG2 cells and human liver samples. Cinacalcet-treated HepG2 cells in the presence of oleic acid exhibited a19% increased TG content. No changes were observed in the expression of lipogenic genes in HepG2 cells, however there was a 50%-300% elevation in the expression of proinflammatory cytokines. CaSR activation in adipocytes may associate with decreased TG storage ability and increased aP2. Hepatic CaSR stimulation may elevate steatosis and proinflammatory factors. We propose that CaSR may contribute to obesity-associated hepatic metabolic consequences.

Obesity is associated with acute inflammation in a sample of adolescents.

BACKGROUND: Obesity is associated with a mild chronic inflammatory response, which has been suggested to be pivotal in the development of cardiometabolic alterations of obesity. However, little is known about the involvement of acute inflammation. OBJECTIVE: To evaluate whether circulating neutrophils, markers of acute inflammation, are associated (quantitatively and qualitatively) with adolescent obesity and whether leptin modulates these associations. SUBJECTS AND METHODS: We assessed 528 adolescents (16.8 yr old, 47% females), without chronic/acute illness. We measured anthropometry and dual energy X-ray absorptiometry and calculated fat mass percentage (FM%). Fasting serum glucose, high-density lipoprotein (HDL)-cholesterol, and triglycerides were used with blood pressure and waist circumference to compute a metabolic z-score. Leukocyte and neutrophil counts were obtained, together with levels of serum leptin. In a subsample of 23 males, flow cytometry was used to assess degranulation (CD66b expression) of neutrophils. RESULTS: Female sex and obesity were positively related to mean neutrophil counts (p < 0.05). When accounting for sex and weight status, leptin was associated with neutrophil counts (p < 0.05), partially explaining the association between obesity and neutrophil counts. Neutrophil counts were related to metabolic risk z-scores, controlling for fat mass. Participants with elevated FM% showed more neutrophil degranulation than controls (p < 0.05). CONCLUSIONS: Participants with increased adiposity had higher circulating neutrophil counts, suggesting acute inflammation. Furthermore, the neutrophils showed more degranulation, indicating inflammation. Obesity-induced alteration of the adipose secretory pattern (i.e., changes in leptin levels) could be involved in acute inflammation.

Effect of Lampaya medicinalis Phil. (Verbenaceae) and Palmitic Acid on Insulin Signaling and Inflammatory Marker Expression in Human Adipocytes.

BACKGROUND: Aging and obesity are associated with insulin resistance (IR) and low-grade inflammation. Molecularly, IR is characterized by a reduction in glucose uptake and insulin signaling (IRS-1/Akt/AS160 pathway), while inflammation may result from upregulated NF-κB pathway after low Tyr-IκBα phosphorylation. Upregulated phosphatase activity of PTP1B is associated with impaired insulin signaling and increased inflammation. Plasma levels of palmitic acid (PA) are elevated in obesity, triggering inflammation and disruption of insulin signaling. Traditional medicine in Northern Chile uses oral infusions of Lampaya medicinalis Phil. (Verbenaceae) to treat inflammatory conditions. Significant amounts of flavonoids are found in the hydroethanolic extract of Lampaya (HEL), which may account for its biological activity. The aim of this work was to study the effect of HEL and PA on insulin signaling and glucose uptake as well as inflammatory marker expression in human adipocytes. METHODS: We studied HEL effects on PA-induced impairment on insulin signaling, glucose uptake and inflammatory marker content in human SW872 adipocytes. HEL cytotoxicity was assessed in adipocytes at different concentrations (0.01 to 10 g/mL). Adipocytes were incubated or not with PA (0.4 mM, 24 h) with or without HEL (2 h pre-incubation), and then stimulated with insulin (10 min, 100 mM) or a vehicle. Phospho-IRS-1, phospho-Akt, phospho-AS160, phospho-NF-κB and phospho-IκBα, as well as protein levels of PTP1B, were assessed using Western blotting, and glucose uptake was evaluated using the 2-NBDG analogue. RESULTS: At the assessed HEL concentrations, no cytotoxic effects were observed. PA decreased insulin-stimulated phospho-Akt and glucose uptake, while co-treatment with HEL increased such markers. PA decreased phospho-IRS-1 and phospho-Tyr-IκBα. On the other hand, incubation with HEL+PA decreased phospho-AS160 and phospho-NF-κB compared with cells treated with PA alone. CONCLUSION: Our results suggest a beneficial effect of HEL by improving PA-induced impairment on molecular markers of insulin signaling, glucose uptake and inflammation in adipocytes. Further studies are necessary to elucidate whether lampaya may constitute a preventive strategy for people whose circulating PA levels contribute to IR and inflammation during aging and obesity.

Adipogenic effect of calcium sensing receptor activation.

We established that human adipose cells and the human adipose cell line LS14 express the calcium-sensing receptor (CaSR) and that its activation induces inflammatory cytokine production. Also, its expression is enhanced upon exposure to obesity-associated proinflammatory cytokines. We have thus proposed that CaSR activation may be associated with adipose dysfunction. Here, we evaluated a possible effect on adipogenesis. We induced adipose differentiation of primary and LS14 human preadipocytes with or without the simultaneous activation of CaSR, by the exposure to the calcimimetic cinacalcet. Activation of the receptor for 24 h decreased by 40 % the early differentiation marker CCAAT/enhancer-binding protein ß. However, upon longer-term (10 day) exposure to the adipogenic cocktail, cinacalcet exerted the opposite effect, causing a dose-response increase in the expression of the mature adipose markers adipocyte protein 2, adiponectin, peroxisome proliferator-activated receptor γ, fatty acid synthase, and glycerol-3-phosphate dehydrogenase. To assess whether there was a time-sensitive effect of CaSR activation on adipogenesis, we evaluated the 10 day effect of cinacalcet exposure for the first 6, 24, 48 h, 6, and 10 days. Our observations suggest that regardless of the period of exposure, 10 day adipogenesis is elevated by cinacalcet. CaSR activation may interfere with the initial stages of adipocyte differentiation; however, these events do not seem to preclude adipogenesis from continuing. Even though adipogenesis (particularly in subcutaneous depots) is associated with insulin sensitivity and adequate adipose function, the implications of our findings in visceral adipocytes, especially in the context of inflamed AT and overnutrition, remain to be established.

Deleterious liver-adipose crosstalk in obesity: Hydroethanolic extract of Lampaya medicinalis Phil. (Verbenaceae) counteracts fatty acid-induced fibrotic marker expression in human hepatocytes.

Elevated circulating fatty acids in obesity may induce hepatic steatosis, leading to liver inflammation, fibrosis and nonalcoholic fatty liver disease (NAFLD). On the other hand, impaired communication between hepatocytes and adipose tissue (AT) in obesity influences adipose lipolysis and fibrosis, negatively affecting metabolic function. Infusions of Lampaya medicinalis Phil. (Verbenaceae) are used in Chilean folk medicine to treat inflammatory diseases. Hydroethanolic extract of lampaya (HEL) contains flavonoids that may explain its anti-inflammatory effect, but it is unknown whether HEL modulates fibrogenic processes in hepatocytes. We studied lipolysis and expression of fibrosis markers after exposure of visceral AT explants from subjects with obesity to HepG2-secreted factors. In addition, we evaluated the effect of HEL on palmitic acid (PA, C16:0) and oleic acid (OA; C18:1)-induced fibrotic marker expression in HepG2 hepatocytes. Results: Exposure to HepG2-secreted factors increased visceral AT lipolysis and expression of CTGF and collagen I. Exposure to OA/PA elevated collagen I, CTGF, fibronectin, α-smooth muscle actin, MMP-2 and MMP-9 expression in HepG2 cells, and these effects were prevented by HEL co-treatment. Conclusion: HEL effect counteracting OA/PA-induced fibrotic marker expression in HepG2 hepatocytes may represent a preventive approach against hepatic fibrosis and deleterious liver-adipose crosstalk in obesity.

"Effect of time-restricted feeding on high-fat diet-induced metabolic dysfunction in Drosophila melanogaster".

BACKGROUND: Metabolic alterations associated with obesity have been related to chronodisruption i.e., the desynchronization of molecular clocks that regulate circadian rhythms. The search for tools that improve the dietary treatment of obesity has recently focused on behaviors related to chronodisruption, and intermittent fasting is increasingly gaining interest. Studies in animal models have identified the benefits of time-restricted feeding (TRF) on metabolic alterations associated with changes in circadian rhythms induced by a high-fat diet. We aimed to evaluate the effect of TRF in flies with metabolic damage and chronodisruption. METHODS: Using high-fat diet fed Drosophila melanogaster as a model of metabolic damage and chronodisruption, we determined the impact of 12-h TRF on metabolic and molecular markers. Flies with metabolic dysfunction were switched to a control diet and randomly assigned to Ad libitum or a TRF regimen for seven days. We evaluated total triglyceride content, glycemia, weight, and 24 h mRNA expression rhythms of Nlaz (insulin resistance marker), clock genes (circadian rhythm molecular markers), and the neuropeptide Cch-amide2. RESULTS: Flies with metabolic damage that received TRF showed lower total triglyceride content, Nlaz expression, circulating glucose, and weight compared to Ad libitum. We observed the recovery of some of the high-fat diet-induced alterations in the amplitude of the circadian rhythm, particularly in the peripheral clock. CONCLUSIONS: TRF produced a partial reversal of metabolic dysfunction and chronodisruption of circadian cycles. GENERAL SIGNIFICANCE: TRF could be a useful tool to help to ameliorate metabolic and chronobiologic damage induced by a high-fat diet.

The Adipocyte-Macrophage Relationship in Cancer: A Potential Target for Antioxidant Therapy.

Obesity has emerged as a major public health concern with a staggering 39% worldwide prevalence as of 2021. Given the magnitude of the problem and considering its association with chronic low-grade systemic inflammation, it does not come as a surprise that obesity is now considered one of the major risk factors for the development of several chronic diseases, such as diabetes, cardiovascular problems, and cancer. Adipose tissue dysfunction in obesity has taken center stage in understanding how changes in its components, particularly adipocytes and macrophages, participate in such processes. In this review, we will initially focus on how changes in adipose tissue upon excess fat accumulation generate endocrine signals that promote cancer development. Moreover, the tumor microenvironment or stroma, which is also critical in cancer development, contains macrophages and adipocytes, which, in reciprocal paracrine communication with cancer cells, generate relevant signals. We will discuss how paracrine signaling in the tumor microenvironment between cancer cells, macrophages, and adipocytes favors cancer development and progression. Finally, as reactive oxygen species participate in many of these signaling pathways, we will summarize the information available on how antioxidants can limit the effects of endocrine and paracrine signaling due to dysfunctional adipose tissue components in obesity.

Assessment of a consensus definition of obesity and metabolic health phenotypes in children at different pubertal stages.

Not all individuals with obesity develop metabolic complications, which has brought about the concepts of metabolically healthy and unhealthy obesity (MHO/MUO). However, inconsistent definitions of these conditions have limited their understanding. We assessed whether a recently-proposed consensus definition for MHO/MUO correlates with adiposity and reflects metabolic risk parameters during puberty. Low-middle income children from the Growth and Obesity Cohort Study (Santiago, Chile) were included (n = 949; 1692 visits at Tanner (T)2, T4 and/or one-year post menarche (1YPM)). Anthropometry, body composition and metabolic parameters were compared between MUO and MHO, and also in children without obesity. The risk for presenting MUO phenotype was significantly elevated with higher waist-height ratio (T2), zBMI (T2, T4), trunk fat, and C-reactive protein (T4). Elevated cardiometabolic indices were important predictors of the "unhealthy" phenotype allocation in children with or without obesity. Our observations suggest that the consensus definition in children at T2, T4 and 1YPM reflects metabolic risk and central obesity. Metabolic health phenotype allocation by this equation enables easy detection of risk factors that call for action to prevent long-term metabolic derangements in children with obesity and, importantly, also those without obesity.

Obesity-associated proinflammatory cytokines increase calcium sensing receptor (CaSR) protein expression in primary human adipocytes and LS14 human adipose cell line.

Obesity-associated health complications are thought to be in part due to the low-grade proinflammatory state that characterizes this disease. The calcium sensing receptor (CaSR), which is expressed in human adipose cells, plays an important role in diseases involving inflammation. To assess the relevance of this protein in adipose pathophysiology, we evaluated its expression in adipocytes under obesity-related proinflammatory conditions. As in primary adipose cells, we established that LS14, a recently described human adipose cell line, expresses the CaSR. Differentiated LS14 and primary adipose cells were exposed overnight to cytokines typically involved in obesity-related inflammation (interleukin (IL)1beta, IL6 and tumor necrosis factor (TNF)alpha). The cytokines increased CaSR abundance in differentiated adipocytes. We incubated LS14 cells with medium previously conditioned (CM) by adipose tissue from subjects with a wide range of body mass index (BMI). Cells exposed to CM from subjects of higher BMI underwent a greater increase in CaSR protein, likely resulting from the greater proinflammatory cytokines secreted from obese tissue. Our observations that proinflammatory factors increase CaSR levels in adipocytes, and the reported ability of CaSR to elevate cytokine levels, open new aspects in the study of obesity inflammatory state pathophysiology, providing a potential novel therapeutic prevention and treatment target.

Energy restriction is associated with lower bone mineral density of the tibia and femur in lean but not obese female rats.

Energy restriction decreases bone mineral density (BMD), and epidemiological studies suggest that the risk of weight loss-induced bone loss is greater in lean than in heavier individuals. Our goal in this study was to determine how bone density and geometry respond to energy restriction in mature obese rats compared with lean rats. At 6 mo of age, 36 diet-induced obese and lean female Sprague-Dawley rats were allocated to control (CTL; ad libitum; n = 18) and energy-restricted (EnR; 40% restriction; n = 18) diets. After 10 wk of dietary intervention, obese EnR rats lost more weight (-91 +/- 34 g) than lean EnR rats(-61 +/- 14 g) (P < 0.02), [corrected] whereas body weight did not change significantly in the 2 CTL groups (14 +/- 23 g). Only the lean EnR (and not obese EnR) rats showed lower BMD compared with CTL rats at the tibia, distal, and proximal femur and femoral neck, and trabecular bone volume (P < 0.05). Serum estradiol declined in lean EnR rats compared with baseline (P < 0.05) but not in the obese EnR rats. In addition, the final serum 25-hydroxyvitamin D (25OHD) concentration was higher (P < 0.05) in obese than in lean EnR rats. Serum parathyroid hormone decreased (P < 0.05) from baseline to final in lean and obese CTL, but not EnR rats. These data support the hypothesis that energy restriction in lean rats compared with obese rats is more detrimental to bone, and it is possible that the greater decline in estrogen and lower levels of 25OHD contribute to this effect.

Protective effect of the hydroalcoholic extract from Lampaya medicinalis Phil. (Verbenaceae) on palmitic acid- impaired insulin signaling in 3T3-L1 adipocytes.

BACKGROUND: Obesity is strongly associated with insulin resistance (IR). IR at the molecular level may be defined as a diminished activation of insulin signaling-related molecules (IRS-1/Akt/AS160) as well as reduced glucose uptake. Subject with obesity have elevated plasma levels of saturated fatty acids, such as palmitic acid (PA), which triggers insulin signaling disruption in vivo and in vitro. Infusions of Lampaya medicinalis Phil. (Verbenaceae) are used in folk medicine of Northern Chile to counteract inflammatory diseases. Hydroethanolic extracts of lampaya (HEL) contain considerable amounts of flavonoids that may explain the biological activity of the plant. The aim of this study was to assess whether HEL exposure protects against PA-disrupted insulin signaling and glucose uptake in adipocytes. METHODS: Cytotoxicity of a range of HEL concentrations (0.01-10 µg/mL) was evaluated in 3T3-L1 adipocytes. Cells were exposed or not to 0.1 µg/mL of HEL before adding 0.65 mM PA or vehicle and incubated with 100 nM insulin (or vehicle) for 15 min. Phosphorylation of Tyr-IRS-1, Ser-Akt, Thr-AS160 was evaluated by Western blot. Glucose uptake was assessed using the 2-NBDG analogue. RESULTS: HEL was not cytotoxic at any concentration assessed. PA-induced reduction in insulin-stimulated phosphorylation of IRS-1, Akt and AS160 and glucose uptake were abolished by co-treatment with HEL. CONCLUSION: These findings give new insights about the effect of HEL ameliorating PA- impaired IRS-1/Akt/AS160 pathway and glucose uptake in adipocytes. More studies should focus on lampaya, since might represent a preventive approach in individuals whose circulating PA levels contribute to IR.

Calcium sensing receptor activation in THP-1 macrophages triggers NLRP3 inflammasome and human preadipose cell inflammation.

Excess adipose tissue (AT) associates with inflammation and obesity-related diseases. We studied whether calcium-sensing receptor (CaSR)-mediated NLRP3 inflammasome activation in THP-1 macrophages elevates inflammation in LS14 preadipocytes, modeling deleterious AT cell crosstalk. THP-1 macrophages exposed to cinacalcet (CaSR activator, 2 µM, 4 h) showed elevated proinflammatory marker and NLRP3 inflammasome mRNA, pro-IL-1ß protein and caspase-1 activity, whereas preincubation with CaSR negative modulators prevented these effects. The key NLRP3 inflammasome component ASC was silenced (siRNA) in THP-1 cells, and inflammasome activation was evaluated (qPCR, Western blot, caspase-1 activity) or they were further cultured to obtain conditioned medium (CoM). Exposure of LS14 preadipocytes to CoM from cinacalcet-treated THP-1 elevated LS14 proinflammatory cytokine expression, which was abrogated by THP-1 inflammasome silencing. Thus, CaSR activation elevates THP-1-induced inflammation in LS14 preadipocytes, via macrophage NLRP3 inflammasome activation. Modulating CaSR activation may prevent deleterious proinflammatory cell crosstalk in AT, a promising approach in obesity-related metabolic disorders.

Hydroethanolic Extract of Lampaya Medicinalis Phil. (Verbenaceae) Decreases Proinflammatory Marker Expression in Palmitic Acid-exposed Macrophages.

BACKGROUND: Obesity is a major health problem associated with increased comorbidities, which are partially triggered by inflammation. Proinflammatory macrophage infiltration in adipose tissue of individuals with obesity increases chronic inflammation. Obesity is associated with elevated plasma levels of saturated fatty acids, such as palmitic acid (PA), which promotes inflammation in vivo and in vitro. Infusions of Lampaya medicinalis Phil. (Verbenaceae) are used in the folk medicine of Northern Chile to counteract inflammation of rheumatic diseases. Hydroethanolic extract of lampaya (HEL) contains spectrophotometrically defined compounds that may contribute to the observed effect on inflammation. METHODS: We evaluated the phytochemical composition of HEL by high-performance liquid chromatography coupled to diode array detection (HPLC-DAD) and liquid chromatography-electrospray ionization- tandem mass spectrometry (LC-ESI-MS/MS). We assessed whether the exposure to HEL affects PA-induced expression of proinflammatory factors in THP-1 macrophages. RESULTS: HPLC-DAD and LC-ESI-MS/MS analyses showed the presence of considerable amounts of flavonoids in HEL. The PA-induced phosphorylation of the inflammatory pathway mediators IKK and NF-κB, as well as the elevated expression and secretion of proinflammatory cytokines (IL-6, TNF-α), were reduced in cells pre-exposed to HEL. CONCLUSION: These findings give new insights about the effect of HEL reducing IKK/NF-κB proinflammatory pathway, likely explained by the number of flavonoids contained in the extract. More studies would be needed to define the possible role of Lampaya as a preventive approach in subjects with obesity whose circulating PA might contribute to chronic inflammation.