Adiponectin Signaling and Impaired GTPase Rab5 Expression in Adipocytes of Adolescents with Obesity.

INTRODUCTION: Abnormalities in adipose tissue AdipoR1; adaptor protein, phosphotyrosine interaction, PH domain, and leucine zipper containing 1 (APPL1); GTPase Rab5; adiponectin; leptin; and visfatin in adults with obesity have been associated with metabolic disturbances. OBJECTIVE: The objective of this study was to examine whether pediatric obesity disrupts elements of the adiponectin signaling pathway and GTPase Rab5 in adipose tissue. METHODS: Primary adipocyte cultures of subcutaneous abdominal tissue were obtained from 96 lean and 66 children and adolescents with obesity (AO). AdipoR1, APPL1, and GTPase Rab5 mRNA levels were measured by RT-PCR and their protein content by Western immunoblotting. Serum total and high-molecular-weight adiponectin, leptin, leptin soluble receptor (sOB-R), and visfatin were measured by ELISA. RESULTS: The mRNA expression and protein content of AdipoR1 and APPL1 did not differ significantly with obesity, age, or puberty. However, GTPase Rab5 protein was increased in the adipocytes of younger prepubertal children with obesity but decreased in AO. Leptin was increased in AO compared to lean adolescents (AL) and in older prepubertal lean (OPL) children and AL compared to younger prepubertal lean and obese children. sOB-R was higher in OPL children and in the AL and AO. Serum visfatin was increased in the younger prepubertal children and AO. CONCLUSIONS: In contrast to adults, obesity did not change the expression of AdipoR1 and APPL1 in cultured adipocytes from biopsies of subcutaneous abdominal adipose tissue of children and adolescents. Similar to adipose tissue studies in adults with obesity and metabolic dysfunction, the AO in our study showed reduced adipocyte GTPase Rab5 expression.

Novel combined insulin-like 3 variations of a single nucleotide in cryptorchidism.

Background Insulin-like 3 hormone (INSL3) is involved in the process of testicular descent, and has been thoroughly studied in cryptorchidism. However, INSL3 allelic variations found in the human genome were heterozygous and only a few of them were found exclusively in patients with cryptorchidism. Under this perspective, we aimed to study the presence of INSL3 allelic variations in a cohort of patients with cryptorchidism and to estimate their potential consequences. Methods Blood samples were collected from 46 male patients with non-syndromic cryptorchidism and from 43 age-matched controls. DNA extraction and polymerase chain reaction (PCR) were performed for exons 1 and 2 of the INSL3 gene in all subjects. Sequencing analysis was carried out on the PCR products. All data were grouped according to testicular location. Results Seven variations of a single nucleotide (SNVs) were identified both in patients with cryptorchidism and in controls: rs2286663 (c.27G > A), rs1047233 (c.126A > G) and rs6523 (c.178A > G) at exon 1, rs74531687 (c.191-30C > T) at the intron, rs121912556 (c.305G > A) at exon 2 and rs17750642 (c.*101C > A) and rs1003887 (c.*263G > A) at the untranslated region (UTR). The allelic variants rs74531687 and rs121912556 were found for the first time in the Greek population. The novel homozygotic combination of the three allelic variants rs1047233-rs6523-rs1003887 seemed to present a stronger correlation with more severe forms of cryptorchidism. Conclusions The combination of specific INSL3 SNVs rather than the existence of each one of them alone may offer a new insight into the involvement of allelic variants in phenotypic variability and severity.

Metabolic Association between Leptin and the Corticotropin Releasing Hormone.

OBJECTIVE: In healthy individuals, leptin is produced from adipose tissue and is secreted into the circulation to communicate energy balance status to the brain and control fat metabolism. Corticotropin- Releasing Hormone (CRH) is synthesized in the hypothalamus and regulates stress responses. Among the many adipokines and hormones that control fat metabolism, leptin and CRH both curb appetite and inhibit food intake. Despite numerous reports on leptin and CRH properties and function, little has been actually shown about their association in the adipose tissue environment. METHODS: In this article, we summarized the salient information on leptin and CRH in relation to metabolism. We also investigated the direct effect of recombinant CRH on leptin secretion by primary cultures of human adipocytes isolated from subcutaneous abdominal adipose tissue of 7 healthy children and adolescents, and measured CRH and leptin levels in plasma collected from peripheral blood of 24 healthy children and adolescents to assess whether a correlation exists between CRH and leptin levels in the periphery. RESULTS AND CONCLUSION: The available data indicate that CRH exerts a role in the regulation of leptin in human adipocytes. We show that CRH downregulates leptin production by mature adipocytes and that a strong negative correlation exists between CRH and leptin levels in the periphery, and suggest the possible mechanisms of CRH control of leptin. Delineation of CRH control of leptin production by adipocytes may explain unknown pathogenic mechanisms linking stress and metabolism.

Epidermal growth factor receptor (EGFR) involvement in successful growth hormone (GH) signaling in GH transduction defect.

BACKGROUND: Growth hormone (GH) transduction defect (GHTD) is a growth disorder with impaired signal transducer and activator of transcription 3 (STAT3) phosphorylation mediated by overexpression of cytokine-inducible SH2-containing protein (CIS), which causes increased growth hormone receptor (GHR) degradation. This study investigated the role of epidermal growth factor (EGF) in the restoration of normal GH signaling in GHTD. METHODS: Protein expression, cellular localization and physical contact of proteins of the GH and EGF signaling pathways were studied by Western immunoblotting, immunofluorescence and co-immunoprecipitation, respectively. These were performed in fibroblasts of one GHTD patient (P) and one control child (C) at the basal state and after induction with human GH (hGH) 200 µg/L (GH200), either with or without silencing of CIS mRNA, and after induction with hGH 1000 µg/L (GH1000) or 50 ng/mL EGF. RESULTS: The membrane availability of the EGF receptor (EGFR) and the activated EGFR (pEGFR) was increased in P only after simultaneous GH200 and silencing of CIS mRNA or with GH1000, whereas this occurred in C after GH200 alone. After EGF induction, the membrane localization of GHR, STAT3 and that of EGFR were increased in P more than in C. CONCLUSIONS: In conclusion, in GHTD, the EGFR seems to participate in successful GH signaling, but induction of GHTD fibroblasts with a higher dose of hGH is needed. The EGF/EGFR pathway, in contrast to the GH/GHR pathway, seems to function normally in P and is more primed compared to C. The involvement of the EGFR in successful GH signaling may explain the catch-up growth seen in the Ps when exogenous hGH is administered.

Protective mechanisms against oxidative stress and angiopathy in young patients with diabetes type 1 (DM1).

OBJECTIVE: Advanced glycation end-products (AGEs) via their receptor, RAGE, are involved in diabetic angiopathy. Soluble RAGE, an inhibitor of this axis, is formed by enzymatic catalysis (sRAGE) or alternative splicing (esRAGE). Malondialdehyde (MDA) is an oxidative stress marker, and ferric reducing ability of plasma (FRAP) is an anti-oxidant capacity marker. METHODS: In isolated mononuclear blood cells from 110 DM1-patients (P) and 124 controls (C) (4-29 years) RAGE mRNA (g) and protein expression (pe) were measured by RT-PCR and Western immunoblotting, respectively. Plasma levels of CML (AGEs) and sRAGE were measured by ELISA, MDA by flurometry and FRAP according to 'Benzie and Strain'. RESULTS: P showed: (i) higher g of RAGE, especially in p>13 years of age and >5 years DM1, (ii) increased pe of esRAGE in DM1>5 years and (iii) increased FRAP and MDA. CONCLUSIONS: The increased esRAGE and FRAP with increased levels of CML and MDA possibly reflects a protective response against the formation of diabetic complications in these young diabetic patients.

Metabolic impact of a ketogenic diet compared to a hypocaloric diet in obese children and adolescents.

BACKGROUND: The effects of carbohydrate-restricted (ketogenic) diets on metabolic parameters in children have been incompletely assessed. OBJECTIVE: To compare the efficacy and metabolic impact of ketogenic and hypocaloric diets in obese children and adolescents. SUBJECTS: Fifty-eight obese subjects were placed on one of the two diets for 6 months. METHODS: Anthropometric measurements, body composition, oral glucose/insulin tolerance test, lipidemic profile, high molecular weight (HMW) adiponectin, whole-body insulin sensitivity index (WBISI), and homeostatic model assessment-insulin resistance (HOMA-IR) were determined before and after each diet. RESULTS: Both groups significantly reduced their weight, fat mass, waist circumference, fasting insulin, and HOMA-IR (p = 0.009 for ketogenic and p = 0.014 for hypocaloric), but the differences were greater in the ketogenic group. Both groups increased WBISI significantly, but only the ketogenic group increased HMW adiponectin significantly (p = 0.025). CONCLUSIONS: The ketogenic diet revealed more pronounced improvements in weight loss and metabolic parameters than the hypocaloric diet and may be a feasible and safe alternative for children's weight loss.

Endocannabinoid (EC) receptor, CB1, and EC enzymes' expression in primary adipocyte cultures of lean and obese pre-pubertal children in relation to adiponectin and insulin.

AIM: The over-expression of CB1 in adult obesity is associated with insulin resistance (IR),but it is not elucidated in childhood obesity. We studied CB1 and endocannabinoid enzymes (EE), Adiponectin (Ad), and Insulin (SI) in lean and obese pre-pubertal (PP) children. METHODS: CB1 mRNA and protein (Pr) expression were studied by RT-PCR, western immunoblotting and immunohistochemistry in primary cultures of adipose tissue. The EE(NAPE-PLD, DAGL-alpha, FAAH, MAGL) expression was assessed by Real-Time PCR. Ad and SI were measured by ELISA and IR by HOMA-IR index. RESULTS: In the older obese vs older lean children: (1) CB1 Pr was decreased, (2) FAAHmRNA and DAGL-alpha mRNA were increased. Ad was decreased and SI and HOMA-IR increased in the older PP children. CONCLUSIONS: Increased CB1 and decreased adiponectin in older lean PP children may facilitate fat deposition and "physiologic" IR necessary for the increased body growth of puberty. The reduced expression of CB1 in the older obese may be an attempt to reduce lipogenesis to avoid greater insulin resistance.