14-3-3ζ coordinates adipogenesis of visceral fat

The proteins that coordinate complex adipogenic transcriptional networks are poorly understood. 14-3-3ζ is a molecular adaptor protein that regulates insulin signalling and transcription factor networks. Here we report that 14-3-3ζ-knockout mice are strikingly lean from birth with specific reductions in visceral fat depots. Conversely, transgenic 14-3-3ζ overexpression potentiates obesity, without exacerbating metabolic complications. Only the 14-3-3ζ isoform is essential for adipogenesis based on isoform-specific RNAi. Mechanistic studies show that 14-3-3ζ depletion promotes autophagy-dependent degradation of C/EBP-δ, preventing induction of the master adipogenic factors, Pparγ and C/EBP-α. Transcriptomic data indicate that 14-3-3ζ acts upstream of hedgehog signalling-dependent upregulation of Cdkn1b/p27Kip1. Indeed, concomitant knockdown of p27Kip1 or Gli3 rescues the early block in adipogenesis induced by 14-3-3ζ knockdown in vitro. Adipocyte precursors in 14-3-3ζKO embryos also appear to have greater Gli3 and p27Kip1 abundance. Together, our in vivo and in vitro findings demonstrate that 14-3-3ζ is a critical upstream driver of adipogenesis.

O besity, a risk factor for many diseases, can result from increased proliferation and/or differentiation of adipocyte precursor cells [1][2][3] . Modulating processes that control the expansion or differentiation of adipose tissue may yield promising drug targets 4,5 , but an incomplete understanding of the complex gene networks that underlie adipogenesis stands in the way of this goal. Precise temporal and spatial control of specific protein-protein and protein-DNA interactions drive the induction of master adipogenic factors 6,7 . The principle events underlying adipogenesis involve the nuclear translocation of CCAAT/enhancer-binding protein (C/EBP)-b and C/EBP-d to initiate the adipogenic programme, leading to the expression of C/EBP-a and peroxisome proliferator-activated receptor-g (Pparg) during terminal adipocyte differentiation 6,7 . Currently, it is not known which proteins ensure the accurate binding and localization of such transcriptional complexes in adipocytes.
Signalling events and networks can be coordinated by adaptor proteins, which facilitate the proper localization of effector molecules, transcription factors and kinases [8][9][10] . Adaptor proteins, such as those of the highly conserved 14-3-3 protein family, remain poorly understood compared with other classes of signalling molecules. These adapters interact with transcription factors harbouring canonical phosphorylated serine and threonine motifs, facilitating their nuclear import or export [9][10][11] . Little is known about the specific adaptor proteins that coordinate the stability and/or nuclear translocation of critical adipogenic factors, but 14-3-3 proteins are ideal candidates. While 14-3-3 isoforms across species display a high degree of homology and may have some functional redundancy, each isoform could perform unique, context-specific functions 12,13 . We have demonstrated that not all 14-3-3 isoforms have equal roles in pancreatic b-cell survival 14 . Whether 14-3-3 isoforms specifically regulate other physiological processes, such as adipogenesis, is still unclear due to the lack of functional studies employing sideby-side comparisons. Aberrant 14-3-3 protein abundance has been proposed to drive the development of various chronic diseases 15,16 . In fact, elevations in 14-3-3b, 14-3-3g and 14-3-3z protein levels have been reported in adipose tissue from obese individuals [17][18][19] . Whether such increases have causal roles in the development of obesity is unclear, but these observations suggest pro-obesogenic roles of this family of adaptor proteins. Given the ability of 14-3-3 proteins to control differentiation in other cell types 20 , it is reasonable to hypothesize that one or more 14-3-3 proteins could play pivotal roles in adipogenesis.
We report herein that out of the seven 14-3-3 isoforms, only 14-3-3z plays an essential role in adipocyte differentiation in vitro. Deletion of 14-3-3z in mice causes marked reductions in adipose tissue within specific depots, as well as metabolic impairments, while 14-3-3z overexpression promotes fat tissue expansion without deleterious metabolic defects. Targeted analysis and unbiased transcriptomics reveals complex mechanisms whereby 14-3-3z regulates a diverse set of parallel and sequential events to drive the adipogenic programme. Loss of 14-3-3z causes the aberrant expression of hedgehog signalling effector, Gli3, and the cyclin-dependent kinase inhibitor, p27 Kip1 , which attenuates adipogenesis. Taken together, our data support the concept that 14-3-3z is a critical upstream regulator of adipocyte differentiation. Therefore, targeting 14-3-3z and components of its interactome may represent novel therapeutic targets for obesity.

14-3-3f regulates adiposity and adipocyte differentiation.
To understand the developmental and physiological roles of 14-3-3z, we examined 14-3-3z-knockout mice . This previously generated mouse model had been used to implicate 14-3-3z in PI3K activation 9 , but characterizations of body composition and/or energy homeostasis had not been reported. Before birth, 14-3-3zKO embryos were smaller and weighed significantly less than wild-type embryos ( Fig. 1a; Supplementary  Fig. 1a). Despite catching up in length to wild-type mice in early adulthood (24 weeks), 14-3-3zKO mice were significantly lighter than wild-type controls due to significantly reduced fat mass as revealed by DEXA body composition analysis (Fig. 1b,c; Supplementary Fig. 1b,c). Analysis of subcutaneous and visceral fat depots showed significant decreases in both gonadal fat and peri-renal fat, but no effect on inguinal fat or brown adipose tissue ( Fig. 1d; Supplementary Fig. 1d). The reduction in fat mass was reflected by significantly reduced fasting and random-fed plasma leptin concentrations, as well as significantly lower triglyceride levels in 14-3-3zKO mice ( Supplementary Fig. 1e). The decrease in adiposity was not associated with alterations in energy expenditure or food intake ( Supplementary Fig. 1f), suggestive of a specific function of 14-3-3z in adipocytes.
Analysis of white adipocyte morphology in gonadal fat pad cross-sections showed significantly smaller adipocytes in 14-3-3zKO mice (Fig. 1e,f), suggesting a less mature cellular phenotype. Protein abundance of Foxo1 and Pparg, markers indicative of mature adipocytes, were significantly reduced in 14-3-3zKO gonadal white adipocytes (Fig. 1g). Pparg mRNA was not altered, suggesting post-transcriptional regulation of this master adipogenesis regulator (Fig. 1h). Of the C/EBP isoforms, only mRNA expression of C/EBP-a was significantly reduced in 14-3-3zKO adipocytes (Fig. 1i), as were Fasn and Atgl mRNA (Fig. 1h). These observations suggest that 14-3-3z deletion results in poorly differentiated, immature adipocytes. The decrease in fat pad size was not associated with increased steady-state apoptosis, quantified by western blot analysis of cleaved caspase-3 (Fig. 1g). Quantitative PCR confirmed that expression levels of remaining 14-3-3 isoforms were unchanged in 14-3-3zKO mice (Fig. 1j), which indicates that any effects in adiposity were specific to changes in 14-3-3z expression and not influenced by alterations in the expression of other 14-3-3 members. Thus, 14-3-3z controls the development and maturity of adipocytes in vivo.
Decreased glucose and insulin tolerance in 14-3-3fKO mice. Decreased 14-3-3z abundance is associated with insulin resistance in humans 21 , but it is unclear if this relationship is causative. Thus, we evaluated glucose homeostasis and insulin sensitivity in 14-3-3zKO mice. No differences in fasting glucose levels were observed between groups (Fig. 1k,m). Intraperitoneal glucose and insulin tolerance tests revealed that 14-3-3zKO mice were mildly glucose intolerant and exhibited mild systemic insulin resistance ( Fig. 1k-n). Significantly higher fasting plasma insulin concentrations were seen in 14-3-3zKO mice (Supplementary Table 1), but the decrease in insulin sensitivity was not due to differences in circulating adiponectin concentrations in 14-3-3zKO mice (Supplementary Table 1). The decrease in insulin sensitivity was associated with decreased Akt activation in livers of 14-3-3zKO mice following an intraperitoneal insulin bolus ( Supplementary Fig. 1j).
Obesity has been associated with increased adipose tissue 14-3-3 protein abundance in several studies [17][18][19] , although it is unknown whether this change alone is sufficient to increase adiposity. To test this hypothesis, we studied transgenic mice with modest global overexpression of 14-3-3z under the control of the ubiquitin-C promoter 22 . Levels of the transgene were equally expressed in insulin-sensitive gonadal white adipose tissue and skeletal muscle (Fig. 2a), as well as other tissues 22 . At 52 weeks of age, 14-3-3z transgenic mice were significantly heavier than their wild-type littermate controls even when fed a normal chow diet (Fig. 2b). Notably, 14-3-3z transgenic mice did not develop glucose intolerance or insulin resistance (Supplementary Fig. 2a-d), suggesting expansion of metabolically neutral adipose tissue. Next, we tested whether overexpression of 14-3-3z promotes increased capacity for adipose expansion in the context of nutrient excess. Indeed, high-fat diet feeding for 8 weeks triggered significantly greater weight gain and fat mass in 14-3-3z overexpressing mice when compared with wild-type littermates ( Fig. 2c-e). Furthermore, these mice had significantly higher Pparg, Lpl and Ap2 expression in gonadal white adipocytes (Fig. 2f). Overexpression of 14-3-3z did not affect expression of other 14-3-3 isoforms in adipose tissue (Fig. 2g), suggesting that these effects are solely due to increased levels of 14-3-3z. High-fat diet promoted the expected glucose intolerance in both wild-type and 14-3-3z-overexpressing mice (Fig. 2h). However, despite markedly greater weight gain, there were no additional deleterious effects on glucose homeostasis in 14-3-3z-overexpressing mice. Similarly, no additional negative effects on insulin sensitivity (1.5 U kg À 1 ) resulted from the increased fat mass in 14-3-3z-overexpressing animals (Fig. 2i). Using a lower dose of insulin (0.75 U kg À 1 ), we found that 14-3-3z-overexpressing mice were actually more insulin sensitive than wild-type littermate controls (Fig. 2k). The degree of hepatic steatosis following high-fat diet exposure was similar between groups, and no differences in circulating plasma free fatty acids and triglycerides were detected (Supplementary Fig. 2e-g). Analysis of genes associated with hepatic lipid metabolism and gluconeogenesis revealed that 14-3-3z-overexpressing mice had decreased transcript abundance of Fasn, Srebp-1c and Acc ( Supplementary  Fig. 2h,i). Collectively, our findings suggest that 14-3-3z is necessary and sufficient to control obesity in vivo.
To further elucidate the molecular mechanisms involved in controlling this master adipogenic network, we tested the possibility that 14-3-3z might control the abundance of C/EBPb and C/EBP-d, regulators of transcription during the early stages of adipogenesis 7 . During the critical first 48 h of 3T3-L1 cell differentiation, we observed parallel increases in the abundance of 14-3-3z together with C/EBP-b and C/EBP-d (Fig. 4a). Pull-down experiments showed that C/EBP-b, but not C/EBP-d, associated with endogenous 14-3-3z in differentiating cells ( Fig. 4b,c). As 14-3-3 proteins participate in the nuclear transport of key adipocyte transcription factors 11 , we tested whether 14-3-3z knockdown might affect the nuclear translocation of C/EBP-b or C/EBP-d during differentiation. Indeed, subcellular fractionation experiments demonstrated that the amount of nuclear-localized 14-3-3z increased during the differentiation process (Fig. 4d). 14-3-3z knockdown had no impact on C/EBP-b nuclear import (Fig. 4d). However, 14-3-3z depletion led to an unexpected and marked degradation of C/EBP-d during differentiation, which reduced its nuclear localization (Fig. 4d). Direct binding to 14-3-3 proteins can prevent protein degradation of target proteins 26 , but we could not detect direct association of C/EBP-d with 14-3-3z (Fig. 4b), suggesting that 14-3-3z regulates the stability of C/EBP-d through indirect mechanisms. We studied the effect of 14-3-3z depletion on C/EBP-d degradation by treating cells with the translation inhibitor cycloheximide and confirmed that 14-3-3z affected the stability of C/EBP-d (Fig. 4e). These findings place 14-3-3z actions at early stages of differentiation that are upstream of canonical master adipogenic transcription factors.
We next sought to determine how 14-3-3z controls C/EBP-d protein stability. To examine the possibility of proteasomemediated degradation of C/EBP-d, we treated 3T3-L1 preadipocytes with MG132 or epoxomicin, during differentiation.  ARTICLE Neither inhibitors affected C/EBP-d stability (Fig. 4f,g) nor were they able to overcome the 14-3-3z siRNA-mediated inhibition of 3T3-L1 differentiation into adipocytes (Fig. 4g). Paradoxically, we also observed a 14-3-3z-dependent increase in the abundance of CHOP, which is known to inhibit C/EBP-b and C/EBP-d 27 and may account for the failure to restore adipogenesis in this context (Fig. 4f). Next we tested the hypothesis that increased autophagy accounted for the decrease in C/EBP-d following knockdown of 14-3-3z, as this isoform has previously been shown to inhibit processes involved in autophagy 28 . Analysis of C/EBP-d protein abundance revealed that inhibition of autophagy with chloroquine during the last 24 h of the induction period was able to maintain C/EBP-d abundance in 14-3-3z-depleted cells. In contrast, inhibition of autophagy during the entire induction period (0-48 h) did not rescue C/EBP-d abundance and actually promoted the loss of 14-3-3z (Fig. 4h). Therefore, autophagy appears to play complex, context-dependent roles in adipogenesis upstream and downstream of 14-3-3z. Inhibition of autophagy itself had inhibitory effects on adipocyte differentiation, and neither 3-methyladenine nor chloroquine rescued the defects in adipocyte differentiation induced by 14-3-3z knockdown (Fig. 4i). While these manipulations are not specific to C/EBP-d, this observation implies that multiple, parallel 14-3-3z-dependent processes are important for adipocyte differentiation and prompted us to broaden the scope of our search for additional mechanisms downstream of 14-3-3z in the context of adipogenesis.
14-3-3f regulates cell cycle progression of pre-adipocytes. Loss of 14-3-3z may impair the nuclear import of critical transcription factors (Fig. 4d) 11,29 and therefore alter the transcriptome of differentiating adipocytes. Thus, we used RNA sequencing to quantitatively measure global changes in the transcriptome and identify downstream effects of 14-3-3z. Over 1,200 genes were significantly altered due to induction of adipocyte differentiation or by knockdown of 14-3-3z (0.05 FDR-adjusted qo0.05) (Fig. 5a), which is not surprising   given the magnitude of the phenotypic differences. Results from the transcriptomic analysis were confirmed by quantitative PCR measurement (Fig. 5b,c). Within the top 25 genes that were significantly changed, we identified genes implicated in adipogenesis, such as Arxes and G0s2 (refs 30,31) (Supplementary Table 2).
To gain a broader understanding of how 14-3-3z depletion affects biological processes within the differentiating adipocyte, we first compared significantly changed genes at t ¼ 0, 24 and 48 h after differentiation (Fig. 5d, Supplementary Fig. 5a,b). Gene ontology classification of significantly differentially expressed genes revealed changes in various biological processes due to 14-3-3z knockdown. Gene-set enrichment analysis 32 revealed that 14-3-3z knockdown significantly modulated multiple cell cycle genes (Supplementary  Tables 3-8). We next investigated how 14-3-3z regulates the cell cycle, a key process in differentiating 3T3-L1 cells 33 , using flow cytometry. Knockdown of 14-3-3z led to an accumulation of cells at G1 phase during the first 48 h of differentiation (Fig. 6a-d). To further understand how depletion of 14-3-3z promoted cell cycle arrest, we examined the expression profiles of cell cycle regulatory genes. Cdkn1b and its product p27 Kip1 were consistently upregulated in 14-3-3z-depleted cells (Fig. 6e,f). p27 Kip1 controls the G1-to S-phase transition in murine pre-adipocytes 33 , and defects in adipogenesis were associated with increased p27 Kip1 abundance during the critical period of differentiation (Fig. 6f). To determine whether the regulation of p27 Kip1 by 14-3-3z during adipogenesis was required for adipogenesis, 3T3-L1 cells were cotransfected with siRNA against 14-3-3z and p27 Kip1 (Fig. 6g,h;  Supplementary Fig. 6a). Simultaneous knockdown of both proteins rescued the defect in adipocyte differentiation, as observed by    Supplementary Fig. 6b). The rescue of differentiation was specific to Cdkn1b/p27 Kip1 knockdown, as depletion of Cdkn1a and Cdkn2c did not rescue the defect in adipogenesis ( Supplementary  Fig. 6d). These observations demonstrate that 14-3-3z functions upstream of the master adipogenic transcriptional programme and is required for the proper maintenance of cell cycle progression during adipogenesis.
14-3-3f inhibits hedgehog signaling to regulate Cdkn1b. Analysis of the Cdkn1b promoter revealed that 14-3-3z knockdown potentiated basal promoter activity of the region between 939 and 554 (Fig. 7a). Within this region, several binding motifs were identified for Gli proteins (Fig. 7b) 34 , which are hedgehog signalling effectors known to regulate adipogenesis 35,36 . Previous studies in other cell types have pointed to physical interactions between the 14-3-3e isoform and Gli proteins 37 . Thus, we assessed whether there are physical and functional links between 14-3-3z and Gli proteins. Using Shh-light2 cells 38 to measure Gli protein-dependent hedgehog activity, we found that 14-3-3z knockdown increased both basal and SAG-induced Gli proteindependent activity (Fig. 7c). The observation that 14-3-3z knockdown increased Cdkn1b promoter activity and Glidependent hedgehog activity (Fig. 7a,c) prompted us to examine whether 14-3-3z regulated the expression of hedgehog effectors themselves. 14-3-3z knockdown decreased Ptch1 expression and significantly increased Smo and Gli3 expression (Fig. 7d). Gli3 can function as an activator or a repressor depending on proteolytic cleavage 39 , and knockdown of 14-3-3z did not promote the expression of the repressor form (Fig. 7e).
Gli3 was found to complex with 14-3-3z during differentiation (Fig. 7f,g), and this was associated with decreased occupancy of Gli3 on the Cdkn1b promoter (Fig. 7h), suggesting that 14-3-3z restricts Gli3-dependent Cdkn1b/p27 Kip1 expression. In undifferentiated cells, transfection with si14-3-3z or siGli3, or co-transfection of si14-3-3z and siGli3, upregulated p27 Kip1 protein abundance. In cells treated with the differentiation cocktail for 48 h, transfection of siGli3 alone potentiated Pparg protein abundance despite increased abundance of p27 Kip1 . Co-transfection of si14-3-3z and siGli3 reduced p27 Kip1 protein abundance and permitted the induction of Pparg and ultimately differentiation into a mature adipocyte (Fig. 7i,j). In contrast, knockdown of the closely related Gli1 and Gli2 genes did not rescue the defect in adipogenesis ( Supplementary Fig. 7). Collectively, these rescue experiments clearly demonstrate that 14-3-3z regulates the expression of p27 Kip1 and the adipocyte progenitor cell cycle through the hedgehog effector Gli3 to control adipocyte differentiation in vitro.
The number of adipocytes is established early in life, with minimal proliferation or apoptosis later in adults 40 . The mechanistic studies described above pointed to an early defect in adipogenesis, perhaps at the level of adipocyte precursors, in 14-3-3zKO mice. Indeed, differences in adiposity could be due to the fact that 14-3-3zKO mice are born either with a reduction in the number of adipose precursor cells or a specific group of precursors that cannot differentiate into mature adipocytes. Thus, we examined e18.5 stage embryos using Pref-1 as a marker for adipocyte precursors 41 . Qualitatively, the number of Pref-1 cells appeared to be slightly decreased (Fig. 8a). We observed striking reductions in the number of lipid-laden 'mature' adipocytes in 14-3-3zKO embryos (Fig. 8a). Adipose precursors in 14-3-3zKO embryos displayed marked increases in Gli3 and p27 Kip1 immunoreactivity (Fig. 8b,c), which complements our in vitro findings where depletion of 14-3-3z increased Gli3 and p27 Kip1 expression in 3T3-L1 pre-adipocytes (Fig. 6f,g; Fig. 7d). Taken together, these findings suggest that in vivo deletion of 14-3-3z alters the expression of Gli3 and p27 Kip1 in adipose precursors in developing embryos before birth and may help set life-long adiposity.
Effects of high-fat feeding in 14-3-3fKO mice. Collectively, the data presented above establish that 14-3-3z plays a critical role in the differentiation of adipocyte progenitors towards a mature state. Next we assessed whether the adipocytes that do develop in 14-3-3zKO mice were capable of expanding in response to a highfat diet challenge. The differences in body weight, body composition and leptin were maintained between 14-3-3zKO and wildtype mice during a 12-week 60% fat diet (Fig. 9a-c).
No differences in fatty acid or triglyceride concentrations were observed (Fig. 9d). High-fat diet-fed 14-3-3zKO mice were modestly glucose intolerant compared with wild-type controls ( Fig. 9e-h). Analysis of white adipocyte morphology from 14-3-3zKO gonadal fat pads revealed an expansion in size similar to that of wild-type mice fed the high-fat diet (Fig. 8i,j). Transcriptome analysis of adult gonadal white adipose tissue revealed only 78 genes that were significantly changed in 14-3-3zKO mice (Supplementary Table 9), suggesting that fat from WT and 14-3-3zKO under these conditions was relatively phenotypically normal. Both genotypes gained weight at a similar rate, suggesting that adaptive weight gain in 14-3-3zKO mice is still possible via hypertrophy of existing adipocytes (Fig. 9i,j). As 14-3-3zKO mice still maintain their differences in adiposity, it implies that the functional capacity of precursor cells to differentiate is reduced early in life.

Discussion
The goal of the present study was to understand the role of a 14-3-3 adaptor protein in obesity and energy homeostasis. We report that the 14-3-3z isoform is uniquely essential for full adipogenesis in vitro and in vivo. Mice lacking 14-3-3z had a visceral adipose depot-specific lean phenotype from birth and mild insulin resistance. Transgenic 14-3-3z overexpression led to the opposite phenotype, exhibiting age-related and high-fat diet-induced obesity without metabolic dysfunction. Mechanistic studies demonstrated that 14-3-3z regulates parallel proximal events underlying adipocyte differentiation, including the control of C/EBP-d stability and cell cycle entry via hedgehog-dependent p27 Kip1 expression (Fig. 10). Collectively, our findings reveal unexpected roles for 14-3-3z in pathways that govern adipocyte differentiation and demonstrate that elevated 14-3-3z expression alone is sufficient to drive obesity.
In the present study, we employed global knockout and overexpression mouse models, which provide information on the systemic effects 14-3-3z. However, without tissue-specific gene manipulation it is not possible to formally rule out potential contributions of non-adipocyte cell types and other tissues in the phenotype of these mice. Notwithstanding, our metabolic cage studies suggested that the decreased adiposity was not due to changes in food intake or whole-body energy expenditure, consistent with a primary role for direct effects on fat tissue. The robust defects in adipogenesis could be recapitulated in mouse embryonic fibroblasts derived from 14-3-3zKO mice and 3T3-L1 and 3T3-F442A pre-adipocytes, which points to cell autonomous effects of 14-3-3z in these in vitro adipocyte models. Comparing the differentiation of control and 14-3-3zKO adipose precursors cells transplanted into wild-type mice 42 would conceivably translate our in vitro observations into an in vivo context, but we estimate that such an experiment would require up to 25 14-3-3zKO donor mice, which represents a nearly insurmountable challenge given the low breeding efficiency in our colony. Moreover, such an experiment would not rule out subtle effects of other organ systems on the overall phenotype of the 14-3-3zKO mice. Collectively, our data point to adipocytecentred effects of 14-3-3z, but it will be important to assess this directly in future studies once adipocyte-specific 14-3-3zKO mice become available.
Another limitation of our study is that we were unable to determine the precise stage(s) when 14-3-3z acts on adipogenesis and glucose homeostasis in vivo without temporal control of our gene manipulations. 14-3-3zKO mice were runted at birth and exhibited rapid catch-up growth. Given that catch-up growth is associated with metabolic disease 43,44 , some of the minor effects on glucose homeostasis and insulin sensitivity in 14-3-3zKO mice may result from aberrant fetal programming. However, the primary phenotype stemming from systemic deletion of 14-3-3z was decreased adiposity, rather than robust changes in glucose homeostasis.
The decreased adiposity from birth observed with in vivo 14-3-3z deletion suggests that 14-3-3z may predetermine the number of maturing adipocyte precursors during development to influence adiposity in adulthood 40 . Subcutaneous adipose tissue is thought to develop embryonically, whereas gonadal adipose tissue is thought to develop postnatally 45,46 , and while we observed striking reductions in the number of mature adipocytes in 14-3-3zKO embryos, it is unclear which depots they will correspond to postnatally. 14-3-3zKO mice still gained weight when challenged with a high-fat diet, suggesting that the developmental effects of 14-3-3z can be uncoupled from pathways controlling adipocyte size in adults. Thus, adipocyte precursors in adult 14-3-3zKO mice, if they play a role in increased adipocyte tissue size, are likely to have intrinsic gene  networks that are independent of the functions of 14-3-3z (refs 3,46,47). It is a limitation of our study that we did not quantify the absolute number of adipocytes in fat pads in our mice, and therefore the relative roles of hyperplasia versus hypertrophy are uncertain in our models.
Molecular adaptors have not been well studied in the context of obesity or glucose homeostasis. Knockdown of 14-3-3b has previously been reported to impair 3T3-L1 differentiation due to defects in lipid storage processes 48 , but we were unable to replicate its requirement using our siRNAs validated not to affect other 14-3-3 isoforms. In contrast, we found a unique role for 14-3-3z in controlling the induction of the master adipogenic transcription factors Pparg and C/EBP-a, which are required for the expression of genes involved in lipid uptake and storage. Pparg and C/EBP-a are both dependent on the actions of and activation of C/EBP-b and C/EBP-d (refs 6,7). Depletion of 14-3-3z promoted autophagy-dependent degradation of C/EBP-d, but additional studies are warranted to understand how 14-3-3z controls C/EBP-d stability, as neither protein was found to direct interaction with the other. We did observe interactions of 14-3-3z with C/EBP-b during differentiation, which suggests a novel mechanism by which 14-3-3z may exert its effects on adipogenesis. Before binding to the Pparg promoter, C/EBP-b is known to form macromolecular complexes consisting of transcription factors, coregulators and 14-3-3y (refs 49,50). As 14-3-3 proteins form heterodimers 9,10 , 14-3-3z may dimerize with 14-3-3y to aid in the formation of these complexes to drive the expression of Pparg. It should be clearly noted that it remains unclear whether the interaction between C/EBP-b and 14-3-3z is direct or indirect, and it is not known whether C/EBP-b harbours the canonical phosphorylation motifs that promote binding 9,10 . We also found that 14-3-3z controls the expression of other genes reported to be required for adipogenesis, such as G0s2 and Arxes 30,31 . Our data point to multiple roles for 14-3-3z in the highly regulated process of adipogenesis.
We also identified a novel role for 14-3-3z in adipocyte progenitor cell cycle progression. Adult human pre-adipocytes, derived from subcutaneous adipose tissue, are not thought to undergo mitotic clonal expansion before undergoing adipogenesis 51,52 , but it remains unclear whether this also applies to fetal pre-adipocytes and/or pre-adipocytes from other depots. Murine pre-adipocytes enter the cell cycle during differentiation via the rapid turnover of p27 Kip1 to promote the expression of C/ EBP-b and C/EBP-d 7,33 . Depletion of 14-3-3z in vitro and in vivo promoted the aberrant expression of Cdkn1b/ p27 Kip1 , which prevented adipogenesis in vitro. Examination of the Cdkn1b promoter revealed binding sites for Gli transcription factors, which are established effectors of the hedgehog signalling pathway and known to interact with 14-3-3 proteins 36,37 . Activation of hedgehog signalling attenuates adipogenesis 35,53 , but the downstream effectors that mediate these effects have yet to be fully elucidated. Gli proteins are required for development and organogenesis 39,54 , and their ability to function as transcriptional activators or repressors 39,54 made them likely candidates to mediate the inhibitory actions of hedgehog signalling on adipogenesis. Our findings directly implicate Gli3 in this process in vitro, as knockdown of 14-3-3z potentiated Gli protein-dependent transcriptional activity and Gli3 depletion was sufficient to restore adipogenesis in 14-3-3z-depleted cells. This places 14-3-3z upstream of hedgehog signalling, p27 Kip1dependent cell cycle progression and ultimately adipogenesis. The in vivo function of Gli3 in the regulation of adipogenesis requires further study, but we observed co-localization of Gli3, in addition to p27 Kip1 , in all Pref-1-marked adipocyte precursor cells in 14-3-3zKO embryos 41 . Taken with our in vitro findings that demonstrate inhibitory actions of Gli3 and p27 Kip1 , this suggests that adipose precursors present in adult 14-3-3zKO mice may have acquired their defect in differentiation during embryogenesis. Furthermore, this raises the possibility of similar defects in human pre-adipocytes during embryo development. Rescue studies employing new transgenic and compound knockout animals will be required to confirm that the Gli3/p27 Kip1 axis is mechanistically downstream of 14-3-3z in vivo.
Pharmacological interventions for obesity have been developed, but their effectiveness and efficacy have been limited 55,56 . In obese individuals, expression of 14-3-3z and other isoforms has been shown to be elevated in visceral and subcutaneous adipose tissue depots [17][18][19] , but whether these changes in 14-3-3 protein expression are causal or associative was not known. Our data suggest that 14-3-3z overexpression exacerbates age-related and diet-induced obesity, independent of changes in glucose tolerance, insulin sensitivity or lipid profile. This suggests that 14-3-3z is a novel factor that may preferentially drive the expansion of metabolically healthy adipocytes 4,5 . It should be noted that 14-3-3zKO and 14-3-3z-overexpressing mice have different genetic backgrounds, precluding direct comparisons between strains. Background strain differences may affect differences in glucose tolerance, insulin sensitivity or weight gain, but do not invalidate the within-model comparisons that involved strict littermate controls. Further studies are required to examine the potential obesogenic effect 14-3-3z overexpression on other genetic backgrounds 57,58 . During the early induction phase of adipocyte differentiation, 14-3-3z functions as a critical upstream regulator of adipogenesis as it controls mitotic clonal expansion through regulation of Gli3-dependent expression of the cyclindependent kinase inhibitor, Cdkn1b/p27 Kip1 . This permits the proper expression of Gli3 and p27 Kip1 in adipose precursor cells in the developing embryo to generate function adipocyte precursors after birth. In the latter stages of the differentiation, 14-3-3z promotes the stability and translocation of C/EBP-d into the nucleus where it is required for inducing the latent expression of the 'master' transcription factors, Pparg and C/EBP-a.
In conclusion, results from this study demonstrate, for the first time, essential roles for 14-3-3z in adipogenesis. Our data add additional levels of complexity to our current understanding of adipocyte differentiation, as one must now consider the function of 14-3-3 proteins and other types of molecular adaptors during adipogenesis. Aside from its ability to enhance the expression of the key adipogenic transcription factors, our transcriptomic analysis revealed the requirement for 14-3-3z in regulating the expression of key genes involved in adipocyte differentiation. This indicates that 14-3-3z has critical roles in the development of mature visceral white adipocytes and that adaptor proteins from the 14-3-3 family can therefore act as specific master regulators of cell differentiation by controlling diverse processes. Our study demonstrates the presence of a 14-3-3z-Gli3-p27 Kip1 axis that regulates adipocyte differentiation and suggest that targeting components of this axis may be a beneficial therapeutic approach for the treatment of obesity.

Methods
Animal husbandry and metabolic analyses. Male 14-3-3z knockout mice on a C57/BL6 background 9 and 14-3-3zTAP transgenic mice on a CD1 background 22 were housed in a specific pathogen-free facility at the University of British Columbia in a 12:12 light: dark, temperature and humidity controlled environment. On a pure C57/BL6 background, heterozygous breeding of mice with 14-3-3z null alleles did not yield progeny at the expected Mendelian ratio. Littermate controls were used in all experiments. For glucose-and insulin-tolerance tests, 14-3-3zKO or 14-3-3zTAP male mice at 10 and 25 weeks or 9 and 52 weeks, respectively, were fasted for 6 h, followed by i.p. injection of 2 g kg À 1 glucose or 1.5 U kg À 1 Humalog insulin (Eli Lilly, Indianapolis, IN), respectively. Tail vein blood glucose levels were measured with a glucometer (OneTouch UltraMini, Life Scan, Milpitas, CA). Commercially available kits were used to measure plasma levels of insulin, adiponectin and corticosterone (Alpco, Salem, NH); leptin (Crystal Chem, Downer's Grove, IL); and triglycerides and free fatty acids (Biovision; Milpitas, CA). Body composition was measured by dual-energy x-ray absorptiometry (DEXA) with a PIXImus Mouse Densitometer (Inside Outside Sales, Madison, WI). Mice were also fed ad libitum a 60% fat diet or its respective 10% control diet (Research Diets, New Brunswick, NJ) for 8 or 12 weeks. Food intake and energy expenditure were measured for 72 h using PhenoMaster metabolic cages (TSE Systems, Bad Homburg, Germany), after 1-week acclimation. Data were averaged from the last two full light:dark cycles. All procedures were approved by the University of British Columbia Committee on Animal Care in accordance with international guidelines.
Measurement of Cdkn1b and Gli-dependent promoter activity. NIH-3T3 cells were co-transfected with plasmids containing Cdkn1b promoter constructs of various lengths upstream of firefly luciferase (provided by Dr D. Everly, Rosalind Franklin University of Medicine and Science, North Chicago, IL) 59 and Renilla luciferase (10:1 dilution), followed by transfection of siRNA against 14-3-3z or the scrambled control. To determine hedgehog-dependent transcriptional activity, Shh-light2 cells were treated with the synthetic Smoothened agonist (SAG; Cayman Chemicals, Ann Arbor, MI) or transfected with siRNA against 14-3-3z or the scrambled control. Luciferase activity was measured after 24 or 48 h with the Dual-Luciferase Reporter Assay system (Promega, Madison, WI). Transcription factor binding sites were analysed with MotifMap 34 . ChIP analysis of Gli3 binding to the Cdkn1b promoter was performed with antibodies against Gli3 and the Pierce Magnetic ChIP kit, as per the manufacturer's protocol (Thermo Scientific, Rockford, IL).
Flow cytometry was performed on 3T3-L1 cells transfected with a scrambled control or siRNA against 14-3-3z, then induced to differentiate with MDI and harvested at 0, 24 and 48 h for, as previously described 62 , on a LSR II-561 Flow Cytometer (BD Biosciences, San Jose, CA). Quantification of cells at various stages of the cell cycle was performed by FlowJo software (v.10, Treestar, Ashland, OR).
RNA isolation, quantitative real-time PCR and transcriptome analysis. RNA was isolated from mouse tissues or 3T3-L1 adipocytes with the RNEasy kit (Qiagen, Mississauga, ON, Canada). Transcript levels of synthesized cDNA (Quanta Biosciences, Gaithersburg, MD) were measured with SYBR green chemistry on a StepOnePlus Real-time PCR System (Life Technologies). All data were normalized to HPRT by the 2 À DCt method as described by Livak and Schmittgen 63 . Libraries for RNA-Seq were generated from isolated RNA, as recommended by the manufacturer's protocol (Illumina, Carlsbad, CA). Following pooling of sequence-indexed libraries, sequencing was performed on a HiSeq 2500 (Illumina) collecting 20 million paired-end reads (150 bp x2). Alignment of reads to the mouse genome (Ensembl NCBIM37) and analysis of differentially expressed genes (0.05 FDR-adjusted qo0.05) were performed by TopHat software (v.2.0.11) and the Cufflinks package (v.2.2.0), respectively 64 . Panther and gene-set enrichment analysis (GSEA) were performed on all RNA-Seq results used to examine gene sets or biological processes that were significantly enriched 32,65 .
Statistical analysis. All data are expressed as the mean ± s.e.m. Data were analysed by ANOVA followed by Dunnett or Bonferroni t-tests, or by Student's t-tests, and significance was achieved when Po0.05. A minimum of n ¼ 3 independent experiments was performed for analysis.