Dlgap1 negatively regulates browning of white fat cells through effects on cell proliferation and apoptosis

Background Obesity is a metabolic imbalance characterized by excessive deposition of white fat. The browning of white fat can effectively treat obesity and related diseases. Although Dlgap1 (Discs, Large (Drosophila) Homolog-Associated Protein 1) is suspected to have an effect on this process, no empirical evidence is available. Methods To understand the role of Dlgap1, we cultured white and brown fat cells, then performed overexpression and knockout experiments. Results We found that Dlgap1 overexpression in brown adipocytes inhibits brown-fat-related gene expression, promotes white-fat-related genes, while also increasing brown-adipocyte proliferation and apoptosis. However, the gene overexpression has no effect on brown adipocyte maturation. Knocking out Dlgap1 in white fat cells promotes the expression and inhibition of brown-fat-related and white-fat-related genes, respectively. Additionally, the knockout inhibits white fat cell proliferation and apoptosis, while also promoting their maturation. Conclusions Dlgap1 negatively regulates the browning of white adipocytes by influencing cell proliferation and apoptosis.


Background
Obesity has become a public health crisis because it is a risk factor for cardiovascular disease, type 2 diabetes, hypertension, stroke, and many cancers [1,2]. Because chronic energy imbalance is the primary cause of most weight gain, nonsurgical therapy must reduce energy intake or increase energy expenditure, or both [3].
Adipocytes (fat cells) form three metabolically distinct categories: white, beige, and brown [4][5][6]. White adipose tissue (WAT) stores excess energy in the form of triglycerides, while brown adipose tissue (BAT) transfers chemical energy into heat as a defense against hypothermia and obesity [7]. Recent findings using a novel radiodiagnosis technique revealed unexpectedly high BAT activity in adult humans [6,[8][9][10]. These studies have also identified complex cell differentiation processes leading to the appearance of active brown adipocytes. Data on rodents suggest that brown adipocytes clustered in defined anatomical BAT depots arise from mesenchymal precursor cells common to the myogenic cell lineage [11]. Brown adipocytes of this origin are now called "classical" or "developmentally programmed" [12]. However, brown adipocytes also appear after thermogenic stimuli at anatomical sites corresponding to WAT, in a process called "browning" [12]. Initial studies suggest that browning of WAT can effectively treat obesity and related diseases [13], but more data are needed to clarify this link.

Cell isolation and culture
Inguinal WAT and interscapular BAT were washed thoroughly with sterile phosphate-buffered saline (PBS) to remove blood cells. The tissues were then minced and shaken in 0.2% collagenase type II at 37°C for 1 h. The cells were centrifuged at 1500 g for 5 min to remove the supernatant, and then plated on a collagen-coated Petri dish for culturing at 37°C in 5% CO 2 . The medium was Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12), 10% fetal bovine serum (FBS), 100 U penicillin, and 100 mg/mL streptomycin.

Construction of Dlgap1 knockout plasmid using CRISPR/Cas9
The Dlgap1 coding region was obtained and submitted to http://crispr.dbcls.jp/ and http://crispr.mit.edu/ to design four sgRNAs that targeted the first 200 bp of the Dlgap1 coding sequence. Targeted sequences were synthesized and annealed to form doublestranded DNA that served as templates for evaluating sgRNAs. These templates were ligated to the skeleton vector pCas-Guide-EF1a-GFP via T4 DNA ligase to form the CRISPR/Cas9 knockout vector.

Liposome-mediated transfection
Adipocytes at 60-70% confluence were transfected using Lipofectamine LTX with PLUS reagent (Invitrogen, Karlsruhe, Germany). Lipofectamine and plasmids were separately diluted in Opti-MEM (Gibco, Munich, Germany). The two solutions were mixed in a 1:1 ratio, incubated for 5 min at 20-25°C, and added to the cells.

RT-qPCR
Total RNA was extracted using RNAiso Plus (TaKaRa Bio, Shiga, Japan) following the manufacturer's protocol, and then treated with gDNA Eraser to eliminate genomic DNA. Next, cDNA was synthesized using Prime Script RT Enzyme Mix I (TaKaRa Bio). Real-time PCR conditions were based on the manufacturer's protocol in the TaKaRa Real-Time PCR Kit. A 7500 Real-Time PCR system was used for the reactions. Relative expression was determined using the comparative Ct (2 -ΔΔCt ) method (n = 3). The primers are listed in Table S1.

Western blotting
Western blotting was performed as described previously [31] Overexpression and knockout cells were washed with PBS and lysed in ice-cold lysis buffer with a protease inhibitor cocktail (Mammalian Protein Extraction Kit, KW Biotechnology, Beijing, China). Protein concentrations were measured using the BCA Protein Assay Kit (23,225, Thermo, USA). Proteins (10 mg) were separated with SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes, and incubated with primary antibodies overnight at 4°C. Signals were then visualized using the Thermo Scientific Pierce ECL western blotting substrate and the Tanon 5200 (Tanon, Shanghai, China) detection system.

Immunofluorescence staining
Cells were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, and stained with primary antibodies (see "Western blotting") overnight at 4°C. After two PBS washes, the cells were incubated with fluorescein isothiocyanate-labeled secondary antibodies (ab7080, Abcam) at room temperature for 1 h. For the negative controls, the primary antibodies were replaced with PBS.

5-Ethynyl-2′-deoxyuridine (EdU) assays
This cellular proliferation assay involved fluorescence detection using EdU (RuiBo, Guangzhou, China), a thymidine analog that can be incorporated into newly synthesized DNA during cell proliferation. At 24 h post-transfection, adipocytes were incubated with EdU (50 μM) for 2 h, fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and labeled with Apollo® fluorescent dye (RuiBo, Guangzhou, China) following the manufacturer's protocol. The EdU-positive cells were imaged with a confocal microscope (Nikon, Tokyo, Japan) to calculate their percentage.

Apoptosis measurements
Cells were digested with trypsin to assay apoptosis using the Cell Cycle and Apoptosis Analysis Kit (7Sea Biotech, Shanghai, China). The treated cells were viewed immediately at room temperature with an inverted fluorescence microscope (Nikon TE2000-U, Japan). Flow cytometry was performed using a BD FACScan system (BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed with FlowJo 7.6 software.

Statistical analysis
All data are shown as means ± SD from three individual experiments. Between-group differences were determined with the two-tailed Student's t-test in GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Significance was set at P < 0.05.

Results
Isolation and identification of white and brown adipocytes in C57BL/6 mice White adipose tissue accumulates under the skin, mainly in the groin, presenting as an even, milky white color from the large amount of oil present. Brown adipose tissue is located between the shoulder blades and gets its reddish-brown coloration from numerous blood cells (Fig. 1a). After 10 d of culturing experimentally isolated adipocytes, we observed that the white fat cells were 80% confluent, while the brown fat cells were only 40% confluent, indicating a significantly slower growth rate of the latter. Fluorescence phase contrast imaging revealed that the white and brown adipocytes did not differ significantly in morphology (Fig. 1a).
We then analyzed RNA expression of marker genes in white and brown adipocytes using RT-qPCR. We found that the white-fat marker genes Asc1, Leptin, and Adipoq [32] were 3.03, 9.33, and 207.03 times higher in white adipocytes than in brown adipocytes, respectively. Additionally, the brown-fat marker genes Cidea, Fndc5, Pgc1α, Prdm16, and Ucp1 [10,33] were expressed at lower levels in white adipocytes, being 0.0034, 0.229, 0.060, 0.582, and 0.0095 times the expression in brown adipocytes, respectively (Fig. 1b). Western blots confirmed the RT-qPCR results (Fig. 1c). Furthermore, immunofluorescence revealed that Ucp1 expression was nearly absent in white adipocytes, but obvious in brown adipocytes. Leptin was expressed in white adipocytes, but not in brown adipocytes (Fig.  1d). Oil red O staining showed that the differentiation of white and brown fat cells occurred at 8 d (Fig. 1e). Cell morphology differed visibly between the two adipocyte types. A mature white adipocyte presented with a large lipid droplet in the center of the cell (single foam fat), whereas a mature brown adipocyte contained many small lipid droplets.

Overexpression of Dlgap1 reduces browning-specific gene expression
Using mouse brain tissue as a template, we amplified the coding sequence of Dlgap1 and generated the pRIRES2-EGFP-Dlgap1 overexpression vector (Fig. 2a). The constructed vector was identified through enzyme digestion and electrophoresis (Fig. 2a). The vector's successful construction was further verified through sequencing.

Overexpression of Dlgap1 inhibits brown adipocyte formation
The results of EdU proliferation assays revealed that Dlgap1 overexpression increased brown adipocyte formation. Fluorescent cells were counted under a confocal laser microscope (Fig. 3a). Oil red O staining showed that Dlgap1 overexpression did not significantly influence adipogenic differentiation (Fig. 3b). Flow cytometry revealed that Dlgap1 overexpression inhibited brown adipocyte apoptosis. As a result, the experimental overexpression group had 4.6 times more than the control group did (Apoptosis rate = Q2 + Q3) (Fig. 3c). These results indicate that Dlgap1 is required for the full activation of a gene program favoring brown adipocytes.

Knocking out Dlgap1 promotes browning-specific gene expression
To knock out Dlgap1 using CRISPR/Cas9 technology, we designed four sgRNA pairs and determined their cutting efficiency. We found that sgRNA1, sgRNA2, and sgRNA3 acted on adipocytes, with sgRNA2 exhibiting the highest cutting efficiency (Fig. 4a). We also investigated how different transfection times affected transfection efficiency, and demonstrated that 48 h post-transfection was optimal (Fig. 4a).
After generating Dlgap1-knockout white adipocytes, our RT-qPCR analysis of fat marker genes showed that Acc, Asc1, Past1, Leptin, Fasn, and Scd expression decreased, while Cidea, Fabp4, Pgc1α, Pparγ, Prdm16, and Ucp1were upregulated (Fig. 4c). Western blots confirmed Dlgap1 downregulation, along with decreases in white-fat marker proteins (ACC, ASC1, PAST1, LEPTIN and FASN) to different degrees and increases in brownfat markers (PGC1α, PRDM16, and UCP1) (Fig. 4d). Consistent with the western blot data, immunofluorescence showed a decrease in LEPTIN expression and increase in UCP1 expression (Fig. 4b). Taken together, these results suggest that Dlgap1 knockout in white adipocytes promotes browning. The results of EdU assays indicated that Dlgap1 knockout in white adipocytes changed proliferation by 0.85 times compared with that of the control group (Fig. 5a). Oil red O staining indicated that Dlgap1 knockout increased adipogenic differentiation (Fig. 5b). Flow cytometry then showed that Dlgap1 knockout slowed adipocyte apoptosis. Furthermore, the knockout group had 0.38 times apoptosis rate as the control group did (Fig. 5c).

Discussion
White adipose tissue has emerged as a key determinant of healthy metabolism and metabolic dysfunction. This realization is paralleled by the finding that adult humans have heat-dissipating BAT, an important contributor to Top, white-fat marker gene; bottom, brown-fat marker gene. *P < 0.05, ** P < 0.01. *** P < 0.001, **** P < 0.0001. d Western blots of proteins in Dlgap1-knockout and control cells. Data are means ± SD energy balance and a possible therapeutic target for treating metabolic disease [34]. We propose that successful targeting of brown and white adipose tissues will depend on research that elucidates their developmental cell-type-specific functional regulators. Here, we explored the effect of Dlgap1 on white fat browning and revealed that Dlgap1 is a negative regulator of this process by influencing cell proliferation and apoptosis.
Genetic Dlgap1 variants are associated with neuropsychiatric disorders, including schizophrenia [35], autism spectrum disorder [36], and obsessive-compulsive disorder [37]. However, the mechanism of Dlgap1 in the browning of white fat remains poorly understood.
Certain Dlgap1 genetic variants are underrepresented in patients with type 2 diabetes in the Netherlands and Korea [28,38]. Because both obesity and type 2 diabetes are characterized by imbalances in energy metabolism in the human body, Dlgap1 may play an unidentified common role in both conditions. Additionally, Dlgap1 functional variants or genetic variants of neighboring genes that have strong linkage disequilibrium with Dlgap1 could be responsible for both browning and type 2 diabetes.
At the molecular level, we detected the effect of the Dlgap1 gene on white and brown fat marker genes by RT-qPCR, western blots, and immunofluorescence, and the results of the three methods were consistent with each other. The Dlgap1 gene was significantly differentially expressed in white and brown fat cells. Knockout of Dlgap1 increased BAT-specific gene expression, while overexpression of Dlgap1 reduced BAT-specific gene expression, especially the expression of UCP1. UCP1 protein plays a key role in the mitochondria in brown fat cells to increase heat production. Viral delivery of irisin, which caused only a moderate increase (~3-fold) in circulating levels, was shown previously to stimulate a 10-20-fold increase in UCP1 levels, increase energy expenditure, and improve glucose tolerance in high-fat fed mice [39].
At the cell level, we examined the effects of Dlgap1 on adipocyte proliferation, differentiation, and apoptosis. We found that Dlgap1 knockout inhibits white adipocyte proliferation, while its overexpression increases dipocyte proliferation. Relevant studies have shown that PPARγ is highly expressed in adipose tissue and is a key factor regulating adipocyte proliferation, differentiation, and increased insulin sensitivity [40]. The mechanisms underlying these patterns may be that Dlgap1 expression affects PPARγ, peroxisome proliferator-activated receptor gamma, which also inhibits cell proliferation. In summary, we successfully constructed the overexpression and knockout vector of the Dlgap1 gene, and verified that Dlgap1 negatively regulates the browning of white fat by affecting cell proliferation and differentiation. This study identifies a new function of the Dlgap1 gene in regulating the browning of white fat, laying a foundation for the alleviation and treatment of obesity.

Dlgap1 overexpression in brown adipocytes inhibits
brown-fat-related gene expression and promotes white-fat-related gene expression, while also increasing brown adipocyte proliferation and apoptosis. 2. Knocking out Dlgap1 in white fat cells promotes the expression and inhibition of brown-fat-related and white-fat-related genes, respectively. Additionally, knockout inhibits white fat cell proliferation and apoptosis. 3. Dlgap1 negatively regulates the browning of white adipocytes by influencing cell proliferation and apoptosis.
Additional file 1: Table S1. The primers of q-PCR.