Examination of the Complex Molecular Landscape in Obesity and Type 2 Diabetes.

The escalating prevalence of metabolic disorders, notably type 2 diabetes (T2D) and obesity, presents a critical global health challenge, necessitating deeper insights into their molecular underpinnings. Our study integrates proteomics and metabolomics analyses to delineate the complex molecular landscapes associated with T2D and obesity. Leveraging data from 130 subjects, including individuals with T2D and obesity as well as healthy controls, we elucidate distinct molecular signatures and identify novel biomarkers indicative of disease progression. Our comprehensive characterization of cardiometabolic proteins and serum metabolites unveils intricate networks of biomolecular interactions and highlights differential protein expression patterns between T2D and obesity cohorts. Pathway enrichment analyses reveal unique mechanisms underlying disease development and progression, while correlation analyses elucidate the interplay between proteomics, metabolomics, and clinical parameters. Furthermore, network analyses underscore the interconnectedness of cardiometabolic proteins and provide insights into their roles in disease pathogenesis. Our findings may help to refine diagnostic strategies and inform the development of personalized interventions, heralding a new era in precision medicine and healthcare innovation. Through the integration of multi-omics approaches and advanced analytics, our study offers a crucial framework for deciphering the intricate molecular underpinnings of metabolic disorders and paving the way for transformative therapeutic strategies.

Salivary Chemical Barrier Proteins in Oral Squamous Cell Carcinoma-Alterations in the Defense Mechanism of the Oral Cavity.

Oral squamous cell carcinoma (OSCC) is one of the most frequent types of head and neck cancer. Despite the genetic and environmental risk factors, OSCC is also associated with microbial infections and/or dysbiosis. The secreted saliva serves as the chemical barrier of the oral cavity and, since OSCC can alter the protein composition of saliva, our aim was to analyze the effect of OSCC on the salivary chemical barrier proteins. Publicly available datasets regarding the analysis of salivary proteins from patients with OSCC and controls were collected and examined in order to identify differentially expressed chemical barrier proteins. Network analysis and gene ontology (GO) classification of the differentially expressed chemical barrier proteins were performed as well. One hundred and twenty-seven proteins showing different expression pattern between the OSCC and control groups were found. Protein-protein interaction networks of up- and down-regulated proteins were constructed and analyzed. The main hub proteins (IL-6, IL-1B, IL-8, TNF, APOA1, APOA2, APOB, APOC3, APOE, and HP) were identified and the enriched GO terms were examined. Our study highlighted the importance of the chemical barrier of saliva in the development of OSCC.

Corrigendum: Human abdominal subcutaneous-derived active beige adipocytes carrying FTO rs1421085 obesity-risk alleles exert lower thermogenic capacity.

[This corrects the article DOI: 10.3389/fcell.2023.1155673.].

Comparative Analysis of Amino Acid and Biogenic Amine Compositions of Fermented Grape Beverages.

Amino acids and biogenic amines are important components of food and beverages. In grape-derived products such as wine and wine vinegar, they can have different origins and can influence the odor and taste of the products. Their concentration is influenced by the grape variety, vintage, and winemaking process. In our study, we carried out an LC-MS-based comparative analysis of 22 grape-derived beverages, including three different wine types and four wine vinegar samples from the Tokaj region in Hungary. The concentrations of 23 amino acids and 10 biogenic amines were examined, and the differences among the sample types were analyzed. The differences in the concentrations of some metabolites between Aszú-Furmint pairs originating from the same wineries and year provide information on the effect of botrytized grape on wine composition. Our data can provide further evidence on how the production process shapes the metabolite content of beverages and highlight the nutritional value of wine vinegar.

Cross-Linking Mass Spectrometry on P-Glycoprotein.

The ABC transporter P-glycoprotein (Pgp) has been found to be involved in multidrug resistance in tumor cells. Lipids and cholesterol have a pivotal role in Pgp's conformations; however, it is often difficult to investigate it with conventional structural biology techniques. Here, we applied robust approaches coupled with cross-linking mass spectrometry (XL-MS), where the natural lipid environment remains quasi-intact. Two experimental approaches were carried out using different cross-linkers (i) on living cells, followed by membrane preparation and immunoprecipitation enrichment of Pgp, and (ii) on-bead, subsequent to membrane preparation and immunoprecipitation. Pgp-containing complexes were enriched employing extracellular monoclonal anti-Pgp antibodies on magnetic beads, followed by on-bead enzymatic digestion. The LC-MS/MS results revealed mono-links on Pgp's solvent-accessible residues, while intraprotein cross-links confirmed a complex interplay between extracellular, transmembrane, and intracellular segments of the protein, of which several have been reported to be connected to cholesterol. Harnessing the MS results and those of molecular docking, we suggest an epitope for the 15D3 cholesterol-dependent mouse monoclonal antibody. Additionally, enriched neighbors of Pgp prove the strong connection of Pgp to the cytoskeleton and other cholesterol-regulated proteins. These findings suggest that XL-MS may be utilized for protein structure and network analyses in such convoluted systems as membrane proteins.

Human abdominal subcutaneous-derived active beige adipocytes carrying FTO rs1421085 obesity-risk alleles exert lower thermogenic capacity.

Introduction: White adipocytes store lipids, have a large lipid droplet and few mitochondria. Brown and beige adipocytes, which produce heat, are characterized by high expression of uncoupling protein (UCP) 1, multilocular lipid droplets, and large amounts of mitochondria. The rs1421085 T-to-C single-nucleotide polymorphism (SNP) of the human FTO gene interrupts a conserved motif for ARID5B repressor, resulting in adipocyte type shift from beige to white. Methods: We obtained abdominal subcutaneous adipose tissue from donors carrying FTO rs1421085 TT (risk-free) or CC (obesity-risk) genotypes, isolated and differentiated their preadipocytes into beige adipocytes (driven by the PPARγ agonist rosiglitazone for 14 days), and activated them with dibutyryl-cAMP for 4 hours. Then, either the same culture conditions were applied for additional 14 days (active beige adipocytes) or it was replaced by a white differentiation medium (inactive beige adipocytes). White adipocytes were differentiated by their medium for 28 days. Results and Discussion: RNA-sequencing was performed to investigate the gene expression pattern of adipocytes carrying different FTO alleles and found that active beige adipocytes had higher brown adipocyte content and browning capacity compared to white or inactive beige ones when the cells were obtained from risk-free TT but not from obesity-risk CC genotype carriers. Active beige adipocytes carrying FTO CC had lower thermogenic gene (e.g., UCP1, PM20D1, CIDEA) expression and thermogenesis measured by proton leak respiration as compared to TT carriers. In addition, active beige adipocytes with CC alleles exerted lower expression of ASC-1 neutral amino acid transporter (encoded by SLC7A10) and less consumption of Ala, Ser, Cys, and Gly as compared to risk-free carriers. We did not observe any influence of the FTO rs1421085 SNP on white and inactive beige adipocytes highlighting its exclusive and critical effect when adipocytes were activated for thermogenesis.

Availability of abundant thiamine determines efficiency of thermogenic activation in human neck area derived adipocytes.

Brown/beige adipocytes express uncoupling protein-1 (UCP1) that enables them to dissipate energy as heat. Systematic activation of this process can alleviate obesity. Human brown adipose tissues are interspersed in distinct anatomical regions including deep neck. We found that UCP1 enriched adipocytes differentiated from precursors of this depot highly expressed ThTr2 transporter of thiamine and consumed thiamine during thermogenic activation of these adipocytes by cAMP which mimics adrenergic stimulation. Inhibition of ThTr2 led to lower thiamine consumption with decreased proton leak respiration reflecting reduced uncoupling. In the absence of thiamine, cAMP-induced uncoupling was diminished but restored by thiamine addition reaching the highest levels at thiamine concentrations larger than present in human blood plasma. Thiamine is converted to thiamine pyrophosphate (TPP) in cells; the addition of TPP to permeabilized adipocytes increased uncoupling fueled by TPP-dependent pyruvate dehydrogenase. ThTr2 inhibition also hampered cAMP-dependent induction of UCP1, PGC1a, and other browning marker genes, and thermogenic induction of these genes was potentiated by thiamine in a concentration-dependent manner. Our study reveals the importance of amply supplied thiamine during thermogenic activation in human adipocytes which provides TPP for TPP-dependent enzymes not fully saturated with this cofactor and by potentiating the induction of thermogenic genes.

Hypercitrullination and anti-citrullinated protein antibodies in chronic apical periodontitis, a laboratory investigation. Does autoimmunity contribute to the pathogenesis?

AIM: The presence of Gram-negative anaerobic bacteria, in particular, Porphyromonas gingivalis (P. gingivalis) in periapical granulomas predicts the generation of citrullinated proteins in the lesion. Citrullination of proteins may lead to the formation of anti-citrullinated autoantibodies (ACPA-s) initiating the formation of an autoimmune loop which may contribute to the perpetuation of inflammatory reactions and tissue damage in chronic apical periodontitis. The objective of this study was to demonstrate the formation of citrullinated proteins in chronic apical periodontitis and whether they can act as autoantigens. METHODOLOGY: Twenty-five periapical granulomas (n = 25) were investigated in the study. Healthy periodontal tissue samples served as normal control tissue (n = 6). The peptidyl-citrulline level was determined with the dot blot method. ACPA levels were analysed using anti-citrullinated cyclic peptide (anti-CCP) EDIA kit. Differences between periapical granuloma and control samples were assessed using Mann-Whitney U tests. p Values <.05 were considered as statistically significant. RESULTS: Protein concentrations, peptidyl-citrulline levels and anti-CCP ratios were compared between periapical granuloma and healthy control groups. Multiple linear regression analysis revealed significant (p = .042) hypercitrullination in periapical granuloma samples. Moreover, there was a significant difference in the ACPA ratios between periapical granuloma (2.03 ± 0.30) and healthy control (0.63 ± 0.17) groups (p = .01). Seventeen of 25 periapical granuloma samples (17/25; 68%), whereas one out of six control samples (1/6; 17%) were shown to be positive for the presence of ACPA. CONCLUSIONS: This is the first study detecting the presence of citrullinated peptides and APCA in periapical granuloma, suggesting the contribution of autoimmune reactions in the pathogenesis and perpetuation of chronic apical periodontitis.

Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico Cleavage Site Prediction.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in its life cycle. The Mpro-mediated limited proteolysis of the viral polyproteins is necessary for the replication of the virus, and cleavage of the host proteins of the infected cells may also contribute to viral pathogenesis, such as evading the immune responses or triggering cell toxicity. Therefore, the identification of host substrates of the viral protease is of special interest. To identify cleavage sites in cellular substrates of SARS-CoV-2 Mpro, we determined changes in the HEK293T cellular proteome upon expression of the Mpro using two-dimensional gel electrophoresis. The candidate cellular substrates of Mpro were identified by mass spectrometry, and then potential cleavage sites were predicted in silico using NetCorona 1.0 and 3CLP web servers. The existence of the predicted cleavage sites was investigated by in vitro cleavage reactions using recombinant protein substrates containing the candidate target sequences, followed by the determination of cleavage positions using mass spectrometry. Unknown and previously described SARS-CoV-2 Mpro cleavage sites and cellular substrates were also identified. Identification of target sequences is important to understand the specificity of the enzyme, as well as aiding the improvement and development of computational methods for cleavage site prediction.

LPS-induced acute neuroinflammation, involving interleukin-1 beta signaling, leads to proteomic, cellular, and network-level changes in the prefrontal cortex of mice.

Neuroinflammation induced by peripheral infections leads to various neuropsychiatric symptoms both in humans and laboratory animals, e.g., to the manifestation of sickness behavior that resembles some features of clinical depression. However, in addition to depression-like behavior, there are other symptoms of acute systemic inflammation that can be associated with the impairment of prefrontal cortex (PFC)-regulated cognitive functions. Thus, we investigated the electrophysiological and proteomic alterations of the PFC using brain slices and the lipopolysaccharide (LPS) model of acute peripheral infection in male mice. Based on the gene expression differences of the coreceptor (Il1rap) of interleukin-1 beta (IL-1ß) between neuron types in our previous single-cell sequencing dataset, we first compared the electrophysiological effects of IL-1ß on PFC pyramidal cells and interneurons. We found that pyramidal cells are more responsive to IL-1ß, as could be presumed from our transcriptomic data. To examine the possible circuit-level correlates of the cellular changes, frontal electroencephalographic (EEG) activity and fronto-occipital functional connectivity were analyzed in LPS-treated mice and significant changes were found in the fronto-occipital EEG correlation and coherence in the delta and high-gamma frequency bands. The upregulation of the prefrontal IL-1 system (IL-1ß and its receptor) after LPS treatment was revealed by immunoassays simultaneously with the observed EEG changes. Furthermore, we investigated the LPS-induced alterations of the synaptic proteome in the PFC using 2-D differential gel electrophoresis and mass spectrometry and found 48 altered proteins mainly related to cellular signaling, cytoskeletal organization, and carbohydrate/energy metabolism. Thus, our results indicate remarkable electrophysiological and molecular changes in the PFC related to acute systemic inflammation that may explain some of the concomitant behavioral and physiological symptoms.

Comparative Analysis of Differential Cellular Transcriptome and Proteome Regulation by HIV-1 and HIV-2 Pseudovirions in the Early Phase of Infection.

In spite of the similar structural and genomic organization of human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2), striking differences exist between them in terms of replication dynamics and clinical manifestation of infection. Although the pathomechanism of HIV-1 infection is well characterized, relatively few data are available regarding HIV-2 viral replication and its interaction with host-cell proteins during the early phase of infection. We utilized proteo-transcriptomic analyses to determine differential genome expression and proteomic changes induced by transduction with HIV-1/2 pseudovirions during 8, 12 and 26 h time-points in HEK-293T cells. We show that alteration in the cellular milieu was indeed different between the two pseudovirions. The significantly higher number of genes altered by HIV-2 in the first two time-points suggests a more diverse yet subtle effect on the host cell, preparing the infected cell for integration and latency. On the other hand, GO analysis showed that, while HIV-1 induced cellular oxidative stress and had a greater effect on cellular metabolism, HIV-2 mostly affected genes involved in cell adhesion, extracellular matrix organization or cellular differentiation. Proteomics analysis revealed that HIV-2 significantly downregulated the expression of proteins involved in mRNA processing and translation. Meanwhile, HIV-1 influenced the cellular level of translation initiation factors and chaperones. Our study provides insight into the understudied replication cycle of HIV-2 and enriches our knowledge about the use of HIV-based lentiviral vectors in general.

Extensive proteome and functional genomic profiling of variability between genetically identical human B-lymphoblastoid cells.

In life-science research isogenic B-lymphoblastoid cell lines (LCLs) are widely known and preferred for their genetic stability - they are often used for studying mutations for example, where genetic stability is crucial. We have shown previously that phenotypic variability can be observed in isogenic B-lymphoblastoid cell lines. Isogenic LCLs present well-defined phenotypic differences on various levels, for example on the gene expression level or the chromatin level. Based on our investigations, the phenotypic variability of the isogenic LCLs is accompanied by certain genetic variation too. We have developed a compendium of LCL datasets that present the phenotypic and genetic variability of five isogenic LCLs from a multiomic perspective. In this paper, we present additional datasets generated with Next Generation Sequencing techniques to provide genomic and transcriptomic profiles (WGS, RNA-seq, single cell RNA-seq), protein-DNA interactions (ChIP-seq), together with mass spectrometry and flow cytometry datasets to monitor the changes in the proteome. We are sharing these datasets with the scientific community according to the FAIR principles for further investigations.

Chemical Barrier Proteins in Human Body Fluids.

Chemical barriers are composed of those sites of the human body where potential pathogens can contact the host cells. A chemical barrier is made up by different proteins that are part of the antimicrobial and immunomodulatory protein/peptide (AMP) family. Proteins of the AMP family exert antibacterial, antiviral, and/or antifungal activity and can modulate the immune system. Besides these proteins, a wide range of proteases and protease inhibitors can also be found in the chemical barriers maintaining a proteolytic balance in the host and/or the pathogens. In this review, we aimed to identify the chemical barrier components in nine human body fluids. The interaction networks of the chemical barrier proteins in each examined body fluid were generated as well.

Relationship between Plasma Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Level and Proteome Profile of Cows.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic and multifunctional neuropeptide; it takes part in the regulation of various physiological processes, such as feeding, reproduction, catecholamine synthesis, thermoregulation, motor activity, brain development and neuronal survival. Since PACAP plays important regulatory roles, we hypothesized that the level of PACAP in blood is associated with expression of other proteins, which are involved in different metabolic pathways. The objective of the present study was to compare plasma protein profiles of cows with high and low plasma PACAP levels. Differential proteome analyses were performed by two-dimensional gel electrophoresis (2D-PAGE) followed by tryptic digestion and protein identification by liquid chromatography−mass spectrometry (LC-MS). A total of 210 protein spots were detected, and 16 protein spots showed statistically significant differences (p < 0.05) in the expression levels between groups. Ten spots showed a higher intensity in the high-PACAP-concentration group, while six spots were more abundant in the low-PACAP-concentration group. The functions of the differentially expressed proteins indicate that the PACAP level of plasma is related to the lipid metabolism and immune status of cattle.

Tissue Transglutaminase Knock-Out Preadipocytes and Beige Cells of Epididymal Fat Origin Possess Decreased Mitochondrial Functions Required for Thermogenesis.

Beige adipocytes with thermogenic function are activated during cold exposure in white adipose tissue through the process of browning. These cells, similar to brown adipocytes, dissipate stored chemical energy in the form of heat with the help of uncoupling protein 1 (UCP1). Recently, we have shown that tissue transglutaminase (TG2) knock-out mice have decreased cold tolerance in parallel with lower utilization of their epididymal adipose tissue and reduced browning. To learn more about the thermogenic function of this fat depot, we isolated preadipocytes from the epididymal adipose tissue of wild-type and TG2 knock-out mice and differentiated them in the beige direction. Although differentiation of TG2 knock-out preadipocytes is phenotypically similar to the wild-type cells, the mitochondria of the knock-out beige cells have multiple impairments including an altered electron transport system generating lower electrochemical potential difference, reduced oxygen consumption, lower UCP1 protein content, and a higher portion of fragmented mitochondria. Most of these differences are present in preadipocytes as well, and the differentiation process cannot overcome the functional disadvantages completely. TG2 knock-out beige adipocytes produce more iodothyronine deiodinase 3 (DIO3) which may inactivate thyroid hormones required for the establishment of optimal mitochondrial function. The TG2 knock-out preadipocytes and beige cells are both hypometabolic as compared with the wild-type controls which may also be explained by the lower expression of solute carrier proteins SLC25A45, SLC25A47, and SLC25A42 which transport acylcarnitine, Co-A, and amino acids into the mitochondrial matrix. As a consequence, the mitochondria in TG2 knock-out beige adipocytes probably cannot reach the energy-producing threshold required for normal thermogenic functions, which may contribute to the decreased cold tolerance of TG2 knock-out mice.

Metabolomic Analysis of Serum and Tear Samples from Patients with Obesity and Type 2 Diabetes Mellitus.

Metabolomics strategies are widely used to examine obesity and type 2 diabetes (T2D). Patients with obesity (n = 31) or T2D (n = 26) and sex- and age-matched controls (n = 28) were recruited, and serum and tear samples were collected. The concentration of 23 amino acids and 10 biogenic amines in serum and tear samples was analyzed. Statistical analysis and Pearson correlation analysis along with network analysis were carried out. Compared to controls, changes in the level of 6 analytes in the obese group and of 10 analytes in the T2D group were statistically significant. For obesity, the energy generation, while for T2D, the involvement of NO synthesis and its relation to insulin signaling and inflammation, were characteristic. We found that BCAA and glutamine metabolism, urea cycle, and beta-oxidation make up crucial parts of the metabolic changes in T2D. According to our data, the retromer-mediated retrograde transport, the ethanolamine metabolism, and, consequently, the endocannabinoid signaling and phospholipid metabolism were characteristic of both conditions and can be relevant pathways to understanding and treating insulin resistance. By providing potential therapeutic targets and new starting points for mechanistic studies, our results emphasize the importance of complex data analysis procedures to better understand the pathomechanism of obesity and diabetes.

Fast and Sensitive Quantification of AccQ-Tag Derivatized Amino Acids and Biogenic Amines by UHPLC-UV Analysis from Complex Biological Samples.

Metabolomic analysis of different body fluids bears high importance in medical sciences. Our aim was to develop and validate a fast UHPLC-UV method for the analysis of 33 amino acids and biogenic amines from complex biological samples. AccQ-Tag derivatization was conducted on target molecules and the derivatized targets were analyzed by UHPLC-UV. The detection of the analytes was carried out with UV analysis and by Selected Reaction Monitoring (SRM)-based targeted mass spectrometry. The method was validated according to the FDA guidelines. Serum and non-stimulated tear samples were collected from five healthy individuals and the samples were analyzed by the method. The method was successfully validated with appropriate accuracy and precision for all 33 biomolecules. A total of 29 analytes were detected in serum samples and 26 of them were quantified. In the tears, 30 amino acids and biogenic amines were identified and 20 of them were quantified. The developed and validated UHPLC-UV method enables the fast and precise analysis of amino acids and biogenic amines from complex biological samples.

Cellular Proteo-Transcriptomic Changes in the Immediate Early-Phase of Lentiviral Transduction.

Lentivirus-based vectors derived from human immunodeficiency viruses type 1 and 2 (HIV-1 and 2) are widely used tools in research and may also be utilized in clinical settings. Like their parental virions, they are known to depend on the cellular machinery for successful gene delivery and integration. While most of the studies on cellular proteomic and transcriptomic changes have focused on the late phase of the transduction, studies of those changes in early time-points, especially in the case of HIV-2 based vectors, are widely lacking. Using second generation HIV-1 and 2 vesicular stomatitis virus G protein (VSV-G) pseudotyped lentiviral vectors, we transduced HEK-293T human embryonic kidney cells and carried out transcriptomic profiling at 0 and 2 h time points, with accompanying proteomic analysis at 2 h following transduction. Significant variations were observed in gene expression profile between HIV-1 and HIV-2 transduced samples. Thrombospondin 1 (THBS1), collagens (COL1A2, COL3A1), and eukaryotic translation factors (EIF3CL) in addition to various genes coding for long non-coding RNA (lncRNA) were significantly upregulated 2 h after HIV-2 transduction compared to HIV-1. Label-free quantification mass spectrometry (MS) indicated that seven proteins involved in RNA binding, mRNA transport, and chaperoning were significantly downregulated. The identification of cellular protein targets of lentiviral vectors and their effect on the cellular transcriptome will undoubtedly shed more light on their complex life cycle and may be utilized against infection by their parental lentiviruses. Furthermore, characterizing the early phase of HIV-2 infection may aid in the understanding of its pathomechanism and long incubation period.

Reduced Level of Tear Antimicrobial and Immunomodulatory Proteins as a Possible Reason for Higher Ocular Infections in Diabetic Patients.

(1) Background: Diabetes mellitus is one of the most common metabolic disorders and a risk factor for bacterial ocular infections. Our aim was to examine the antibacterial activity of tears from patients with diabetes mellitus with and without diabetic retinopathy and to link this activity to the level of tear proteins. (2) Methods: Non-stimulated basal tears were collected from 39 eyes of 35 subjects. The antibacterial activity of tear pools was tested against pathogenic Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 26922 and Pseudomonas aeruginosa ATCC 27853 strains. The levels of 10 antimicrobial and immunomodulatory proteins were analyzed in the individual tear samples of the studied groups by SRM-based targeted mass spectrometry analysis. (3) Results: Disease stage-specific antimicrobial effect was observed in case of Staphylococcus aureus ATCC 29213 strain, and a non-disease specific inhibitory effect was observed in case of Pseudomonas aeruginosa ATCC 27853 strain. Changes in the levels of the studied antimicrobial and immunomodulatory proteins in the tears of the studied groups were also observed. (4) Conclusions: The higher ocular infection rate observed in diabetic patients may be the consequence of the decreased antimicrobial activity of tears possibly caused by the changes in the levels of antimicrobial and immunomodulatory proteins.

ASC-1 transporter-dependent amino acid uptake is required for the efficient thermogenic response of human adipocytes to adrenergic stimulation.

Brown and beige adipocytes dissipate energy by uncoupling protein 1 (UCP1)-dependent and UCP1-independent thermogenesis, which may be utilized to develop treatments against obesity. We have found that mRNA and protein expression of the alanine/serine/cysteine transporter-1 (ASC-1) was induced during adipocyte differentiation of human brown-prone deep neck and beige-competent subcutaneous neck progenitors, and SGBS preadipocytes. cAMP stimulation of differentiated adipocytes led to elevated uptake of serine, cysteine, and glycine, in parallel with increased oxygen consumption, augmented UCP1-dependent proton leak, increased creatine-driven substrate cycle-coupled respiration, and upregulation of thermogenesis marker genes and several respiratory complex subunits; these outcomes were impeded in the presence of the specific ASC-1 inhibitor, BMS-466442. Our data suggest that ASC-1-dependent consumption of serine, cysteine, and glycine is required for efficient thermogenic stimulation of human adipocytes.