ABSTRACT Recent studies have focused on identifying novel genes involved in the browning process of inguinal white adipose tissue (iWAT). In this context, we propose that the mitochondrial

ATPase gene lactation elevated 1 (Lace1) utilizes lactate to regulate the browning capacity of iWAT, specifically in response to challenge with CL-316,243 (CL), a beta3-adrenergic receptor

(β3-AR) agonist. The mice were injected with CL over a span of 3 days and exposed to cold temperatures (4–6 °C) for 1 week. The results revealed a significant increase in Lace1 expression

levels during beige adipogenesis. Additionally, a strong positive correlation was observed between _Lace1_ and _Ucp1_ mRNA expression in iWAT under browning stimulation. To further explore

browning in iWAT under CL challenge compared with those of the controls. These unique phenomena stem from increased lactate release due to the inactivation of pyruvate dehydrogenase (PDH) in

the hearts of Lace1 KO mice. SIMILAR CONTENT BEING VIEWED BY OTHERS _FAS_ MUTATION REDUCES OBESITY BY INCREASING IL-4 AND IL-10 EXPRESSION AND PROMOTING WHITE ADIPOSE TISSUE BROWNING

Article Open access 20 July 2020 _EGR1_ LOSS-OF-FUNCTION PROMOTES BEIGE ADIPOCYTE DIFFERENTIATION AND ACTIVATION SPECIFICALLY IN INGUINAL SUBCUTANEOUS WHITE ADIPOSE TISSUE Article Open

access 28 September 2020 PERILIPIN 5 LINKS MITOCHONDRIAL UNCOUPLED RESPIRATION IN BROWN FAT TO HEALTHY WHITE FAT REMODELING AND SYSTEMIC GLUCOSE TOLERANCE Article Open access 03 June 2021

INTRODUCTION Stimuli such as cold exposure, treatment with β3-adrenergic receptor (β3-AR) agonizts such as CL-316,243 (CL), and exercise can induce the conversion of inguinal white adipose

tissue (iWAT) into beige adipose tissue1. This phenomenon is referred to as the “browning” of white adipocytes2. Beige adipocytes are similar to brown adipocytes and present characteristics

such as multilocular lipid droplets and abundant mitochondria. Their primary role involves the dissipation of energy to generate heat. Therefore, the browning of iWAT is recognized as an

important mechanism in obesity research. CL, an agonist of β3-AR, stimulates β3-AR by binding to its G protein α-subunit. This activation initiates the protein kinase A (PKA) pathway within

adipocytes by triggering adenylate cyclase and cyclic AMP (cAMP) production3. Consequently, this process induces lipolysis, which converts triacylglycerol (TG) into fatty acids (FAs) via the

phosphorylation of hormone-sensitive lipase (HSL) in adipocytes. The generated free FAs then activate the induction of uncoupling protein 1 (Ucp1) in mitochondria, which is the thermogenic

pathway4. UCP1 is a mitochondrial protein and a key gene that is upregulated during the browning process5. Recent studies have revealed novel forms of Ucp1-independent thermogenesis, but

these processes have been poorly explored6,7,8. These current research trends have ignited the quest to identify new genes involved in the browning process. Hence, we conducted an RNA-seq

analysis of iWAT following CL treatment. The aim of this study was to assess the genes whose expression is upregulated in beige adipocytes compared with white adipocytes. From the pool of

292 genes whose expression was upregulated in the CL-injected group, 97 mitochondrial genes were selected for further investigation. Within this subset, we identified 11 genes that were

enriched in BAT, leveraging the BioGPS database. Among these candidates, we chose to focus on lactation elevated 1 (Lace1), a gene with an as-yet-undetermined function and role, for our

study. Lace1 is a human homolog of the yeast mitochondrial ATPase family gene 1 (Afg1), a member of the SEC18-NSF, PAS1, CDC48-VCP, and TBP families9. ATPases are categorized into F-ATPase,

P-ATPase, and V-ATPase, of which Lace1 is classified as an F0/F1 ATPase10. F0/F1 ATP synthase is an enzyme localized in the inner membrane of mitochondria, where it catalyzes the synthesis

of ATP from ADP11. Recent studies have reported that ATPases regulate thermogenesis in a Ucp1-dependent or Ucp1-independent manner12,13. Lace1 is also a mitochondrial integral membrane

protein that controls mitochondrial protein homeostasis9. Cesnekova et al. reported that Lace1 mediates the degradation of the nuclear-encoded complex IV subunits cytochrome c oxidase 4

(COX4), COX5A, and COX6A and is required for the normal activity of complexes III and IV of the respiratory chain9. Consequently, Lace1, identified as a mitochondrial gene, has increased

expression due to CL or cold exposure. These observations strongly suggest that Lace1 might serve as a potential novel marker for browning. These findings prompted us to further explore the

role of Lace1 in white fat browning and ascertain its importance. Our approach involved the generation of Lace1 knockout (KO) mice. Our findings revealed that the Lace1 gene was upregulated

during the browning process of iWAT. Strikingly, Lace1 KO mice displayed an increased capacity for browning. This intriguing outcome prompted us to meticulously document and analyze the

intricate mechanisms behind this phenomenon. In this study, we investigated the role of Lace1 in white adipose tissue browning. MATERIALS AND METHODS ANIMALS AND EXPERIMENTAL DESIGN All

animal experiments were performed according to the “Guide for Animal Experiments” (edited by the Korean Academy of Medical Sciences). These animal experiments were approved by the

Institutional Animal Care and Use Committee of Seoul National University, Seoul, Korea (permission number: SNU-210217-9). Lace1 KO mice were used to delete exon 2 of the Lace1 gene via the

CRISPR-Cas9 system by Macrogen (Korea). All the animals were housed under a 12-h light/dark cycle at 22 ± 1 °C and 50–60% humidity with a 12 h light/dark cycle in a specific pathogen-free

facility. PHYSIOLOGICAL MEASUREMENT The fat mass and lean mass of the mice were measured via nuclear magnetic resonance (NMR) methods (Minispec LF-50, Bruker, Germany). The rectal

temperature of the mice was recorded via a BAT-12 microprobe thermometer (Physitemp, USA). PROTEIN EXTRACTION AND WESTERN BLOTTING For protein expression analysis, all proteins were

extracted via RIPA buffer (BR002, Biosolution, Korea) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (P3100-001, P3200-001, GeneDEPOT, USA). Equal amounts

of proteins were separated on SDS‒PAGE gels and transferred to PVDF membranes. Primary antibodies targeting the following proteins were used: LACE1 (NBP1-89215, Novus Biologicals, CO, USA),

UCP1 (ab10983, Abcam), MCT1 (ab250131, Abcam), MCT4 (ab74109, Abcam), LDHA (2012, CST), phospho-PDH (31866, CST), PDH (3205, CST), α-actin (A2066, Sigma‒Aldrich), and β-tubulin (ab2146,

Abcam). RNA EXTRACTION AND QUANTITATIVE REAL-TIME PCR For mRNA expression analysis, total RNA was extracted via TRIzol reagent (Invitrogen), and 1 µg of RNA was synthesized into cDNA via RT

premix (K-2044-B, Bioneer, Korea). qPCR was performed via the SYBR Green Kit (BIO-92005, Meridian Bioscience, OH, USA) according to the manufacturer’s instructions. The fold change for all

the samples was calculated via the 2−ΔΔCt method. The primer sequences are listed in Table 1. HISTOPATHOLOGY All the tissues were fixed with 4% paraformaldehyde (HP2031, Biosesang) at room

temperature overnight, embedded in paraffin, and sectioned into 3–5 μm sections. For H&E staining, all the slides were stained following the manufacturer’s instructions. For

immunohistochemical staining, the slides were stained with an anti-UCP1 antibody (ab10983, Abcam). INDIRECT CALORIMETRY VO2 and energy expenditure were estimated by indirect calorimetry

during CL challenge for 3 days and cold exposure (4 °C) for 7 days. During indirect calorimetry, all the mice were housed in a single cage with free access to food and water under a 12 h

light/dark cycle. All the mice were monitored by PhenoMaster 7.5.6 (TSE system, Germany) during the measurement of VO2 and energy expenditure. L-LACTATE MEASUREMENT Serum L-lactate levels

were measured with an L-lactate assay kit (ab65331, Abcam, UK). All protocols were performed following the manufacturer’s instructions. ANALYSIS OF BULK-RNA SEQUENCING AND BIOINFORMATICS

DATA lllumina’s TruSeq Stranded mRNA LT Sample Prep Kit was used to prepare RNA-sequencing libraries and high-throughput sequencing was performed with Illumina’s NovaSeq 6000 Platform for

each sample. The sequenced reads were mapped to the GRCm39 mouse reference genome via STAR v2.7.4a. Differential expression analysis was performed via the R packages DESeq2 v.1.32.0 and

apeglm v.1.14.0 via the shrinkage method. Differentially expressed genes were identified with cutoffs of adjusted _P_ values < 0.01 and log2-fold changes > 1 in the CL-challenged

experimental group and adjusted _P_ values < 0.05 and log2-fold changes > 0.58 in the Lace1 KO experimental group. All graphical visualizations were implemented in R via ggplot2 v3.3.5

BROWN ADIPOCYTE CULTURE When the brown preadipocyte cells reached 90–100% confluency, the cells were induced with induction medium (DMEM with 10% FBS, 0.5 mM isobutylmethylxanthine (I7018,

Sigma, USA), 0.5 μM dexamethasone (D1756, Sigma), 125 μM indomethacin (I7378, Sigma), 1 nM T3 (T2877, Sigma) and 20 nM insulin (sc-360248, Santa Cruz)). After induction for 2 days, the

medium was changed to insulin medium (DMEM with 10% FBS, 1 nM T3 and 20 nM insulin). Five days after differentiation, brown adipocytes were transfected with 50 nM Lace1 siRNA (30 nM) or

negative control siRNA for 36 h via Lipofectamine RNAiMAX reagent (13778075, Thermo Scientific, USA) to generate Lace1-knockdown adipocytes. The predesigned siRNA directed against murine

Lace1 mRNA (accession no. NM_001359297.1) was purchased from Bioneer (Daejeon, Korea). BEIGE ADIPOCYTE CULTURE Seven-week-old male C57BL/6N mice were sacrificed, and the stromal vascular

fraction (SVF) was harvested from iWAT. The tissues were digested with 1.5 U/ml collagenase D (11088882001, Roche) in a 37 °C shaker for 30 min. The SVF pellets were incubated in

collagen-coated plates with maintenance medium (DMEM/F12 with 10% FBS and 1X P/S) for 1 hr. After that, the medium was changed to fresh medium for removing immune cells, etc. Beige

adipogenesis of preadipocytes of iWAT was induced according to a previous protocol14,15. MITOCHONDRIAL ISOLATION Mitochondrial and cytosolic isolation from differentiated brown adipocytes

was performed with a mitochondrial isolation kit (ab110170, Abcam). All protocols were performed following the manufacturer’s instructions. ATP ASSAY ATP levels in fully differentiated brown

adipocytes were analyzed via an ATP assay kit (K354-100, BioVision, CA, USA). All protocols were performed following the manufacturer’s instructions. OXYGEN CONSUMPTION RATIO (OCR) The

oxygen consumption rate (OCR) of differentiated brown adipocytes from the control siRNA and _siLace1_ knockdown (KD) groups was measured via a Seahorse Fe Extracellular Flux Analyzer

(Agilent Technologies, CA, USA). For the measurement of the OCR, differentiated brown adipocytes were incubated at 37 °C in a non-CO2 incubator for 1 h with Seahorse XF DMEM (103575–100,

Agilent) supplemented with 1 mM pyruvate (103577–100, Agilent), 2 mM glutamine (103579–100, Agilent), and 10 mM glucose (103578–100, Agilent). After incubation, the brown adipocytes were

treated with 1.5 μM oligomycin, 2.0 μM carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 µM rotenone and antimycin (Rot/AA). All reagents used were from the Agilent

Seahorse XF Cell Mito Stress Test Kit (103015-100, Agilent). The OCR of brown adipocytes was normalized to the protein amount. STATISTICAL ANALYSIS All the statistical data were analyzed via

GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA). All the data are presented as the means ± standard errors of the means (SEMs). Statistical significance was analyzed by an

unpaired _t-_test. Statistical significance was determined at *_p_ < 0.05, **_p_ < 0.01 and ***_p_ < 0.001. RESULTS LACE1 IS A MITOCHONDRIAL ATPASE THAT IS HIGHLY EXPRESSED IN BAT

To investigate novel genes involved in beige adipogenesis, we compared the CL-treated and untreated mice via the bulk RNA-seq technique. Among the 292 genes upregulated by CL challenge

(adjusted _P_ value < 0.01 and a fold change > 2 as the cutoff), 97 genes were identified as nonbrowning mitochondrial genes; these genes were annotated as mitochondrial genes

(GO:0005739) and unannotated for browning-related biological processes (GO:1990845, GO:0070342, GO:0050873, KEGG PATHWAY: mmu04714). Using the BioGPS database to identify candidate genes of

interest, we selected 11 genes highly expressed in BAT. In this research, we present Lace1, a highly enriched nonbrowning mitochondrial-annotated gene in BAT, as a novel gene involved in

beige adipogenesis (Fig. 1a and Supplementary Fig. 3). To clarify the involvement of this novel gene, Lace1, in beige adipogenesis, we first measured the expression level of Lace1 in the

whole tissues of 7-week-old C57BL/6N male mice. The expression of LACE1 protein was highest in BAT, followed by the heart, soleus, and kidney (Fig. 1b). Compared with that in other tissues,

_Lace1_ mRNA was highly expressed in BAT (Fig. 1c). Next, as Lace1 expression is highest in BAT, we measured Lace1 expression during brown adipogenesis in immortalized brown preadipocytes.

As a result, LACE1 protein levels were found to be increased during brown adipogenesis, similar to those of Ucp1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha

(Pgc1α) (Supplementary Fig. 1a). _Lace1_ mRNA was also increased during brown adipogenesis in a _Ucp1-_, cell death-inducing DNA fragmentation factor alpha-like effector A _(Cidea)_- and

_Pgc1α_-dependent manner (Supplementary Fig. 1b–e). During beige adipogenesis, LACE1 protein levels, similar to those of UCP1, increased (Supplementary Fig. 1f). Additionally, _Lace1_ mRNA

expression increased during beige adipogenesis in a _Ucp1_ mRNA expression-dependent manner (Supplementary Fig. 1g, h). These data suggest that Lace1 is highly expressed in the BAT of mice

and that its expression increases during brown adipogenesis and beige adipogenesis. To determine the localization of Lace1 expression, we confirmed the expression of Lace1 in the cytosol and

mitochondria of preadipocytes and fully differentiated brown adipocytes via immortalized brown preadipocytes15. As expected, the LACE1 protein was expressed only in the mitochondria of

fully differentiated brown adipocytes (Supplementary Fig. 2a). Consistent with these findings, we transfected fully differentiated brown adipocytes with siRNAs targeting the Lace1 gene.

After transfection with _siLace1_, we attempted to confirm mitochondrial function in fully differentiated brown adipocytes by directly measuring the oxygen consumption rate (OCR). We found

that the OCR of the _Lace1_-knockdown (KD) group of brown adipocytes was lower than that of the control siRNA group (Supplementary Fig. 2b–f). In particular, basal respiration and maximal

respiration were lower in the Lace1-KD brown adipocytes than in the WT brown adipocytes. Lace1 is also known as a mitochondrial ATPase, which synthesizes ATP9,16. As with previous studies,

we confirmed that ATP levels were lower in the _Lace1_-KD brown adipocytes than in the WT brown adipocytes (Supplementary Fig. 2g). LACE1 IS INCREASED IN IWAT FOLLOWING CL CHALLENGE AND COLD

EXPOSURE Our transcriptomic data revealed that the _Lace1_ gene is one of the genes upregulated in iWAT by CL challenge. Therefore, we hypothesized that Lace1 is increased in beige fat in a

Ucp1 expression-dependent manner and is upregulated during beige adipogenesis. As expected, we confirmed that LACE1 protein levels, similar to those of Ucp1, are increased in CL-induced

beige fat (Fig. 1d). Additionally, _Lace1_ mRNA levels increased, similar to those of thermogenic genes (Fig. 1e and f). Next, we investigated Lace1 expression under cold exposure. Cold

exposure induces browning of iWAT via the release of NE and β3-adrenergic stimulation17,18. Consequently, LACE1 protein levels were greater in cold-induced beige fat than in white fat (Fig.

1g). Similarly, _Lace1_ mRNA expression increased, similar to that of thermogenic genes (Fig. 1h and i). We found that Lace1 was increased in a Ucp1-dependent manner in response to CL

challenge and cold exposure; therefore, we performed a correlation analysis between _Lace1_ and _Ucp1_ mRNA expression. Interestingly, _Lace1_ and _Ucp1_ mRNA levels were positively

correlated under CL challenge and cold exposure (CL challenge: _r_ = 0.904, _p_ = 0.000/cold exposure: _r_ = 0.961, _p_ = 0.000) (Fig. 1j and k). These data suggested that Lace1 is increased

in iWAT following CL challenge and cold exposure. LACE1 DEFICIENCY INCREASES BROWNING OF IWAT FOLLOWING CL INJECTION To investigate the role of Lace1 in beige fat, we generated Lace1 null

knockout (KO) mice via the CRISPR-CAS9 system. A scheme of the construction of Lace1 KO mice is presented in Supplementary Fig. 4. We administered CL to Lace1 KO mice according to this

experimental design (Fig. 2a). First, we confirmed that _Lace1_ protein and mRNA expression is downregulated in the iWAT of the Lace1 KO mice compared with the WT mice despite CL injection

(Figs. 2b and c). Next, we investigated the browning capacity of iWAT in the Lace1 KO mice under CL challenge. Surprisingly, histological analysis revealed that the Lace1 KO mice had a

greater beige fat content than the WT mice when challenged with CL (Fig. 2d). Together, the results of the UCP1 immunostaining analysis revealed that the Lace1 KO mice had more Ucp1-positive

adipocytes than the WT mice did under CL challenge (Fig. 2e). Additionally, thermogenic marker gene levels were greater in the CL-induced beige fat of the Lace1 KO mice than in that of

their control littermates (Fig. 2f). Ucp1 is critical for maintaining core temperature and increasing energy expenditure19. CL challenge and cold exposure-dependent oxygen consumption rate

(VO2) are known to be derived from Ucp1 activity20. To explore the systemic metabolic effects of heightened browning in white adipose tissue of Lace1 KO mice, we conducted indirect

calorimetry during the CL challenge. The Lace1 KO mice exhibited increased oxygen consumption (Fig. 2g) and heat generation after CL challenge (Fig. 2h). We hypothesized that the browning

capacity of iWAT is decreased by Lace1 gene deletion compared with that of WT iWAT because Lace1 is upregulated in iWAT under browning stimuli such as Ucp1; however, surprisingly, we found

that Lace1 deficiency increases the browning capacity of iWAT under CL challenge. On the basis of the above results, we explored whether this phenotype occurred in the Lace1 KO mice upon

exposure to another stimulus, cold, as with the CL challenge. LACE1 DEFICIENCY RESULTS IN INCREASED BROWNING OF IWAT FOLLOWING COLD EXPOSURE For analysis of the phenotype of Lace1 KO mice in

response to cold stimuli, the mice were exposed to cold at 4–6 °C for 7 days (Fig. 3a). As shown in Fig. 3b and c, _Lace1_ protein and mRNA expression was downregulated in the iWAT of the

Lace1 KO mice compared with that of the WT mice despite cold exposure. Histological analysis revealed that the Lace1 KO mice had greater beige fat content than the WT mice when challenged

with cold (Fig. 3d). Additionally, Ucp1 immunostaining analysis revealed that the iWAT of the Lace1 KO mice had more Ucp1-positive adipocytes than did that of the WT mice upon cold exposure

(Fig. 3e). Additionally, thermogenesis-related gene expression was greater in the iWAT of the Lace1 KO mice than in that of the WT mice (Fig. 3f). Compared with those of WT mice, we

subsequently analyzed the systemic metabolic characteristics of Lace1 KO mice after cold exposure and observed significant increases in both VO2 (Fig. 3g) and thermogenesis (Fig. 3h).

Furthermore, we found that the Lace1 KO mice have higher rectal temperatures than the WT mice under cold conditions (Fig. 3i). These findings indicate that the Lace1 KO mice exhibit elevated

energy expenditure when subjected to cold exposure, demonstrating an increased cold tolerance and capacity to sustain core body temperature through heightened thermogenesis. This augmented

thermogenic response strongly aligns with the histological and molecular results, confirming that deficiency of the Lace1 gene promotes browning of iWAT. LACE1 LOSS PROMOTES LACTATE/GPR81

SIGNALING IN CL-INDUCED IWAT To investigate why Lace1 deficiency accelerates the browning of iWAT, we compared the genes whose expression was upregulated in iWAT between the WT and Lace1 KO

mice subjected to CL challenge via bulk RNA sequencing (Supplementary Fig. 5). The RNA-seq data showed that hydroxycarboxylic acid receptor 1 (_Hcar1_) was upregulated in the iWAT of the

Lace1 KO mice upon CL compared with that in the wild-type mice was (Fig. 4a). Hcar1 is a well-known G protein-coupled receptor gene that functions as a receptor for lactate21,22. Hcar1, also

known as Gpr81, is expressed in adipose tissue, liver, and muscle, and its upregulation in iWAT has been reported in response to CL challenge23. On the basis of our sequencing results and

literature, we hypothesized that the Lace1 KO mice exhibit greater browning capacity than the WT mice through lactate utilization. Lactate, a well-known promoter of browning, serves as the

foundation for this hypothesis24. Initially, we quantified the serum L-lactate levels in response to CL treatment. Intriguingly, we observed an elevated amount of circulating L-lactate in

the CL-treated Lace1 KO mice compared with the WT mice (Fig. 4b). We proceeded to assess the expression levels of monocarboxylate transporter 1 (Mct1), which is responsible for lactate

influx, to gauge the lactate uptake capacity in the iWAT of the Lace1 KO mice. First, we analyzed _Mct1_ mRNA levels in the entire tissues of the CL-treated Lace1 KO mice. Notably, _Mct1_

mRNA expression increased more than 10-fold exclusively in the iWAT of the Lace1 KO mice upon CL treatment, with no concurrent alterations in other tissues (Fig. 4c). Additionally, we

observed a substantial increase in MCT1 protein levels, specifically in the iWAT of the Lace1 KO mice subjected to CL treatment (Fig. 4d). Conversely, the expression of the lactate efflux

transporter monocarboxylate transporter 4 (Mct4) remained unaltered in the iWAT of the Lace1 KO mice subjected to CL challenge (Fig. 4e). These findings collectively indicate that, in

comparison with their control counterparts, the Lace1-deficient mice exhibit increased lactate uptake while concurrently impeding lactate efflux in iWAT during CL challenge. Lactate can be

converted to pyruvate to increase Ucp1 expression via lactate dehydrogenase (LDH) in the mitochondria of iWAT and BAT24. LDHB functions to convert lactate to pyruvate; in contrast, LDHA

converts pyruvate to lactate25. Hence, we proceeded to assess the mRNA expression of _Ldhb_ and _Ldha_ in iWAT following CL challenge. The results revealed elevated _Ldhb_ mRNA expression in

the Lace1 KO mice, whereas _Ldha_ mRNA expression remained unchanged compared with that in the CL-treated controls (Fig. 4f). Under cold conditions for 7 days, _Mct1_ mRNA levels, but not

_Mct4 levels_, in the iWAT of the Lace1 KO mice showed a trend toward increased expression, as in CL conditions (Supplementary Fig. 6a). In addition, _Ldhb_ but not Ldha mRNA expression was

increased (Supplementary Fig. 6b and c). Collectively, these findings suggest that Lace1-deficient mice exhibit increased lactate uptake in their iWAT following CL challenge or cold

exposure, with subsequent active conversion to pyruvate. For comprehensive verification, we performed Lace1 gene knockdown in a differentiated BAT cell line, followed by the induction of

adrenergic receptor activity through isoproterenol stimulation. Intriguingly, this manipulation resulted in elevated Mct1 gene expression, mirroring the in vivo observations (Fig. 4g and h).

Furthermore, when L-lactate was administered once daily for 3 days to 7-week-old male C57BL/6N mice, we noted a significant increase in _Ucp1_ protein and mRNA expression within the iWAT

(Fig. 4i–k). L-lactate, a well-recognized metabolite produced from pyruvate during anaerobic glycolysis26, has gained prominence as a novel metabolic regulator, with recent studies

highlighting its role in white adipose tissue browning23,24. Lace1 knockout in mice is thought to regulate lactate metabolism, potentially through the induction of white fat browning. LOSS

OF LACE1 RESULTS IN INACTIVATION OF PYRUVATE DEHYDROGENASE, WHICH PROMOTES LACTATE EFFLUX IN CL-INDUCED HEARTS To investigate why Lace1 KO mice have a high amount of circulating L-lactate

upon CL challenge, we measured _Mct4_ mRNA expression, which is related to a lactate efflux, in whole tissues (Fig. 5b). Lace1 KO mice presented highly increased _Mct4_ mRNA and protein

expression in the heart (Fig. 5b–d). However, MCT4 protein expression, which was significantly increased in the hearts of the CL-treated KO mice, did not differ in the soleus, gas, or kidney

(Supplementary Fig. 7). On the basis of the _Mct4_ mRNA level in the hearts of the Lace1 KO mice subjected to CL challenge, we hypothesized that lactate released from the heart contributes

to lactate uptake and utilization for browning in Lace1 KO mice subjected to CL challenge. Lactate release through Mct4 activation in heart tissue is a cardiac hypertrophic phenotype27.

Cardiac hypertrophy can progress to heart failure through increasing cardiomyocyte and heart size27,28. However, hypertrophic morphology was not observed in the hearts of the Lace1 KO mice

subjected to CL challenge. Nevertheless, we found that the hearts of the Lace1 KO mice displayed pyruvate dehydrogenase (PDH) inactivation compared with those of the WT mice under CL

challenge (Fig. 5e and f). When PDH is inactive, pyruvate from glycolysis cannot be converted to acetyl-CoA, thereby converting pyruvate to lactate. With an unbalanced pyruvate‒lactate axis,

lactate is released into the blood27 (Fig. 5a). Additionally, _Ldha_ mRNA levels were increased in the hearts of the Lace1 KO mice under CL challenge (Fig. 5g). This finding is in stark

contrast to the observed increase in MCT1 and LDHB levels following CL stimulation in iWAT. Taken together, our data revealed the inactivation of PDH in the hearts of the Lace1 KO mice

during CL challenge, alongside increased lactate efflux through MCT4 via increased LDHA expression. Moreover, we propose that these mechanisms may have contributed to lactate uptake-induced

browning in iWAT (Fig. 6). DISCUSSION The current study examined the role of Lace1 in the browning of iWAT. Lace1 expression was substantially increased in iWAT by browning stimuli such as

CL injection and cold exposure. However, the browning capacity of the Lace1-deficient mice was not disrupted; rather, the Lace1-deficient mice presented increased browning capacity. To

understand these conflicting consequences, we investigated the mechanisms by which Lace1 deficiency promotes browning capacity in mice. Here, we observed that CL-induced upregulation of

browning in Lace1-deficient mice was stimulated by lactate uptake in iWAT, suggesting for the first time that this phenomenon is due to increased lactate secretion induced by PDH

phosphorylation in the hearts of Lace1 KO mice. First, our data clearly revealed the expression of Lace1 in the browning process of iWAT, such as CL stimulation and cold exposure, which was

significantly correlated with the Ucp1 level. Ucp1 is the most representative marker of adipose tissue browning and is predominantly expressed in cells where many mitochondria are found,

such as BAT3,29. Thus, accumulating evidence has revealed the mechanism of Ucp1 expression in brown adipocytes and beige adipocytes with a high content of mitochondria and its effect on

whole-body energy homeostasis. Therefore, we concluded that Lace1 is a gene that plays an important role in the browning process. β3-ARs are the predominant regulators of WAT browning, and

CL is a potent and highly selective β3-AR agonist30. According to many studies related to WAT browning, CL treatment significantly increased browning marker gene expression, reduced lipid

droplet size, and increased heat generation and energy expenditure in both BAT and iWAT31,32, which was more evident over time. Since we identified Lace1 as an important gene for browning,

we administered CL to Lace1 KO mice and investigated the importance of Lace1 in the browning process. Interestingly, the Lace1 KO mice presented smaller lipid droplets and greater Ucp1

expression following CL treatment. Furthermore, oxygen uptake capacity and heat generation were significantly greater in the KO mice than in the WT mice. This phenomenon was the same with

cold exposure. To identify the cause or mediator of these unexpected results, we performed RNA-seq to investigate which gene had altered expression in CL-induced iWAT in Lace1 KO mice. We

found that the expression of the _Hcar1_ gene was the highest in iWAT when the CL was administered to Lace1 KO mice, which prompted us to establish a new hypothesis in this study. Hcar1,

also known as GPR81, is a cell-surface receptor for lactate and is highly expressed in adipose tissues but is also found in the kidney, skeletal muscle, and liver in mammals23.

Lactate-induced GPR81 effectively suppresses lipolysis via modulation of the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, which contributes to hormone-sensitive

lipase (HSL) downregulation in adipose tissue32,33. Furthermore, the activation of the GPR81-induced cAMP‒PKA pathway is synergistically dependent on antilipolytic effects and insulin

actions34. These results suggest that lactate can modulate metabolic processes via Gpr81-dependent mechanisms in adipose tissue. Lactate may play a role in browning as a metabolite. Recent

studies reported that lactate plays a crucial role in the browning of WAT through its receptor GPR81 and transporter Mct1 in a β3-AR stimulation-dependent manner23,35. On the basis of a

previous study and our RNA-seq data, we hypothesized that CL-induced Hcar1 gene expression in the iWAT of the Lace1 KO mice could be due to increased circulating lactate levels. As expected,

the serum lactate level was significantly greater in the CL-induced Lace1 KO mice than in the WT mice, and the _Hcar1_ receptor level was significantly elevated in the Lace KO mice. In

addition, we confirmed the lactate uptake ability of the Lace1 KO mice by CL stimulation and confirmed that Mct1, a lactate influx transporter, was specifically expressed only in iWAT.

Lagarde et al. reported a positive correlation between Mct1 and thermogenic marker gene levels in iWAT. These researchers also revealed that Ucp1-positive cells were highly expressed in the

subpopulation of adipocytes expressing Mct1 after cold exposure. Lactate is first converted to pyruvate by the LDH reaction for oxidation, indicating that lactate consumption is directly

dependent on mitochondrial pyruvate utilization26,36. Compared with the WT mice, the CL-induced Lace1 KO mice presented significantly elevated Ldhb expression levels in iWAT. These results

suggest that CL-stimulated WAT from Lace1 KO mice takes up higher levels of lactate via upregulation of Mct1 than does that from WT mice and increases Ucp1 expression by increasing oxidative

phosphorylation in mitochondria through a high rate of conversion to pyruvate. We believe that these data provide essential insight into the role of lactate in β3-AR-induced browning of

iWAT. Although we found that Lace1 KO mice displayed increased browning capacity through lactate uptake upon CL stimulation, lactate concentrations did not differ in iWAT between WT and KO

mice. Therefore, we aimed to determine where and why lactate increases, and we measured Mct4, a lactate exporter, in whole tissues of the CL-stimulated WT mice and KO mice via the same

method as that used for Mct1 in iWAT. Interestingly, in contrast to the Mct1 results in iWAT, the CL-induced Lace1 KO mice exhibited exclusively increased Mct4 expression in the heart

compared with the WT mice. Under normal conditions, cardiomyocytes express low levels of Mct4, whose levels are increased by hypertrophy or heart failure27,28. However, we did not observe

hypertrophic morphology in the CL-induced Lace1 KO mice. Unlike previous reports, there was no difference in the cardiac hypertrophic phenotype according to elevated Mct4. Nevertheless, we

noted that PDH phosphorylation was significantly increased only in the Lace1 KO mice upon CL treatment. PDH is the rate-limiting enzyme for glucose oxidation, and cardiac-specific deletion

of PDH impairs glucose oxidation in mice37. Our results suggest that the increase in MCT4 in the CL-induced Lace1 KO mice is due to impaired pyruvate flux into mitochondria caused by PDH

phosphorylation. The role of β3-ARs in the heart has long been debated. This receptor could have either a protective effect or a detrimental effect on the heart38,39. However, studies on the

effect of CL-specific β3-AR activation on the heart have not been conducted, which is a limitation of the discussion in our study. Further studies are needed to investigate the molecular

mechanisms that may explain how LACE1 KO blocks PDH in a cardiac-specific manner and the mechanisms that increase MCT4 expression. These results would ultimately provide an important basis

for explaining the lactate-induced browning of iWAT observed in this study and could be the start of a new line of research to elucidate the relationship between cardiac metabolism and

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fate and maintenance. Second, beige fat is relevant to adult humans and may be a therapeutic target in the treatment of metabolic disorders40. We first suggested that Lace1 is a new gene

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which we used as a representative tool in browning research, regulates the browning of white fat by modulating the molecular and physiological functions of the heart through the Lace1 gene.

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PubMed  PubMed Central  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the Korea Mouse Phenotyping Project (NRF-2013M3A9D5072550) of the Ministry of

Science and ICT through the National Research Foundation. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Laboratory of Developmental Biology and Genomics, Research Institute for Veterinary

Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea Youn Ju Kim, Sang Gyu Lee, Hye Jin Kim & Je Kyung Seong * Korea Model animal Priority Center

(KMPC), Seoul National University, Seoul, Republic of Korea Youn Ju Kim, Sang Gyu Lee, Su In Jang, Hye Jin Kim & Je Kyung Seong * Division of Endocrine and Kidney Disease Research,

Department of Chronic Disease Convergence Research, Korea National Institute of Health, Korea Disease Control and Prevention Agency, Cheongju, Republic of Korea Youn Ju Kim *

Interdisciplinary Program in Bioinformatics, Seoul National University, Seoul, Republic of Korea Sang Gyu Lee & Je Kyung Seong * Metabolic Regulation Research Center, Korea Research

Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea Won Kon Kim, Kyoung-Jin Oh & Kwang-Hee Bae * Department of Functional Genomics, KRIBB School of Bioscience,

Korea University of Science and Technology (UST), Daejeon, Republic of Korea Won Kon Kim, Kyoung-Jin Oh & Kwang-Hee Bae * Interdisciplinary Program for Bioinformatics, Program for Cancer

Biology, BIO-MAX/N-Bio Institute, Seoul National University, Seoul, Republic of Korea Je Kyung Seong Authors * Youn Ju Kim View author publications You can also search for this author

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author inPubMed Google Scholar * Won Kon Kim View author publications You can also search for this author inPubMed Google Scholar * Kyoung-Jin Oh View author publications You can also

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can also search for this author inPubMed Google Scholar * Je Kyung Seong View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Youn Ju Kim:

Methodology, Data curation, Formal analysis, Investigation, Validation, Visualization, Writing—original draft. Sang Gyu Lee: Data curation, Formal analysis. Su In Jang: Methodology. Hye Jin

Kim: Data curation, Investigation, Validation, Visualization, Writing—review & editing. Je Kyung Seong: Funding acquisition, Project administration, Supervision. CORRESPONDING AUTHORS

Correspondence to Hye Jin Kim or Je Kyung Seong. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature

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Lactate utilization in Lace1 knockout mice promotes browning of inguinal white adipose tissue. _Exp Mol Med_ 56, 2491–2502 (2024). https://doi.org/10.1038/s12276-024-01324-w Download

citation * Received: 04 January 2024 * Revised: 02 June 2024 * Accepted: 02 July 2024 * Published: 07 November 2024 * Issue Date: November 2024 * DOI:

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