Circulating microRNA signatures in patients with chronic Urticaria.

Background: Chronic Urticaria (CU) is a complex skin disease that appears as recurrent raised itchy rash/angioedema or both for more than six weeks. The pathophysiology of CU is complex and has yet to be understood entirely. It is predominantly a mast cell-driven disease with the possible involvement of type 2 inflammation. Current evidence largely favors mast cell activation by an IgE-mediated autoallergic mechanism or an autoimmune mechanism by IgG autoantibodies to IgE/ high-affinity receptor of IgE. MicroRNAs (miRNA) are small coding RNAs regulating gene expression at the post-transcription level. This study aimed to investigate the circulating miRNA as potential biomarkers in CU patients compared to healthy controls. Methods: The miRNA gene expression was done in seven patients with CU and seven healthy controls. The expression of miRNA is done using TaqMan openArray human advanced miRNA Panel. ExpressionSuite Software (Thermo Fischer Scientific, Waltham, MA, USA) is used for data analysis to quantify the miRNA expressions. P<0.05 is considered to be statistically significant. Results: A significant upregulation (p<0.05) in the miR-451a, miR-9-5p, miR-150-5p, miR-296-5p, and miR-182-5p was observed in CU compared to controls. Dysregulation of miR-451a is identified as an early biomarker in allergic diseases. Functional enrichment analysis with the KEGG pathway and disease ontology databases showed that these miRNAs were associated with skin diseases and inflammation. The differentially expressed miRNAs contribute to determining the genes regulated in CU. miRNA-based therapies that target different genes in a given pathway might be a potential candidate for treating CU. Conclusion: miRNA field has grown steadily over the past few years, but the role of circulating miRNAs in CU remains relatively unexplored. This study showed that the upregulated circulating miRNA might play an essential role in CU pathogenesis and inflammation. Also, our study highlights the importance of miRNAs as a future biomarker and potential therapeutic target to be investigated.

Severe iatrogenic hypoglycaemia modulates the fibroblast growth factor protein response.

INTRODUCTION: There is evidence that fibroblast growth factor (FGF) levels may be implicated in hypoglycaemia, with FGF19 being a potential contributor to insulin-independent pathways driving postprandial hypoglycaemia following bariatric surgery and basic FGF (FGF2) being elevated following mild hypoglycaemia occurring after the glucose tolerance test. However, their response following severe iatrogenic hypoglycaemia is unknown and therefore this pilot exploratory study was undertaken. METHODS: A case-control study of aged-matched type 2 diabetes (T2D; n = 23) and control (n = 23) subjects who underwent a hyperinsulinaemic clamp, initially to euglycaemia in T2D (5 mmol/L; 90 mg/dl), and then to hypoglycaemia (<2 mmol/L; <36 mg/dl) with subsequent follow-up time course to 24 h. FGF and FGF receptor proteins were determined by Slow Off-rate Modified Aptamer (SOMA)-scan plasma protein measurement. RESULTS: At baseline, FGF12 (p = .006) was higher and FGF20 (p = .004) was lower in T2D versus controls. At hypoglycaemia, FGF7 was lower in T2D. Post-hypoglycaemic levels of FGF18, FGF19, FGF20 and FGF23 were lower while FGF12 and FGF16 were higher in T2D versus control at different time points. No differences between T2D and controls were seen for FGF1, FGF2, FGF4, FGF6, FGF8, FGF9, FGF10, FGF21 or any of the FGF receptors. At 24 h post-hypoglycaemia, FGF20 (p = .01) differed between controls and T2D, while the levels for the other proteins measured returned to baseline. None of the FGF proteins altered from baseline to euglycaemia when clamped in T2D subjects. FGF23 negatively correlated with fasting blood glucose, but no FGFs correlated with body mass index in T2D. CONCLUSION: Severe transient hypoglycaemia modulated FGF7, 16, 19, 20 and 23 (known to be associated with diabetes), together with FGF18 and 12, not previously reported to be associated with diabetes but that may be important in the pathophysiology of hypoglycaemia; FGF20 remained low at 24 h. Taken together, these data suggest that recurrent hypoglycaemia may contribute to the development of complications through changes in FGF proteins.

MicroRNA Changes Up to 24 h following Induced Hypoglycemia in Type 2 Diabetes.

Hypoglycemia, as a complication of type 2 diabetes (T2D), causes increased morbidity and mortality but the physiological response underlying hypoglycemia has not been fully elucidated. Small noncoding microRNA (miRNA) have multiple downstream biological effects. This pilot exploratory study was undertaken to determine if induced miRNA changes would persist and contribute to effects seen 24 h post-hypoglycemia. A parallel, prospective study design was employed, involving T2D (n = 23) and control (n = 23) subjects. The subjects underwent insulin-induced hypoglycemia (2 mmol/L; 36 mg/dL); blood samples were drawn at baseline, upon the induction of hypoglycemia, and 4 h and 24 h post-hypoglycemia, with a quantitative polymerase chain reaction analysis of miRNA undertaken. The baseline miRNAs did not differ. In the controls, 15 miRNAs were downregulated and one was upregulated (FDR < 0.05) from the induction of hypoglycemia to 4 h later while, in T2D, only four miRNAs were altered (downregulated), and these were common to both cohorts (miR-191-5p; miR-143-3p; let-7b-5p; let-7g-5p), correlated with elevated glucagon levels, and all were associated with energy balance. From the induction of hypoglycemia to 24 h, 14 miRNAs were downregulated and 5 were upregulated (FDR < 0.05) in the controls; 7 miRNAs were downregulated and 7 upregulated (FDR < 0.05) in T2D; a total of 6 miRNAs were common between cohorts, 5 were downregulated (miR-93-5p, let-7b-5p, miR-191-5p, miR-185-5p, and miR-652-3p), and 1 was upregulated (miR-369-3p). An ingenuity pathway analysis indicated that many of the altered miRNAs were associated with metabolic and coagulation pathways; however, of the inflammatory proteins expressed, only miR-143-3p at 24 h correlated positively with tumor necrosis factor-α (TNFa; p < 0.05 and r = 0.46) and negatively with toll-like receptor-4 (TLR4; p < 0.05 and r = 0.43). The MiRNA levels altered by hypoglycemia reflected changes in counter-regulatory glucagon and differed between cohorts, and their expression at 24 h suggests miRNAs may potentiate and prolong the physiological response. Trial registration: ClinicalTrials.gov NCT03102801.

Circulatory EVs as a predictor of chronic urticarial activity.

Background: Chronic urticaria (CU) is a common and complex disorder that occurs without any identifiable provoking factor. The mechanisms underlying CU pathogenesis are still not fully understood. The autoimmune theory of IgG autoantibodies to IgE/high-affinity receptor of IgE on mast cells and mast cell activation and autoallergy (IgE-mediated disease) might contribute to CU pathogenesis. Extracellular vesicles (EVs) are membranous vesicles released from apoptotic or activated cells of different types. In this study, we aimed to investigate circulating EVs as potential biomarkers in patients with CU compared with that in healthy controls. Methods: We studied 15 patients with CU and 16 healthy controls. Circulatory EVs (plasma) were characterized by the presence of externalized phosphatidylserine (annexin V staining). An unpaired t-test was used, and P < 0.05 was considered statistically significant. Results: We did not find significant differences in the total number of EVs in patients with CU. A significant decrease in the levels of T-cells (CD3) and endothelial cells (CD146) (P < 0.05) in these patients than in controls was found. No significant differences were observed between patients with CU and healthy controls in terms of platelets, macrophages, PECAM-1, B cells, and tissue factors. Conclusion: Endothelial cells have been shown to contribute to the pathogenesis of CU and are also targeted by mediators released by mast cells and other cellular infiltrates. We identified that circulatory endothelial and T-cell EVs might play an important role in CU pathogenesis. In addition, our study highlights the importance of EVs as future therapeutic targets to be investigated.

Regulation of circulating CTRP-2/CTRP-9 and GDF-8/GDF-15 by intralipids and insulin in healthy control and polycystic ovary syndrome women following chronic exercise training.

BACKGROUND: Polycystic ovary syndrome (PCOS) is associated with obesity, diabetes, and insulin resistance. The circulating C1Q/TNF-related proteins (CTRP-2, CTRP-9) and growth differentiation factors (GDF-8, GDF-15) contribute to glucose and lipid homeostasis. The effects of intralipids and insulin infusion on CTRP-2, CTRP-9, GDF-8 and GDF-15 in PCOS and control subjects before and after chronic exercise training were examined. METHODS: Ten PCOS and nine healthy subjects were studied at baseline status and after moderate-intensity chronic exercise training (1 h exercise, 3 times per week, 8 weeks). All participants were infused with 1.5 mL/min of saline or intralipids (20%) for 5 h, and during the last 2 h of saline or intralipids infusion hyperinsulinemic-euglycemic clamp (HIEC) was performed. CTRP-2, CTRP-9, GDF-8 and GDF-15 levels were measured at 0, 3 and 5 h. RESULTS: Intralipids dramatically increased CTRP-2 levels in PCOS (P = 0.02) and control (P = 0.004) subjects, which was not affected by insulin infusion or by exercise. Intralipids alone had no effects on CTRP-9, GDF-8, or GDF-15. Insulin increased the levels of GDF-15 in control subjects (P = 0.05) during the saline study and in PCOS subjects (P = 0.04) during the intralipid infusion. Insulin suppressed CTRP9 levels during the intralipid study in both PCOS (P = 0.04) and control (P = 0.01) subjects. Exercise significantly reduced fasting GDF-8 levels in PCOS (P = 0.03) and control (P = 0.04) subjects; however, intralipids infusion after chronic exercise training increased GDF-8 levels in both PCOS (P = 0.003) and control (P = 0.05) subjects and insulin infusion during intralipid infusion reduced the rise of GDF-8 levels. CONCLUSION: This study showed that exogenous lipids modulate CTRP-2, which might have a physiological role in lipid metabolism. Since chronic exercise training reduced fasting GDF-8 levels; GDF-8 might have a role in humoral adaptation to exercise. GDF-15 and CTRP-9 levels are responsive to insulin, and thus they may play a role in insulin responses.

Metabolic profiling of pre-gestational and gestational diabetes mellitus identifies novel predictors of pre-term delivery.

BACKGROUND: Pregnant women with gestational diabetes mellitus (GDM) or type 2 diabetes mellitus (T2DM) are at increased risks of pre-term labor, hypertension and preeclampsia. In this study, metabolic profiling of blood samples collected from GDM, T2DM and control pregnant women was undertaken to identify potential diagnostic biomarkers in GDM/T2DM and compared to pregnancy outcome. METHODS: Sixty-seven pregnant women (21 controls, 32 GDM, 14 T2DM) in their second trimester underwent targeted metabolomics of plasma samples using tandem mass spectrometry with the Biocrates MxP® Quant 500 Kit. Linear regression models were used to identify the metabolic signature of GDM and T2DM, followed by generalized linear model (GLMNET) and Receiver Operating Characteristic (ROC) analysis to determine best predictors of GDM, T2DM and pre-term labor. RESULTS: The gestational age at delivery was 2 weeks earlier in T2DM compared to GDM and controls and correlated negatively with maternal HbA1C and systolic blood pressure and positively with serum albumin. Linear regression models revealed elevated glutamate and branched chain amino acids in GDM + T2DM group compared to controls. Regression models also revealed association of lower levels of triacylglycerols and diacylglycerols containing oleic and linoleic fatty acids with pre-term delivery. A generalized linear model ROC analyses revealed that that glutamate is the best predictors of GDM compared to controls (area under curve; AUC = 0.81). The model also revealed that phosphatidylcholine diacyl C40:2, arachidonic acid, glycochenodeoxycholic acid, and phosphatidylcholine acyl-alkyl C34:3 are the best predictors of GDM + T2DM compared to controls (AUC = 0.90). The model also revealed that the triacylglycerols C17:2/36:4 and C18:1/34:1 are the best predictors of pre-term delivery (≤ 37 weeks) (AUC = 0.84). CONCLUSIONS: This study highlights the metabolite alterations in women in their second trimester with diabetes mellitus and identifies predictive indicators of pre-term delivery. Future studies to confirm these associations in other cohorts and investigate their functional relevance and potential utilization for targeted therapies are warranted.

Lipids and insulin regulate mitochondrial-derived peptide (MOTS-c) in PCOS and healthy subjects.

OBJECTIVE: Polycystic ovarian syndrome (PCOS) is a heterogeneous endocrine disorder associated with mitochondrial dysfunction and insulin resistance (IR). MOTS-c, a mitochondrial peptide, promotes insulin sensitivity (IS) through activating AKT and AMPK-dependent pathways. The current study was designed to examine the response of MOTS-c to lipids (intralipid) followed by insulin in PCOS and healthy subjects. METHODS: All subjects underwent 5-hour intralipid/saline infusion with a hyperinsulinemic-euglycaemic clamp in the final 2 hours. Plasma samples were collected to measure circulating MOTS-c using a commercial ELISA kit. Subsequently, this was repeated following an eight-week exercise intervention. RESULTS: Intralipid significantly increased plasma MOTS-c both in controls and PCOS subjects, whilst the insulin infusion blunted the intralipid-induced response seen for both lipids and MOT-c. Intralipid elevated plasma MOTS-c to 232 ± 124% of basal in control (P < 0.01) and to 349 ± 206% of basal in PCOS (P < 0.001) subjects. Administration of insulin suppressed intralipid-induced MOTS-c from 232 ± 124% to 165 ± 97% (NS) in control and from 349 ± 206% to 183 ± 177% (P < 0.05) in PCOS subjects, respectively. Following exercise, intralipid elevated plasma MOTS-c to 305 ± 153% of basal in control (P < 0.01) and to 215 ± 103% of basal in PCOS (P < 0.01) subjects; insulin suppressed intralipid-induced MOTS-c only in controls. CONCLUSIONS: In conclusion, this is the first study to show increased lipid enhanced circulating MOTS-c whilst insulin attenuated the MOTS-c response in human. Further, eight weeks of moderate exercise training did not show any changes in circulating MOTS-c levels in healthy controls and in women with PCOS.

Effects of visfatin on brown adipose tissue energy regulation using T37i cells.

The role of brown adipose tissue (BAT) in pathological states of energy homeostasis and impaired adipocyte function, such as obesity has been a major area of research interest in recent years. Herein, we sought to determine the direct effects of adipokines, visfatin and leptin on BAT thermogenesis. The effects of mouse recombinant visfatin, nicotinamide mononucleotide (NMN) and leptin with or without FK866 were studied on differentiated T37i cells. Treated cells were analyzed for key genes and proteins regulating BAT [UCP-1, PRD1-BF1-RIZ1 homologous domain-containing 16 (PRDM-16), PPARgamma-coactivator-1alpha (PGC-1α) and receptor-interacting protein 140 (RIP-140)] using quantitative PCR and western blot analysis. Data is presented as mean P-values. Both visfatin and leptin had significant concentration dependent effects on thermogenesis in brown pre-adipocytes and at physiological levels, increased uncoupling protein-1 (UCP-1) levels in brown adipocytes. These effects of visfatin were similar to that of nicotinamide mononucleotide (NMN), further strengthening the enzymatic role of visfatin. We also showed that leptin induced UCP-1 mRNA expression and protein production appears to be mediated by visfatin. High concentrations of both visfatin and leptin led to a dramatic decrease in UCP-1 protein levels, supporting the notion that visfatin levels are raised in obesity and that obese people have reduced BAT activity, plausibly through a reduction in UCP-1 levels. Additionally, we found differential regulation of key brown adipogenic genes, specifically, PRD1-BF1-RIZ1 homologous domain-containing 16 (PRDM-16), PPARgamma-coactivator-1alpha (PGC-1α) and receptor-interacting protein 140 (RIP-140) by visfatin. Our observations provide novel insights in the potential actions of visfatin in BAT.

Effect of ACTH and hCG on the Expression of Gonadotropin-Inducible Ovarian Transcription Factor 1 (Giot1) Gene in the Rat Adrenal Gland.

Gonadotropin-inducible ovarian transcription factor-1 (Giot1) belongs to a family of fast-responsive genes, and gonadotropins rapidly induce its expression in steroidogenic cells of ovaries and testes of rats. Gonadal Giot1 gene expression is regulated by cyclic adenosine monophosphate (cAMP) -dependent protein kinase A pathway, with essential role of orphan nuclear receptor NR4A1 transcription factor (nuclear receptor subfamily 4, group A, member 1). A recent study reports that Giot1 is also expressed in adrenals, however, the mechanism of its regulation in adrenal gland is yet to be identified. Therefore, the aim of this study was to characterise the changes in Giot1 gene expression in male and female rat adrenals using wide range of in vivo and in vitro experimental models. Special emphasis was directed at the Giot1 gene regulation by ACTH and gonadotropin. In our study, we found that ACTH rapidly stimulates Giot1 expression both in vivo and in vitro. However, gonadotropin does not affect the adrenal Giot1 gene expression, presumably due to the low expression of gonadotropin receptor in adrenals. Both testosterone and estradiol administered in vivo had inhibitory effect on Giot1 gene expression in the adrenals of post-gonadectomized adult rats. Further, our studies revealed that the intracellular mechanism of Giot1 gene regulation in rat adrenals is similar to that of gonads. As in the case of gonads, the expression of Giot1 in adrenal gland is regulated by cAMP-dependent signaling pathway with essential role of the NR4A1 transcription factor. The results of our studies suggest that Giot1 may be involved in the regulation of rat adrenocortical steroidogenesis.

Circulatory changes of the novel adipokine adipolin/CTRP12 in response to metformin treatment and an oral glucose challenge in humans.

OBJECTIVE: Adipolin/CTRP12 is a novel adipokine with anti-inflammatory and glucose-lowering properties in rodents. We sought to investigate the effects of metformin treatment (850 mg twice daily for 6 months) and a 2 h 75 g oral glucose tolerance test (OGTT) on serum adipolin concentrations in humans. DESIGN: Cross-sectional study [PCOS (n = 83) and control (n = 39) subjects]. Serum adipolin was measured by ELISA. Metformin treatment (850 mg twice daily for 6 months) was offered to all women with PCOS, 34 women participated but 21 women completed 6 months of metformin therapy. Reasons for subjects not completing the study were nausea and gastrointestinal side effects (n = 4), pregnancies (n = 5), noncompliance (n = 2) and loss of contact (n = 2). RESULTS: Metformin treatment (850 mg twice daily for 6 months) substantially increased serum adipolin concentrations (P < 0·05) in women with polycystic ovary syndrome (PCOS), a pro-inflammatory state associated with obesity, diabetes, dyslipidaemia and atherosclerosis. Furthermore, changes in waist-hip ratio, glucose, triglycerides, CRP and carotid intima media thickness showed significant negative associations with changes in adipolin levels (P < 0·05, P < 0·01); in multiple regression analyses, only changes in glucose were predictive of changes in adipolin levels (ß = -0·570, P = 0·009). Serum adipolin decreased significantly in response to the OGTT in PCOS and control subjects at 90 min (P < 0·05) and 120 min (P < 0·01). CONCLUSIONS: Adipolin and/or novel pharmacologic agents that increase adipolin's circulating concentrations might constitute a novel approach in the treatment of insulin resistant states.

QRFP induces aldosterone production via PKC and T-type calcium channel-mediated pathways in human adrenocortical cells: evidence for a novel role of GPR103.

Hormonal regulation of adrenal function occurs primarily through activation of GPCRs. GPCRs are central to many of the body's endocrine and neurotransmitter pathways. Recently, it was shown that activation of GPR103 by its ligand QRFP induced feeding, locomotor activity, and metabolic rate, and QRFP is bioactive in adipose tissue of obese individuals. Given that the adrenal gland is a pivotal organ for energy balance and homeostasis, we hypothesized that GPR103 and QRFP are involved in steroidogenic responses. Using qRT-PCR and immunohistochemistry, we mapped both GPR103 and QRFP in human fetal and adult adrenal gland as well as rat adrenals. Both were primarily localized in the adrenal cortex but not in the medulla. Activation of GPR103 in human adrenocortical H295R cells led to a decrease in forskolin-increased cAMP and an increase of intracellular Ca(2+) levels. In addition, treatment of H295R cells with QRFP induced aldosterone and cortisol secretion as measured by ELISA. These increases were accompanied by increased expression and activity of StAR, CYB11B1, and CYP11B2 as assessed by qRT-PCR and luciferase reporter assay, respectively. Using specific inhibitors, we also demonstrated that aldosterone induction involves MAPK, PKC, and/or T-type Ca(2+) channel-dependent pathways. These novel data demonstrate that QRFP induces adrenal steroidogenesis in vitro by regulating key steroidogenic enzymes involving MAPK/PKC and Ca(2+) signaling pathways.

Clinical improvement may not reflect metabolic homeostasis normalization in subjects with and without Roux-En-Y bariatric surgery after 12 years: comparison of surgical subjects to a lean cohort.

Background: A 12-year study comparing clinical outcomes following Roux-en-Y bariatric surgery showed long-term weight loss with remission/prevention of type-2-diabetes (T2D), hypertension and dyslipidemia. However, it is unknown whether the underlying homeostatic metabolic processes involving hepatokines, adipokines and myokines also normalize. Using this 12-year study, we determined whether metabolic indices improved in post-surgical (BMI:34.4kg/m2) versus non-surgical comparator-subjects-with-obesity (BMI:43.8kg/m2) at 12-year follow-up (both cohorts with baseline diabetes), and if post-surgical subjects normalized their metabolic processes to those of a normal-weight cohort without diabetes. Methods: Cross-sectional design. Plasma from a cohort of Roux-en-Y bariatric surgery (n=50) and non-surgery (n=76) comparator-subjects-with-obesity (both cohorts at 12-year follow-up) plus a normal-weight cohort (n=39) was assayed by Luminex immunoassay or ELISA for hepatokines [angiopoietin-like proteins-(ANGPTL3; ANGPTL4; ANGPTL6); fibroblast growth factors-(FGF19; FGF21; FGF23)]; adipokines [adipsin; adiponectin; FGF19] and myonectin. Results: After age and gender adjustment, surgery versus comparator-subjects-with-obesity had lower BMI (34.4 ± 1.0 vs 43.8 ± 0.9kg/m2; p<0.0001), HbA1c (6.2 ± 0.3 vs 7.7 ± 0.2%; p<0.0001), insulin resistance (HOMA-IR, 2.0 ± 1.5 vs 10.8 ± 1.4; p<0.0001) fat mass (45.6 ± 2.2 vs 60.0 ± 2.0; p<0.0001), HDL-C (55.4 ± 2.6 vs 42.6 ± 2.3mg/dL; p<0.0001), triglycerides (130 ± 14 vs 187 ± 12mg/dL; p<0.0001) and higher adiponectin (25.9 ± 2.3 vs 15.7 ± 2.0µg/ml; p<0.001); Adipsin, ANGPTL3, ANGPTL4, ANGPTL6, FGF19, FGF21, FGF23 and myonectin did not differ. Surgery versus normal-weight group: higher ANGPTL4 (156 ± 6 vs 119 ± 7ng/mL; p<0.0001), higher FGF23 (96.4 ± 10.1 vs 50.9 ± 11.5pg/mL; p=0.007) and lower myonectin (744 ± 55 vs 969 ± 66ng/mL; p=0.002); adiponectin, adipsin ANGPTL3, ANGPTL6, FGF19, FGF21 did not differ. Non-surgery comparator-subjects-with-obesity versus normal-weight group: higher adipsin (1859 ± 94 vs 1314 ± 133ng/mL; p=0.0001), higher FGF23 (84.6 ± 8.5 vs 50.9 ± 11.5pg/mL; p<0.0001) and higher ANGPTL4 (171 ± 5 vs 119 ± 7ng/mL; p<0.0001); adiponectin ANGPTL3, ANGPTL6, FGF19, FGF21 and myonectin did not differ. Conclusion: Bariatric surgery markedly improved anthropometric and metabolic features versus comparator-subjects-with-obesity at 12-year follow-up, indicating benefit of weight loss. However, despite weight loss, these patients still had class-1 obesity, as reflected in the adipokine, hepatokine and myokine markers of body homeostasis that did not completely normalize to indicative values of normal-weight subjects, suggesting either that this is the new normal for these patients or that weight loss to a BMI<25kg/m2 is needed for normalization of these parameters.

Sphingolipids in Childhood Asthma and Obesity (SOAP Study): A Protocol of a Cross-Sectional Study.

Asthma and obesity are two of the most common chronic conditions in children and adolescents. There is increasing evidence that sphingolipid metabolism is altered in childhood asthma and is linked to airway hyperreactivity. Dysregulated sphingolipid metabolism is also reported in obesity. However, the functional link between sphingolipid metabolism, asthma, and obesity is not completely understood. This paper describes the protocol of an ongoing study on sphingolipids that aims to examine the pathophysiology of sphingolipids in childhood asthma and obesity. In addition, this study aims to explore the novel biomarkers through a comprehensive multi-omics approach including genomics, genome-wide DNA methylation, RNA-Seq, microRNA (miRNA) profiling, lipidomics, metabolomics, and cytokine profiling. This is a cross-sectional study aiming to recruit 440 children from different groups: children with asthma and normal weight (n = 100), asthma with overweight or obesity (n = 100), overweight or obesity (n = 100), normal weight (n = 70), and siblings of asthmatic children with normal weight, overweight, or obesity (n = 70). These participants will be recruited from the pediatric pulmonology, pediatric endocrinology, and general pediatric outpatient clinics at Sidra Medicine, Doha, Qatar. Information will be obtained from self-reported questionnaires on asthma, quality of life, food frequency (FFQ), and a 3-day food diary that are completed by the children and their parents. Clinical measurements will include anthropometry, blood pressure, biochemistry, bioelectrical impedance, and pulmonary function tests. Blood samples will be obtained for sphingolipid analysis, serine palmitoyltransferase (SPT) assay, whole-genome sequencing (WGS), genome-wide DNA methylation study, RNA-Seq, miRNA profiling, metabolomics, lipidomics, and cytokine analysis. Group comparisons of continuous outcome variables will be carried out by a one-way analysis of variance or the Kruskal-Wallis test using an appropriate pairwise multiple comparison test. The chi-squared test or a Fisher's exact test will be used to test the associations between categorical variables. Finally, multivariate analysis will be carried out to integrate the clinical data with multi-omics data. This study will help us to understand the role of dysregulated sphingolipid metabolism in obesity and asthma. In addition, the multi-omics data from the study will help to identify novel genetic and epigenetic signatures, inflammatory markers, and mechanistic pathways that link asthma and obesity in children. Furthermore, the integration of clinical and multi-omics data will help us to uncover the potential interactions between these diseases and to offer a new paradigm for the treatment of pediatric obesity-associated asthma.

Adiponectin (15-36) stimulates steroidogenic acute regulatory (StAR) protein expression and cortisol production in human adrenocortical cells: role of AMPK and MAPK kinase pathways.

Adiponectin is an abundantly circulating adipokine, orchestrating its effects through two 7-transmembrane receptors (AdipoR1 and AdipoR2). Steroidogenesis is regulated by a variety of neuropeptides and adipokines. Earlier studies have reported adipokine mediated steroid production. A key rate-limiting step in steroidogenesis is cholesterol transportation across the mitochondrial membrane by steroidogenic acute regulatory protein (StAR). Several signalling pathways regulate StAR expression. The actions of adiponectin and its role in human adrenocortical steroid biosynthesis are not fully understood. The aim of this study was to investigate the effects of adiponectin on StAR protein expression, steroidogenic genes, and cortisol production and to dissect the signalling cascades involved in the activation of StAR expression. Using qRT-PCR, Western blot analysis and ELISA, we have demonstrated that stimulation of human adrenocortical H295R cells with adiponectin results in increased cortisol secretion. This effect is accompanied by increased expression of key steroidogenic pathway genes including StAR protein expression via ERK1/2 and AMPK-dependent pathways. This has implications for our understanding of adiponectin receptor activation and peripheral steroidogenesis. Finally, our study aims to emphasise the key role of adipokines in the integration of metabolic activity and energy balance partly via the regulation of adrenal steroid production. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

NUCB2/Nesfatin-1 Reduces Obesogenic Diet Induced Inflammation in Mice Subcutaneous White Adipose Tissue.

BACKGROUND: Excess adipose tissue accumulation and obesity are characterised by chronic, low-grade, systemic inflammation. Nestfatin-1 is a neuropeptide derived from the precursor protein nucleobindin-2 (NUCB2), which was initially reported to exert anorexigenic effects. The present study aimed to investigate the effects of an obesogenic diet (OD; high-fat, high-sugar) in NUCB2 knockout (KO) mice and of nesfatin-1 treatment in LPS-stimulated 3T3-L1 preadipocytes. METHODS: Subcutaneous white adipose tissue (Sc-WAT) samples from wild type (WT) and NUCB2 KO mice that were fed a normal diet (ND), or the OD for 12 weeks were used for RNA and protein extraction, as well as immunohistochemistry. 3T3-L1 cells were treated with 100 nM nesfatin-1 during differentiation and stimulated with 1 µg/mL LPS for measuring the expression and secretion of pro-inflammatory mediators by qPCR, western blotting, immunofluorescence, Bioplex, and ELISA. RESULTS: Following the OD, the mRNA, protein and cellular expression of pro-inflammatory mediators (Tnfα, Il-6, Il-1ß, Adgre1, Mcp1, TLR4, Hmbgb1 and NF-kB) significantly increased in the ScWAT of NUCB2 KO mice compared to ND controls. Adiponectin and Nrf2 expression significantly decreased in the ScWAT of OD-fed NUCB2 KO, without changes in the OD-fed WT mice. Furthermore, nesfatin-1 treatment in LPS-stimulated 3T3-L1 cells significantly reduced the expression and secretion of pro-inflammatory cytokines (Tnfα, Il-6, Il-1ß, Mcp1) and hmgb1. CONCLUSION: An obesogenic diet can induce significant inflammation in the ScWAT of NUCB2 KO mice, involving the HMGB1, NRF2 and NF-kB pathways, while nesfatin-1 reduces the pro-inflammatory response in LPS-stimulated 3T3-L1 cells. These findings provide a novel insight into the metabolic regulation of inflammation in WAT.

Differences in protein expression, at the basal state and at 2 h of insulin infusion, in muscle biopsies from healthy Arab men with high or low insulin sensitivity measured by hyperinsulinemic euglycemic clamp.

Background: Skeletal muscle is the main site for insulin-dependent glucose disposal. The hyperinsulinemic euglycemic clamp (HIEC) is the gold standard for the assessment of insulin sensitivity (IS). We have previously shown that insulin sensitivity, measured by HIEC, varied widely among a group of 60 young healthy men with normoglycemia. The aim of this study was to correlate the proteomic profile of skeletal muscles to insulin sensitivity. Methods: Muscle biopsies from 16 subjects having the highest (M ≥ 13; n = 8, HIS) and lowest (M ¾ 6, n = 8, LIS) IS were obtained at baseline and during insulin infusion after stabilization of the blood glucose level and glucose infusion rate at the end of the HIEC. The samples were processed using a quantitative proteomic analysis approach. Results: At baseline, 924 proteins were identified in the HIS and LIS groups. Among the 924 proteins detected in both groups, three were suppressed and three were increased significantly in the LIS subjects compared with the HIS subjects. Following insulin infusion, 835 proteins were detected in both groups. Among the 835 proteins, two showed differential responsiveness to insulin; ATP5F1 protein was decreased, and MYLK2 was higher in the LIS group compared with that in the HIS group. Our data suggest that alteration in mitochondrial proteins and an increased number of proteins involved in fast-twitch fiber correlate to insulin sensitivity in healthy young Arab men. Conclusions: These results suggest a change in a small number of differentially expressed proteins. A possible reason for this small change could be our study cohorts representing a homogeneous and healthy population. Additionally, we show differences in protein levels from skeletal muscle in low and high insulin sensitivity groups. Therefore, these differences may represent early events for the development of insulin resistance, pre-diabetes, and type 2 diabetes.

MiRNA and associated inflammatory changes from baseline to hypoglycemia in type 2 diabetes.

Objective: Hypoglycemia in type 2 diabetes (T2D) increases morbidity and mortality but the underlying physiological response is still not fully understood, though physiological changes are still apparent 24 hours after the event. Small noncoding microRNA (miRNA) have multiple downstream biological effects that may respond rapidly to stress. We hypothesized that hypoglycemia would induce rapid miRNA changes; therefore, this pilot exploratory study was undertaken. Methods: A pilot prospective, parallel study in T2D (n=23) and controls (n=23). Insulin-induced hypoglycemia (2mmol/l: 36mg/dl) was induced and blood sampling performed at baseline and hypoglycemia. Initial profiling of miRNA was undertaken on pooled samples identified 96 miRNA that were differentially regulated, followed by validation on a custom designed 112 miRNA panel. Results: Nine miRNAs differed from baseline to hypoglycemia in control subjects; eight were upregulated: miR-1303, miR-let-7e-5p, miR-1267, miR-30a-5p, miR-571, miR-661, miR-770-5p, miR-892b and one was downregulated: miR-652-3p. None of the miRNAs differed from baseline in T2D subjects. Conclusion: A rapid miRNA response reflecting protective pathways was seen in control subjects that appeared to be lost in T2D, suggesting that mitigating responses to hypoglycemia with blunting of the counter-regulatory response in T2D occurs even in patients with short duration of disease. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT03102801?term=NCT03102801&draw=2&rank=1, identifier NCT03102801.

Elevated Adipsin and Reduced C5a Levels in the Maternal Serum and Follicular Fluid During Implantation Are Associated With Successful Pregnancy in Obese Women.

Introduction: Complement factors mediate the recruitment and activation of immune cells and are associated with metabolic changes during pregnancy. The aim of this study was to determine whether complement factors in the maternal serum and follicular fluid (FF) are associated with in vitro fertilization (IVF) outcomes in overweight/obese women. Methods: Forty overweight/obese (BMI = 30.8 ± 5.2 kg/m2) female patients, 33.6 ± 6.3 years old, undergoing IVF treatment for unexplained infertility were recruited. Baseline demographic information, including biochemical hormonal, metabolic, and inflammatory markers, and pregnancy outcome, was collected. Levels of 14 complement markers (C2, C4b, C5, C5a, C9, adipsin, mannose-binding lectin, C1q, C3, C3b/iC3b, C4, factor B, factor H, and properdin) were assessed in the serum and FF and compared to IVF outcome, inflammatory, and metabolic markers using multivariate and univariate models. Results: Out of 40 IVF cycles, 14 (35%) resulted in pregnancy. Compared to women with failed pregnancies, women with successful pregnancies had higher levels of adipsin in the serum and FF (p = 0.01) but lower C5a levels (p = 0.05). Serum adipsin levels were positively correlated with circulating levels of vitamin D (R = 0.5, p = 0.02), glucagon (R = 0.4, p = 0.03), leptin (R = 0.4, p = 0.01), resistin (R = 0.4, p = 0.02), and visfatin (R = 0.4, p = 0.02), but negatively correlated with total protein (R = -0.5, p = 0.03). Higher numbers of top-quality embryos were associated with increased levels of C3, properdin, C1q, factors H and B, C4, and adipsin, but with reduced C2 and C5a levels (p ≤ 0.01). Conclusions: Higher adipsin and lower C5a levels in the maternal serum during implantation are potential markers of successful outcome in obese women undergoing IVF-assisted pregnancies.

Sputum and Plasma Neutrophil Elastase in Stable Adult Patients With Cystic Fibrosis in Relation to Chronic Pseudomonas Aeruginosa Colonization.

Background and purpose Neutrophil elastase (NE) has been implicated in the pathogenesis of airway inflammation in cystic fibrosis (CF) patients and it impairs defenses against Pseudomonas aeruginosa (PA) infection or colonization. Sputum NE may act as a biomarker of neutrophilic inflammation in CF patients. This study aimed to determine sputum and plasma total NE levels in clinically stable adult CF patients and control subjects, and their correlation to PA colonization and lung functions. Methods This is a cross-sectional study. Total NE was measured on spontaneously expectorated sputum and plasma obtained from 21 CF patients, aged 18-40 years, during routine visits to the adult CF clinic. This was compared to plasma obtained from 22 matching healthy controls. The levels of NE were measured by the magnetic bead-based multiplex assay. Results Sputum and plasma NE levels had a significant positive correlation (Pearson r=0.533, P=0.013) with PA colonization. Sixteen CF patients (76.2%) were chronically colonized with PA. Both median sputum and plasma NE were found to be higher in CF patients with PA as compared with non-PA patients, even though this difference was statistically insignificant. Sputum and plasma NE levels did not correlate with the percentage predicted forced expiratory volume in one second (FEV1), the forced vital capacity (FVC), and FEV1/FVC and no association with PA. Conclusion The findings suggest that clinically stable adult CF patients colonized with PA may have higher NE levels in both plasma and sputum as compared to non-PA CF patients and probably total NE does not influence lung functions.

High Endurance Elite Athletes Show Age-dependent Lower Levels of Circulating Complements Compared to Low/Moderate Endurance Elite Athletes.

Introduction: Aerobic exercise activates the complement system in the peripheral blood. However, the effect of age and high intensity endurance training on the levels of circulating complements and sassociated inflammatory cytokines, oxidative stress markers and cellular aging remains unknown. Methods: In this study, serum samples from 79 elite athletes who belong to high (n = 48) and low/moderate (n = 31) endurance sports and two age groups (below 30 years old, n = 53, and above 30 years old, n = 26) were profiled for 14 complements. Linear models were used to assess differences in complements levels between sport and age groups. Spearmann's correlation was used to assess the relationship among detected complements and proinflammatory cytokines, oxidative stress markers and telomere lengths. Results: High endurance elite athletes exhibited significantly lower levels of circulating C2, C3b/iC3b and adipsin complements than their age-matched low/moderate endurance counterparts. Levels of C2, adipsin and C3b/iC3b were positively correlated with most detected complements, the pro-inflammatory cytokines TNF-alpha and IL-22 and the anti-oxidant enzyme catalase. However, they were negatively correlated with telomere length only in younger elite athletes regardless of their sport groups. Furthermore, high endurance elite athletes showed significantly lower concentrations of C3b/iC3b, C4b, C5, C5a, C1q, C3, C4, factor H and properdin in younger athletes compared to their older counterparts. Conclusion: Our novel data suggest that high endurance elite athletes exhibit age-independent lower levels of circulating C2, C3b/iC3b and adipsin, associated with lower inflammatory, oxidative stress and cellular aging, as well as lower levels of 10 other complements in younger athletes compared to older counterparts. Assessing the effect of various levels of endurance sports on complements-based immune response provides a better understanding of exercise physiology and pathophysiology of elite athletes.