AHNAK deficiency promotes browning and lipolysis in mice via increased responsiveness to β-adrenergic signalling

In adipose tissue, agonists of the β3-adrenergic receptor (ADRB3) regulate lipolysis, lipid oxidation, and thermogenesis. The deficiency in the thermogenesis induced by neuroblast differentiation-associated protein AHNAK in white adipose tissue (WAT) of mice fed a high-fat diet suggests that AHNAK may stimulate energy expenditure via development of beige fat. Here, we report that AHNAK deficiency promoted browning and thermogenic gene expression in WAT but not in brown adipose tissue of mice stimulated with the ADRB3 agonist CL-316243. Consistent with the increased thermogenesis, Ahnak−/− mice exhibited an increase in energy expenditure, accompanied by elevated mitochondrial biogenesis in WAT depots in response to CL-316243. Additionally, AHNAK-deficient WAT contained more eosinophils and higher levels of type 2 cytokines (IL-4/IL-13) to promote browning of WAT in response to CL-316243. This was associated with enhanced sympathetic tone in the WAT via upregulation of adrb3 and tyrosine hydroxylase (TH) in response to β-adrenergic activation. CL-316243 activated PKA signalling and enhanced lipolysis, as evidenced by increased phosphorylation of hormone-sensitive lipase and release of free glycerol in Ahnak−/− mice compared to wild-type mice. Overall, these findings suggest an important role of AHNAK in the regulation of thermogenesis and lipolysis in WAT via β-adrenergic signalling.

Previous studies suggest that β -adrenergic signals produced by norepinephrine and β -adrenergic agonists can enhance lipolysis and adaptive thermogenesis 14,15 . Three adrenergic receptors mediate thermogenesis via sympathetic activation; among them, β 3-adrenergic receptor (ADRB3) has been shown to alleviate obesity and diabetes and is abundant in rodent adipose tissue 16,17 . CL-316243, an ADRB3 agonist, has been utilized to mimic the browning effect in WAT of rodents 18 . In adipocytes, CL-316243 stimulates the production of cyclic AMP (cAMP), an activator of protein kinase A (PKA) that upregulates UCP1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α ), which is involved in thermogenesis 19,20 .
The neuroblast differentiation-associated protein AHNAK is a ubiquitously expressed high-molecular-weight protein essential for cell migration; it has been shown to regulate the actin cytoskeleton by functioning as a molecular linker for cardiac calcium channels 21 . We recently reported that Ahank knockout (KO) mice exhibit enhanced insulin sensitivity and higher energy expenditure under a high-fat diet 22 . The levels of UCP1 mRNA were increased in epididymal (e)WAT from high fat diet-fed Ahnak −/− mice compared with that from wild-type mice. Although AHNAK is highly expressed in adipose tissue and up-regulated in murine models of obesity 23,24 , the thermogenic function of AHNAK in adipose tissue has not been determined.
In the present study, we demonstrate that loss of AHNAK leads to browning of WAT depot and promotes energy expenditure by β -adrenergic stimulation. AHNAK gene ablation upregulates mitochondria biogenesis and enhances lipolysis via induction of PKA-mediated gene expression in WAT in response to β -adrenergic signalling.

Results
Regulation of AHNAK in obesity. AHNAK is involved in adipocyte differentiation and plays a crucial role in body fat accumulation 22 . To determine the expression of AHNAK in different fat depots, adipose tissues were classified as visceral (visc), subcutaneous (subQ), and brown fat (BAT). Notably, AHNAK expression was higher in visceral tissues compared to SubQ fat (Fig. 1A). AHNAK gene expression was significantly elevated in BAT and epididymal (eWAT) and inguinal (iWAT) fat depots in mice fed a high-fat diet (HFD) compared to mice fed a regular chow (RC) diet (Fig. 1B). In agreement with our previous data 22 , the AHNAK KO mice on the HFD exhibited decreased body weight when compared with wild-type and heterogygote knockout mice (Fig. 1C). Furthermore, AHNAK KO mice had significantly reduced body fat contents (Fig. 1E,F). Gene profiling of eWAT from HFD fed AHNAK KO mice showed increased insulin signalling and glucose metabolism. In contrast, inflammation-related genes were downregulated in HFD-fed AHNAK KO mice compared with their HFD-fed WT littermates (Fig. 1D). Consistently, HFD-induced macrophage infiltration in adipose tissue was less abundant in AHNAK KO mice (Fig. 1G). Expression of inflammatory genes, such as CD68, F4/80, monocyte chemoattractant protein-1 (MCP1), IL6, and TNF-α , was significantly decreased in eWAT of HFD-fed AHNAK KO mice with upregulation of M2 macrophage specific transcripts, including those encoding macrophage galactose N-acetyl-galactosamine specific lectin 2 (Mgl2), mannose receptor, C type 2 (Mrc2) and IL10 (Fig. 1H). Serum inflammatory parameters were also decreased in AHNAK KO mice (Fig. 1I).
We previously reported that AHNAK ablation increased energy expenditure in HFD-fed animals 22 . Consistently, thermogenic genes including Ucp1, Pgc-1α , Cidea, Cpt2, and Adrb3 were significantly upregulated in the eWAT and iWAT of HFD-fed AHNAK KO mice (Fig. 1J,K). However, there were no differences in BAT ( Supplementary Fig. 1A). Notably, ablation of AHANK leads to upregulation of Adrb3, which is essential for the regulation of adaptive thermogenesis and oxidative metabolism 25 . These findings suggest that AHNAK ablation in WAT protects mice from obesity and its related complications accompanied by elevation of energy expenditure. AHNAK ablation promotes a thermogenic gene program and browning in WAT but not in BAT via β-adrenergic stimulation. To evaluate the role of AHNAK in cold-induced adaptive thermogenesis, mice were exposed to an ambient temperature of 4 °C for 3 days and compared to the animals kept at thermoneutral conditions (30 °C). AHNAK expression was selectively decreased in eWAT and iWAT (p < 0.07) ( Supplementary Fig. 1B). At 30 °C, AHNAK KO mice had few UCP1-positive adipocytes and their morphology was similar to that of the wild-type mice. However, after cold exposure, AHNAK KO mice demonstrated an increase in multilocular UCP1-expressing adipocytes in eWAT and inguinal iWAT (Supplementary Fig. 2A). Both eWAT and iWAT but not BAT from AHNAK-deficient cold-exposed mice tended to have higher mRNA levels of thermogenic genes (Pgc1α , Cidea, Dio2, Cpt1); in addition, eWAT in these mice was enriched for beige adipocyte markers (CD137, Tmem26) compared to the wild-type mice after cold exposure (Supplementary Fig.  2B-D). These results suggest that AHNAK genetic ablation promotes adaptive browning of WAT under environmental stimulation. The sympathetic nervous system plays a critical role in BAT activation and adaptive thermogenesis mediated by β -adrenergic receptors 25,26 . To examine whether β -adrenergic signalling influenced thermogenesis, AHNAK KO mice were administered CL-316243 (CL), an ADRB3 agonist. When CL was administered to wild-type mice, we found significantly lower expression of AHNAK in the eWAT and iWAT, but not BAT ( Fig. 2A). Histological analysis revealed numerous multilocular, UCP1-expressing adipocytes in the WAT of CL-treated AHNAK KO mice relative to wild-type controls. (Fig. 2B,C). We also examined cell proliferation in adipose tissue after CL treatment by BrdU labelling ( Supplementary Fig. 3A). The frequency of BrdU + cells expressing PDGFRα , a marker of adipocyte progenitors, was similar between WT and AHNAK KO mice under non-CL treatment ( Supplementary Fig. 3B,C). We observed that 6.18 ± 0.77% of dividing cells expressed detectable adiponectin, a mature adipocyte marker that is constitutively expressed in non-CL-treated WAT of WT mice. This proportion was comparable to that of KO mice (6.64% ± 0.39) (Supplementary Fig. 3B.C). CL treatment increased the number of UCP1 + cells that incorporated BrdU (Supplement Fig. 3D,E). However, more UCP1 + cells were positive for BrdU in the CL-treated WAT of AHNAK KO mice than in that of WT mice ( Supplementary Fig. 3D,E). These results indicate that the increase in UCP1 + adipocytes in the WAT of AHNAK KO mice is derived from increased proliferation of cells under β 3 adrenergic stimulation. At the mRNA level, the expression of both brown Scientific RepoRts | 6:23426 | DOI: 10.1038/srep23426 adipocyte-related genes (Ucp1, Cidea, Dio2, and Pgc1α ) and mitochondrial genes (Cpt1, Cpt2, and Cox8b) in CL-stimulated eWAT and iWAT was higher in AHNAK KO mice than in WT mice (Fig. 2D). Western blot analysis showed that UCP1, PGC1α , and CPT2 levels were significantly elevated in eWAT and iWAT from CL-stimulated AHNAK KO mice compared to that from WT mice (Fig. 2E,F). However, there were no obvious changes in the histological morphology and activity of BAT between WT and AHNAK KO mice (Fig. 2B, Supplementary Fig.  4A,B). Furthermore, AHNAK KO mice exhibited a higher core body temperature than wild-type mice after CL injection (Fig. 2G). Consistent with a previous report 27 , oxygen consumption (VO 2 ) and energy expenditure were significantly elevated in AHNAK KO mice compared to WT mice after CL stimulation (Fig. 2H,I). These results suggest that β 3-adrenergic stimulation mediates the formation of multilocular adipocytes and enhances adaptive thermogenesis through upregulation of thermogenic genes in the AHNAK-deficient WAT depots, leading to increased energy expenditure.
In addition, ADRB3 stimulation has been associated with reduction of fat mass and induction of mitochondrial biogenesis 28 . To determine whether the increase in energy expenditure and UCP1 expression in AHNAK-deficient WAT was associated with mitochondrial activation, we investigated the changes in mitochondrial biogenesis in adipose tissues. Expression of the OXPHOS genes in mitochondrial respiratory complexes I-V was remarkably increased in the WAT of CL-treated AHNAK KO mice compared to that of wild-type animals (Fig. 2J,K); however, no significant effect was observed for BAT ( Supplementary Fig. 4C). Thus, the increased Increased levels of eosinophils and type 2 cytokines in AHNAK-deficient WAT. We observed phenotypic change into brown-like adipocytes in CL-stimulated WAT, even though we used mice with a null mutation. We therefore hypothesize that this enhanced thermogenesis through β -adrenergic signalling can be induced by non-adipose progenitor cell types. Recent studies have focused on the eosinophil-mediated regulation of beige fat development [29][30][31] . To investigate whether or not eosinophils mediate WAT browning induced by β -adrenergic stimulation, we isolated eWAT and identified cell lineages that express sialic acid-binding immunoglobulin receptor (Siglec-F) and CD11b as molecular markers for eosinophils (Fig. 3A). CL exposure increased the frequency and number of Siglec-F + /CD11b + cells, representing eosinophils, in AHNAK-deficient eWAT (Fig. 3B,C). Consistently, mRNA levels of eosinophil (Siglec-F and Ccr3), type 2 cytokines (IL-4, IL-4ra, and IL-13), and an alternatively activated M2-specific gene (IL-10) were also significantly increased in the eWAT and iWAT of CL-treated AHNAK KO mice (Fig. 3D,E), but not in the BAT (Fig. 3F). Eosinophils and type 2 cytokines are implicated in regulation of the biogenesis of thermogenic beige fat 30,32 . Together, these results indicate that genetic ablation of AHNAK augments eosinophil content and type 2 cytokine signals in CL-treated WAT, which could promote WAT browning upon β 3-adrenergic stimulation. Ablation of AHNAK increases fat lipolysis. Stimulation by a β 3-adrenergic receptor agonist induces activation of protein kinase A (PKA), which then leads to lipolysis mediated by phosphorylation of hormone-sensitive lipase (HSL) 33 . Therefore, we investigated catecholamine signalling in the adipose tissue of CL-treated AHNAK KO mice. There was no difference in the serum concentration of free glycerol between AHANK KO and wild-type mice after CL injection, probably because of a decrease in body fat in AHNAK KO mice (Fig. 4A). However, exposure to CL316243 resulted in a higher fold increase in glycerol release in AHNAK KO mice than in wild-type mice (Fig. 4B). Importantly, AHNAK-deficient WAT also displayed increased expression of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of catecholamines (Fig. 2E,F). Moreover, circulating levels of epinephrine were also increased in KO mice (Fig. 4C), suggesting that white fat browning may be attributed to enhanced sympathetic input to WAT in AHNAK-deficient mice. Furthermore, Adrb3 mRNA expression was significantly increased after CL stimulation in WAT but not in BAT of AHNAK KO mice (Fig. 4D). However, the expression of other adrenergic receptor subtypes, i.e., Adrb1 and Adrb2, was not altered (Supplementary Fig.  5A,B). As previously decribed 34 , the levels of Rgs2, a negative regulator of adrenergic signalling, were also not different between the two groups ( Supplementary Fig. 5C). These results suggest that robust sympathetic tone and enhanced catecholamine sensitivity may alter β -adrenergic responsiveness in AHNAK-deficient WAT. In support of this notion, PKA activity in WAT was substantially elevated in AHNAK KO mice as evidenced by the increase in the level of phosphorylated PKA substrates and hormone sensitive lipase (HSL) in both eWAT and iWAT of AHNAK-deficient mice after CL injection (Fig. 4E,F); however, no changes were observed in BAT ( Supplementary Fig. 5D). Furthermore, AHNAK KO mice exhibited a higher CL-stimulated lipolytic response after HFD feeding (Fig. 4G,H). Thus, AHNAK may regulate catecholamine sensitivity in WAT.
Mice lacking AHNAK exhibited enhanced β -adrenergic signalling in WAT; therefore, we examined the effect of AHNAK on acute adrenergic signalling in murine adipose tissue and adipocytes. Isolated adipose tissues were incubated with CL for 2 h at 37 °C, and the release of FFA and glycerol to media was determined and normalized to the fat tissue weight. In both eWAT and iWAT of AHNAK-deficient mice under either RC or HFD feeding, CL treatment led to significantly increased lipolysis, as indicated by FFA and free glycerol release (Fig. 5A-D). Taken together, these results suggest that fat increased lipolysis in AHNAK ablation is mediated by β 3-adrenergic signalling through induction of Adrb3.

Discussion
Understanding of signalling pathways that regulate thermogenesis and enhance energy expenditure and identification of their potential targets is important for the development of measures to control obesity. It has been suggested that activation of brown adipocytes and increased BAT mass may constitute effective strategies to counteract obesity 7,8 . Recent studies have reported that catecholamine resistance in adipose tissue could be associated with obesity 35,36 . AHNAK-deficient mice were previously shown to display increased energy expenditure during HFD challenge, indicating resistance to obesity 22 . Here, we revealed a critical role of AHNAK in regulating browning of WAT in response to β -adrenergic stimulation. AHNAK deficiency promotes an increase in the number of functional and proliferating brown-like adipocytes in WAT depots, which results in higher energy ADRB3 agonists have been recognized as critical mediators of lipolysis and thermogenesis. ADRB3 activation stimulates cAMP and PKA activation, which promotes adipocyte lipolysis via HSL phosphorylation 33 . ADRB3-activated PKA stimulates the transcriptional coactivator PGC1α , leading to increased UCP1 expression and thermogenesis 19,37 . Efforts have been made to develop therapeutic approaches targeting ADRB3 to treat or prevent obesity and metabolic disorders, and CL-316243 has been used for generating brown-like adipocytes in WAT 17,38 . However, the development of such drugs has been challenging because of insufficient oral bioavailability and cross-reactivity with ADRB1 39,40 . Nevertheless, new ADRB3 agonists have been investigated in clinical trials and have shown improved bioavailability and binding affinity [41][42][43] . In this study, AHNAK-deficient CL-316243-stimulated mice demonstrated PKA activation in WAT, which was accompanied by increased phosphorylation of HSL and FFA mobilization, indicating enhanced lipolysis. Moreover, genetic ablation of AHNAK increased the expression of genes involved in fat browning (Ucp1, Pgc1a, and Cpt2) and mitochondrial oxidative phosphorylation in response to β -adrenergic signalling. Our results suggest that AHNAK deficiency may contribute to improved catecholamine sensitivity by enhancing ADRB3 expression and signalling in WAT.
Several factors have been reported to be involved in fat browning and thermogenesis. PGC1α has been recognized as a key regulator of energy metabolism and mitochondrial biogenesis. PGC1α activation regulated by mitogen-activated protein kinase p38 enhances the expression of thermogenic genes in adipose tissues in response to β -adrenergic signaling 44 . It has been shown that Pgc1α −/− mice exhibit an abnormal thermogenic phenotype, suggesting that PKA activation, which regulates PGC1α expression, mediates thermogenesis 45 . CPT2 is a crucial regulator of long chain fatty acid transport and is involved in initiation of β -oxidation in mitochondria. Mice overexpressing the vitamin D receptor were previously shown to have reduced energy metabolism, which correlated with decreased fatty acid oxidation and lipolysis, in addition to suppression of CPT2 in WAT 46 . Mice with adipose tissue-specific CPT2 ablation are defective in cold-or CL-induced thermogenesis because of deregulation of fatty acid oxidation, suggesting that the increase in CPT2 underlies the enhanced lipolysis and thermogenesis in AHNAK KO mice 47 .
A local thermogenic circuit involving eosinophils and type 2 cytokines activates WAT browning and induces thermogenesis 29,30 , whereas inhibition of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis, or IL-4/13 signalling disrupts browning-related responses 30,48 . Here, we investigated the process of generating functional beige cells in AHNAK-deficient WAT. AHNAK ablation led to robust recruitment of eosinophils into WAT and induced the secretion of eosinophil-derived cytokines after β -adrenergic stimulation, resulting in the production of epinephrine accompanied by increased expression of IL-10 and tyrosine hydroxylase, which promotes WAT browning and enhances mitochondrial biogenesis. It is still unclear whether eosinophils are regulated in an AHNAK-dependent manner because these parameters are also increased in unstimulated AHNAK-deficient eWAT. These results suggest that the increase in eosinophils and cytokine (IL-4/IL-13) secretion by adipocyte precursors could lead to catecholamine-induced WAT browning in AHNAK KO mice.
Collectively, our results demonstrate that the enhanced response to β -adrenergic signalling in AHNAK KO mice induces the development of beige fat, which promotes thermogenic effects with minimal alteration in BAT activity and mass. Although the precise molecular mechanism underlying AHNAK involvement in β -adrenergic signalling remains to be determined, downregulation of AHNAK may be a new approach to increase the response to β -adrenergic stimulation and energy expenditure in adipose tissue, thereby treating obesity.

Methods
Animals. AHNAK KO mice were generated by disruption of exon 5 in the Ahnak gene, as previously Indirect calorimetry. Oxygen consumption (VO 2 ), carbon dioxide production (VCO 2 ), respiratory exchange ratios (RER), and heat production were measured using an indirect calorimetry system (TSE Systems, Core body temperature measurement. Body temperature was measured using telemetry transmitters (TSE Systems). A transmitter was implanted into the peritoneal cavity under anaesthesia.
Quantitative real-time PCR. Total RNA was extracted using an RNA purification system (Invitrogen) following the manufacturer's protocol. mRNA was reverse-transcribed using AccuPower CycleScript RT PreMix (Bioneer, Daejeon, Korea) and quantitative (q)PCR was performed using SYBR Green and an ABI StepOne Real-Time PCR instrument (Applied Biosystems, Cheshire, UK). Target gene expression was normalized to that of the control gene (36B4), and relative expression was quantified by the comparative Ct method (ΔΔCt). The primers used for PCR are listed in Supplementary Table 1. Lipolysis assay. To assess in vivo lipolysis, serum free fatty acids (FFAs) were measured in cardiac blood obtained from non-fasted mice before and 20 min after i.p. injection of CL316243 (1 mg/kg). Ex vivo and in vitro lipolysis was measured as described previously 36,50 with some modifications. Briefly, for ex vivo analysis, adipose tissues were surgically removed from male and female mice, weighed, and washed with cold PBS; fat depots were incubated with CL316243 (1 μM) or vehicle in Krebs-Ringer Bicarbonate Buffer (KRBH, Sigma) containing 1% fatty acid-free BSA (GenDEPOT) and glucose (2.5 mM) for 2 h at 37 °C with mild shaking. After incubation, FFA and glycerol were measured using a glycerol-free reagent (Sigma) and the FFA quantification kit (BioVision, Mountain View, CA, USA) and normalized to the weight of adipose tissue samples.

Statistics.
All values were expressed as the mean ± SEM. Statistical analysis was performed using Student's t-test between two groups. The Kruskal-Wallis test was used for flow cytometric analysis. P < 0.05 was considered significant.