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Pentraxin 3 (PTX3) is a soluble pattern recognition receptor playing an important role in immune response and inflammation. Lipopolysaccharide (LPS) stimulation can significantly induce PTX3 expression and secretion in adipocytes. Appropriate regulation of PTX3 secretion is critical for inflammatory homeostasis. Using chemical inhibitors of conventional and unconventional protein secretion, we explored the mechanisms that control LPS-stimulated PTX3 secretion in 3T3-L1 adipocytes. Inhibiting the conventional protein secretion blocked LPS-stimulated PTX3 secretion, resulting in cellular PTX3 accumulation in adipocytes. We also detected PTX3 in exosomes from LPS-treated adipocytes; inhibiting exosome trafficking attenuated PTX3 secretion. However, only 4.3% of secreted PTX3 was detected in exosomes compared to 95.7% in the non-exosomal fractions. The fractionation of isolated exosomes by the iodixanol density gradient centrifugation confirmed that a small portion of secreted PTX3 overlapped with exosomal markers in small extracellular-vesicle fractions. We conclude that PTX3 is secreted mainly through conventional protein secretion, and a small percentage of PTX3 is released in exosomes from LPS-stimulated adipocytes.

Pathogenic variants in the transcription factor CCCTC-binding factor (CTCF) are associated with mental retardation, autosomal dominant 21 (MRD21, MIM#615502). Current studies supported the strong relationship between CTCF variants and short stature, yet the mechanism of CTCF mutation leading to short stature is not known. Clinical information, treatment regimens and follow-up outcomes of a patient with MRD21 were collected. The possible pathogenic mechanisms of CTCF variants leading to short stature were investigated using immortalized lymphocyte cell lines (LCLs), HEK-293T and immortalized normal human liver cell lines (LO2). This patient received a long-term treatment with recombinant human growth hormone (rhGH) and resulted in an increased height of 1.0 SDS. She had low IGF-1 levels before the treatment and the IGF-1 level was not significantly increased during the treatment (-1.38±0.61 SDS). The finding suggested that the CTCFR567W variant could have impaired the insulin growth factors 1 (IGF-1) signaling pathway. We further demonstrated that the mutant CTCF had a reduced ability binding to the promoter region of IGF-1, consequently significantly reducing the transcriptional activation and expression of IGF-1. Our results demonstrated a direct positive regulation of CTCF on the transcription of the IGF1 promoter for the first time. The impaired IGF-1 expression due to CTCF mutation could explain the poor effect of rhGH treatment on MRD21 patients. This study provided novel insights to the molecular basis of CTCF associated disorder.

Lipocalin-2 (LCN2) has been previously characterized as an adipokine regulating thermogenic activation of brown adipose tissue and retinoic acid (RA)-induced thermogenesis in mice. The objective of this study was to explore the role and mechanism for LCN2 in the recruitment and retinoic acid-induced activation of brown-like or ‘beige’ adipocytes. We found LCN2 deficiency reduces key markers of thermogenesis including uncoupling protein-1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) in inguinal white adipose tissue (iWAT) and inguinal adipocytes derived from Lcn2 ^−/− mice. Lcn2 ^−/− inguinal adipocytes have attenuated insulin-induced upregulation of thermogenic gene expression and p38 mitogen-activated protein kinase (p38MAPK) signaling pathway activation. This is accompanied by a lower basal and maximal oxidative capacity in Lcn2 ^−/− inguinal adipocytes, indicating mitochondrial dysfunction. Recombinant Lcn2 was able to restore insulin-induced p38MAPK phosphorylation in both WT and Lcn2 ^−/− inguinal adipocytes. Rosiglitazone treatment during differentiation of Lcn2 ^−/− adipocytes is able to recruit beige adipocytes at a normal level, however, further activation of beige adipocytes by insulin and RA is impaired in the absence of LCN2. Further, the synergistic effect of insulin and RA on UCP1 and PGC-1α expression is markedly reduced in Lcn2 ^−/− inguinal adipocytes. Most intriguingly, LCN2 and the retinoic acid receptor-alpha (RAR-α) are concurrently translocated to the plasma membrane of adipocytes in response to insulin, and this insulin-induced RAR-α translocation is absent in adipocytes deficient in LCN2. Our data suggest a novel LCN2-mediated pathway by which RA and insulin synergistically regulates activation of beige adipocytes via a non-genomic pathway of RA action.

Lactoferrin (LF) is an iron-binding glycoprotein that plays an important role in promoting bone formation and inhibiting bone resorption; however, its effects on senile osteoporosis remain unknown. This study aimed to investigate the effects and mechanism of LF intervention using a senile osteoporosis model (SAMP6 mice) and senescent osteoblasts. Micro-CT and hematoxylin and eosin staining demonstrated that the intragastric administration (2 g/kg/day) of LF could improve the bone mass and microstructure of SAMP6 mice. Furthermore, LF treatment improved bone metabolism and increased insulin-like growth factor 1 (Igf1) mRNA expression and activated phosphorylation status of AKT. Using osteoblasts passaged for ten generations as an in vitro senescence model, various markers associated with osteoblast formation and differentiation, as well as related indices of oxidative stress were analyzed. Our results revealed that after multiple generations, osteoblasts entered senescence, in conjunction with increased oxidative stress damage, reduced bone metabolism and enhanced expression of aging-related markers. While inhibiting oxidative stress, LF improved osteoblast proliferation by promoting the expression of osteogenesis markers, including alkaline phosphatase (ALP) activity, Igf1, bone gla protein (Bglap) and osteoprotegerin/receptor activator of nuclear factor-kB ligand (Opg/Rankl) mRNA and delayed senescence by decreasing the level of p16 and p21 expression. RNAI-mediated downregulation of IGF1 attenuated the effect of LF on osteogenesis. Therefore, the findings of the present study indicate that LF may promote osteogenesis via IGF1 signaling, thereby preventing senile osteoporosis.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease with complicated pathogenesis and targeting gluconeogenesis inhibition is a promising strategy for anti-diabetic drug discovery. G protein-coupled receptors (GPCRs) are classified as distinct families by heterotrimeric G proteins, primarily including Gαs, Gαi and Gαq. Gαs-coupled GPCRs function potently in the regulation of hepatic gluconeogenesis by activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and Gαi-coupled GPCRs exhibit inhibitory effect on adenylyl cyclase and reduce intracellular cAMP level. However, little is known about the regulation of Gαq-coupled GPCRs in hepatic gluconeogenesis. Here, small-molecule 2-(2,4-dimethoxy-3-methylphenyl)-7-(thiophen-2-yl)-9-(trifluoromethyl)-2,3-dihydropyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(1H)-one (DMT) was determined to suppress hepatic glucose production and reduce mRNA levels of gluconeogenic genes. Treatment of DMT in db/db mice decreased fasting blood glucose and hemoglobin A1C (HbA1c) levels, while improved glucose tolerance and pyruvate tolerance. Mechanism study demonstrated that DMT-inhibited gluconeogenesis by regulating the Gαq/phospholipase C (PLC)/inositol-1,4,5-triphosphate receptor (IP3R)-mediated calcium (Ca²⁺)/calmodulin (CaM)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT)/forkhead box protein O1 (FOXO1) signaling pathway. To our knowledge, DMT might be the first reported small molecule able to suppress hepatic gluconeogenesis by regulating Gαq signaling, and our current work has also highlighted the potential of DMT in the treatment of T2DM.

Although implantation types differ between species, the interaction between blastocyst trophectoderm and apical surface of the uterine epithelium is a common event during the implantation process. In this study, uterine luminal epithelium at implantation and inter-implantation sites was isolated by enzymatic digestion and used for microarray analysis. In a mouse microarray containing 12 345 unigenes, we found 136 genes upregulated more than twofold at the implantation site, while 223 genes were downregulated by at least twofold. Reverse transcription-PCR was used to verify the differential expression of seven upregulated and six downregulated genes chosen randomly from our microarray analysis, and the expression trends were similar. The differential expression patterns of eight upregulated genes were verified by in situ hybridization. Compared with the inter-implantation site on day 5 of pregnancy and the uterus on day 5 of pseudopregnancy, protease, serine, 12 neurotrypsin, endothelin-1, γ-glutamyl hydrolase, Ras homolog gene family, member U, T-cell immunoglobulin, and mucin domain containing 2, proline–serine–threonine phosphatase-interacting protein 2, 3-monooxgenase/tryptophan 5-monooxgenase activation protein, γ-polypeptide, and cysteine-rich protein 61 (Cyr61) were upregulated in the luminal epithelium at implantation site on day 5 of pregnancy. These genes may be related to embryo apposition and adhesion during embryo implantation. Cyr61, a gene upregulated at the implantation site, was chosen to examine its expression and regulation during the periimplantation period by in situ hybridization. Cyr61 mRNA was specifically localized in the luminal epithelium surrounding the implanting blastocyst at day 4 midnight and on day 5 of pregnancy, and in the activated uterus, but not expressed in inter-implantation sites and under delayed implantation, suggesting a role for Cyr61 in mediating embryonic–uterine dialog during embryo attachment. Our data could be a valuable source for future study on embryo implantation.

More aggressive thyroid cancer cells show a higher activity of glycometabolism. Targeting cancer cell metabolism has emerged as a novel approach to prevent or treat malignant tumors. Glucose metabolism regulation effect of metformin in papillary thyroid cancer was investigated in the current study. Human papillary thyroid carcinoma (PTC) cell lines BCPAP and KTC1 were used. Cell viability was detected by CCK8 assay. Glucose uptake and relative gene expression were measured in metformin (0–10 mM for 48 h)-treated cells by ¹⁸F-FDG uptake assay and western blotting analysis, respectively. MicroPET/CT imaging was performed to detect ¹⁸F-FDG uptake in vivo. After treatment with metformin at 0, 2.5, 5 and 10 mM for 48 h, the ratio of p-AMPK to total AMPK showed significant rising in a dose-dependent manner in both BCPAP and KTC1, whereas p-AKT and p-mTOR expression level were downregulated. ¹⁸F-FDG uptake reduced after metformin treatment in a dose-dependent manner, corresponding to the reduced expression level of HK2 and GLUT1 in vitro. Xenograft model of PTC using BCPAP cells was achieved successfully. MicroPET/CT imaging showed that in vivo ¹⁸F-FDG uptake decreased after treatment with metformin. Immunohistochemistry staining further confirmed the reduction of HK2 and GLUT1 expression in the tumor tissue of metformin-treated PTC xenograft model. In conclusion, metformin could reduce glucose metabolism of PTC in vitro and in vivo. Metformin, by targeting glycometabolism of cancer cells, could be a promising adjuvant therapy alternative in the treatment modality of advanced thyroid carcinoma.

WNT/β-catenin signalling is involved in regulating adipogenesis, and its dysregulation occurs in obesity. Secreted frizzled-related protein 5 (SFRP5) is a WNT protein inhibitor; however, its role in adipogenesis and obesity is controversial. In this study, we observed that SFRP5 mRNA levels were increased in the fat tissues of obese humans and mice. Sfrp5 expression was gradually induced during differentiation of white and brown adipocytes and was highly increased in mature adipocytes rather than preadipocytes. However, the effects of the exogenous overexpression of Sfrp5 indicated that Sfrp5 may not directly regulate adipogenesis in vitro under the conditions studied. Moreover, SFRP5 did not inhibit the canonical WNT/β-catenin signalling pathway in preadipocytes. Subsequently, we measured the levels of circulating SFRP5 in obese patients and non-obese subjects using ELISA and did not find any significant difference. Collectively, these findings indicate that Sfrp5 represents a candidate for a mature adipocyte marker gene. Our data provide new evidence concerning the role of SFRP5 in adipogenesis of white and brown adipocytes and obesity.

Aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia are the two characteristic types of primary aldosteronism. Dysregulation of adrenal cortical cell proliferation contributes to both diseases. We previously demonstrated that APA expressed less dopamine D2 receptor than the respective non-tumor tissue and might contribute to the overproduction of aldosterone. As activation of D2 receptor inhibits the proliferation of various cells, downregulation of D2 receptor in APA may play a role in the tumorigenesis of APA. In this study, we demonstrate that D2 receptor plays a role in angiotensin II (AII)-stimulated adrenal cortical cell proliferation. The D2 receptor agonist, bromocriptine, inhibited AII-stimulated cell proliferation in primary cultures of the normal human adrenal cortex and APA through attenuating AII-induced phosphorylation of PK-stimulated cyclin D1 protein expression and cell proliferation. D2 receptor also inhibited AII-induced ERK1/2 phosphorylation. Our results demonstrate that, in addition to inhibiting aldosterone synthesis/production, D2 receptor exerts an anti-proliferative effect in adrenal cortical and APA cells by attenuating PKCμ and ERK phosphorylation. The lower level of expression of D2 receptor in APA may augment cell proliferation and plays a crucial role in the tumorigenesis of APA. Our novel finding suggests a new therapeutic target for primary aldosteronism.