Gonadal Adipose Tissue Research Articles

Pre-Clinical Evidence for the Anti-Obesity Potential of Quercetin and Curcumin Loaded Chitosan/PEG Blended PLGA Nanoparticles

This research aimed to formulate quercetin (Qu) and curcumin (CUR)-loaded PLGA NPs coated with chitosan (CS) & PEG and to explore their therapeutic effect against obesity in rats. Qu and CUR nanostructures were prepared and characterized by Zetasizer and TEM. Then, the formulated nanoparticles and their free couterparts were employed for mitigation of obesity in female rats. The size of NPs was in nanometer range with an average size distribution 307.9 nm for Qu NPs and 322.5 nm for CUR NPs. The Qu NPs and CUR NPs were appeared in the TEM image containing core in which the Qu or CUR was localized and surrounded by the coat of PLGA-CS-PEG. The Qu NPs exhibited negative zeta potential at -8.5 mV, while, CUR NPs exhibited positive zeta potential at +0.916 mV. Treatment with orlistat, free Qu, Qu NPs, free CURor CUR NPs elicited significant decline in body weight, BMI and Lee index. Orlistat and CUR NPs significantly diminished liver, heart and visceral adipose tissue weights. Furthermore, the suggested treatments significantly reduced the gonadal and subcutaneous adipose tissue weights. Orlistat significantly lessened kidney and adrenal weights. All treatments significantly minimized serum Chol., TG, LDL, glucose, INS, HOMA-IR, LH, MDA, TLR4 and NF-κB levels and elevated serum HDL, E2 and TAC levels. Orlistat significantly enhanced serum IL-10 level. Conclusively, Qu and CUR nanoformulations offer anti-obesity potency through their hypolipidemic, hypoglycemic,antioxidative and anti-inflammatory effects. Both Qu and CUR NPs manifested superior effect than their free counterparts, may be because of solubility elevation as well as bioavailability of the nanoencapsulation.

18:0 Lyso PC Derived by Bioactivity-Based Molecular Networking from Lentil Mutant Lines and Its Effects on High-Fat Diet-Induced Obese Mice.

Lentil (Lens culinaris; Fabaceae), one of the major pulse crops in the world, is an important source of proteins, prebiotics, lipids, and essential minerals as well as functional components such as flavonoids, polyphenols, and phenolic acids. To improve crop nutritional and medicinal traits, hybridization and mutation are widely used in plant breeding research. In this study, mutant lentil populations were generated by γ-irradiation for the development of new cultivars by inducing genetic diversity. Molecular networking via Global Natural Product Social Molecular Networking web platform and dipeptidyl peptide-IV inhibitor screening assay were utilized as tools for structure-based discovery of active components in active mutant lines selected among the lentil population. The bioactivity-based molecular networking analysis resulted in the annotation of the molecular class of phosphatidylcholine (PC) from the most active mutant line. Among PCs, 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (18:0 Lyso PC) was selected for further in vivo study of anti-obesity effect in a high-fat diet (HFD)-induced obese mouse model. The administration of 18:0 Lyso PC not only prevented body weight gain and decreased relative gonadal adipose tissue weight, but also attenuated the levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol, and leptin in the sera of HFD-induced obese mice. Additionally, 18:0 Lyso PC treatment inhibited the increase of adipocyte area and crown-like structures in adipose tissue. Therefore, these results suggest that 18:0 Lyso PC is a potential compound to have protective effects against obesity, improving obese phenotype induced by HFD.

Diet-induced obesity in mice impairs host defense against Klebsiella pneumonia in vivo and glucose transport and bactericidal functions in neutrophils in vitro.

Obesity impairs host defense against Klebsiella pneumoniae, but responsible mechanisms are incompletely understood. To determine the impact of diet-induced obesity on pulmonary host defense against K. pneumoniae, we fed 6-wk-old male C57BL/6j mice a normal diet (ND) or high-fat diet (HFD) (13% vs. 60% fat, respectively) for 16 wk. Mice were intratracheally infected with Klebsiella, assayed at 24 or 48 h for bacterial colony-forming units, lung cytokines, and leukocytes from alveolar spaces, lung parenchyma, and gonadal adipose tissue were assessed using flow cytometry. Neutrophils from uninfected mice were cultured with and without 2-deoxy-d-glucose (2-DG) and assessed for phagocytosis, killing, reactive oxygen intermediates (ROI), transport of 2-DG, and glucose transporter (GLUT1-4) transcripts, and protein expression of GLUT1 and GLUT3. HFD mice had higher lung and splenic bacterial burdens. In HFD mice, baseline lung homogenate concentrations of IL-1β, IL-6, IL-17, IFN-γ, CXCL2, and TNF-α were reduced relative to ND mice, but following infection were greater for IL-6, CCL2, CXCL2, and IL-1β (24 h only). Despite equivalent lung homogenate leukocytes, HFD mice had fewer intraalveolar neutrophils. HFD neutrophils exhibited decreased Klebsiella phagocytosis and killing and reduced ROI to heat-killed Klebsiella in vitro. 2-DG transport was lower in HFD neutrophils, with reduced GLUT1 and GLUT3 transcripts and protein (GLUT3 only). Blocking glycolysis with 2-DG impaired bacterial killing and ROI production in neutrophils from mice fed ND but not HFD. Diet-induced obesity impairs pulmonary Klebsiella clearance and augments blood dissemination by reducing neutrophil killing and ROI due to impaired glucose transport.

Long Term Sodium Nitrate Administration Positively Impacts Metabolic and Obesity Indices in Ovariectomized Rats

In postmenopausal women, nitric oxide (NO) deficiency is associated with obesity and type 2 diabetes (T2D). This study aims at determining the long-term effects of low-dose nitrate administration on metabolic and obesity indices in ovariectomized (OVX) rats. OVX rat model was induced using the two dorsolateral skin incision method. Two months after ovariectomy, rats were divided into three groups (n=10/group): Control, OVX, and OVX+nitrate, and the latter received sodium nitrate at a dose of 100 mg/L in their drinking water for nine months. Fasting serum glucose and lipid profile were measured every month. A glucose tolerance test was performed at months 1, 3, and 9 (the end of the study). Obesity indices were calculated, and histological analyses were performed on the gonadal adipose tissues at month 9. OVX rats had impaired fasting glucose, glucose intolerance, and dyslipidemia with higher obesity indices at month 9. Nitrate improved glucose and lipid metabolism in OVX rats and decreased body weight (6.9%), body mass index (12.5%), Lee index (5.4%), adiposity index (23.9%), abdominal circumference (10.5%), and thoracic circumference (17.1%). Also, nitrate decreased adipocyte area by 49% and increased adipocyte density by 193% in gonadal adipose tissue. Long-term low-dose nitrate administration improves glucose and lipid metabolism in OVX rats in association with decreasing OVX-induced adiposity, increasing adipocyte density, and decreasing adipocyte area. These findings provide support for a potential therapeutic role of nitrate in postmenopausal women with some features of metabolic syndrome.

Identification of four novel QTL linked to the metabolic syndrome in the Berlin Fat Mouse

BackgroundThe Berlin Fat Mouse Inbred line (BFMI) is a model for obesity and the metabolic syndrome. This study aimed to identify genetic variants associated with impaired glucose metabolism using the obese lines BFMI861-S1 and BFMI861-S2, which are genetically closely related, but differ in several traits. BFMI861-S1 is insulin resistant and stores ectopic fat in the liver, whereas BFMI861-S2 is insulin sensitive.MethodsIn generation 10, 397 males of an advanced intercross line (AIL) BFMI861-S1 × BFMI861-S2 were challenged with a high-fat, high-carbohydrate diet and phenotyped over 25 weeks. QTL-analysis was performed after selective genotyping of 200 mice using the GigaMUGA Genotyping Array. Additional 197 males were genotyped for 7 top SNPs in QTL regions. For the prioritization of positional candidate genes whole genome sequencing and gene expression data of the parental lines were used.ResultsOverlapping QTL for gonadal adipose tissue weight and blood glucose concentration were detected on chromosome (Chr) 3 (95.8–100.1 Mb), and for gonadal adipose tissue weight, liver weight, and blood glucose concentration on Chr 17 (9.5–26.1 Mb). Causal modeling suggested for Chr 3-QTL direct effects on adipose tissue weight, but indirect effects on blood glucose concentration. Direct effects on adipose tissue weight, liver weight, and blood glucose concentration were suggested for Chr 17-QTL. Prioritized positional candidate genes for the identified QTL were Notch2 and Fmo5 (Chr 3) and Plg and Acat2 (Chr 17). Two additional QTL were detected for gonadal adipose tissue weight on Chr 15 (67.9–74.6 Mb) and for body weight on Chr 16 (3.9–21.4 Mb).ConclusionsQTL mapping together with a detailed prioritization approach allowed us to identify candidate genes associated with traits of the metabolic syndrome. In addition, we provided evidence for direct and indirect genetic effects on blood glucose concentration in the insulin-resistant mouse line BFMI861-S1.

Impact of Dietary Sodium Reduction on the Development of Obesity and Type 2 Diabetes in db/db Mice.

The impact of dietary sodium reduction on mouse models of type 2 diabetes is not well understood. Therefore, we analyzed the effect of a low-salt diet on obesity and parameters of type 2 diabetes in db/db mice. Five-week-old male db/db and lean db/m mice were fed a normal salt (0.19% Na+, NS) or a low-salt diet (<0.03% Na+, LS) for 5 weeks. Body and organ weight and parameters of glucose and insulin tolerance were analyzed. Plasma levels of steroids were determined by liquid chromatography tandem mass spectrometry. Body weight, glucose, and insulin tolerance were not affected by LS. The amount of gonadal adipose tissue showed a trend to be increased by LS whereas liver, pancreas, kidney, heart, and adrenal weight remained unaffected. LS reduced urinary sodium-to-creatinine ratio but did not affect plasma Na+ levels in both genotypes. Plasma and urinary potassium-to-creatinine ratio did not differ in all groups of mice. Aldosterone as a major determinant of changes in dietary sodium remained unaffected by LS in db/db mice as well as further investigated steroid hormones. The present study showed reduced sodium-to-creatinine ratio, but no additional effects of dietary sodium reduction on major metabolic parameters and steroid levels in obese and hyper-glycemic db/db mice.

Trypanosoma brucei triggers a broad immune response in the adipose tissue.

Adipose tissue is one of the major reservoirs of Trypanosoma brucei parasites, the causative agent of sleeping sickness, a fatal disease in humans. In mice, the gonadal adipose tissue (AT) typically harbors 2-5 million parasites, while most solid organs show 10 to 100-fold fewer parasites. In this study, we tested whether the AT environment responds immunologically to the presence of the parasite. Transcriptome analysis of T. brucei infected adipose tissue revealed that most upregulated host genes are involved in inflammation and immune cell functions. Histochemistry and flow cytometry confirmed an increasingly higher number of infiltrated macrophages, neutrophils and CD4+ and CD8+ T lymphocytes upon infection. A large proportion of these lymphocytes effectively produce the type 1 effector cytokines, IFN-γ and TNF-α. Additionally, the adipose tissue showed accumulation of antigen-specific IgM and IgG antibodies as infection progressed. Mice lacking T and/or B cells (Rag2-/-, Jht-/-), or the signature cytokine (Ifng-/-) displayed a higher parasite load both in circulation and in the AT, demonstrating the key role of the adaptive immune system in both compartments. Interestingly, infections of C3-/- mice showed that while complement system is dispensable to control parasite load in the blood, it is necessary in the AT and other solid tissues. We conclude that T. brucei infection triggers a broad and robust immune response in the AT, which requires the complement system to locally reduce parasite burden.

The B56α subunit of PP2A is necessary for mesenchymal stem cell commitment to adipocyte.

Adipose tissue plays a major role in maintaining organismal metabolic equilibrium. Control over the fate decision from mesenchymal stem cells (MSCs) to adipocyte differentiation involves coordinated command of phosphorylation. Protein phosphatase 2A plays an important role in Wnt pathway and adipocyte development, yet how PP2A complexes actively respond to adipocyte differentiation signals and acquire specificity in the face of the promiscuous activity of its catalytic subunit remains unknown. Here, we report the PP2A phosphatase B subunit B56α is specifically induced during adipocyte differentiation and mediates PP2A to dephosphorylate GSK3β, thereby blocking Wnt activity and driving adipocyte differentiation. Using an inducible B56α knock-out mouse, we further demonstrate that B56α is essential for gonadal adipose tissue development invivo and required for the fate decision of adipocytes over osteoblasts. Moreover, we show B56α expression is driven by the adipocyte transcription factor PPARγ thereby establishing a novel link between PPARγ signaling and Wnt blockade. Overall, our results reveal B56α is a necessary part of the machinery dictating the transition from pre-adipocyte to mature adipocyte and provide fundamental insights into how PP2A complex specifically and actively regulates unique signaling pathway in biology.

CTRP-3 Regulates NOD1-mediated Inflammation and NOD1 Expression in Adipocytes and Adipose Tissue

The anti-inflammatory adipokine CTRP-3 might affect innate immune reactions such as NOD1. The impact of CTRP-3 on NOD1-mediated inflammation in adipocytes and monocytic cells as well as on NOD1 expression was investigated. Murine 3T3-L1 pre-adipocytes and adipocytes as well as human THP-1 monocyte-like cells were co-stimulated with the synthetic NOD1 agonist Tri-DAP and recombinant CTRP-3. Gonadal adipose tissue and primary adipocytes were obtained from a murine model carrying a knockout (KO) of CTRP-3 in adipocytes but not in stroma-vascular cells. Wildtype mice with lipopolysaccharide (LPS)-induced elevated NOD1 expression were treated with CTRP-3. Secreted inflammatory cytokines in cell supernatants were measured by ELISA and mRNA levels were quantified by RT-PCR. Pro-inflammatory chemokine and cytokine secretion (MCP-1, RANTES, TNFα) was induced by NOD1 activation in adipocytes and monocyte-like cells, and MCP-1 and RANTES release was effectively inhibited by pre-incubation of cells with CTRP-3. CTRP-3 also antagonized LPS-triggered induction of NOD1 gene expression in murine adipose tissue, whereas adipocyte CTRP-3 deficiency upregulated NOD1 expression in adipose tissue. CTRP-3 is an effective antagonist of peptidoglycan-induced, NOD1-mediated inflammation and of LPS-induced NOD1 expression. Since basal NOD1 expression is increased by adipocyte CTRP-3 deficiency, there have to be also inflammation-independent mechanisms of NOD1 expression regulation by CTRP-3.

Rodents on a High-Fat Diet Born to Mothers with Gestational Diabetes Exhibited Sex- Specific Lipidomic Changes in Reproductive Organs, Hormonal Imbalances and Insulin Resistance

Background: Maternal gestational diabetes mellitus (GDM) and offspring high-fat diet (HFD) have shown to have sex-specific detrimental effects on the health of offspring. Methods: A maternal GDM mice model was established by feeding maternal C57BL/6 mice HFD and then their offspring were exposed to either HFD or low-fat diet. Testes, ovaries, liver and gonadal adipose tissue were collected from offspring at 20 weeks, and their reproductive lipidomic profiles were characterized using gas chromatography-mass spectrometry. Findings: Male offspring following HFD had a substantially elevated body weight. In reproductive organs and hormones, male offspring from GDM mothers had decreased testes weights and testosterone levels, while female offspring from GDM mothers had increased ovary weights and estrogen levels. AKT pathway were increased in the offspring liver and gonadal adipose tissue of those fed with HFD. The fatty acid profiling showed that testes were prone to the effect of maternal GDM, whereas ovarian metabolite profiles were upregulated in maternal GDM and downregulated in offspring following HFD. Eicosapentaenoic acid were attenuated in both maternal serum and offspring testes under the impact of maternal GDM and in offspring following HFD. Interpretation: Maternal GDM and offspring HFD have different metabolic effects on offspring reproductive organs, and PUFAs may protect against detrimental outcomes in offspring such as obesity. Funding: the National Natural Science Foundation of China, The 111 Project, The National Key Research and Development Program of Reproductive Health & Major Birth Defects Control and Prevention, Chongqing Health Commission, Kuanren Talents Programs, and Chongqing Science & Technology Commission. Declaration of Interest: Andi Wang, Xinyang Yu, Zhu Chen, Yinyin Xia, Chang Chen, Hongbo Qi, Philip N. Baker, Richard Saffery, Ting-Li Han and Hua Zhang declare that they have no conflict of interest. Ethical Approval: All WT C57BL/6 mice in this study were purchased from the Model Animal Research Center of Nanjing University. All animal experiments complied with the ARRIVE guidelines and approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (Batch number: 2020-41).

Adipocyte calcium sensing receptor is not involved in visceral adipose tissue inflammation or atherosclerosis development in hyperlipidemic Apoe\u2212/\u2212 mice

The calcium sensing receptor (CaSR) is a G-protein coupled receptor that especially plays an important role in the sensing of extracellular calcium to maintain its homeostasis. Several in-vitro studies demonstrated that CaSR plays a role in adipose tissue metabolism and inflammation, resulting in systemic inflammation and contributing to atherosclerosis development. The aim of this study was to investigate whether adipocyte CaSR plays a role in adipose tissue inflammation in-vivo and atherosclerosis development. By using a newly established conditional mature adipocyte specific CaSR deficient mouse on a hyperlipidemic and atherosclerosis prone Apoe−/− background it could be shown that CaSR deficiency in adipocytes does neither contribute to initiation nor to progression of atherosclerotic plaques as judged by the unchanged lesion size or composition. Additionally, CaSR deficiency did not influence gonadal visceral adipose tissue (vAT) inflammation in-vivo, although a small decrease in gonadal visceral adipose cholesterol content could be observed. In conclusion, adipocyte CaSR seems not to be involved in vAT inflammation in-vivo and does not influence atherosclerosis development in hyperlipidemic Apoe−/− mice.

Histomorphological testicular changes and decrease in the sperm count in pubertal rats induced by a high-sugar diet

BackgroundDuring childhood and adolescence, excessive food consumption stimulates adipose tissue expansion promoting overweight in humans, and mice. A high-sucrose diet is related to obesity and metabolic syndrome. Infertility is commonly related to these pathologies. We aim to evaluate possible histomorphological testicular changes induced by a high-sucrose diet on sperm count during the post-weaning period. MethodsWistar male rats aged 21 days, weaned, were randomly assigned into two groups: control (fed and hydrated normally) and sugar group (fed normally but hydrated with a solution containing 30% of diluted sucrose during 30 days). At the pubertal age of 51 days, animals were killed and blood samples were taken to measure testosterone and leptin. Testicles were collected and gonadal adipose tissue and semen samples from the epididymis were excised. Testicle samples were used for morphological description using H&E staining, as well as to quantify the triacylglycerol content and the lactate dehydrogenase (LDH) expression. Semen samples were used to assess motility, viability, and sperm count. ResultsThe sugar group presented an increase in the testicular weight, but a reduction in the cross-sectional area of seminiferous tubules. Moreover, disorganization of Sertoli cells and spermatogonia, an increase in the LDH expression within the entire seminiferous tubule, and a reduced sperm count and spermatozoid motility were found. These alterations were accompanied by high serum levels of testosterone and leptin. ConclusionsOur results indicate strong damage of testis by sugar consumption during early life that may lead to the onset of infertility in adulthood.

Peripancreatic adipose tissue protects against high-fat-diet-induced hepatic steatosis and insulin resistance in mice

Background/objectivesVisceral adiposity is associated with increased diabetes risk, while expansion of subcutaneous adipose tissue may be protective. However, the visceral compartment contains different fat depots. Peripancreatic adipose tissue (PAT) is an understudied visceral fat depot. Here, we aimed to define PAT functionality in lean and high-fat-diet (HFD)-induced obese mice.Subjects/methodsFour adipose tissue depots (inguinal, mesenteric, gonadal, and peripancreatic adipose tissue) from chow- and HFD-fed male mice were compared with respect to adipocyte size (n = 4–5/group), cellular composition (FACS analysis, n = 5–6/group), lipogenesis and lipolysis (n = 3/group), and gene expression (n = 6–10/group). Radioactive tracers were used to compare lipid and glucose metabolism between these four fat depots in vivo (n = 5–11/group). To determine the role of PAT in obesity-associated metabolic disturbances, PAT was surgically removed prior to challenging the mice with HFD. PAT-ectomized mice were compared to sham controls with respect to glucose tolerance, basal and glucose-stimulated insulin levels, hepatic and pancreatic steatosis, and gene expression (n = 8–10/group).ResultsWe found that PAT is a tiny fat depot (~0.2% of the total fat mass) containing relatively small adipocytes and many “non-adipocytes” such as leukocytes and fibroblasts. PAT was distinguished from the other fat depots by increased glucose uptake and increased fatty acid oxidation in both lean and obese mice. Moreover, PAT was the only fat depot where the tissue weight correlated positively with liver weight in obese mice (R = 0.65; p = 0.009). Surgical removal of PAT followed by 16-week HFD feeding was associated with aggravated hepatic steatosis (p = 0.008) and higher basal (p < 0.05) and glucose-stimulated insulin levels (p < 0.01). PAT removal also led to enlarged pancreatic islets and increased pancreatic expression of markers of glucose-stimulated insulin secretion and islet development (p < 0.05).ConclusionsPAT is a small metabolically highly active fat depot that plays a previously unrecognized role in the pathogenesis of hepatic steatosis and insulin resistance in advanced obesity.

Sexual dimorphism in insulin resistance in a metabolic syndrome rat model.

ObjectiveWe assessed the sex-specific differences in the molecular mechanisms of insulin resistance in muscle and adipose tissue, in a MS rat model induced by a high sucrose diet.MethodsMale, female, and ovariectomized female Wistar rats were randomly distributed in control and high-sucrose diet (HSD) groups, supplemented for 24 weeks with 20% sucrose in the drinking water. At the end, we assessed parameters related to MS, analyzing the effects of the HSD on critical nodes of the insulin signaling pathway in muscle and adipose tissue.ResultsAt the end of the treatment, HSD groups of both sexes developed obesity, with a 15, 33 and 23% of body weight gain in male, female, and OVX groups respectively, compared with controls; mainly related to hypertrophy of peripancreatic and gonadal adipose tissue. They also developed hypertriglyceridemia, and liver steatosis, with the last being worse in the HSD females. Compared to the control groups, HSD rats had higher IL1B and TNFA levels and insulin resistance. HSD females were more intolerant to glucose than HSD males. Our observations suggest that insulin resistance mechanisms include an increase in phosphorylated AKT(S473) form in HSD male and female groups and a decrease in phosphorylated P70S6K1(T389) in the HSD male groups from peripancreatic adipose tissue. While in gonadal adipose tissue the phosphorylated form of AKT decreased in HSD females, but not in HSD males. Finally, HSD groups showed a reduction in p-AKT levels in gastrocnemius muscle.ConclusionA high-sucrose diet induces MS and insulin resistance with sex-associated differences and in a tissue-specific manner.

Adipose tissue of female Wistar rats respond to Ilex paraguariensis treatment after ovariectomy surgery

Background and aimMetabolic disturbances are known for their increasing epidemiological importance. Ilex paraguariensis presents a potential option for mitigating lipid metabolism imbalance. However, most of the literature to date has not considered sex bias. This study aimed to evaluate the effect of Ilex paraguariensis on the metabolism of different adipose tissue depots in males and females. Experimental procedureAfter ovariectomy, female Wistar rats received daily treatment with the extract (1 g/kg) for forty-five days. Biochemical serum parameters and tissue metabolism were evaluated. Oxidation, lipogenesis and lipolysis were evaluated in brown, white visceral, retroperitoneal and gonadal adipose tissues. Results and conclusionThe results showed that treatment with the extract led to a reduced weight gain in ovariectomised females in comparison to control. The triglyceride concentration was decreased in males. Glucose oxidation and lipid synthesis in visceral and retroperitoneal adipose tissues were restored in ovariectomised females after treatment. The response to epinephrine decreased in visceral adipose tissue of control males; however, lipolysis in females did not respond to ovariectomy or treatment. These findings highlight the enormous potential effects of I. paraguariensis on lipid metabolism, modulating lipogenic pathways in females and lipolytic pathways in males. Furthermore, the sex approach applied in this study contributes to more effective screening of the effects of I. paraguariensis bioactive substances.

3094 – ROLE OF BONE MARROW INJURY IN PROMOTING TET2-MEDIATED CLONAL HEMATOPOIESIS OF INDETERMINATE POTENTIAL (CHIP)

CHIP is characterized by selective outgrowth of hematopoietic stem/progenitor cell (HSPC) in the bone marrow (BM) which frequently harbors oncogenic mutations in genes such as TET2. This leads to aberrant expansion of hyperinflammatory immune cells that can contribute to cardiovascular disease and other comorbidities. Prior genotoxic treatments such as chemotherapy and radiation reportedly correlate with the expansion of CHIP mutant HSPC. However, the mechanism(s) remain poorly understood. To address this question, we modeled human CHIP via adoptively transferring Tet2-/- mouse BM into wild-type (WT) recipient mice. In this setting, Tet2-/- HSPC and their progeny preferentially expanded in the BM and blood. Interestingly, Tet2-/- HSPC also colonized non-hematopoietic organs including lung, liver and gonadal adipose tissue. To test whether genotoxic stress caused by radiation accelerated this process, we subjected Tet2-/- CHIP mice to 2.5Gy radiation. Indeed, radiation accelerated Tet2-/- HSPC expansion in nearly all tissues. To assess the impact of microenvironmental factors induced by radiation on CHIP progression, we modeled radiation-induced inflammation and tissue oxygenation using in vitro HSPC culture. Surprisingly, inflammatory cytokines were not sufficient to drive selective expansion of Tet2-/- HSPC. On the other hand, Tet2-/- HSPC displayed a significant proliferative advantage over WT when cultured at high oxygen tension (20%O2) but not in hypoxic conditions representative of healthy BM (1%O2), suggesting high O2 tension caused by BM injury may play a vital role in the selective expansion of Tet2-/- HSPC. Ongoing studies are aimed at understanding the impact of increased oxygen levels in the BM using non-genotoxic approaches and identifying mechanism(s) by which Tet2-/- HSPC harness high O2 tension to proliferate. Such investigations could provide a basis for preventing CHIP and its associated comorbidities.

Maternal high-fat diet stimulates proinflammatory pathway and increases the expression of Tryptophan Hydroxylase 2 (TPH2) and brain-derived neurotrophic factor (BDNF) in adolescent mice hippocampus

Maternal high-fat diet (HFD) consumption can promote a systemic inflammatory condition that may impair the offspring brain development, damaging memory and learning, when it reaches the hippocampus. This study aims to evaluate maternal HFD effects, during pregnancy and lactation, upon dams/mice offspring nutritional status, protein and gene expression of inflammatory pathway (JNK, pJNK and TNF-α), serotonin system molecules (Tryptophan Hydroxylase 2 (TPH2), key-enzyme of serotonin synthesis, serotonin transporter (SERT); 5-HT1A serotonergic receptor (5-HT1A)) and brain derived neurotrophic factor (BDNF) on recently weaned mice offspring hippocampus. Female Swiss mice were fed a control diet (CD, 11,5% fat) or a HFD (45.0% fat) from pre-mating to lactation. After weaning, the offspring received CD up to 28 post-natal days (PND28). Body weight and visceral adiposity (retroperitoneal and gonadal adipose tissue) of dams and offspring were measured. After euthanasia, the offspring hippocampus was dissected for evaluations of BDNF, inflammatory pathway and serotonergic system molecules protein and gene expression, through the techniques of Western Blotting, RTqPCR and ELISA. Our findings show that, during pregnancy, HFD-dams and HFD-offspring exhibited an increase in body weight gain and visceral adipose tissue compared to control animals. The hippocampus of HFD-offspring showed increased protein expression of TPH2, BDNF, pJNK and increased mRNA levels of TNF-α. However, the TPH2 increase in HFD-offspring did not alter hippocampal serotonin levels quantified through ELISA. Maternal HFD promoted an obesity phenotype in its offspring with increased body weight and visceral adiposity, increased protein and gene expression of the pro-inflammatory proteins pJNK and TNF-α. These changes were accompanied by increased TPH2 and BDNF protein expression. Thus, our findings show that maternal HFD during gestation and lactation increased pJNK and TNF-α expression in their offspring hippocampus indicating a pro-inflammatory state, with increased BDNF expression and alterations in its serotonergic system reflected by increased TPH2 expression.

P0685RENAL SUBCAPSULAR ADIPOSE-DERIVED MESENCHYMAL STEM CELLS (ASC) ADMINISTRATION, ASSOCIATED TO LOSARTAN TREATMENT, PREVENTED THE PROGRESSION OF RENAL DAMAGE IN AN EXPERIMENTAL MODEL OF CHRONIC KIDNEY DISEASE (CKD)

Abstract INTRODUCTION / AIMS Chronic kidney disease (CKD) is considered a public health problem with epidemic proportions worldwide. Overactivity of the renin-angiotensin-aldosterone system (RAAS), associated to the establishment of a chronic self-sustained renal inflammation, are the main factors which contribute to the progression of CKD, leading to tissue fibrosis and loss of function. Consequently, RAAS inhibitors, such as the AT-1 receptor antagonist, Losartan, are among the most currently employed therapeutic strategies to slow down CKD progression, although the renoprotection afforded by these drugs is not complete. The association of anti-inflammatory and immunosuppressive drugs to RAAS inhibition is limited to the treatment of specific nephropathies, and its safety is still not entirely known. In this context, experimental application of mesenchymal stem cells (mSC) as a treatment to control renal inflammation and fibrosis have been showing promising results in studies with animal models of CKD. The aim of the present study was to analyze the renoprotective effects of subcapsular application of adipose-tissue derived mSC (ASC), associated to Losartan treatment, in rats submitted to 5/6 nephrectomy, after the establishment of CKD. METHODS ASC were isolated from gonadal adipose tissue from male Wistar rats, cultured until P4 and characterized by flow cytometry and in vitro differentiation. Male Wistar rats underwent 5/6 nephrectomy and were followed for 15 days to confirm the establishment of the nephropathy (CKD 15d). After this period, animals underwent a new surgery in which they received a subcapsular injection of 10 μL of sterile PBS (vehicle), or 2x106 ASC diluted in 10 μL of sterile PBS. Part of these animals were than treated orally with 50 mg/kg/day of Losartan (LOS) for further 15 days (groups CKD + LOS 30d and CKD + LOS + ASC 30d). A group of animals that received only subcapsular vehicle were kept untreated for the same period (CKD 30d). Sham-operated rats, euthanized at days 15 (Sham 15d) and 30 (Sham 30d), were used as controls. Survival rate, body weight (BW), systolic blood pressure (BP), 24h urinary protein (24h UPE) and albumin (24h UAE) excretion, serum creatinine (SCr) and blood urea nitrogen (BUN) concentrations, percentage (GS%) and index (GSI) of glomerulosclerosis, percentage of tubulointerstitial fibrosis (INT%) and renal infiltration by macrophages (CD68) were assessed 15 and 30 days after 5/6 nephrectomy. The obtained results are presented as Mean ± SE. Differences among groups were analyzed by one-way ANOVA. RESULTS The renal subcapsular application of ASC after the establishment of severe CKD improved the renoprotection obtained with the classic treatment with LOS. The association of LOS + ASC significantly improved animal’s survival, reduced blood pressure, proteinuria and albuminuria, prevented the development of glomerular structural damage and interstitial fibrosis, and inhibited renal inflammation by normalizing renal macrophage infiltration in the CKD + LOS + ASC group. CONCLUSIONS Our preliminary results demonstrate that the application of ASC, associated to the oral administration of LOS exerted additional renoprotection in the 5/6 renal ablation model, compared to LOS monotherapy. These findings suggest that ASC therapy could be employed as a therapeutic additive together with the traditional RAAS blockade, to detain the progression of CKD.

P0678RENAL SUBCAPSULAR ADMINISTRATION OF ADIPOSE DERIVED MESENCHYMAL STEM CELLS PREVENTED THE PROGRESSION OF RENAL DAMAGE IN AN EXPERIMENTAL MODEL OF CKD

Abstract INTRODUCTION / AIMS Chronic kidney disease (CKD) is a progressive, debilitating condition of high lethality, which prevalence have been increasing considerably in recent decades. CKD can be triggered by many different factors, such as genetic predisposition, systemic hypertension, diabetes mellitus and autoimmune diseases. It is characterized by the gradual loss of renal function, leading to kidney failure and the need for renal replacement therapy for the maintenance of life. Regardless of the etiology of CKD, the establishment of local renal inflammation, with leucocyte recruitment, cell proliferation, extracellular matrix accumulation, glomerular and tubulointerstitial fibrosis, contribute significantly to its establishment and evolution. Due to its known pathophysiology, the primary aim when clinically treating CKD is to slow the progression of renal function loss and the advance of inflammation. However, until the present moment, there is no efficient pharmacological treatment to completely arrest the aggravation of renal inflammation and, specially, renal fibrosis. This motivates the scientific community to develop experimental research in order to test new therapeutic approaches to stunt renal fibrosis. In this context, experimental application of mesenchymal stem cells (mSC) as a treatment to control renal inflammation have been showing promising results in studies with animal models of CKD. The aim of the present study was to analyze the renoprotective effects of subcapsular application of Adipose Tissue-derived mSC (ASC), in rats submitted to 5/6 nephrectomy, after the establishment of the disease (15 days after CKD induction), in order to more closely resemble the clinical settings in humans. METHODS ASC were obtained from gonadal adipose tissue from healthy male Wistar rats. These cells were cultured until P4 when characterization by flow cytometry and in vitro differentiation were performed. Male Wistar rats underwent 5/6 nephrectomy and were followed for 15 days until the complete establishment of CKD (group CKD 15d). At this time, animals underwent a new surgery in which they received a subcapsular injection of 2x106 ASC diluted in 10 μL of sterile PBS (group CKD + ASC 30d), or only 10 μL of sterile PBS (group CKD 30d). Sham-operated rats, euthanized at day 15 (Sham 15d) and 30 (Sham 30d) were used as controls. Survival rate, body weight (BW), 24h urinary protein (24h UPE) and albumin (24h UAE) excretion serum creatinine (SCr) and blood urea nitrogen (BUN) concentration, percentage (GS%) and index (GSI) of glomerulosclerosis, tubulointerstitial fibrosis (INT%) and renal infiltration by macrophages (CD68) were studied at 15 and 30 days after 5/6 nephrectomy. Our results are presented as Mean ± SE. Differences among groups were analyzed by one-way ANOVA. RESULTS ASC injection significantly improved the survival rate of CKD + ASC 30d animals, compared to the observed in the untreated group. Moreover, ASC treatment markedly reduced protein and albumin urinary excretion, prevented the development of glomerulosclerosis, both the percentage of sclerotic glomeruli and the index of glomerular damage, numerically reduced interstitial fibrosis and significantly avoided renal inflammation by halting the progression of renal cortical macrophage infiltration. CONCLUSIONS According to our results, subcapsular ASC application promoted considerable renoprotection in the 5/6 renal ablation model, even after the complete establishment of severe CKD, suggesting that experimental therapy with these cells could be associated to the current pharmacological treatments employed to detain the progression of CKD. Figure

Dietary Supplementation with a Low Dose of Finasteride Does Not Alter the Lipid Profile of Ldlr-Deficient Mice

Abstract Objectives Androgenic alopecia is characterized by a receding hairline and hair loss from the frontal scalp. This genetic condition affects approximately 80% of men and 42% of women. Finasteride is the only oral treatment currently approved by the FDA to prevent and treat androgenic alopecia, and it is currently prescribed to over nine million males in the US. Recent claims suggest that long-term use of finasteride could have detrimental health effects by affecting the lipid profile in people. Finasteride prevents the conversion of testosterone to its active metabolite dihydrotestosterone by inhibiting the type II 5 alpha-reductase, which is predominantly present in the hair follicle and the prostate. As a result, finasteride alters the systemic levels of testosterone and its derivative estradiol. Hormone homeostasis affects plasma lipid levels, therefore we hypothesize that supplementation with finasteride will affect systemic lipid profile in atheroprone low-density lipoprotein receptor (Ldlr−/−) mice fed a Western diet (high cholesterol, high fat). Methods We fed six-week-old male Ldlr−/-mice (10/group) fed a Western diet (control) or a Western diet supplemented with 10 mg Finasteride/kg diet for 12 weeks. We monitored body weight progression and food intake during the entire experiment. A week before harvesting the tissues, mice were fasted overnight to perform a glucose tolerance test. At the moment of sacrifice, final body weight and several tissues were collected such as the inguinal, retroperitoneal, and gonadal adipose tissues, skeletal muscle (gastrocnemius), and liver. Plasma was harvested to determine total cholesterol and triglyceride levels. Results Our data show that 12-week supplementation with a low dose of finasteride does not have a significant impact on food intake, body weight, or adiposity index. We did not observe any significant change in plasma lipids or glucose tolerance. Conclusions A low dose of finasteride supplemented for 12 weeks does not alter the lipid profile in the atheroprone Ldlr−/-mouse model, nor alters adiposity index. These data indicate that finasteride does not have detrimental effects on these metabolic parameters. Funding Sources U.S. Department of Agriculture (Multi-State grant project W4002).