Zbtb16 has a role in brown adipocyte bioenergetics

ABSTRACT OBJECTIVE: A better understanding of the processes influencing energy expenditure could provide new therapeutic strategies for reducing obesity. As the metabolic activity of the

brown adipose tissue (BAT) and skeletal muscle is an important determinant of overall energy expenditure and adiposity, we investigated the role of genes that could influence cellular

bioenergetics in these two tissues. DESIGN: We screened for genes that are induced in both the BAT and skeletal muscle during acute adaptive thermogenesis in the mouse by microarray. We used

C57BL/6J mice as well as the primary and immortalized brown adipocytes and C2C12 myocytes to validate the microarray data. Further characterization included gene expression, mitochondrial

density, cellular respiration and substrate utilization. We also used a Hybrid Mouse Diversity Panel to assess _in vivo_ effects on obesity and body fat content. RESULTS: We identified the

transcription factor _Zbtb16_ (also known as _Plzf_ and _Zfp14_) as being induced in both the BAT and skeletal muscle during acute adaptive thermogenesis. _Zbtb16_ overexpression in brown

adipocytes led to the induction of components of the thermogenic program, including genes involved in fatty acid oxidation, glycolysis and mitochondrial function. Enhanced _Zbtb16_

expression also increased mitochondrial number, as well as the respiratory capacity and uncoupling. These effects were accompanied by decreased triglyceride content and increased

carbohydrate utilization in brown adipocytes. Natural variation in _Zbtb16_ mRNA levels in multiple tissues across a panel of >100 mouse strains was inversely correlated with body weight

and body fat content. CONCLUSION: Our results implicate Zbtb16 as a novel determinant of substrate utilization in brown adipocytes and of adiposity _in vivo_. SIMILAR CONTENT BEING VIEWED BY

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RESPONSE Article Open access 20 November 2024 INTRODUCTION The increasing prevalence of obesity worldwide reflects changes in lifestyle, including a combination of increased food intake and

reduced physical activity.1, 2, 3 This combination contributes to the accumulation of an energy surplus in the form of adipose tissue. Because obesity develops when energy intake exceeds

energy expenditure, increasing energy expenditure is an attractive strategy to reduce body weight and fat storage. There has been considerable interest in modulating thermogenesis,

particularly in the skeletal muscle and brown adipose tissue (BAT), as a target for the treatment of obesity.4, 5, 6 Skeletal muscle is one of the central tissues involved in shivering

thermogenesis and also contributes to non-shivering thermogenesis.5, 7, 8 Skeletal muscle is responsible for at least 30% of the basal metabolic energy expenditure and 80% of whole-body

glucose uptake.9 Because skeletal muscle accounts for approximately 30–40% of total body mass, it may contribute substantially to adaptive thermogenesis in humans.5 Recently, the existence

of BAT in humans has been reappraised and there is good evidence that brown fat depots are active in adults and are capable of energy dissipation through adaptive thermogenesis.10, 11, 12,

13 Furthermore, body-mass index and percentage of body fat are inversely correlated with the BAT in humans12, suggesting that the BAT contributes to metabolic rate and is an important

regulator of body fat accumulation. Further evidence for a shared physiological function between brown adipocytes and myocytes is the fact that they are derived from a common cell

lineage.14, 15 Increased heat production through decreased coupling between fatty acid oxidation and ATP synthesis has been observed during both diet-induced and cold-induced adaptive

thermogenesis.16, 17 During adaptive thermogenesis, a significant amount of energy stored as fat and glycogen is converted to heat instead of being efficiently converted to ATP, suggesting

that induction of mitochondrial respiration or the level of uncoupling activity could promote caloric dissipation. Because of its potential for energy dissipation, it is of great interest to

identify the genes that orchestrate the changes occurring during adaptive thermogenesis and to determine their mechanisms of action. One key factor in adaptive thermogenesis of the BAT is

the mitochondrial _uncoupling protein-1_ (_Ucp1_). _Ucp1_ is responsible for enabling the proton leak in mitochondria that dissipates energy produced through oxidative metabolism to generate

heat.17, 18, 19 Previous systematic studies have identified genes that are induced in BAT after cold exposure in mouse, rat and squirrel,20, 21, 22, 23, 24, 25, 26 and a recent

transcriptome-profiling study identified several genes that are induced in the skeletal muscle and BAT in mice.27 We hypothesized that understanding the function of genes promoting energy

expenditure would reveal new targets for modulation of energy balance. Genes differentially expressed in both the BAT and skeletal muscle could be of great importance because they are

indicative of a general thermogenic response not limited to the BAT-specific uncoupling or skeletal muscle-specific shivering. We performed transcriptional profiling of the BAT and skeletal

muscle during acute cold exposure in mice, a model of adaptive thermogenesis. We identified genes that were induced in both the BAT and skeletal muscle following cold exposure. One of the

significantly upregulated genes is the transcription factor _Zbtb16_, which belongs to the POZ/domain and Krüpel zinc finger family and generally acts as a repressor.28, 29, 30 Here, we

demonstrate that _Zbtb16_ levels influence cellular bioenergetics _in vitro_ and inversely correlate with adiposity _in vivo_. MATERIALS AND METHODS MICE Animal studies were performed under

approved UCLA animal research protocols and according to guidelines established in the Guide for Care and Use of Laboratory Animals. C57BL/6J mice were maintained in 12-h light/dark

conditions and fed a regular chow diet (Purina 50010, Lab Diet, Richmond, IN, USA). Cold exposure was performed as described.31 After euthanasia, tissues were collected and frozen until use.

MICROARRAY ANALYSIS Total RNA was isolated from mouse tissues by extraction with TRIzol (Invitrogen, Carlsbad, CA, USA). Samples were then hybridized to Illumina 24K BeadChips using

standard protocols (The Southern California Genotyping Consortium at UCLA). Differentially expressed genes were identified by applying a Student’s _t_-test and fold-change thresholds. The

microarray data can be accessed from the NCBI Gene Expression Omnibus (GEO) database. CELL CULTURE AND OVER-EXPRESSION Primary brown adipocytes were cultured as described previously.31 C2C12

cells (ATCC, Manassas, VA, USA) were grown in complete DMEM (Dulbecco’s modified Eagle’s medium; 10% fetal bovine serum, 25 mM glucose, 1 mM pyruvate and 1 mM glutamine). For

differentiation, serum was switched to 2% horse serum for 6 days. The immortalized brown adipocyte cell line was a gift from Bruce Spiegelman and was cultured as described.32 Mouse Zbtb16

cDNA was cloned into pcDNA3.1/V5-His vector (Invitrogen) and transfected with BioT (Bioland, La Palma, CA, USA). Empty vector was used as control. For transduction, an adenovirus was

constructed from the Zbtb16 cDNA and the AdEasy XL Adenoviral Vector System (Stratagene, Santa Clara, CA, USA). Viral particles were purified with Adeno-X Maxi Purification Kit (Clontech,

Mountain View, CA, USA) and titered with Adeno-X Rapid Titer Kit (Clontech). Transduction (MOI 50) was performed at day 3 post-confluence and gene expression analyzed at day 6. QUANTITATIVE

REAL-TIME (QRT-PCR) GENE EXPRESSION ANALYSES Total RNA was isolated from mouse tissues by extraction with TRIzol (Invitrogen). For human tissues, a cDNA panel was purchased from Clontech.

cDNA synthesis, real-time PCR, and qRT-PCR were performed as described previously.33 Values are expressed in arbitrary units, normalized to housekeeping genes. All primer sequences used in

this study are presented in Supplementary Table S1. IMMUNOBLOTTING Cells were lysed in lysis buffer containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Proteins (50 

μg/lane) were resolved by 7% Tris-Acetate pre-cast gel (Invitrogen), transferred to nitrocellulose membrane and incubated for 3 h with 5% milk TBS-T (Tris-buffered saline with Tween 20),

overnight with primary antibody in 1% milk TBS-T, then 1 h with a horseradish peroxidase-conjugated secondary antibody in 1% milk TBS-T. ECL Plus (GE Healthcare, Piscataway, NJ, USA) was

used for chemiluminescent detection. Antibodies included ZBTB16 (OP128, Calbiochem, Billerica, MA, USA), UCP1 (662045, Calbiochem), β-ACTIN (AC-15, Sigma, Dallas, TX, USA) and LAMIN A/C

(sc-6215, Santa Cruz Biotechnology, Santa Cruz, CA, USA). TRIGLYCERIDE (TAG) CONTENTS AND LIPOLYSIS ASSAY Cells were homogenized by sonification and TAG contents determined with L-Type

triglyceride M kit (Waco Chemicals, Richmond, VA, USA). Lipolysis experiments from cell medium were performed after 10 h incubation with the Adipolysis Assay Kit (AB100, Millipore,

Billerica, MA, USA). TAG contents and glycerol concentrations were normalized to protein. CELLULAR OXYGEN CONSUMPTION AND EXTRACELLULAR ACIDIFICATION RATES Cellular metabolic rates were

measured using a XF24 Analyzer (Seahorse Bioscience, Billerica, MA, USA). Immediately before the measurement, cells were washed with unbuffered DMEM as described.34 Plates were placed into

the XF24 instrument for measurement of oxygen consumption and extracellular acidification (ECAR) rates. Mixing, waiting and measurement times were 3, 2, and 3 min for C2C12 and 5, 2 and 1.5 

min for brown adipocyte cells, respectively. The measures were normalized per protein. Test compounds were obtained from Sigma and injected during the assay at the following final

concentration: 0.75 or 0.5 μM oligomycin, 0.5 or 0.4 μM FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone), 0.75 or 0.15 μM rotenone/myxothiazol (differentiated brown adipocyte

cells or C2C12, respectively), 25 mM glucose, 100 μM 2,4-dinitrophenol, 50 mM Na-oxamate, and 200 mM 2-deoxyglucose. The basal and percentage of oxygen consumption rate and ECAR levels as

well as area under the curve were obtained with the XF24 Analyzer software. MITOCHONDRIAL DNA CONTENT Total genomic DNA from the BAT tissue was isolated by phenol/chloroform/isoamyl alcohol

extraction. Mitochondrial and nuclear DNA were amplified by RT-PCR with 25 ng of DNA and primers in the D-Loop region and Tert gene, respectively (sequences in Supplementary Table S1).

SUBSTRATE UTILIZATION WITH BIOLOG MICROPLATES PM-M TOX1 microplates (Biolog, Hayward, CA, USA), consisting of eight different substrates in replicate, were used. Differentiated brown

adipocyte cells were seeded at 60 000 cells per well in complete medium without pyruvate, glucose or phenol red. After 24 h, Biolog Redox Dye MA was added and rates measured every hour at

590 nm to detect the metabolic activity. Time 0 was subtracted for each time point. The reductase activity (NADH) is proportional to the energy produced from each substrate. HYBRID MOUSE

DIVERSITY PANEL Mouse strains, RNA isolation and expression profiling were described previously.35 Briefly, at 16 weeks of age, 4–6 mice per strain were assessed for body composition using

an EchoMRI instrument (EchoMRI, Houston, TX, USA). Mice were fasted overnight before plasma and tissue collection. Tissues were weighed and then snap frozen in liquid nitrogen until RNA,

cRNA and cDNA were prepared. Biotin-labeled cRNA was generated from the cDNA and used to probe by using Affymetrix Mouse Genome HT_MG430A arrays (4–6 mice per strain). Expression data (probe

1442025_a_at) and phenotypic traits are presented in Supplementary Table S2. STATISTICAL ANALYSIS The data are expressed as the mean±s.d., except for the XF24 Seahorse Bioscience analysis

where s.e. was used. Unpaired Student’s _t_-test was used to compare the difference between the groups. RESULTS IDENTIFYING GENES DIFFERENTIALLY REGULATED IN ADAPTIVE THERMOGENESIS We

assayed the transcriptomes of mice exposed to cold using gene expression microarrays to identify genes differentially expressed during thermogenesis. In the BAT, 124 genes were upregulated

and 176 were downregulated at least twofold in response to cold exposure. In the skeletal muscle (quadriceps), 69 genes were upregulated and 67 were downregulated at least twofold in

response to cold exposure. We detected 11 genes that were altered by cold exposure in both the BAT and skeletal muscle, 5 of which were coordinately upregulated and 6 downregulated (Table

1). The transcription factor _Zbtb16_ (also known as _Plzf_ and _Zfp145_) exhibited a robust induction in both the BAT (5.6-fold) and skeletal muscle (4.8-fold). _Zbtb16_ has been associated

with white adipogensis36 but has not previously been implicated in the BAT or muscle metabolism. We selected this gene to further characterize its function. VALIDATION OF _ZBTB16_ INDUCTION

IN ADAPTIVE THERMOGENESIS We first confirmed that _Zbtb16_ expression is consistently upregulated by cold exposure at the mRNA and protein levels in an independent group of mice. As shown

in Figure 1a, _Zbtb16_ mRNA levels were increased 15.3-fold in the BAT and 2.1-fold in the muscle as detected by qPCR. _Zbtb16_ mRNA induction by cold was observed after 4 or 8 h and whether

mice were fasted or not during the cold exposure (data not shown). Western blot analysis confirmed a concomitant increase in ZBTB16 protein levels (Figure 1a, bottom inserts). Thus, the

upregulation of _Zbtb16_ during adaptive thermogenesis was validated at both the transcript and protein levels. TISSUE EXPRESSION PATTERN OF _ZBTB16_ IN MOUSE AND HUMAN We then investigated

whether the BAT and skeletal muscle were the most biologically relevant tissues for studying _Zbtb16_ function by comparing mRNA expression levels in mouse and human tissue panels by qPCR.

The highest _Zbtb16_ expression levels were detected in the skeletal muscle and heart for both mouse and human (Figure 1b). In contrast to what Mikkelsen, _et al._36 reported, we detected a

lower expression level in the mouse BAT than in the skeletal muscle. However, the levels of ZBTB16 protein measured by immunoblot in the white adipose tissue and BAT were similar to those of

the heart and skeletal muscle (Figure 1b, insert). Within the muscle, the expression of _Zbtb16_ was similar in fast twitch (extensor digitorum longus and tibialis), slow twitch (soleus)

and mixed (quadriceps and gastronecmius) muscles (Figure 1c), suggesting that Zbtb16 may function across muscle fiber types. EXPRESSION OF _ZBTB16_ DURING BROWN ADIPOCYTE AND MYOCYTE

DIFFERENTIATION We then studied Zbtb16 function _in vitro_ using a mouse brown adipocyte cell line32 and the mouse C2C12 myocyte cell line. We first assessed _Zbtb16_ expression dynamics

during differentiation of brown adipocyte precursors into mature brown adipocytes. _Zbtb16_ expression was dramatically upregulated 60-fold during brown adipocyte differentiation, whereas

_Ucp1_ showed a 30-fold induction (Figure 1d). By contrast, Zbtb16 mRNA levels did not change during differentiation of C2C12 myoblasts to myotubes, whereas myogenin, used as control, was

dramatically induced. The increased mRNA levels in brown adipocytes were mirrored by a substantial increase in ZBTB16 protein levels (Figure 1d, bottom inserts). The induction of _Zbtb16_

during brown adipogenesis is consistent with a role for this protein in mature brown adipocytes. _ZBTB16_ EXPRESSION ACTIVATES THE BROWN ADIPOCYTE GENE EXPRESSION PROGRAM To begin to

elucidate the effect of _Zbtb16_ induction during thermogenesis, we overexpressed Zbtb16 in brown adipocyte cells using an adenoviral vector carrying _Zbtb16_ cDNA. We achieved roughly

sixfold overexpression of _Zbtb16_ mRNA (Figure 2a), which led to alterations in expression of genes involved in brown adipocyte differentiation and mitochondrial function. Indeed, compared

with cells infected with a control lacZ-containing adenovirus, cells overexpressing _Zbtb16_ exhibited significant upregulation of brown fat-enriched markers such as _Ucp1_, _Ppargc1a_,

_Ppara_ and _Prdm16_ (_PR domain containing 16_; Figure 2b). Enhanced _Zbtb16_ expression also stimulated the expression of genes promoting fatty acid oxidation, such as _Fabp3 (fatty

acid-binding protein 3_), _Fabp5_ (also known as _Mal1_) and _Cpt1b_ (_carnitine palmitoyltransferase 1b_). _Zbtb16_ expression also elevated expression levels of mitochondrial gene markers

such as _Tfam_ (_mitochondrial transcription factor A_), _Elovl3_ (_elongation of very long chain fatty acids-like 3_), _Cidea_ (_cell death-inducing DFFA-like effector a_), _Cox7a1_

(_cytochrome c oxidase_, _subunit VIIa 1_) and _Cox8b_ (_cytochrome c oxidase_, _subunit VIIIb_). To confirm these effects observed in an immortalized brown adipocyte cell line, we performed

similar studies in primary brown adipocytes. Zbtb16 adenoviral expression induced UCP1 protein levels (Figure 2c) and increased expression of brown fat, fatty acid oxidation and

mitochondrial genes (Figure 2d). Thus, _Zbtb16_ expression promotes a gene expression signature characteristic of active brown adipocytes and enhanced fatty acid oxidation. ZBTB16 ENHANCES

MITOCHONDRIAL RESPIRATION AND MITOCHONDRIAL BIOGENESIS The effect of Zbtb16 on mitochondrial gene expression described above suggested that it may promote changes in brown adipocyte

energetics. To assess this, we measured cellular respiration in brown adipocytes by real-time detection of oxygen consumption. Using a XF24 Seahorse Bioscience instrument, we determined the

level of different components of mitochondrial respiration contributing to the oxygen consumption using specific effectors. First, we determined the basal and coupling state of mitochondrial

respiration with sequential injections of oligomycin and rotenone/myxothiazol. Oligomycin (F1F0-ATP synthase inhibitor) allows the measurement of ATP-linked oxygen consumption (ATP

turnover) and rotenone/myxothiazol (complex I and III inhibitor, respectively) by blocking mitochondrial respiration infers the non-mitochondrial respiration and the mitochondrial uncoupling

(difference between oligomycin and rotenone/myxothiazol injections). Cells were ‘activated’ 2 h before the first measurement with 10 nM of the adrenoceptor agonist CL316,243. In both the

immortalized and primary brown adipocytes, _Zbtb16_ overexpression did not alter basal mitochondrial respiration, but showed higher mitochondrial uncoupling at the expense of coupling

respiration (Figures 3a and b). C2C12 cells can also be ‘activated’ by beta-adrenergic stimulation when they are differentiated in myotubes.37 When we pretreated the myotubes for 1 h with

100 nM isoproterenol, we also observed a significant increase in mitochondrial uncoupling owing to _Zbtb16_ expression (Figure 3c). We also assessed mitochondrial reserve capacity by

injecting the electron transport uncoupler FCCP. Brown adipocytes (primary and immortalized) and C2C12 cells over-expressing _Zbtb16_ exhibited a higher FCCP response, indicating more

mitochondrial respiratory capacity (Figure 3d). Altogether, these results demonstrate that Zbtb16 is able to promote proton leak during mitochondrial respiration and increase mitochondrial

respiration capacity. The enhanced expression of mitochondrial gene markers and mitochondrial respiration capacity suggested a potential effect of Zbtb16 on mitochondrial biogenesis. To

assess mitochondrial density, we measured the relative abundance of mitochondrial-encoded gene _D-loop_ to the nuclear-encoded telomerase reverse transcriptase (_Tert_) by qPCR. _Zbtb16_

expression resulted in increased mitochondrial DNA by threefold in brown adipocytes and by 1.7-fold in C2C12 myotubes (Figure 3e). We next asked whether the increased mitochondrial

respiration owing to _Zbtb16_ overexpression affects lipolysis or lipid content in brown adipocytes. Lipolysis rates in brown adipocytes were not affected by _Zbtb16_ expression, whereas

forskolin elicited the expected increase (Figure 3f). However, intracellular TAG content was decreased by 40% in _Zbtb16_ overexpressing cells (Figure 3g). Together, these data demonstrate

that _Zbtb16_ promotes biogenesis and respiration of mitochondria, and that higher expression of _Zbtb16_ reduces cellular TAG levels in brown adipocytes. ZBTB16 INCREASES GLUCOSE OXIDATION

We then investigated whether _Zbtb16_ affects substrate utilization. Control brown adipocytes or Zbtb16 overexpressing cells were plated in Biolog microplates in which each well contains one

of eight specific energy substrates. All the eight substrates were metabolized by brown adipocytes (Figure 4a and Supplementary Figure S1). Overexpression of _Zbtb16_ specifically increased

utilization of glucose (glycolysis pathway) and xylitol (pentose phosphate pathway), while metabolism of other substrates was not affected by _Zbtb16_. The preference in carbohydrate

utilization suggests that _Zbtb16_ may influence glucose metabolism. Therefore, we assayed the expression of genes controlling glycolysis in response to _Zbtb16_ overexpression using qPCR.

Levels of _Glut4_ mRNA were not changed, but _Hexkin2_ (_hexokinase II_) and _Pkm2_ (_pyruvate kinase_) expression levels were increased by _Zbtb16_ (Figure 4b). In addition, expression of

_Pdk4_ was decreased. Increased glucose utilization and the differential expression of glycolysis-specific enzymes support a role for _Zbtb16_ in glucose consumption. We then assessed the

effects of _Zbtb16_ overexpression on glucose metabolism by quantitating a measure of glycolysis, the ECAR. Addition of glucose or the mitochondrial uncoupler 2,4-dinitrophenol increased

ECAR in differentiated brown adipocytes. This response was amplified in cells with increased _Zbtb16_ expression, suggesting a greater ability to increase glycolysis (Figure 4c). We

therefore exposed the brown adipocytes to different modulators of glycolysis and measured the ECAR response. Cells overexpressing _Zbtb16_ had a greater response to treatment with oligomycin

which inhibits mitochondrial ATP production, suggesting an increased glycolytic capacity when ATP demand increases (Figure 4d). When treated with the glucose analog 2-deoxyglucose, which

inhibits glycolysis, the effect was slightly less pronounced in cells expressing _Zbtb16_ (Figure 4d). We also performed experiments with Na-oxamate, an inhibitor of lactate dehydrogenase,

which catalyzes the conversion of pyruvate to lactic acid. Na-oxamate did not significantly inhibit ECAR in brown adipocytes (∼7%), suggesting that the final step of glycolysis does not

account for a substantial amount of glycolysis detected by the ECAR and that anerobic glycolysis is not very active in these cells. Nevertheless, this modest inhibition was abrogated when

_Zbtb16_ was overexpressed (Figure 4d). Together, these results suggest that _Zbtb16_ promotes glucose oxidation, consistent with the increased mitochondrial respiration and lower _Pdk4_

expression. ZBTB16 EXPRESSION CORRELATES WITH BODY WEIGHT, FAT MASS AND DIABETES _IN VIVO_ As described above, our data indicate that _Zbtb16_ is involved in the adaptive thermogenesis

response and promotes mitochondrial respiration and carbohydrate utilization in brown adipocytes. To assess the role of Zbtb16 on energy balance _in vivo_, we assessed whether genetic

variations in _Zbtb16_ gene expression levels are correlated with clinical traits. For these studies, we examined _Zbtb16_ expression levels in a Hybrid Mouse Diversity Panel.35 This

resource consists of more than 100 highly diverse inbred and recombinant inbred mouse strains, which allow the analysis of association between gene expression levels and metabolic traits

such as plasma lipids, plasma hormones and different body fat parameters.35, 38 We investigated the correlations between _Zbtb16_ expression levels and obesity traits in two tissues

available in this panel that are most relevant to our study: as the BAT and skeletal muscle were not available in the hybrid mouse diversity panel, we examined the white adipose tissue and

heart. The most robust associations for _Zbtb16_ were inverse correlations with body weight and fat mass (measured by nuclear magnetic resonance and weight) in both the tissues (Table 2 and

Supplementary Figure S2). The inverse correlation for fat mass included gonadal, retroperitoneal, femoral and mesenteric fat pads. This finding demonstrates that common, natural variations

in _Zbtb16_ expression levels are correlated with body fat content in the mouse. Given the association of Zbtb16 mRNA levels with body weight and fat mass, we also assessed whether _ZBTB16_

expression levels are associated with fat mass in humans as well. Data mining from previously reported microarray analyses revealed that in visceral adipose tissue of obese diabetic women,

_ZBTB16_ expression was significantly lower than age- and body-mass index-matched normal glucose-tolerant controls (_t_-test _P_-value=0.0056; GEO accession GDS3665). This result

demonstrates that in humans, _ZBTB16_ expression levels are associated with glucose levels, suggesting a complex interrelationship between _ZBTB16_ expression, glucose and obesity.

DISCUSSION Manipulation of cellular bioenergetics is an attractive approach to increase cellular energy expenditure with the ultimate goal to decrease obesity at the whole body level.

Mitochondrial respiration, primarily in the BAT and skeletal muscle, can be modified in order to respond to physical activity, diet or ambient temperature. In the current work, we identified

_Zbtb16_ as a novel gene involved in the cold response in both the BAT and skeletal muscle. _Zbtb16_ was among a few genes that were transcriptionally induced by several-fold in both the

BAT and skeletal muscle during acute cold exposure. Previous works on Zbtb16 have shown potential involvement in cancer and development. The human _ZBTB16_ gene has been implicated in acute

promyelocytic leukemia, through chromosomal translocation and fusion to the retinoic receptor alpha.39, 40 Mice lacking _Zbtb16_ function exhibit limb defects and loss of spermatogonial stem

cells.41, 42 Interestingly, Zbtb16 is induced during differentiation of 3T3-L1 preadipocytes.36 The role of Zbtb16 as a transcription factor provides a potential mechanism by which it may

influence thermogenesis, through effects on expression of other genes. Indeed, we found that induction of _Zbtb16_ expression in brown adipocytes enhances the expression of many genes of the

thermogenic program, such as genes involved in fatty acid oxidation and mitochondrial respiration. This effect was accompanied by an increase in mitochondrial content and maximal

respiration capacity, and a decrease in intracellular TAG levels, consistent with a role for _Zbtb16_ in fatty acid metabolism in brown adipocytes. When differentiated brown adipocytes or

myotubes were activated by adrenoceptor agonists, _Zbtb16_ increased mitochondrial uncoupling, in agreement with the increased _Ucp1_ gene expression. Another aspect of the role of _Zbtb16_

is its function in glucose metabolism. _Zbtb16_ expression drives the expression of glycolysis-related genes (_Hexkn2_, _Pkm2_), and is able to increase glycolytic capacity. Consistent with

this role, _Zbtb16_ expression increases carbohydrate utilization _in vitro_. The glucose utilization seems to be directed to mitochondrial oxidation as observed by the decrease in _Pdk4_

expression and lower Na-oxamate-ensitive ECAR levels. Altogether, these results indicate that Zbtb16 promotes oxidative metabolism. Ultimately, the goal is to identify genes that influence

energy metabolism in BAT that translates to whole-body effects on energy balance and adiposity. To evaluate the potential effect of variations in _Zbtb16_ expression levels _in vivo_, we

assessed the relationship between _Zbtb16_ expression levels and body mass and fat pad mass across a set of >100 mouse strains for which gene expression and body fat traits have been

determined.35 In this panel, we observed a striking association between _Zbtb16_ mRNA levels in two different tissues and body fat parameters. Higher levels of _Zbtb16_ expression were

associated with a decrease in body fat storage in four major white adipose tissue depots and overall body weight. We also detected a correlation between decreased _ZBTB16_ expression levels

in human visceral adipose tissue and diabetic state among obese women. These results are consistent with a role for _Zbtb16_ in increasing mitochondrial respiration and substrate

utilization. Interestingly, a congenic rat strain harboring a mutation in _Zbtb16_ affected total body weight and adiposity, but it was not possible to rule out other mutations in the

congenic strain.43 Although global loss of _Zbtb16_ function in mice41 and humans44, 45 causes skeletal defects, more subtle variation in Zbtb16 expression levels observed among individuals

may be a determinant of adiposity. In the future, cell-type–specific knockout or transgenic models could be useful in determining further the role of _Zbtb16_ in adipose tissue function,

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hypoplasia. _J Med Genet_ 2008; 45: 731–737. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Dr Bruce Spiegelman for the kind gift of the mouse brown adipocyte

cell line and Dr Andrea Hevener for providing cDNA for mouse skeletal muscle subtypes. This work was supported by the American Heart Association Grant 09BGIA2080363 (to LV), National

Institutes of Health P01 HL28481 (to KR), and the National Center for research Resources Grant S10RR026744 (to KR). AUTHOR INFORMATION Author notes * C L Plaisier Present address: 6Current

address: Institute for Systems Biology, Seattle, WA 98109, USA, * B J Bennett Present address: 7Current address: Department of Genetics, University of North Carolina, Chapel Hill, NC 28081,

USA, AUTHORS AND AFFILIATIONS * Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA, USA C L Plaisier, B J Bennett, A J Lusis, K Reue 

& L Vergnes * Department of Applied Genomics, Bristol Myers-Squibb, Pennington, NJ, USA A He & B Guan * Department of Medicine, University of California, Los Angeles, CA, USA A J

Lusis & K Reue * Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA, USA A J Lusis * Department of

Molecular Biology Institute, University of California, Los Angeles, CA, USA A J Lusis & K Reue Authors * C L Plaisier View author publications You can also search for this author

inPubMed Google Scholar * B J Bennett View author publications You can also search for this author inPubMed Google Scholar * A He View author publications You can also search for this author

inPubMed Google Scholar * B Guan View author publications You can also search for this author inPubMed Google Scholar * A J Lusis View author publications You can also search for this

author inPubMed Google Scholar * K Reue View author publications You can also search for this author inPubMed Google Scholar * L Vergnes View author publications You can also search for this

author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to L Vergnes. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION

Supplementary Information accompanies the paper on the Nutrition and Diabetes website SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE S1 (PDF 57 KB) SUPPLEMENTARY FIGURE S2 (PDF 112 KB)

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Bennett, B., He, A. _et al._ Zbtb16 has a role in brown adipocyte bioenergetics. _Nutr & Diabetes_ 2, e46 (2012). https://doi.org/10.1038/nutd.2012.21 Download citation * Received: 16

August 2012 * Accepted: 18 August 2012 * Published: 17 September 2012 * Issue Date: September 2012 * DOI: https://doi.org/10.1038/nutd.2012.21 SHARE THIS ARTICLE Anyone you share the

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Nature SharedIt content-sharing initiative KEYWORDS * Zbtb16 * Plzf * brown adipose tissue * adaptive thermogenesis * bioenergetics