Modified lipoproteins can negatively affect beta cell function and survival. However, the mechanisms behind interactions of modified lipoproteins with beta cells – and in particular, relationships to increased uptake – are only partly clarified. By over-expressing the scavenger receptor CD36 (Tet-on), we increased the uptake of fluorescent low-density modified lipoprotein (oxLDL) into insulin-secreting INS-1 cells. The magnitude of uptake followed the degree of CD36 over-expression. CD36 over-expression increased concomitant efflux of 3H-cholesterol in proportion to the cellular contents of 3H-cholesterol. Exposure to concentrations of oxLDL from 20 to 100 µg/mL dose-dependently increased toxicity (evaluated by MTT) as well as apoptosis. However, the increased uptake of oxLDL due to CD36 over-expression did not exert additive effects on oxLDL toxicity – neither on viability, nor on glucose-induced insulin release and cellular content. Reciprocally, blocking CD36 receptors by Sulfo-N-Succinimidyl Oleate decreased the uptake of oxLDL but did not diminish the toxicity. Pancreatic islets of CD36−/− mice displayed reduced uptake of 3H-cholesterol-labeled oxLDL vs wild type but similar toxicity to oxLDL. OxLDL was found to increase the expression of CD36 in islets and INS-1 cells. In summary, given the experimental conditions, our results indicate that (1) increased uptake of oxLDL is not responsible for toxicity of oxLDL, (2) increased efflux of the cholesterol moiety of oxLDL counterbalances, at least in part, increased uptake and (3) oxLDL participates in the regulation of CD36 in pancreatic islets and in INS-1 cells.
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Z Ma, D F J Ketelhuth, T Wirström, T Ohki, M J Forteza, H Wang, V Grill, C B Wollheim and A Björklund
Qi Zhang, Qin Zhu, Ruyuan Deng, Feiye Zhou, Linlin Zhang, Shushu Wang, Kecheng Zhu, Xiao Wang, Libin Zhou and Qing Su
Fibroblast growth factor 21 (FGF21) plays an important role in the regulation of lipid and glucose metabolism. MS-275, as a class I-specific histone deacetylase (HDAC) inhibitor, has also been reported to affect energy metabolism. In this current study, we investigated the effects of MS-275 on hepatic FGF21 expression in vitro and in vivo and explored whether cAMP-responsive element-binding protein H (CREBH) was involved in the action of MS-275. Our results showed that MS-275 stimulated hepatic FGF21 mRNA and protein expressions in a dose- and time-dependent manner, as well as FGF21 secretion in primary mouse hepatocytes. Serum concentration and hepatic expression of FGF21 were elevated after injection of MS-275, along with increased expressions of genes involved in fatty acid oxidation and ketogenic production (peroxisome proliferator-activated receptor gammacoactivator1α, PGC-1α; carnitine palmitoyl-transferase 1a, CPT1a; 3-hydroxy-3-methylglutaryl-CoA synthase 2, Hmgcs2) as well as improved blood lipid profile. As a proved transcription factor of FGF21, the expression of CREBH was initiated by MS-275, with increased histone H3 lysine 18 acetylation (H3K18ac) signals and hepatocyte nuclear factor 4 alpha (HNF-4α) recruitment in CREBH promoter. Adenovirus-mediated knockdown of CREBH abolished MS-275-induced hepatic FGF21 and lipid metabolism-related gene expressions. These results suggest that MS-275 induces hepatic FGF21 by H3K18ac-mediated CREBH expression.
Lan Xu, Wenting Wang, Xinyue Zhang, Hanni Ke, Yingying Qin, Li You, Weiping Li, Gang Lu, Wai-Yee Chan, Peter C K Leung, Shidou Zhao and Zi-Jiang Chen
Obesity is a worldwide health problem with rising incidence and results in reproductive difficulties. Elevated saturated free fatty acids (FFAs) in obesity can cause insulin resistance (IR) in peripheral tissues. The high intra-follicular saturated FFAs may also account for IR in ovarian granulosa cells (GCs). In the present study, we investigated the relationship between saturated FFAs and IR in GCs by the use of palmitic acid (PA). We demonstrated that the glucose uptake in cultured GCs and lactate accumulation in the culture medium were stimulated by insulin, but the effects of insulin were attenuated by PA treatment. Besides, insulin-induced phosphorylation of Akt was reduced by PA in a dose- and time-dependent manner. Furthermore, PA increased phosphorylation of JNK and JNK blockage rescued the phosphorylation of Akt which was downregulated by PA. These findings highlighted the negative effect of PA on GCs metabolism and may partially account for the obesity-related reproductive disorders.
Nadia Islam, Ugwoke Sunday Paul, Rana Alhamdan, Juan Hernandez-Medrano, Bruce K Campbell, Peter Marsters and Walid E Maalouf
Ovarian cortical tissue cryopreservation is a relatively novel approach to preserving fertility in women diagnosed with cancer. However, the effects of freezing-thawing are not fully understood, mainly due to the lack of suitable methods to assess tissue’s survival after thawing. Disparities in steroid production have been associated with ovarian failure by disrupting folliculogenesis, ovulation and oocyte apoptosis. Moreover, specific miRNAs, identified in human ovarian follicles, are thought to play a fundamental role in folliculogenesis. In this study, we investigated the possible interplay between the ovarian steroidal production and miRNA expression patterns in spent culture media, as potential non-invasive markers for ovarian tissue damage after cryopreservation. Cryopreservation of ovarian cortical tissue decreased (P < 0.05) both steroid production (oestradiol and progesterone) and expression of miRNA-193b and 320A in spent culture media over 5 days; however, expression of miRNA-24 increased (P < 0.05). The number of primordial follicles was also reduced (P < 0.05) in fresh-cultured and cryopreserved-cultured cortical tissues when compared with fresh tissues. Downregulation of miRNA-193b and miRNA-320A together with upregulation of miRNA-24 could have a synergistic role in cell apoptosis, and consequently leading to reduced oestradiol and progesterone production. Thus, there appears to be an interplay between these miRNAs, ovarian steroid production and cell damage, which can be further explored as novel non-invasive markers of cell damage following cryopreservation.
Sogol Gachkar, Sebastian Nock, Cathleen Geissler, Rebecca Oelkrug, Kornelia Johann, Julia Resch, Awahan Rahman, Anders Arner, Henriette Kirchner and Jens Mittag
It is well established that thyroid hormones are required for cardiovascular functions; however, the molecular mechanisms remain incompletely understood, especially the individual contributions of genomic and non-genomic signalling pathways. In this study, we dissected how thyroid hormones modulate aortic contractility. To test the immediate effects of thyroid hormones on vasocontractility, we used a wire myograph to record the contractile response of dissected mouse aortas to the adrenergic agonist phenylephrine in the presence of different doses of T3 (3,3′,5-triiodothyronine). Interestingly, we observed reduced vasoconstriction under low and high T3 concentrations, indicating an inversed U-shaped curve with maximal constrictive capacity at euthyroid conditions. We then tested for possible genomic actions of thyroid hormones on vasocontractility by treating mice for 4 days with 1 mg/L thyroxine in drinking water. The study revealed that in contrast to the non-genomic actions the aortas of these animals were hyperresponsive to the contractile stimulus, an effect not observed in endogenously hyperthyroid TRβ knockout mice. To identify targets of genomic thyroid hormone action, we analysed aortic gene expression by microarray, revealing several altered genes including the well-known thyroid hormone target gene hairless. Taken together, the findings demonstrate that thyroid hormones regulate aortic tone through genomic and non-genomic actions, although genomic actions seem to prevail in vivo. Moreover, we identified several novel thyroid hormone target genes that could provide a better understanding of the molecular changes occurring in the hyperthyroid aorta.
Jennifer Miller-Gallacher, Paul Sanders, Stuart Young, Andrew Sullivan, Stuart Baker, Samuel C Reddington, Matthew Clue, Katarzyna Kabelis, Jill Clark, Jane Wilmot, Daniel Thomas, Monika Chlebowska, Francesca Cole, Emily Pearson, Emma Roberts, Matthew Holly, Michele Evans, Ricardo Núñez Miguel, Michael Powell, Jane Sanders, Jadwiga Furmaniak and Bernard Rees Smith
The crystal structures of the thyroid-stimulating hormone receptor (TSHR) leucine-rich repeat domain (amino acids 22–260; TSHR260) in complex with a stimulating human monoclonal autoantibody (M22TM) and in complex with a blocking human autoantibody (K1-70™) have been solved. However, attempts to purify and crystallise free TSHR260, that is not bound to an autoantibody, have been unsuccessful due to the poor stability of free TSHR260. We now describe a TSHR260 mutant that has been stabilised by the introduction of six mutations (H63C, R112P, D143P, D151E, V169R and I253R) to form TSHR260-JMG55TM, which is approximately 900 times more thermostable than wild-type TSHR260. These six mutations did not affect the binding of human TSHR monoclonal autoantibodies or patient serum TSHR autoantibodies to the TSHR260. Furthermore, the response of full-length TSHR to stimulation by TSH or human TSHR monoclonal autoantibodies was not affected by the six mutations. Thermostable TSHR260-JMG55TM has been purified and crystallised without ligand and the structure solved at 2.83 Å resolution. This is the first reported structure of a glycoprotein hormone receptor crystallised without ligand. The unbound TSHR260-JMG55TM structure and the M22 and K1-70 bound TSHR260 structures are remarkably similar except for small changes in side chain conformations. This suggests that neither the mutations nor the binding of M22TM or K1-70TM change the rigid leucine-rich repeat domain structure of TSHR260. The solved TSHR260-JMG55TM structure provides a rationale as to why the six mutations have a thermostabilising effect and provides helpful guidelines for thermostabilisation strategies of other soluble protein domains.
Ayse Basak Engin, Atilla Engin and Ipek Isik Gonul
Adipose tissue is the primary source of many pro-inflammatory cytokines in obesity. Macrophage numbers and pro-inflammatory gene expression are positively associated with adipocyte size. Free fatty acid and tumor necrosis factor-α involve in a vicious cycle between adipocytes and macrophages aggravating inflammatory changes. Thereby, M1 macrophages form a characteristic ‘crown-like structure (CLS)’ around necrotic adipocytes in obese adipose tissue. In obese women, CLSs of breast adipose tissue are responsible for both increase in local aromatase activity and aggressive behavior of breast cancer cells. Interlinked molecular mechanisms between adipocyte–macrophage–breast cancer cells in obesity involve seven consecutive processes: Excessive release of adipocyte- and macrophage-derived inflammatory cytokines, TSC1–TSC2 complex–mTOR crosstalk, insulin resistance, endoplasmic reticulum (ER) stress and excessive oxidative stress generation, uncoupled respiration and hypoxia, SIRT1 controversy, the increased levels of aromatase activity and estrogen production. Considering elevated risks of estrogen receptor (E2R)-positive postmenopausal breast cancer growth in obesity, adipocyte–macrophage crosstalk is important in the aforementioned issues. Increased mTORC1 signaling in obesity ensures the strong activation of oncogenic signaling in E2Rα-positive breast cancer cells. Since insulin and insulin-like growth factors have been identified as tumor promoters, hyperinsulinemia is an independent risk factor for poor prognosis in breast cancer despite peripheral insulin resistance. The unpredictable effects of adipocyte-derived leptin–estrogen–macrophage axis, and sirtuin 1 (SIRT1)–adipose-resident macrophage axis in obese postmenopausal patients with breast cancer are unresolved mechanistic gaps in the molecular links between the tumor growth and adipocytokines.
Cristina Velasco, Sara Comesaña, Marta Conde-Sieira, Jesús M Míguez and José L Soengas
We hypothesize that cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) are involved in the modulation of metabolic regulation of food intake by fatty acids in fish. Therefore, we assessed in rainbow trout (Oncorhynchus mykiss) the effects of intracerebroventricular treatment with 1 ng/g of CCK-8 and with 2 ng/g of GLP-1 on food intake, expression of neuropeptides involved in food intake control and the activity of fatty acid-sensing systems in hypothalamus and hindbrain. Food intake decreased up to 24 h post-treatment to 49.8–72.3% and 3.1–17.8% for CCK-8 and GLP-1, respectively. These anorectic responses are associated with changes in fatty acid metabolism and an activation of fatty acid-sensing mechanisms in the hypothalamus and hindbrain. These changes occurred in parallel with those in the expression of anorexigenic and orexigenic peptides. Moreover, we observed that the activation of fatty acid sensing and the enhanced anorectic potential elicited by CCK-8 and GLP-1 treatments occurred in parallel with the activation of mTOR and FoxO1 and the inhibition of AMPKα, BSX and CREB. The results are discussed in the context of metabolic regulation of food intake in fish.
Shu-Ching M Wang, Dennis H Dowhan and George E O Muscat
Breast cancer is a heterogeneous disease, and the complexity of breast carcinogenesis is associated with epigenetic modification. There are several major classes of epigenetic enzymes that regulate chromatin activity. This review will focus on the nine mammalian protein arginine methyltransferases (PRMTs) and the dysregulation of PRMT expression and function in breast cancer. This class of enzymes catalyse the mono- and (symmetric and asymmetric) di-methylation of arginine residues on histone and non-histone target proteins. PRMT signalling (and R methylation) drives cellular proliferation, cell invasion and metastasis, targeting (i) nuclear hormone receptor signalling, (ii) tumour suppressors, (iii) TGF-β and EMT signalling and (iv) alternative splicing and DNA/chromatin stability, influencing the clinical and survival outcomes in breast cancer. Emerging reports suggest that PRMTs are also implicated in the development of drug/endocrine resistance providing another prospective avenue for the treatment of hormone resistance and associated metastasis. The complexity of PRMT signalling is further underscored by the degree of alternative splicing and the scope of variant isoforms (with distinct properties) within each PRMT family member. The evolution of PRMT inhibitors, and the ongoing clinical trials of PRMT inhibitors against a subgroup of solid cancers, coupled to the track record of lysine methyltransferases inhibitors in phase I/II clinical trials against cancer underscores the potential therapeutic utility of targeting PRMT epigenetic enzymes to improve survival outcomes in aggressive and metastatic breast cancer.
Junye Chen, Yi Lu, Mengyuan Tian and Qiren Huang
Forkhead box-O1 (FOXO1) is a downstream target of AKT and plays crucial roles in cell cycle control, apoptosis, metabolism and adipocyte differentiation. It is thought that FOXO1 affects adipocyte differentiation by regulating lipogenesis and cell cycle. With the deepening in the understanding of this field, it is currently believed that FOXO1 translocation between nuclei and cytoplasm is involved in the regulation of FOXO1 activity, thus affecting adipocyte differentiation. Translocation of FOXO1 depends on its post-translational modifications and interactions with 14-3-3. Based on these modifications and interactions, FOXO1 could regulate lipogenesis through PPARγ and the adipocyte cell cycle through p21 and p27. In this review, we aim to provide a comprehensive FOXO1 regulation network in adipocyte differentiation by linking together distinct functions mentioned above to explain their effects on adipocyte differentiation and to emphasize the regulatory role of FOXO1. In addition, we also focus on the novel findings such as the use of miRNAs in FOXO1 regulation and highlight the improvable issues, such as RNA modifications, for future research in the field.