A Novel PPARG R212W Variant Causes Familial Partial Lipodystrophy Type 3: Clinical Presentation and Functional Characterization

TL;DR

A new genetic variant in PPARG causes a rare fat distribution disorder, leading to metabolic issues and potential treatment with rosiglitazone.

Contribution

Identifies a novel PPARG R212W variant causing FPLD3 and reveals a multi-faceted pathogenic mechanism involving protein instability and mitochondrial dysfunction.

Findings

The PPARG R212W variant shows partial loss of transcriptional activity and reduced protein stability.

The mutant PPARG variant causes mitochondrial dysfunction and downregulation of key metabolic genes in adipocytes.

Rosiglitazone partially rescues the functional deficits caused by the PPARG R212W variant.

Abstract

Familial partial lipodystrophy type 3 (FPLD3) is a rare autosomal dominant disorder caused by mutations in peroxisome proliferator-activated receptor gamma(PPARG), which encodes the key adipogenic transcription factor peroxisome proliferator-activated receptor gamma(PPARγ). Clinical diagnosis is challenging due to phenotypic overlap with common metabolic syndromes. We identified a novel PPARG variant in a Chinese family and performed comprehensive functional characterization to elucidate its pathogenic mechanism. The proband, a 15-year-old boy presenting with atypical fat distribution, severe insulin resistance, hypertriglyceridemia, and pancreatitis, underwent clinical evaluation and whole-exome sequencing. The identified variant was confirmed by Sanger sequencing. Its functional impact was assessed through in silico modeling, luciferase reporter assays, protein stability analysis (cycloheximide chase), and evaluation of mitochondrial function (JC-1 staining) and adipocyte gene expression in cellular models. A heterozygous PPARG c.634C>T (p.Arg212Trp, R212W) variant was identified and segregated with the phenotype. Functional studies revealed that the R212W mutant exhibits a partial loss of transcriptional activity (~40% of wild-type) while retaining ligand sensitivity. Crucially, we demonstrated that the mutant protein has significantly reduced stability due to accelerated degradation. In adipocyte models, R212W expression led to impaired mitochondrial membrane potential, depleted cellular ATP levels, and downregulated expression of key metabolic genes (glucose transporter 4[GLUT4], adiponectin[ADIPOQ], fatty acid binding protein 4[FABP4], lipoprotein lipase[LPL], perilipin 1[PLIN1]). These functional deficits were partially rescued by treatment with the PPARγ agonist rosiglitazone. We report a novel pathogenic PPARG R212W variant associated with FPLD3. Our data extend beyond a simple loss-of-function model by establishing a multi-faceted pathogenic mechanism involving protein destabilization, mitochondrial dysfunction, and cellular bioenergetic failure. The partial rescue by rosiglitazone suggests a potential therapeutic avenue. This study underscores the importance of integrating clinical phenotyping with deep functional analysis to diagnose and understand rare monogenic lipodystrophies.

Figures

Structural and Hydropathic Analysis of the PPARγ R212W Mutant Protein. (A,B) Predicted three-dimensional structures of the wild-type (WT, (A)) and mutant (B) PPARγ proteins. These structures highlight the global architectural differences introduced by the R212W mutation. (C,D) Close-up views of the local protein structure surrounding amino acid position 212. The substitution of arginine (in WT, (C)) with tryptophan (in mutant, (D)) leads to a distinct change in the side-chain conformation, which may affect protein–protein or protein–ligand interactions. (E,F) Hydropathy plots of the WT (E) and mutant (F) PPARγ proteins.

Transcriptional and Functional Profiling of WT and R212W Mutant PPARγ. (A) Transcriptional activity in COS7 cells transfected with WT, R212W mutant, or empty vector PPARG cDNA, followed by 24 h incubation with DMSO (left) or 10 μM rosiglitazone (Rosi). (B) PPARγ function after co-transfection of vector (control), WT, and R212W mutant plasmids. COS7 cells were treated with DMSO or rosiglitazone post-transfection. Mean ± SD from 3 experiments; no significant differences indicate the absence of dominant-negative effects. (C) Rosiglitazone dose–response (0.5, 5, 10, 20 μM) in COS7 cells transfected with WT, R212W mutant, or empty vector. (D,E) Immunoblot analysis of PPARγ protein expression. Whole-cell lysates from undifferentiated Chub-S7 transfected as indicated (D) or subjected to a cycloheximide (CHX) chase assay (E) were immunoblotted for PPARγ. GAPDH serves as a loading control. (D) The steady-state expression level of the R212W mutant is notably lower than that of the WT protein. (E) demonstrates the degradation kinetics following CHX treatment, showing a significantly shorter half-life for the R212W mutant. (* p < 0.05, p < 0.01, * p < 0.001, p < 0.0001).

Full text

1. Introduction

Monogenic forms of diabetes often present diagnostic challenges due to overlapping features with both type 1 and type 2 diabetes mellitus (T1DM and T2DM) [1]. Among these, familial partial lipodystrophy (FPLD) constitutes a distinctive syndromic subtype, characterized by selective loss of subcutaneous fat in the limbs and gluteal regions, frequently accompanied by severe insulin resistance and dyslipidemia [2,3,4].

Mutations in PPARG, encoding peroxisome proliferator-activated receptor gamma (PPARγ), underlie FPLD type 3 (FPLD3) [5,6]. FPLD3 is considered an extremely rare disorder, with precise prevalence difficult to determine but estimated to be in the range of a few per million, and only several dozen families reported worldwide to date [7,8]. Most reported PPARG variants are heterozygous missense mutations that impair the receptor’s transcriptional activity, leading to defective adipogenesis and systemic metabolic dysfunction [9,10]. Previous functional studies have established that pathogenic PPARG mutations can operate through diverse mechanisms, including simple haploinsufficiency, partial loss-of-function, or dominant-negative effects that disrupt the activity of the wild-type allele [11]. For instance, the well-characterized F388L mutation exerts a strong dominant-negative effect, while other mutations primarily reduce transcriptional activation without such interference.

The specific variant c.634C>T (p.Arg212Trp, R212W) investigated in our study has indeed been previously documented in patients with familial partial lipodystrophy. As summarized in a recent review [12], patients carrying the R212W variant typically present with lipoatrophy affecting the gluteal region and limbs, mild hypertriglyceridemia, diabetes, hypertension, and metabolic-dysfunction-associated fatty liver disease [12]. Functional studies have classified this variant as pathogenic, with evidence suggesting it exerts a dominant-negative effect [13]. However, the precise molecular mechanisms by which specific mutations cause disease—beyond simple loss of transactivation—remain incompletely understood. In particular, whether mutations affect protein stability, induce cellular organelle dysfunction (e.g., in mitochondria), or alter response to pharmacological ligands, and whether these defects can be functionally rescued, are critical questions that have not been systematically addressed for many rare variants. This mechanistic gap limits both prognostic insight and the development of targeted therapeutic strategies.

Here, we identify a novel PPARG missense variant (c.634C>T; p.Arg212Trp, R212W) in a 15-year-old proband presenting with classic FPLD3 features: early-onset diabetes with negative autoantibodies, severe hypertriglyceridemia, pancreatitis, and partial lipodystrophy with a maternal family history of diabetes. Moving beyond genetic diagnosis, we performed a comprehensive functional characterization of the R212W mutant. We demonstrate that this variant not only reduces PPARγ transcriptional activity but also significantly decreases protein stability by accelerating its degradation. Furthermore, we show that R212W disrupts mitochondrial membrane potential and cellular bioenergetics in adipocytes, leading to profound deficits in ATP production and the expression of key metabolic genes. Importantly, these defects are partially reversible upon treatment with the PPARγ agonist rosiglitazone. Our study thus elucidates a multi-layered pathogenic mechanism for a novel PPARG mutation and provides experimental support for the potential utility of PPARγ-targeted therapy in this genetic form of lipodystrophy-associated diabetes.

2. Results

2.1. Pedigree and Clinical Phenotype of the Proband

A two-generation pedigree was constructed based on the clinical and genetic evaluation of the proband and his family (Figure 1A). The proband (II-1), a 15-year-old male, had experienced persistent polydipsia, hyperphagia, polyuria, abdominal pain, and significant weight loss over the preceding two years. He was diagnosed with diabetes mellitus complicated by ketoacidosis at initial presentation.

Notable clinical features included abnormal fat distribution, characterized by peripheral lipoatrophy involving the limbs and gluteal regions, alongside central fat accumulation. Acanthosis nigricans was observed in the neck, axillae, and lumbosacral areas (Figure 1B,C). Urine testing revealed glucosuria and ketonuria. Arterial blood gas analysis showed metabolic acidosis (pH 7.29, bicarbonate 12 mmol/L, base excess −13 mmol/L). Laboratory findings also revealed marked hypertriglyceridemia (15.62 mmol/L), elevated total cholesterol (8.37–13.83 mmol/L), and elevated pancreatic enzymes (serum amylase 165 U/L, urine amylase 706 U/L). Glycemic control was poor, with HbA1c at 14.9%, plasma glucose at 18.3 mmol/L, low fasting insulin (4.69 µIU/mL), and reduced C-peptide (0.357 ng/mL). All diabetes-related autoantibodies tested negative.

Initial insulin therapy corrected ketoacidosis, but blood glucose remained unstable (6.5–15 mmol/L) during follow-up. Six months later, the patient developed yellowish papular skin lesions on the elbows and knees, which were confirmed by histopathology as fibroxanthomas (Figure 1B,C). At the time of re-evaluation, metabolic parameters remained significantly deranged: random glucose 20.85 mmol/L, HbA1c 14.3%, triglycerides 13.97 mmol/L, and total cholesterol 6.06 mmol/L. Insulin (29.6 µIU/mL) and C-peptide (1.31 ng/mL) levels had increased, but remained inadequate for glycemic compensation. Autoantibody testing remained negative. The proband’s mother (I-1) had a history of type 2 diabetes, raising the possibility of a hereditary metabolic disorder.

2.2. Genetic Mutation Identification and Validation

Given the clinical suspicion of a monogenic form of diabetes, WES was performed. A heterozygous missense variant in the PPARG gene (c.634C>T, p.R212W) was identified in the proband. Sanger sequencing confirmed that this variant was also present in his mother (I-1), but absent in his father (I-2), consistent with maternal inheritance (Figure 1A).

Sanger sequencing electropherograms demonstrated a heterozygous C>T substitution at nucleotide position 634 within exon 5 of the PPARG2 isoform (NM_015869), resulting in an arginine-to-tryptophan substitution at codon 212 (p.Arg212Trp, R212W) (Figure 2). Given the significant phenotypic overlap between FPLD3 and the LMNA-associated FPLD1, sequencing of the LMNA gene was performed, and no pathogenic mutations were detected [14]. The segregation pattern further supported a maternally inherited autosomal dominant variant.

2.3. Structural and Functional Implications of the PPARG R212W Mutation

To evaluate the potential structural impact of the R212W substitution, we conducted in silico modeling using AlphaFold2 (version 2.3.0) and PyMOL (version 2.2; Schrödinger, LLC) (Figure 3A–D). The mutation replaces a hydrophilic arginine residue with a bulky hydrophobic tryptophan, located in a region important for conformational stability. Comparison of the Kyte-Doolittle hydropathy profiles for the wild-type and mutant proteins showed that the overall curves are largely superimposable (Figure 3E,F), indicating that the mutation does not drastically alter the global hydrophobic character of the protein.

Functional prediction using the PROVEAN tool yielded a score of −7.019, well below the pathogenicity threshold of −2.5, indicating a likely deleterious effect. Collectively, these in silico analyses suggest that the R212W variant primarily causes a localized structural and physicochemical perturbation at the mutation site, which may compromise protein stability or function. This prediction formed the basis for our subsequent experimental investigations into the mutant’s protein stability and transcriptional activity.

2.4. Functional Analyses of PPARG R212W Mutation

To investigate the functional consequences of the R212W mutation on PPARγ-mediated transcriptional regulation, we performed luciferase reporter assays in 293T cells. Under basal conditions (DMSO-treated), cells expressing the R212W mutant exhibited significantly reduced transcriptional activity compared to wild-type (WT) PPARγ (Figure 4A). Although treatment with the PPARγ agonist rosiglitazone enhanced activity in both groups, the R212W mutant remained functionally impaired, suggesting a partial loss-of-function phenotype (Figure 4A). Co-transfection of WT and mutant plasmids in equal ratios did not significantly reduce transcriptional output compared to WT alone, excluding a dominant-negative interaction (Figure 4B). A rosiglitazone dose–response assay demonstrated preserved ligand sensitivity, but the maximal activity of the R212W mutant remained approximately 40% lower than WT (Figure 4C).

Structural and Hydropathic Analysis of the PPARγ R212W Mutant Protein. (A,B) Predicted three-dimensional structures of the wild-type (WT, (A)) and mutant (B) PPARγ proteins. These structures highlight the global architectural differences introduced by the R212W mutation. (C,D) Close-up views of the local protein structure surrounding amino acid position 212. The substitution of arginine (in WT, (C)) with tryptophan (in mutant, (D)) leads to a distinct change in the side-chain conformation, which may affect protein–protein or protein–ligand interactions. (E,F) Hydropathy plots of the WT (E) and mutant (F) PPARγ proteins.

Transcriptional and Functional Profiling of WT and R212W Mutant PPARγ. (A) Transcriptional activity in COS7 cells transfected with WT, R212W mutant, or empty vector PPARG cDNA, followed by 24 h incubation with DMSO (left) or 10 μM rosiglitazone (Rosi). (B) PPARγ function after co-transfection of vector (control), WT, and R212W mutant plasmids. COS7 cells were treated with DMSO or rosiglitazone post-transfection. Mean ± SD from 3 experiments; no significant differences indicate the absence of dominant-negative effects. (C) Rosiglitazone dose–response (0.5, 5, 10, 20 μM) in COS7 cells transfected with WT, R212W mutant, or empty vector. (D,E) Immunoblot analysis of PPARγ protein expression. Whole-cell lysates from undifferentiated Chub-S7 transfected as indicated (D) or subjected to a cycloheximide (CHX) chase assay (E) were immunoblotted for PPARγ. GAPDH serves as a loading control. (D) The steady-state expression level of the R212W mutant is notably lower than that of the WT protein. (E) demonstrates the degradation kinetics following CHX treatment, showing a significantly shorter half-life for the R212W mutant. ( p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).*

To explore the mechanism underlying the functional impairment of the R212W mutant, we first examined its protein expression level. Immunoblotting revealed a marked reduction in the steady-state level of the R212W mutant compared to the wild-type (WT) protein (Figure 4D), suggesting potential instability. To directly test this hypothesis, we performed a cycloheximide (CHX) chase assay. The results demonstrated that the R212W mutant had a significantly shorter half-life than WT (Figure 4E), indicating accelerated protein degradation. Thus, the loss-of-function phenotype observed in the R212W mutant can be attributed, at least in part, to reduced protein stability and abundance.

2.5. The R212W Mutation Induces Mitochondrial and Bioenergetic Dysfunction in Adipocytes

PPARγ is a master regulator of adipogenesis, and its activity is intrinsically linked to cellular energy metabolism. As adipocytes differentiate, they undergo extensive mitochondrial biogenesis to meet the high energetic demands of lipid storage and endocrine function. Given that PPARγ directly regulates the expression of key genes involved in mitochondrial function and biogenesis [15,16], we hypothesized that the R212W mutation would impair mitochondrial integrity and cellular bioenergetics.

To test this, we first assessed mitochondrial health. Analysis of mitochondrial membrane potential using JC-1 staining revealed a severe defect in R212W-expressing (MUT) adipocytes, as evidenced by a marked reduction in red fluorescence (J-aggregates) and a concomitant increase in green fluorescence (J-monomers) compared to wild-type (WT) cells (Figure 5A), indicating mitochondrial depolarization.

This mitochondrial dysfunction precipitated a profound bioenergetic crisis. Real-time monitoring of intracellular ATP levels showed that while WT cells maintained stable ATP content over 36 h, MUT cells exhibited a sharp and progressive decline, depleting nearly all measurable ATP by the end point (Figure 5B).

Consistent with this energy deficit and loss of PPARγ activity, the expression of a panel of key adipocyte-specific genes was broadly suppressed. Quantitative PCR analysis demonstrated significant downregulation in MUT cells of genes involved in glucose uptake (GLUT4), adipokine secretion (ADIPOQ), fatty acid binding (FABP4), lipid metabolism (LPL), and lipid droplet formation (PLIN1) (Figure 5C).

Importantly, the functional impairments induced by the R212W mutation were pharmacologically targetable. Treatment with the PPARγ agonist rosiglitazone (Rosi) robustly ameliorated these defects. Rosiglitazone treatment substantially attenuated the ATP depletion in MUT cells and restored the expression of all five adipogenic and metabolic genes. The rescued expression levels approached those observed in untreated WT cells (Figure 5C).

To directly visualize the impairment in adipogenesis, we performed Oil Red O staining on differentiated adipocytes. Consistent with the gene expression data, lipid droplet accumulation was substantially reduced in R212W-expressing (MUT) cells compared to wild-type (WT) cells (Figure 5D), confirming a defect in terminal adipogenic differentiation. Collectively, these data establish that the R212W mutation induces a partial loss of PPARγ function, leading to a cascade of mitochondrial dysfunction, bioenergetic failure, and impaired adipogenesis—as evidenced by reduced lipid droplet accumulation and suppression of key adipogenic genes—which can be significantly mitigated by receptor agonist treatment.

2.6. Clinical Follow-Up and Therapeutic Outcomes

Following the genetic diagnosis of FPLD3, the patient was placed on a comprehensive, pathophysiology-targeted treatment regimen. Initial glycemic control was achieved with a basal-bolus insulin regimen (insulin glargine 15 U/day and preprandial aspart 14 U/day), which resolved ketoacidosis but resulted in significant glucose fluctuations, as shown by continuous glucose monitoring (Figure 6). Metformin (0.25 g TID) was added on day 5, which improved glycemic stability and permitted gradual insulin dose reduction.

Subsequently, given the confirmed PPARG loss-of-function mutation, the PPARγ agonist rosiglitazone (4 mg/day) was introduced alongside metformin. This combination led to a marked improvement in insulin sensitivity and sustained glycemic stability, enabling the complete discontinuation of insulin therapy. The patient was ultimately maintained on dual oral therapy: metformin (0.5 g at breakfast, 0.25 g at lunch and dinner) and rosiglitazone (4 mg/day).

Concurrently, atorvastatin (10 mg/day) was prescribed for severe hypertriglyceridemia, which normalized during follow-up. Repeat abdominal CT showed resolution of prior pancreatic abnormalities. The patient and family were instructed to continue home glucose monitoring and return for regular evaluation of HbA1c and lipid profiles.

3. Discussion

In this study, we identified and characterized a novel heterozygous PPARG missense variant (c.634C>T; p.Arg212Trp, R212W) in a Chinese family with clinical features of FPLD3. Our functional analysis demonstrates that the R212W mutation compromises PPARγ function via several interconnected mechanisms: reduced protein stability, impaired mitochondrial integrity, and consequent cellular energy deficit. These defects jointly contribute to the observed metabolic dysregulation.

The proband’s clinical presentation—including partial lipodystrophy, severe insulin resistance, hypertriglyceridemia, and pancreatitis—aligns with the spectrum of PPARγ-related disorders [17,18,19]. This case further expands the mutational and phenotypic heterogeneity of FPLD3, reinforcing the necessity of genetic testing in atypical diabetes with lipodystrophic features [18,19,20,21,22,23,24,25].

Mechanistically, we demonstrate that the R212W variant causes a partial loss of transcriptional activity without dominant-negative interference, consistent with a hypomorphic allele [22,26]. However, moving beyond conventional reporter assays, we uncovered a previously unreported pathogenic dimension: the R212W mutant exhibits markedly reduced protein stability due to accelerated degradation. This finding provides a direct molecular explanation for the decreased steady-state protein levels observed in vitro and may contribute to the in vivo deficiency of functional PPARγ. This defect also extends to the function of cellular organelles. We found that the R212W variant impairs mitochondrial membrane potential, precipitating a severe bioenergetic crisis in adipocytes characterized by progressive ATP depletion. This mitochondrial dysfunction is likely responsible for the broad downregulation of key adipogenic and metabolic genes (GLUT4, ADIPOQ, FABP4, LPL, PLIN1), thereby linking impaired cellular energetics to defective adipose tissue function.

Therapeutically, our data offer a mechanistic rationale for considering PPARγ agonists in similar cases. While the mutant’s maximal transcriptional output is reduced, it retains ligand sensitivity. Critically, treatment with rosiglitazone not only partially restored transcriptional activity but also rescued gene expression deficits and improved cellular bioenergetics in R212W-expressing adipocytes. This in vitro rescue suggests that pharmacological activation of the residual functional pool of PPARγ may yield clinical benefit, aligning with observations from other hypomorphic variants [26,27]. In our patient, the subsequent introduction of rosiglitazone alongside metformin contributed to sustained glycemic stability, underscoring the potential of targeted therapy informed by functional characterization [27,28,29].

Several limitations warrant consideration. Our findings are derived from a single family, and broader cohort studies are needed to define penetrance and phenotypic variability. The functional studies, though comprehensive, were conducted in heterologous systems and immortalized preadipocytes. Future work employing patient-derived iPSC-differentiated adipocytes would provide a more physiologically relevant model [22,26,30]. Furthermore, aspects such as chromatin occupancy and interactions with coregulators in adipocytes remain to be explored [26,31]. Additionally, a future important direction will be to determine the precise degradation pathway responsible for the accelerated turnover of the R212W mutant, such as the ubiquitin–proteasome system or autophagy–lysosome pathway. Long-term clinical follow-up will be essential to fully evaluate the therapeutic response and natural history associated with this variant [24,28,29].

In conclusion, we have delineated a novel pathogenic pathway for a PPARG mutation in FPLD3, encompassing protein instability, mitochondrial dysfunction, and cellular energy deficit. This study underscores that the pathogenicity of PPARγ mutations can extend beyond simple transcriptional repression to involve fundamental cellular homeostatic processes. Integrating such multi-level functional insights with genetic diagnosis is crucial for understanding disease mechanisms and guiding personalized therapeutic strategies in monogenic lipodystrophies.

4. Materials and Methods

4.1. Subjects and Ethical Compliance

This study involved two generations of a Chinese family. The proband, a 15-year-old male, was referred to the Pediatric Outpatient Department of Tongji Hospital (Wuhan, China) with a clinical suspicion of type 2 diabetes mellitus and FPLD3. Detailed clinical information and medical history were obtained from all participating family members.

The study protocol was approved by the Ethics Committee of Tongji Hospital, affiliated with Tongji Medical College, Huazhong University of Science and Technology (approval number: TJ-IRB20201016). Written informed consent was obtained from all adult participants, and from the parents or legal guardians of individuals under the age of 18. All procedures conformed to the principles outlined in the Declaration of Helsinki.

4.2. DNA Sequencing & Variant Analysis

Genomic DNA extraction and exome sequencing: Peripheral blood samples were collected in EDTA-containing tubes, and genomic DNA was extracted for whole-exome sequencing (WES). Library preparation was conducted using the GenCap Whole Exons Capture Kit (MyGenostics, Beijing, China), and sequencing was performed on the DNBSEQ-T7 platform (MGI Tech, Shenzhen, China) with 150-bp paired-end reads. The target region included coding sequences and exon–intron boundaries of approximately 23,000 genes [32].

Bioinformatics analysis: Raw data were processed through a standard BWA-GATK pipeline for alignment, quality control, and variant calling. Functional annotation was performed using ANNOVAR (2024-05-01), incorporating multiple public databases. The pathogenicity of the candidate variant was evaluated using the PROVEAN tool (http://provean.jcvi.org/index.php) (accessed on 20 May 2024) [33]. Predicted protein structures were generated using AlphaFold2 [31] and visualized with Swiss-PDB Viewer (SPDBV, http://www.genebee.msu.su/spdbv/text/getpc.htm accessed on 20 May 2024).

Variant validation: Targeted Sanger sequencing of all coding exons of the PPARG gene (NM_015869) was conducted using PCR amplification followed by sequencing on an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). A restriction fragment length polymorphism (RFLP) assay using MnII was also employed to confirm the identified variant [33].

4.3. Plasmid Construction & Cell Transfection

Plasmid generation: Full-length human PPARG cDNAs (wild-type and c.634C>T mutant, p.R212W) were cloned into the pCMV-MCS-3 × Flag vector (Vigene Biosciences, Jinan, China). The integrity of all constructs was confirmed by PCR, double enzyme digestion, and Sanger sequencing to exclude off-target mutations.

Cell culture and transfection: HEK293T and Cos7 cells were cultured at 37 °C in a humidified 5% CO_2_ incubator using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; KeyGEN BioTECH, Nanjing, China). Cells were seeded in 6-well plates at 5 × 10^5^ cells per well and transfected with expression vectors using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions [34]. Cos7 cells were transfected with either the empty vector or the mutant PPARG plasmid for subsequent immunoblotting experiments [35,36].

4.4. Culture, Transfection, and Adipogenic Differentiation of Immortalized Preadipocytes

The immortalized human preadipocyte cell line Chub-S7 was cultured and maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO_2_ atmosphere.

For experiments, cells were seeded in 6-well plates. Upon reaching 70–80% confluence, cells were transfected with either the wild-type (WT) or R212W mutant (MUT) PPARγ expression plasmids using Lipofectamine 3000, according to the manufacturer’s instructions.

To induce adipogenic differentiation, 48 h post-transfection, the culture medium was replaced with adipogenic induction medium. The induction medium consisted of the growth medium supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone, 10 μg/mL insulin, and 1 μM rosiglitazone. After 3 days of induction, the medium was replaced with adipogenic maintenance medium the growth medium supplemented with 10 μg/mL insulin, which was refreshed every 2–3 days.

4.5. Reporter Gene Assay

To assess transcriptional activity, 293T cells were used in two experimental setups: (1) Basal and agonist-stimulated activity: Cells were co-transfected with 1 μg of pGL3-basic reporter plasmid containing the ACSL3 promoter (WZ Biosciences, Jinan, China), 0.1 μg of pPL-TK Renilla luciferase plasmid (internal control), and 1 μg of wild-type, mutant (PPARG R212W), or empty vector plasmids. After 24 h, cells were treated with either DMSO (vehicle) or rosiglitazone (0.5–20 μM; APExBIO Technology, Houston, TX, USA) for an additional 24 h [34,37]. (2) Co-expression assay: Cells were transfected with ① 2.4 μg of either empty vector or 1.2 μg of wild-type plasmid, or ② 1.2 μg of wild-type and 1.2 μg of mutant plasmids to examine potential dominant-negative interactions.

Luciferase detection: Dual-luciferase activity was measured using the Promega Luciferase Assay System (Promega, Madison, WI, USA). Firefly luciferase signals were normalized to Renilla luciferase to account for transfection efficiency. Relative transcriptional activity was expressed as fold-change compared to vehicle-treated wild-type controls. All experiments were independently repeated three times, and results were reported as mean ± standard deviation (SD).

4.6. Immunoblotting

At 24 h post-transfection, Cos7 cells were lysed in RIPA buffer supplemented with protease inhibitors. Total protein was quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (30 µg per lane) were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. After blocking in 5% non-fat milk in TBST, membranes were incubated overnight at 4 °C with primary antibody against PPARγ (Cell Signaling Technology, Danvers, MA, USA), followed by incubation with HRP-conjugated goat anti-rabbit IgG secondary antibody. Signal detection was performed using enhanced chemiluminescence (ECL; Thermo Fisher Scientific), and band intensity was quantified using ImageJ software 1.54p (NIH, Bethesda, MD, USA) [35,36].

4.7. Measurement of Mitochondrial Membrane Potential

The mitochondrial membrane potential (ΔΨm) was assessed using the lipophilic cationic dye JC-1 (Beyotime Biotechnology, China). In brief, differentiated adipocytes (WT and R212W mutant) were washed with PBS and incubated with JC-1 staining solution (5 µg/mL) at 37 °C for 20 min in the dark. After washing twice with JC-1 staining buffer, cells were immediately imaged using a fluorescence microscope (Nikon Eclipse Ti2) with appropriate filter sets: green fluorescence (excitation/emission: 514/529 nm) for JC-1 monomers and red fluorescence (excitation/emission: 585/590 nm) for JC-1 aggregates. For quantitative analysis, the integrated fluorescence intensity of red and green channels from at least five random fields per group was measured using ImageJ software, and the red/green fluorescence ratio was calculated.

4.8. Intracellular ATP Measurement

Intracellular ATP levels were monitored in real-time using a CellTiter-Glo^®^ Luminescent Cell Viability Assay kit (Promega, USA) according to the manufacturer’s protocol. WT and R212W mutant adipocytes were seeded in a white-walled 96-well plate at equal density. At the indicated time points (0, 2, 6, 12, 24, 36 h), an equal volume of CellTiter-Glo^®^ reagent was added to each well. The plate was incubated on an orbital shaker for 2 min to induce cell lysis, followed by a 10 min incubation at room temperature to stabilize the luminescent signal. Luminescence was recorded using a microplate reader (BioTek Synergy H1; Agilent Technologies, Santa Clara, CA, USA). ATP levels were normalized to the cell number at the 0 h time point and expressed as relative luminescence units (RLU). Three independent experiments were performed, each with six technical replicates.

4.9. RNA Extraction, Reverse Transcription, and Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from adipocytes using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. RNA concentration and purity were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). One microgram of total RNA was reverse-transcribed into cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan). Quantitative PCR was performed on a QuantStudio 6 Pro Real-Time PCR System (Applied Biosystems, USA) using TB Green Premix Ex Taq II (Takara, Japan). The reaction mixture (20 µL) contained 10 µL TB Green mix, 0.8 µL each of forward and reverse primers (10 µM), 2 µL cDNA template, and 6.4 µL nuclease-free water. The thermal cycling conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Gene expression was normalized to the endogenous control GAPDH, and the relative expression level was calculated using the 2−ΔΔCt method. Primer sequences used in this study are listed in Table 1. Experiments were conducted in triplicate and repeated at least three times independently.

4.10. Statistical Analysis

Statistical analyses were conducted using SPSS version 21.0 (IBM Corp., Armonk, NY, USA). Continuous variables were summarized as mean ± SD. Between-group comparisons were made using Student’s t-test. A two-tailed p value < 0.05 was considered statistically significant. All in vitro experiments were performed in triplicate unless otherwise noted.

5. Conclusions

In conclusion, we have identified a novel pathogenic PPARG variant (p.R212W) in a Chinese family with FPLD3. Our study moves beyond genetic association by providing a comprehensive functional characterization, revealing that the mutation causes disease through a multi-faceted mechanism involving protein destabilization, mitochondrial dysfunction, and bioenergetic failure in adipocytes. Importantly, we demonstrate that these defects are amenable to pharmacological rescue with rosiglitazone, providing a mechanistic rationale for PPARγ agonist therapy in similar cases. Our findings underscore the importance of integrating functional studies with genetic diagnosis to unravel disease pathophysiology and guide personalized treatment in monogenic lipodystrophies and diabetes.