Experimental models

Human scWAT

Tissue samples were obtained from subjects undergoing abdominoplasty surgery in accordance with institutional human ethics guidelines and informed consent was obtained from all subjects. Experimental procedures for the isolation of scWAT from human samples were approved by the University of Alberta Biosafety Office. The scWAT was excised from the overlaying skin tissue (up to 1 cm of scWAT depth from the skin was used). Non-fat tissue was removed, and the scWAT was then minced and rinsed several times. The scWAT was digested for 0.5 h at 37 °C in 1.5 mg/mL of collagenase type 1 (Worthington, Lakewood, NJ, USA) in KRH buffer, filtered through a 250 µm cell strainer and centrifuged at 1000 RPM for 3 min. The pre-adipocyte pellet was plated in DMEM culture media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Two days post-confluency, differentiation was induced on differentiation day 0 by the addition of 172 nM human insulin, 1 µM dexamethasone, 0.5 mM isobutyl-methylxanthine, and 1 µM rosiglitazone supplemented with 10% FBS and 1% P/S. On differentiation day 3, the media was changed to media containing 172 nM human insulin, 10% FBS and 1% P/S. The cells were cultured in this media for the duration of their use as well as during experimental protocols.

3T3-L1 adipocytes

3T3-L1 adipocytes were initially cultured as pre-adipocytes in DMEM supplemented with 10% bovine calf serum and 1% P/S. The differentiation protocol for 3T3-L1 cells was the same as for human primary adipocytes, except bovine insulin was used instead of human insulin.

Mouse scWAT

All animal study protocols for tissue isolation were approved by University of Alberta Animal Care and Use Committee (protocol #s AU00286 and AU01417). Inguinal scWAT was isolated from male C57BL/6 mice (ages 3–6 mo.). The scWAT was then digested for 75 min at 37 °C in 1 mg/mL of type 1 collagenase (Worthington, Lakewood, NJ, USA) in KRH buffer. Digested scWAT was filtered through a 250 µm cell strainer, centrifuged at 1500 RPM for 10 min, and the pre-adipocyte-containing pellet was re-suspended in DMEM with 10% FBS and 1% P/S and subsequently filtered through a 40 µm cell strainer. Pre-adipocytes were plated in DMEM with 10% FBS and 1% P/S and then differentiated using the same protocol for 3T3-L1 adipocytes.

SGBS adipocytes

The Simpson-Golabi-Behmel Syndrome (SGBS) human pre-adipocyte cell strain was provided by Dr. Martin Wabitsch (Ulm University, Germany) and cultured and differentiated as described previously (Wabitsch et al., 2001).

Method details

Electrophysiology

The whole-cell patch-clamp technique was used to record currents from differentiated adipocytes. Prior to recording, adipocytes were incubated in an extracellular solution containing 1 µM all-trans retinal. All manipulations and visualizations of the recording chamber were conducted under low levels of red light to avoid inactivation of the current. Cells were superfused with bath solution containing (in mM) 140 NaCl, 5 CsCl, 1 MgCl 2 , 2 CaCl 2 , 10 glucose, 10 HEPES and 1 μM all-trans-retinal (pH 7.4).The pipette solution contained (in mM) 130 CsCl, 2 NaCl, 7 KCl, 0.2 Na-GTP, 1 MgCl 2 , 0.4 EGTA, 10 HEPES (pH 7.2). Cells were maintained at a holding potential of −50 mV. Blue (470 nm) light stimulation was delivered using an LED (M470F3, ThorLabs, Newton NJ, USA). Opsinamide (#509267, EMDMillipore, Etobicoke, ON, Canada), U73122 (#U6756, Sigma-Aldrich), and clemizole hydrochloride (#5371, Tocris, Avonmouth, UK) were all dissolved in DMSO prior to use. Light intensity was measured using X-Cite XR2100 Power meter (Excelitas Technologies, Mississauga, ON, Canada). A DeltaRam X monochromator was used to apply a spectral ramp of 400–600 nm wavelengths to cells (Photon Technology International, Edison NJ, USA). All recordings were performed using an Axon Instruments 200B patch-clamp amplifier, and Clampex 9.2 and Clampfit 9.2 software (Molecular Devices, Sunnyvale CA, USA) for data acquisition and analysis.

Reverse transcriptase PCR

Total RNA was isolated from all samples using TRIzol Reagent (ThermoFisher Scientific, Waltham, MA, USA) and then reverse transcribed using qScript cDNA SuperMix, (Quanta Biosciences, Beverly, MA, USA). Q5® High-Fidelity DNA Polymerase was used for RT-PCR and Phusion High-Fidelity DNA polymerase (NEB, Ipswich, MA, USA) was used for the nested PCR. PCR was performed with the cDNA reverse transcribed from 1 µg total RNA using the primer sets detailed in Table 1.

Table 1 Primer sets used for the PCR and nested PCR experiments detailed in Fig. 2. Full size table

Nested PCR

As multiple PCR products were obtained by RT-PCR of OPN4 cDNA due to the presence of multiple OPN4 transcripts in 3T3-L1 differentiated adipocytes, nested PCR was performed to analyze OPN4 transcripts between Exon3 and Exon7. A forward primer that spans Exon3 and Exon4, (5′GGAGACAGGTTGCGAGTTCTATGCCTTC3′) and a reverse primer that spans Exon6 and Exon7 (5′GATGTGCGAGTATCCAGC AAAGGCCAC3′) were used to amplify OPN4 transcripts. The PCR fragments were then separated by a 0.8% agarose gel in TBE buffer. Two bands, approximately 550 bp and 900 bp, were excised and each PCR fragment was extraced from the gel using QIAquick Gel extraction kit (QIAGEN, Valencia, CA, USA). Second round of PCR was carried out using a PCR fragment as a template, an Exon5 forward primer (5′TGCTGACATCCTGCTCCTG3′) and an Exon6 reverse primer (5′TGACAATCAGTGCGACCTTGGC3′). The PCR fragments were separated by a 0.8% agarose gel and then a main PCR product of each template was extracted from the gel as described above. Nested PCR products were sequenced by The Applied Genomics Core, University of Alberta.

Chronic blue light exposure

Starting on differentiation day 7, 3T3 L1 differentiated adipocytes were exposed to a chronic blue light protocol for 4 h per day for 13 consecutive days. The cells were kept in DMEM media containing 172 nM bovine insulin, as outlined above for differentiated adipocytes. Every day prior to light stimulation, beginning with the first day, a 1 mL sample of the cell culture supernatant was taken. Blue light (460 nm) stimulation (2.9 mW/cm2 at bottom of culture dish) was delivered using a Solis460A LED (Thorlabs, Newton NJ, USA). The LED was positioned at a distance of 76 cm above a platform containing six 35 mm culture dishes arranged in a circle to ensure equal light intensity distribution (Fig. 4B). The stimulation protocol was as follows: light on for 1 h, 15 min break, light on for 1 h, 30 min break, light on for 1 h, 15 min break, light on for 1 h—for a total daily exposure time of 4 h.

Lipid and glycerol analysis

Oil Red-O lipid (ORO) staining was used to assess changes in lipid homeostasis on day 14 of the chronic light stimulation protocol after the last day of light exposure (day 13). Cells were fixed in Z-fix for 15 min at room temperature, and then washed in ddH 2 O. They were then incubated in 60% isopropanol for 5 min, followed by incubation in 3 parts ORO stock solution (in 99% isopropanol) and 2 parts ddH 2 O for 10 min, and then washed in ddH 2 O. ORO-stained adipocytes were imaged using the Evos XL core inverted microscope (ThermoFisher Scientific, Waltham, MA, USA). A glycerol assay kit (#MAK117, Sigma-Aldrich) was used to quantify glycerol release by analyzing 3T3-L1 cell culture supernatant samples from days 1, 5, 8, 11, and 14 of the chronic blue light treatment and control (dark) groups. The assay was used as per the manufacturer's instructions.

Adipokine secretion

Leptin and adiponectin levels were quantified in 3T3-L1 cell culture supernatant samples from days 1, 5, 8, 11, and 14 of the chronic blue light protocol using electrochemiluminescent assays (K15124C-1 and K152BXC-1, MesoScale Discovery, Rockville, MD, USA) as per the manufacturer’s instructions.

Quantification and statistical analysis

Lipid droplet size analysis was performed using the Image Processing Toolbox in MatLab software (R2017a), with a custom written code to detect spherical, ORO-stained objects and obtain droplet area in pixels2 2. Area values were then converted to µm2 using a manually determined conversion factor. Following image processing, GraphPad Prism 7.03 software was used to plot and analyze data as well as all statistical analysis (GraphPad Software, La Jolla, CA, USA). Details of the statistical analysis performed and definition of n numbers can be found in the figure legends. All values provided are mean ± S.E.M, with the exception of Fig. 4F where values are the means of the median values ± S.E.M.