Cell culture
Human adrenal cortex cell line [H295R, CRL-2128™] was purchased from ATCC. This cell line was derived from the NCI-H295 pluripotent adrenocortical carcinoma cells [ATCC CRL-10296]. The cells were grown as per the protocols provided by the supplier in 10 cm cell culture dish containing DMEM:F12 Medium [cat no. 30-2006, ATCC] supplemented with 1% ITS + Premix [cat no. 354352, Corning, Canada] and 2.5% Nu-serum [cat no. 355100, Corning, Canada], at 37 ◦C in 5% CO2 and 95% humidified atmosphere. When cells reached confluency, they were split at the desired density and used for specific experiments.
Immunocytochemistry
Localization of NUCB1/NLP and NUCB2/NESF-1 in H295R cells was carried out using immunocytochemistry [ICC] based on previously reported protocols [1]. The primary antibodies used in this study were rabbit anti-mouse NUCB1 [1:200, custom synthesized, cat no. 1312-PAC-02, Pacific Immunology, USA], rabbit anti-mouse NUCB2 [1:200, RRID: AB_2891124, cat no. 1312-PAC-01, Pacific Immunology, USA]. Secondary antibodies in this study were goat anti-rabbit Alexa Fluor 488 [1:200, RRID: AB_2630356, cat no. ab150077, ABCAM, UK]. No primary antibody was considered as a negative control group. The imaging was conducted using an Olympus DP70 camera and an Olympus BX51 microscope. Immunostaining obtained is referred to as NUCB1/NLP and NUCB2/NESF-1 to represent precursors and the encoded peptides detected by the antibodies used.
Effects of NLP and NESF-1 on steroidogenic enzymes and endogenous Nucb mRNAs in H295R cells
H295R cells at a confluency of 90% were treated with mouse NLP or rat NESF-1 at different concentrations [1–10 nM] for different time points [6 h and 12 h]. Human ACTH [cat no. 001–01, Phoenix Pharmaceuticals Inc., USA] at 100 nM was used as a positive control group. After the incubation period, cells were harvested, and the abundance of mRNAs for steroidogenic enzymes was assessed using qPCR. The abundance of endogenous Nucb mRNAs was also measured. The effective dose and time points were selected based on pilot studies [data not shown] and previous research [1].
Mechanism of action of NLP and NESF-1 on H295R cells
We reported that NLP and NESF-1 act through AC/PKA/CREB pathway to regulate POMC synthesis in mouse corticotrophs [1]. ACTH stimulates the production of GCs through MC2R-cAMP/PKA/CREB pathway in the adrenal cortex cell line, including Y1 mouse adrenocortical cells [16]. Therefore, we hypothesized that NLP and NESF-1 modulate cortisol synthesis through the CREB-mediated pathway in human adrenal cortex cells. To determine the cells signalling mediators, H295R cells were incubated with fresh media [control group] or NLP/NESF-1 [experimental groups] at effective doses [10 nM for peptides and 100 nM for ACTH] for 90 min, and then phosphorylated [P]-CREB/total [T]-CREB ratio was assessed in the cell lysate.
In the second part of this study, H295R cells were preincubated with NLP or NESF-1 to block the CREB-mediated pathway for 12 h, then forskolin [cat no. 11018-5, Cayman Chemical Co, USA] as a specific adenylate cyclase stimulator [10 µM] was added to the media for 12 h. The cortisol level in cell lysate or cell culture media was measured using a cortisol ELISA kit as explained below. The dissolved forskolin in DMSO was less than 0.1% in cell culture media. The concentration and time point for forskolin incubation were chosen based on the recommended dose range in the supplier catalogue and were independently validated in pilot studies [data not shown]. ACTH treated cells were used as a positive control group.
Cortisol measurement
Cortisol was measured in cell lysate and cell culture media. For this purpose, H295R cells were seeded in 6 well plates and treated with NLP/NESF-1/ACTH [10 nM for peptides and 100 nM for the positive control group] for different time points, including 6 h, 12 h and 24 h. Then, cells were washed with cold PBS and trypsinized. After centrifugation at 15,000 g at 4 ºC, trypsin-EDTA was removed, and cells were suspended in cold PBS and sonicated 3 times for 1–2 min. Cell culture media from all groups were collected to measure the secreted cortisol level in the media using human cortisol ELISA kit [cat no. COR31-K01, Eagle Bioscience, Inc, USA] according to manufacturer’s instructions. All cell lysate samples were diluted with cold PBS. The assay sensitivity and dynamic range test were 0.4 µg/dL and 0.5–60 µg/dL, respectively.
Intraperitoneal NLP injection in wildtype mice and Nucb1 knockout [KO] mice
For the first study, C57BL/6J wildtype [WT] mice [6–7 total number; both males and females] were used to study whether intraperitoneal [IP] injection of NLP affects HPA-related hormones and genes. All mice were fasted for 4 h [from 9 am] before the commencement of experiment. NLP was dissolved in sterile saline [0.9% NaCl] and then diluted to 100 µg/kg BW. It was administered to mice using insulin syringes [BD®, cat no. 324911] attached to a 27G needle in the lower right quadrant of abdomen to avoid damage to internal organs. The selected dose was validated in a previous study [14]. After 30 min, mice were euthanized, and blood samples and internal organs were collected.
To characterize the effects of disruption of Nucb1 in the second study, breeding pairs of homozygous C57BL/6NCrl-Nucb1em1[IMPC]Mbp/Mmucd mice (global Nucb1 disrupted mice) were purchased from the University of California [generated by Dr. Kent Lloyd, Mouse Biology Program, University of California-Davis, USA]. These breeding pairs were used to establish a colony of homozygous Nucb1 disrupted mice. In these mice, the exon 4 and flanking splicing regions of Nucb1 were constitutively deleted using CRISPR Cas9 gene editing technology in C57BL/6J mouse zygotes. All the animals were housed under 12 h light:12 h dark cycle [7 am − 7 pm], humidity [30–60%], and temperature [18–22 ºC] controlled vivarium located in the College of Medicine Lab Animal Services Unit, University of Saskatchewan. All protocols were prepared based on guidelines of the Canadian Council for Animal Care and were approved by the University of Saskatchewan Animal Care Committee [2012-0033]. The experiments conducted adhered to the ARRIVE guidelines on animal use and care. Age-matched mice were chosen and fed a standard rodent chow diet [cat no. 500I, LabDiet, 7.94% carbohydrate, 28.67% protein, 13.38% fat, Energy density = 4.09 Kcal/g]. Mice were anesthetized using 3% isoflurane inhalation and were euthanized by cervical dislocation. Different tissues, including the adrenal gland and pituitary, from these mice were collected after cervical dislocation, total RNA was extracted, and the expression of stress-related genes was assessed in the cell lysate.
Total RNA extraction, cDNA synthesis, and real-time quantitative PCR
This section was carried out as described in previously reported protocols [1]. Briefly, RNA was extracted using Ribozol [cat no. N580, VWR, USA], and then RNA’s quantity and quality were determined by NanoDrop 2000 [Thermo Fisher Scientific]. The RNA was reverse transcribed to cDNA using iScript Reverse Transcription Supermix for RT-qPCR [cat no. 170884, Bio-Rad, USA] followed by the quantitative measurement of mRNA expression by qPCR in a CFX Connect Optic module [Bio-Rad, USA] following the requirements of the MIQE guidelines [17] and using SensiFAST™ SYBR No-ROX MIX [cat no. BIO-98050, Bioline, UK]. The primers sequences and annealing temperatures are listed in Table S1 [Supplementary Information] purchased from Integrated DNA Technologies [IDT]. Three negative controls, including no template DNA [NTC control], no reverse transcriptase control from cDNA synthesis process [RTC control], and a nuclease-free water sample [PCR control], were also included for each gene expression study. Thermal cycling set-up for all genes was the following: denaturation [95 ºC for 5 s], annealing [specific for each primer for 25 s] and elongation [60 ºC for 25 s], 35 cycles. At least 3 independent experiments with triplicates for in vitro studies [final samples ≥ 9] and more than 6 mice/group/sex were considered. The abundance of mRNA was calculated based on the Pfaffl method and gene-specific efficiencies [18], relative to the geometric means of the 2 housekeeping genes.
Western blot analysis
The Western blot protocol followed was described previously [1]. Briefly, total protein was extracted using RIPA Lysis buffer [cat no. 89901, Thermo Fisher Scientific, USA] and the protein concentration was determined by Bradford assay. An equal amount of crude protein [40 µg] was electrophoresed on 8–16% Mini-Protean TGX gels [cat no. 456–1104, Bio-Rad, USA], and the protein bands were transferred to nitrocellulose membranes [cat no. 1704158, Bio-Rad, USA] using the Trans-Blot Turbo Transfer System [Bio-Rad, USA]. After blocking the membranes, they were incubated with primary antibodies overnight at 4 ºC followed by washing steps, incubation with secondary antibodies for 1 h and final washing. Then the membranes were visualized by ChemiDoc MP Imaging System [Bio-Rad, USA]. The band intensity was analyzed by ImageJ [National Institutes of Health, Bethesda, MD, USA].
Primary antibodies used in this study were monoclonal mouse anti-beta-actin antibody [1:1000, RRID: AB_528068, cat no. JLA20, Developmental Studies Hybridoma Bank, DSHB, University of Iowa, USA], polyclonal rabbit anti-phospho [Ser 133]-CREB antibody [1:1000, RRID: AB_2561044, cat no. 9198, Cell Signalling, USA], monoclonal rabbit anti-CREB antibody [1:1000, RRID: AB_331277, cat no. 9197, Cell Signalling, USA]. The secondary antibodies used were goat anti-rabbit IgG [H + L]-HRP conjugate antibody [1:5000, RRID: AB_11125142, cat no. 170–6515, Bio-Rad, USA] and goat anti-mouse IgG [H + L]-HRP conjugate antibody [1:5000, RRID: AB_11125547, cat no. 170–6516, Bio-Rad, USA].
Statistical analysis
Statistical analysis was conducted using the SPSS statistical software [IBM SPSS Statistics for Windows, Version 23.0, USA]. The normality of data distribution was analyzed by Shapiro-Wilk’s test and all data were normally distributed. The homogeneity of variances was checked by Levene’s test. The significance level was set at p < 0.05. The single comparison was performed by Student’s t-test, and multiple comparisons were performed by one-way ANOVA followed by Tukey’s multiple comparison test. All graphs were plotted by GraphPad Prism [GraphPad Software, Inc., Prism 8 for Windows, Version 8.4.2, USA].