Nesfatin-1 inhibits proliferation and enhances apoptosis of human adrenocortical H295R cells

  1. Harpal S Randeva1,7,8⇑
  1. 1Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK
    2Interim Translational Research Institute, Academic Health System, Hamad Medical Corporation, Doha, Qatar
    3Department of Obstetrics and Gynaecology, Birmingham Heartlands Hospital, Heart of England NHS Foundation Trust, Birmingham, UK
    4Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland
    5Department of Endocrinology, Metabolism and Internal Medicine, University of Medical Sciences, 16 Karol Marcinkowski, Poznan, Poland
    61st Medical Department, University of Lübeck Medical School, Lübeck, Germany
    7Department of Diabetes, Endocrinology and Metabolism, University Hospital, Coventry CV2 2DX, UK
    8Aston Medical Research Institute, School of Life and Health Sciences, Aston University, Birmingham, UK
  1. Correspondence should be addressed to H S Randeva; Email: Harpal.Randeva{at}warwick.ac.uk
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    Figure 1

    Expression of NUCB2 mRNA, nesfatin-1 protein expression in H295R cells, human adrenal gland and rat adrenal gland. (A) RT-PCR showing the presence of NUCB2 mRNA, (B) western blotting showing nesfatin-1 protein expression in human adipose tissue (h-AT), H295R cells and human adrenal gland (h-AG). Expression data shown in the figure is based on our findings from a sample size of 6. (C) Immunofluorescence of nesfatin-1 in the H295R cell line. (D, E, F and G) Immunohistochemistry showing the presence of nesfatin-1 protein localization in rat adrenal gland. Immunopositive substances are visible in cytoplasm of adrenocortical cells and in endothelial cells of the cortex. D: cross section through the entire rat adrenal gland. (E) Control section. (F) Higher magnification of the zona glomerulosa (ZG) and zona fasciculata (ZF) (region shown in D), CT – connective tissue capsule. Arrowheads show intense positive reaction in endothelial cells of the capillary bed; G: higher magnification of the zona reticularis (ZR) and medulla (M) (region shown in D). Sections counterstained with haematoxylin. Magnifications defined by scale bars.

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    Figure 2

    Effects of nesfatin-1 on (A) cortisol and (B) aldosterone secretion from H295R cells. H295R cells (four replicates) were stimulated with nesfatin-1 (100 nM), forskolin (10 μM) or angiotensin II (Ang II) (100 nM) at 6, 24, 36 and 48 h. At various timepoints media was collected for cortisol and aldosterone assay, and protein lysates from each treatment group were collected for normalization of steroid estimation. (C) Effects of nesfatin-1 on STAR protein (StAR) mRNA levels in H295R cells. Cells were cultured for the indicated periods in the presence or absence of nesfatin-1 (100 nM). StAR mRNA expression levels were normalized against GAPDH mRNA expression. Data are expressed as mean±s.e.m., from four independent experiments. *P<0.05 and **P<0.01.

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    Figure 3

    Following overnight serum depletion, H295R cells were stimulated with nesfatin-1 (Nes) (5, 50, 100 nM) and/or camptothecin (CAM) (2 μM) for 24 h to study the effects of nesfatin-1 on H295R (A) cell viability, (B) and YO-PRO-1 staining (apoptotic cells labeled green), (C) shows signal intensity of the YO-PRO-1 staining in control and nesfatin-1 stimulated cells quantified by ImageJ. Signal intensity of control cells was set to 1. (D) Concentration dependent effects of nesfatin-1 on DNA fragmentation data in H295R cells. (E) Concentration dependent effects of nesfatin-1 on Y1 cell viability. (F) Concentration dependent effects of nesfatin-1 on DNA fragmentation data in mouse Y1 cells. Data are expressed as mean±s.e.m., from three independent experiments, each performed in quadruplicate samples. **P<0.01 and ***P<0.001.

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    Figure 4

    Effects of nesfatin-1 (100 nM) stimulation for 4 h on pro and anti-apoptotic genes expression in H295R cells; mRNA expression levels were normalized against GAPDH mRNA expression. Data are expressed as mean±s.e.m., from four independent experiments. *P<0.05 and **P<0.01.

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    Figure 5

    Effects of nesfatin-1 (Nes) (1, 10, 100 nM) and/or angiotensin II (ANG) (100 nM) on intracellular calcium levels in H295R cells. H295R cells were cultured as described in the methods section. Data are expressed as mean±s.e.m., from four independent experiments. Time in (s). A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-14-0496.

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    Figure 6

    Effects of nesfatin-1 (100 nM) stimulation on ERK1/2, p38 and JNK1/2 protein phosphorylation and corresponding total protein expressions in H295R cells at 2.5, 5, 15, 30 and 60 min. Western blot analysis of protein extracts from H295R cells demonstrate that the antibody against p-ERK1/2, total ERK1/2, p-p38, total p38, p-JNK1/2 and total JNK1/2 recognized bands with apparent molecular weights of 42, 44, 43, 43, 46 and 54 kDa, respectively. Data are expressed as mean±s.e.m., from three independent experiments. **P<0.01 and ***P<0.001. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-14-0496.

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  1. Published online before print April 13, 2015, doi: 10.1530/JOE-14-0496 J Endocrinol 226 1-11
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