Reagents and antibodies

Unless otherwise specified, all reagents and chemicals were purchased from Sigma-Aldrich. Anti-rabbit DBC1 antibody was purchased from Bethyl Laboratories (Cat. # A300-434A), and mouse monoclonal anti-B-actin was from Sigma-Aldrich (Cat. # A2228).

Animal handling and experiments

All mice used in this study were bred and maintained at the Institut Pasteur Montevideo Animal facility (UBAL). The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Institut Pasteur Montevideo (CEUA; protocol numbers 70153-000839-17, 003-19, and 006-19). All studies described were performed according to the methods approved in the protocol and following all international guidelines and national legal regulations (Law 18.611). Mice received a standard chow or high-fat diet (42% fat and 0.25% cholesterol, AIN93G, LabDiet, USA) and water ad libitum. Weight gain was monitored during obesity experiments in Dbc1^LoxP/LoxP;CRE (n = 24 females, n = 18 males) and in control littermates Dbc1^LoxP/LoxP (n = 30 females, n = 17 males). In chow diet experiments, weight gain was assessed in Dbc1^LoxP/LoxP (n = 7 females, n = 6 males) and Dbc1^LoxP/LoxP;CRE (n = 6 females, n = 9 males).

Tolfenamic acid was administered subcutaneously (1 mg/kg, Tolfedine, Vetoquinol, Madrid, Spain) in order to provide analgesia and anti-inflammatory effects, when necessary. At the end of the experiment, animals were sacrificed under deep anesthesia (a mixture of ketamine (150 mg/kg, Pharmaservice, Ripoll Vet, Montevideo, Uruguay) and xylazine (15 mg/kg, Seton 2%; Calier, Montevideo, Uruguay), followed by cervical dislocation.

Generation of the Dbc1 conditional knock-out mice

All animal procedures to generate the mutant line were performed at the SPF animal facility of the Laboratory Animal Biotechnology Unit of Institut Pasteur de Montevideo. Experimental protocols were opportunely approved by the Institutional Animal Ethics Committee (protocol number 007–18), in accordance with National Law 18.611 and international animal care guidelines (Guide for the Care and Use of Laboratory Animal) [11] regarding laboratory animal protocols. Mice were housed in individually ventilated cages (Tecniplast, Milan, Italy) containing chip bedding (Toplit 6, SAFE, Augy, France), in a controlled environment at 20 ± 1°C with a relative humidity of 40–60%, in a 14/10 h light-dark cycle. Autoclaved food (Labdiet 5K67, PMI Nutrition, IN, USA) and autoclaved filtered water were administered ad libitum.

Cytoplasmic microinjection was performed on B6D2F2 or C57BL/6J zygotes using a mix of 100 ng/µl Cas9 mRNA, 25 ng/µl of each sgRNA, and 50 ng/µl ssDNA oligo diluted in microinjection buffer. Viable embryos were cultured overnight in M16 medium microdrops under embryo-tested mineral oil, containing the ligase IV inhibitor SCR7, in 5% CO₂ in air at 37ºC. The next day, 2-cell embryos were transferred into the oviduct of B6D2F1 0.5 days post coitum (dpc) pseudopregnant females (an average of 20 embryos/female), following surgical procedures [12]. For surgery, recipient females were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). Tolfenamic acid was administered subcutaneously (1 mg/kg, Tolfedine, Vetoquinol, Madrid, Spain) in order to provide analgesia and anti-inflammatory effects [13]. Pregnancy diagnosis was determined by visual inspection by an experienced animal caretaker two weeks after embryo transfer, and litter size was recorded on day 7 after birth.

Pups were tail-biopsied and genotyped 21 days after birth, and mutant animals were maintained as founders. The first round of microinjections resulted in the insertion of only the 3’ LoxP site. Dbc1 3’ LoxP F1 animals were used to produce embryos by in vitro fertilization using the Center for Animal Resources and Development (CARD) protocol [14]. Three hours after fertilization, zygotes were microinjected into the cytoplasm with CRISPR reagents at the same aforementioned concentrations, to insert the lacking 5’ LoxP site. Viable two-cell embryos were transferred to pseudopregnant females as detailed above. Founders with both 3’ and 5’ LoxP sites were used to generate the final mutant line.

The mice used in this study were backcrossed with C57BL/6J mice to homogenize the genetic background. They were then bred to homozygosity for the floxed Dbc1 allele. To generate adipocyte-specific Dbc1 knockout mice, the homozygous floxed Dbc1 mice were crossed with B6.FVB-Tg(AdipoQ-CRE)1Evdr/J mice obtained from Jackson Laboratories.

The experimental colony was established by crossing AdipoQ-CRE Dbc1^LoxP/LoxP hemizygous mice with Dbc1^LoxP/LoxP mice. For the experiments, groups with a sample size (N) of ten or more mice per genotype were used.

Blood glucose measurements

Mice were fasted for 16 hours prior to fasting glucose measurements and glucose tolerance tests (GTT). For the GTT, mice received an intraperitoneal injection of a glucose solution at a dose of 1.5 g/kg of body weight. Plasma glucose concentrations were measured from tail blood using a handheld glucometer (Accu-Chek, Roche). Under the normal diet (ND) condition, six Dbc1^LoxP/LoxP and six Dbc1^LoxP/LoxP;CRE mice were used for each sex. In the high-fat diet (HFD) condition, the male groups consisted of seven Dbc1^LoxP/LoxP and nine Dbc1^LoxP/LoxP;CRE mice, while the female groups included six Dbc1^LoxP/LoxP and six Dbc1^LoxP/LoxP;CRE mice. Area under the curve (AUC) values were calculated for the GTT, and comparisons were performed using unpaired t-tests.

Hepatic and renal function

To assess liver and kidney function, whole blood from Dbc1^LoxP/LoxP (n = 5 males, n = 8 females) and from Dbc1^LoxP/LoxP;CRE (n = 10 males, n = 8 females) mice was analyzed using the Pointcare V2 Chemistry Analyzer (Tianjin MNCHIP Technologies Co., China). The parameters determined were total proteins (TP), albumin (ALB) level, globulin (GLO) level, ALB/GLO ratio, total bilirubin (BIL T) level, alanine aminotransferase (ALT) level, aspartate aminotransferase (AST) level, gamma-glutamyl transpeptidase (GGT) level, blood urea nitrogen (BUN) level, creatinine (CRE) level, BUN/CRE ratio, and glucose (GLU) level.

Isolation of WAT adipocytes

Fat depots were dissected and weighed. A collagenase digestion solution (collagenase type II, GIBCO 1 mg/ml in Hank’s Balanced Salt Solution containing 2% BSA) was added at a 3:1 ratio of collagenase solution to fat tissue, and the tissue was finely minced and incubated for 1 hour at 37°C in a shaking incubator. Then, the adipocyte suspension was centrifuged at 100g for 10 minutes at room temperature (RT). Subsequently, floating mature adipocytes were washed twice with cold PBS.

Western blotting

Adipocytes were lysed using radioimmunoprecipitation assay buffer (at a 1:10 volume ratio) supplemented with 5 mM NaF, 5 mM nicotinamide, 50 mM β-glycerophosphate, 1 μM trichostatin A (catalog no.: 647925; Sigma), a protease inhibitor cocktail, and then sonicated in five cycles of 10 seconds each, followed by intervals of 5 seconds. Homogenates were incubated for 20–30 minutes at 4 °C under constant agitation and then centrifuged at 10,000g for 10 minutes. Protein concentration in the supernatants were determined using the Bradford protein assay reagent. Samples were resuspended in a Laemmli buffer, separated by SDS-PAGE, and transferred to polyvinylidene fluoride membranes. After blocking (Tris-buffered saline containing 0.2% Tween-20 and 5% nonfat milk), the membranes were incubated overnight with the appropriate antibodies. Secondary antibodies were incubated for 1 hour and detected using SuperSignal West Pico Chemiluminescent Kit (catalog no.: 34080; Pierce).

Histology

Liver sections were obtained from paraffin-embedded tissue and stained with hematoxylin and eosin (H&E) following standard procedures. For each mouse, at least three sections were analyzed. In both the normal diet (ND) and high-fat diet (HFD) groups, three Dbc1^LoxP/LoxP and three Dbc1^LoxP/LoxP;CRE mice were used.

Plasma NEFA quantification

In males, 8 Dbc1^LoxP/LoxP and 9 Dbc1^LoxP/LoxP;CRE mice were used in the high-fat diet (HFD) group, while 5 Dbc1^LoxP/LoxP and 5 Dbc1^LoxP/LoxP;CRE mice were used in the normal diet (ND) group. In females, 6 Dbc1^LoxP/LoxP and 6 Dbc1^LoxP/LoxP;CRE mice were used in both ND and HFD groups. Non-esterified fatty acids were measured using the NEFA-HR Assay (Wako) according to the manufacturer’s instructions. Statistical analysis was performed using an unpaired t-test.

Measurements of lipid accumulation in the liver

Liver triglycerides were measured according to [15]. Liver samples from Dbc1^LoxP/LoxP (n = 5 males, n = 5 females) and Dbc1^LoxP/LoxP;CRE (n = 5 males, n = 5 females) were homogenized in lysis buffer, as described above. Lipid content was measured using the TG Color GPO/PAP AA kit (Wiener Lab, USA) following the manufacturer’s instructions.

Adipocyte Lysis and RNA Extraction

Total RNA from adipocytes was extracted following lysis with TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, adipocytes were harvested and resuspended in 1 mL of TRIzol reagent. The cells were lysed by pipetting up and down several times and then incubated at room temperature for 5 minutes to ensure complete dissociation of nucleoprotein complexes. Following lysis, 0.2 mL of chloroform was added per 1 mL of Trizol reagent. The mixture was vigorously shaken for 15 seconds and then incubated at room temperature for 2–3 minutes. The samples were centrifuged at 12,000 x g for 15 minutes at 4°C. The aqueous phase was then processed using the Direct-zol RNA Miniprep Kit (Zymo Research) according to the manufacturer’s protocol.

RNA quality control, library and sequencing

An Infinite 200 PRO (Tecan) spectrophotometer was used to evaluate the concentration and the 260/280 ratio of the RNA obtained from mature adipocytes. The RNA quality was evaluated using the RNA integrity number (RIN), determined with a Bioanalyzer RNA 6000 Nano chip (Agilent, Cat# 5067–1511). All samples demonstrating high RNA integrity, with an average RIN of 9.0, were used for further studies. RNA-seq was performed on samples from individual animals (n ≥ 3) of each experimental group. All libraries were prepared and sequenced by Genewiz (https://www.genewiz.com/) using the TruSeq mRNA Sample Prep Kit v2 protocol (poly(A)+ selection) and Illumina HiSeq 2x150 bp sequencing..

RNA-seq analysis

Raw reads (FASTQ files) obtained from Genewiz (https://www.genewiz.com/) were used as the input for miARma-Seq pipeline, a comprehensive tool for miRNA and mRNA analysis [16], following the user manual (v 1.7.2). Low quality reads and adapter sequences were removed with Cutadapt software [17], allowing a minimum read Phred quality score of 20. Filtered high-quality reads (Ave. 54,995,205 ± SD 6,123,659 reads, S1 Table in S1 File) were aligned to Mus musculus reference genome (GRCm38/mm10, indexed from http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) using HISAT2 [18] with default parameters (Overall genome mapping %: Ave. 94.9 ± SD 0.3, S1 Table in S1 File). Gene counts were assessed with featureCounts [19] using default parameters (S1 Table in S1 File). For quantification, we use the annotation coordinates of Ensembl Mus_musculus.GRCm38.96 GTF file. Normalization and Differential Expression Genes (DEGs) analysis were conducted using SARTools pipeline [20] in RStudio, selecting the edgeR algorithm [21], Trimmed Mean of the M-values (TMM) normalization, and CPM (Counts Per Million) ≥ 1 as the cut-off (S1 Table and S4 Fig in S1 File). A significance threshold of log2 fold change > |0.58 | and p-value <0.05 was applied for DEGs, instead of the adjusted p-value, in order to have a sufficient number of genes to perform over-representation analysis.

RNA-seq validation by qPCR

Expression levels of candidate DEGs involved in identified enriched pathways were confirmed by qPCR. Genes not directly involved in such pathways but significantly up or down regulated according to RNA-seq data, were also included. Tissues were homogenized in Trizol for RNA extraction according to the manufacturer’s protocol. DNase I treatment was used to eliminate genomic DNA contamination. Reverse transcription was done using SuperScript II RT, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using a Fast SYBR Green. Relative quantification of changes in gene expression were expressed in relation to housekeeping genes. Expression was calculated as fold increase with respect to control. The primers were from IDT and their sequences are provided in S2 Table in S1 File.

Gene set enrichment analysis

Gene Set Enrichment Analysis (GSEA) was conducted using the gseGO function from the clusterProfiler R package (v4.8.2) [22]. This analysis examined Gene Ontology (GO) terms related to biological processes by considering the complete list of genes, ranked by their fold change in expression, rather than focusing solely on differentially expressed genes (DEGs). The terms with significance threshold of adjusted p-value <0.05 were selected.

Statistics

Unless stated otherwise, values are shown as the mean ± standard deviation (SD) from three to five experiments. Statistical analyses were conducted using GraphPad Prism for most experiments, while RNA-seq data were analyzed using R (version 3.6) with the enrichplot, xlsx, ggplot2, and heatmap.2 libraries (employing Euclidean distance and Ward clustering algorithms). Group differences were evaluated using either ANOVA or a two-tailed Student’s t-test, and a p-value below 0.05 was considered statistically significant.

Generation of Dbc1^LoxP/LoxP conditional mice by CRISPR/Cas9 and adipocyte-specific deletion

The mouse Dbc1 gene, located on chromosome 14, comprises 21 exons forming an open reading frame that extends from exon 2 to exon 21. It is predicted to have only one full-length transcript encoding a protein of 922 amino acids. To create a conditional knockout (cKO) murine line, we targeted exon 3 to be floxed. We designed two guide RNAs (gRNAs) to target intron 2 and intron 3 using CRISPR/Cas9 and assessed their effectiveness by transiently transfecting a murine NIH3T3 cell line that stably expresses Cas9, followed by heteroduplex analysis (Fig S1 in S1 File).

Additionally, we generated two single-stranded DNA (ssDNA) donors for knock-in of LoxP sites. These sequences comprise part of intron 2 and intron 3, LoxP sites, and two restriction enzyme sites, which allow for the identification of the insertion. In the first step, we inserted the 3’ LoxP site. A second round of genetic manipulation was necessary to insert the 5’ LoxP site.

We crossed a male mouse carrying the selected mutation with C57BL/6J females to establish the colony and performed five rounds of backcrossing before beginning the characterization of the line. This process should be sufficient to segregate any off-target or unwanted modifications of the genome produced during genetic manipulation. From that point onward, we maintained the line by crossing homozygous mutant animals. To achieve knockout in mature adipocytes, we crossed the floxed Dbc1 animals with the AdipoQ-CRE mouse line (Fig 1A).

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Fig 1. Generation of an Adipocyte-Specific Dbc1 Knockout Mouse Model.

(A) Breeding strategy used to generate adipocyte-specific Dbc1 knockout mice. The combination of the AdipoQ-CRE transgene with Dbc1 floxed alleles led to the deletion of Dbc1 specifically in adipocytes. (B) Schematic representation of Dbc1 gene targeting, showing LoxP sites flanking exon 3. (C) Genotyping by PCR confirming recombination at the Dbc1 floxed locus in adipocytes from mice carrying both the AdipoQ-CRE transgene and Dbc1 floxed alleles. (D) Western blot analysis of DBC1 protein expression in different adipose tissue depots (inguinal, iWAT; epididymal, eWAT), isolated adipocytes, stromal vascular fraction (SVF), brain, and liver.

https://doi.org/10.1371/journal.pone.0322732.g001

First, we sequenced the region where the genetic manipulation was performed to confirm the expected sequence. We used primers located near the 5’ and 3’ ends of exon 3 of Dbc1 to sequence the PCR product. The expected mutations were detected, and no unwanted mutations were found (Fig 1B). Next, to verify the construct, we performed PCR on genomic DNA from cKO MEF cells transiently expressing Cre recombinase. The presence of a 300 bp PCR product confirmed that DNA recombination occurred successfully (Fig 1C).

Next, we evaluated Dbc1 at the protein level by western blot. The ablation of exon 3 (300 bp) by Cre recombinase was expected to cause a shift in the open reading frame of the transcript and the introduction of a premature stop codon. The mutant allele could therefore produce a 35-amino acid peptide. In agreement with that prediction, the ~ 100 kDa Dbc1 band was absent in adipocytes from different fat tissue depots. In addition, in whole tissue extracts from white adipose tissue (WAT) and brown adipose tissue (BAT), Dbc1 protein levels decreased. In contrast, there was no difference in protein expression in the liver and brain (Fig 1D). Although an apparent increase in Dbc1 expression was observed in the isolated stromal vascular fraction (SVF) from CRE + fat tissue (Fig 1D), its quantification using replicates (N = 4), revealed no statistically significant differences (S1 Fig in S1 File).

Metabolic characterization of Dbc1^LoxP/LoxP;CRE KO male and female mice on a normal diet

In previous studies, we and others showed that whole-body Dbc1 knockout mice do not develop obesity when fed a regular chow diet [15,23]. However, they present abnormal glucose management that is mainly due to altered liver gluconeogenesis [23]. This effect was a consequence of dysregulation of hepatic REV-ERBα activity and increased PEPCK expression [18,23]. Consistently, the specific deletion of Dbc1 only in mature adipocytes, did not affect body weight or glucose homeostasis, measured by GTT in normal chow conditions, in both male and female mice (Fig 2). Free-fatty acid levels were also comparable among genotypes in both males and females (Fig 2). In conclusion, the absence of Dbc1 specifically in mature adipocytes failed to influence body weight, glucose management, or free-fatty acid levels in mice under normal chow conditions.

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Fig 2. Similar Weight, Glucose, and Blood Free-Fatty Acid Levels Across Different Genotypes under a Normal Diet (ND).

(A) Mean body weight of female (gray and purple) and male mice (black and yellow) from different genotypes. (B) Glucose Tolerance Test (GTT). Glucose response evaluation in male and female mice from different genotypes. (C) Blood Free-Fatty Acid Levels of male and female mice from different genotypes. Data represent the mean ± standard deviation (SD) for each group. Statistical analysis was performed using a t-test, and no statistically significant differences were observed.

https://doi.org/10.1371/journal.pone.0322732.g002

Metabolic characterization of Dbc1^LoxP/LoxP;CRE KO male and female mice on high-fat diet

Next, we evaluated the effect of diet-induced obesity on the metabolic phenotype of Dbc1^LoxP/LoxP;CRE KO mice. Our previous work, conducted in mice with a mixed genetic background, showed that whole-body Dbc1 KO mice fed with high-fat diet for 20 weeks, develop morbid obesity with prevention of fatty-acid spillover and metabolic damage [3]. This phenotype was also observed in mice with pure C57BL/6J genetic background (S2 Fig in S1 File).

When Dbc1^LoxP/LoxP;CRE mice were fed a high-fat diet (HFD), both males and females gained weight at a rate similar to that of their control littermates (Fig 3A). Weight gain was monitored for up to 20 weeks on HFD, and no significant differences were observed between genotypes. Furthermore, we did not detect any differences in glucose tolerance (Fig 3B) or in plasma free fatty acid levels (Fig 3C), which are key indicators of the healthy obesity phenotype we previously described [3].

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Fig 3. Development of Diet-Induced Obesity (DIO) in the Dbc1^LoxP/LoxP;CRE mouse model.

(A) Comparable weight gain was observed across both sexes and genotypes during HFD administration, with no significant differences detected. (B) Glucose tolerance test was assessed after 8 weeks on the HFD regimen. No significant differences were found in glucose tolerance between the experimental groups. (C) Plasma Free-Fatty Acid Levels of serum samples collected after 8 and 15 weeks of HFD administration were analyzed for free fatty acid concentrations.

https://doi.org/10.1371/journal.pone.0322732.g003

We measured several markers of liver and renal function after 15 weeks of a high-fat diet, in both males and females. Despite a clear effect of diet on the levels of several markers, we found no differences among genotypes (Fig 4). Kidney function markers, such as the albumin to globulin ratio (A/G) and blood urea nitrogen to creatinine ratio (BUN/CRE), indicated that renal function was normal compared to levels in chow-fed mice. Hepatic enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) showed a significant increase in blood levels compared to lean mice, indicating an effect of the high-fat diet on hepatic physiology. However, bilirubin levels and gamma-glutamyl transferase (GGT) activity remained unchanged relative to controls, suggesting that liver damage was moderate. Under HFD, both genotypes accumulated triglycerides in the liver without significant differences.

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Fig 4. Renal and Hepatic Function in Male and Female Mice of Both Genotypes.

Graphs illustrating liver and kidney function data obtained from blood analysis of female (gray and purple) and male mice (black and yellow) following 15 weeks of high-fat diet (HFD) administration. The data represent the evaluation of renal and hepatic markers, including total bilirubin (BIL T), BUN/CRE, total protein (TP), albumin and globulin levels (ALB and GLOB), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alanine transaminase (ALT), albumin-to-globulin ratio (A/G), and non-fasting glucose (GLU). The dashed line shows biomarker levels from normal chow-fed mice. Results are shown for both genotypes. Data represent the mean ± standard deviation (SD) for each group. Statistical analysis was performed using a t-test.

https://doi.org/10.1371/journal.pone.0322732.g004

Histological analysis of liver samples confirmed the presence of steatosis, as expected in the diet-induced obesity (DIO) condition (Fig 5). Overall, Dbc1^LoxP/LoxP;CRE KO mice did not show significant differences in weight gain, glucose management, or liver and kidney function compared to controls under a high-fat diet treatment, indicating that adipocyte-specific Dbc1 deletion does not replicate the protective effects observed in whole-body Dbc1 knockout models.

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Fig 5. Hepatic Responses to Diet-induced Obesity

(A) Liver triglyceride content in both sexes and genotypes under Normal Diet and High-Fat Diet (HFD). Data represent mean ± standard deviation (SD). Statistical analysis was performed using two-way ANOVA. (B) Hematoxylin and eosin staining of liver sections from male mice under Normal Diet and High-Fat Diet. Scale Bar: 50 μm.

https://doi.org/10.1371/journal.pone.0322732.g005

RNA-seq analysis of isolated white adipocytes in WT and Dbc1^LoxP/LoxP;CRE KO obese mice

In an effort to determine the role Dbc1 is playing in mature adipocytes during obesity, we performed RNA-seq analysis of isolated adipocytes from Dbc1^LoxP/LoxP;CRE mice and their control littermates after 20 weeks of high-fat diet. Whole-transcriptome sequencing was conducted on adipocytes isolated from five Dbc1^LoxP/LoxP;CRE knockout mice and three control mice. We performed transcriptome analyses to identify differentially expressed genes (DEGs) and functional pathways using bioinformatics approaches. RNA-Seq revealed 60 DEGs with log₂FC | 0.58 | and p-value < 0.05 between control and Dbc1 KO adipocytes (Fig 6A). Among these, 42 genes were upregulated in Dbc1 KO adipocytes, while 18 genes were downregulated in control adipocytes, with a 1.5-fold change (Fig 6B). Overall, the data indicate that Dbc1 has a moderate influence on the transcriptional landscape of adipocytes during diet-induced obesity, potentially affecting their function. To explore the functions of the identified DEGs, we analyzed which pathways and cellular processes were enriched for each comparison using Enrichr (https://maayanlab.cloud/Enrichr/) and the Reactome 2022 database [24]. We found significant pathways enriched only for the upregulated DEGs, with the top three pathways being related to immune system response: Immune System, Cytokine Signaling in Immune System, and Signaling of Interleukins (Fig 6C). To surpass the constraints of over-representation analysis based on a limited number of DEGs, we employed Gene Set Enrichment Analysis (GSEA) involving the entire set of genes in the transcriptome. This is a well-known strategy that permits exploring the complete list of genes ranked based on the FC of expression between conditions [22]. We conducted this analysis for Gene Ontology terms using the ClusterProfiler algorithm. This approach identified significantly enriched Gene Ontology terms (Biological Process). According to the GO dataset, Dbc1 knockout (KO) adipocytes showed global upregulation in several inflammatory response pathways (Fig 6D). Particularly noticeable are the positive enriched terms related with cytokines production and Nf-κb transcriptional activity (Fig 6E).

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Fig 6. Transcriptomic data of mature adipocytes from Dbc1 KO vs WT mice

(A) Heatmap of differentially expressed protein coding genes (DEGs) with log₂FC | 0.58 | and p-value < 0.05. (B) Volcano plot highlighting upregulated (red) and downregulated (blue) genes. (C) Reactome-enriched pathways identified for upregulated DEGs. (D) Emapplot of the top 50 significantly enriched Gene Ontology terms (Biological Process), with increased terms in red and decreased terms in blue, in Dbc1 KO compared to Dbc1^LoxP/LoxP. (E) Gene Set Enrichment Analysis (GSEA) of selected Gene Ontology terms from (C). The x-axis represents the ranked genes based on log2 (Fold Change); the y-axis (upper panel) shows the Enrichment Score value, while the y-axis (bottom panel) represents the value log₂FC of the ranking metric.

https://doi.org/10.1371/journal.pone.0322732.g006

In addition, selected genes identified in the RNA-seq analysis were validated by qPCR, showing changes that were consistent to those observed in the transcriptomic data. Moreover, qPCR was performed to assess inflammatory markers such as Il1β and Tgfβ. Although these markers did not display significant differences in the RNA-seq analysis of isolated adipocytes, their expression in whole adipose tissue suggested a trend toward increased inflammation in the knockout mice. These qPCR data are shown in S5 Fig in F1 File. Globally, this analysis underscores the involvement of Dbc1 in regulating adipocyte inflammatory responses.