Nutrient overload, particularly in combination with a sedentary lifestyle, is the main cause of the increasing incidence of obesity we are facing today. In most cases, chronic obesity provokes the development of metabolic diseases like insulin resistance, type 2 diabetes (T2D), non-alcoholic fatty liver disease (NFALD), and non-alcoholic steatohepatitis (NASH) (Longo et al., 2019). The surplus of fat is stored in the white adipose tissue (WAT) largely without an increase in adipocyte number, resulting in an increase in fat cell size. This hypertrophy is accompanied with reduced vascularization and oxygen supply, and an increase in macrophage infiltration, and inflammation (Frasca et al., 2017; Tchkonia et al., 2010). Since the storage capacity of the hypertrophic cells is limited, fat starts to accumulate in ectopic tissues like liver, heart and skeletal muscle (Frasca et al., 2017). This steatosis, also referred to as ‘lipotoxicity’ further promotes metabolic disorders. However, there is an exception from this scenario as individuals exist that are chronically obese but stay – at least transiently – metabolically healthy. Evidence from mouse and a few human studies suggest that storing surplus of nutrients as fat through adipocyte hyperplasia (increasing number) is associated with metabolic health (White and Ravussin, 2019). As the fat storage can be distributed over more fat cells, the individual fat cells stay smaller, are metabolically more active, and less inflamed (Ghaben and Scherer, 2019). So far not much is known about the underlying molecular mechanisms and involved regulators that drive fat storage in either the hypertrophic or the hyperplastic direction. Such regulators may be attractive targets to therapeutically switch the adipocytes from a hypertrophic into a hyperplasic state in order to prevent metabolic complications associated with obesity.

CCAAT/Enhancer Binding Protein beta (C/EBPβ) is a transcription factor known to regulate adipocyte differentiation together with other C/EBPs and peroxisome proliferator-activated receptor gamma (PPARγ) (Siersbæk and Mandrup, 2011). In all cases of C/EBPβ controlled cellular processes, it is important to consider that different protein isoforms of C/EBPβ exist. The two long C/EBPβ isoforms, LAP1 and LAP2 (Liver-enriched activating proteins) differ slightly in length and both function as transcriptional activators. The N-terminally truncated isoform LIP (Liver-enriched inhibitory protein) acts inhibitory because it lacks transactivation domains yet binds to DNA in competition with LAP1/2 (Descombes and Schibler, 1991). We have shown earlier that LIP expression is stimulated by mTORC1 signaling involving a cis-regulatory short upstream open reading frame (uORF) in the Cebpb-mRNA (Calkhoven et al., 2000; Zidek et al., 2015).

Mutation of the uORF in mice (Cebpb^ΔuORF mice) results in loss of LIP expression, unleashing LAP transactivation function, resulting in C/EBPβ super-function. (Müller et al., 2018; Wethmar et al., 2010; Zidek et al., 2015) In Cebpb^ΔuORF mice, metabolic and physical health is preserved and maintained during ageing with features also observed under calorie restriction (CR), including leanness, enhanced fatty acid oxidation, prevention of steatosis, better insulin sensitivity and glucose tolerance, preservation of motor coordination, delayed immunological ageing and reduced interindividual variation in gene expression of particularly metabolic genes (Müller et al., 2018; Zidek et al., 2015).

Here, we show that C/EBPβ is critically involved in dictating the adipocyte phenotype and the metabolic outcome in response to high-fat diet (HFD) feeding in mice. C/EBPβ super-function in Cebpb^ΔuORF mice causes fat to accumulate in hyperplastic rather than hypertrophic depots. In addition, Cebpb^ΔuORF male mice store the surplus of fat more efficiently in subcutaneous fat stores. Accordingly, the Cebpb^ΔuORF mice are protected against the development of steatosis and better maintain glucose tolerance, indicating a healthy obese phenotype.

Separate cohorts of male and female Cebpb^ΔuORF mice and wild-type (wt) control littermates on a C57BL/6 J background were fed a high-fat diet (HFD; 60% fat) or standard chow (10% fat) for a period of 19 weeks. In males, both genotypes gained weight over the whole experimental period yet with significantly lower body weights for Cebpb^ΔuORF mice, suggesting that they are partially protected from HFD induced weight gain (Figure 1A). Both the food intake and the energy efficiency (energy that was extracted from the food during digestion) were similar in Cebpb^ΔuORF and wt males (Figure 1—figure supplement 1A, B). Lean mass and fat mass body composition was determined by computer tomography (CT) after 19 weeks of HFD feeding. On standard chow, fat mass of Cebpb^ΔuORF males was reduced compared to wt males (Figure 1B) as we showed before (Zidek et al., 2015). Unexpectedly, we observed a slight but significantly increased overall fat mass volume in the Cebpb^ΔuORF males compared to wt controls (Figure 1B), which correlates with a relative reduction in lean mass (Figure 1—figure supplement 1C). There was no significant difference in the amount of visceral fat mass between wt and Cebpb^ΔuORF males under HFD (Figure 1C). However, the HFD fed Cebpb^ΔuORF males had a significantly increased subcutaneous fat mass compared to the wt controls (Figure 1D). Taken together, the data show that Cebpb^ΔuORF male mice store more fat upon HFD feeding compared to wt littermates, and that this extra fat is mainly stored in the subcutaneous fat depot. Similar to male mice, the body weight gain of Cebpb^ΔuORF females after 19 weeks on HFD was smaller compared to the weight gain of wt females (Figure 1E). However, different from the male mice, in females this was associated with a reduced accumulation of fat particularly in the subcutaneous depot in Cebpb^ΔuORF compared to wt females as was determined by fat tissue weight (Figure 1F, G).

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Next, we compared histological sections from visceral fat of HFD fed Cebpb^ΔuORF mice and wt littermates and observed that the adipocyte size in the Cebpb^ΔuORF mice for both sexes is significantly smaller compared to the wt mice (Figure 2A, B). Calculated from visceral fat volume (CT analysis) of males or visceral fat weight of females and the average adipocytes area (histology) per mouse, the number of adipocytes is approximately 3 times higher in Cebpb^ΔuORF males and 1.5 times higher in Cebpb^ΔuORF females compared to their wt littermates.

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Adipose tissue composed of small adipocytes is metabolically more active and better supplied with oxygen, and its inflammatory state is usually lower than that of enlarged adipocytes (Ghaben and Scherer, 2019). We therefore analyzed the inflammatory state of visceral white adipose tissue (WAT) by determining the expression of the macrophage marker CD68 and the inflammatory cytokines TNFα, MCP1, IL-1 and IL-6 using quantitative PCR (qPCR) and immunohistochemistry (anti-CD68). Male wt mice on HFD show a strong increase in CD68 mRNA expression compared to wt males on ND indicating increased macrophage infiltration. In contrast, CD68 mRNA expression is much lower in the visceral fat from HFD fed Cebpb^ΔuORF males and not significantly different from ND fed mice (Figure 3A). Accordingly, histological staining of visceral WAT derived from three different mice shows more pronounced macrophage infiltration in wt mice on HFD (19 weeks) compared to Cebpb^ΔuORF males (Figure 3B). In addition, expression of the inflammatory markers TNFα, MCP1, IL-1β, and IL-6 was significantly induced in the visceral fat of wt males on HFD (Figure 3C). In contrast, in Cebpb^ΔuORF males, HFD feeding did not significantly induce TNFα and IL-1β expression, and the induction of MCP1 was significantly lower compared to HFD fed wt males. Solely IL-6 levels were comparable between the two genotypes on HFD feeding. For female wt mice the mean value of induction of CD68 expression in response to HFD is high but strongly varies between the mice. Therefore, the lower value of CD68 expression in HFD fed Cebpb^ΔuORF mice is not statistically significant yet shows much less variation (Figure 4A). Notwithstanding, the anti-CD68 staining of visceral WAT did show infiltration by macrophages in wt females on HFD although to a lower extend compared to wt males, while in Cebpb^ΔuORF females hardly any staining was observed (Figure 4B). The inflammatory markers TNFα, MCP1 and IL-1 are all induced in wt and Cebpb^ΔuORF mice on HFD feeding to similar extends and thus no differences are measured between the genotypes. No induction was observed for IL6 expression in both genotypes upon HFD feeding (Figure 4C).

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Together, these data demonstrate that Cebpb^ΔuORF mice on HFD feeding store extra fat through an increase in adipocyte numbers (hyperplasia), which results in smaller sized adipocytes and reduced fat tissue inflammation in males. In Cebpb^ΔuORF females, adipocytes are also smaller but consistent differences in the inflammation state of the visceral fat were not measured.

Adipocyte hypertrophy under obese conditions is associated with a limit in fat storage capacity of the adipocytes and enhanced lipolysis (Khan et al., 2009; Laurencikiene et al., 2011) The resulting increase in the concentration of fatty acids in the circulation causes lipid accumulation in non-fat tissues like liver, muscle and heart (Longo et al., 2019). In a previous study, we showed that Cebpb^ΔuORF males on a C57Bl/6 genetic background are protected against age-related steatosis on a ND, compared to wt mice that do accumulate fat in the liver at an age of 8 months (Zidek et al., 2015). To investigate possible differences in HFD-induced steatosis, we stained histological sections of liver for fat accumulation. Livers of both male and female wt mice showed massive fat accumulation (steatosis) on HFD. Compared to the wt mice, fat accumulation was much lower in Cebpb^ΔuORF mice of both sexes (Figure 5A, B). In agreement with the differences in steatosis, the livers of wt females on HFD are significantly heavier than livers of Cebpb^ΔuORF females while in males a trend towards heavier wt livers is visible (Figure 5C, D). Fat accumulation on HFD was also lower in the heart and skeletal muscle of male mice (Figure 5—figure supplement 1A) and for both males and females the weight of the heart on HFD is higher in wt compared to Cebpb^ΔuORF mice (Figure 1—figure supplement 1B). Altogether, these data show that Cebpb^ΔuORF mice are protected against steatosis in the liver and other organs in response to HFD.

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Chronic obesity often results in the loss of glucose homeostasis (Abranches et al., 2015). We therefore analyzed glucose tolerance and insulin sensitivity in the HFD fed Cebpb^ΔuORF mice and wt littermates. Glucose clearance from the circulation measured by intraperitoneal glucose tolerance test (IPGTT) was impaired in response to 7 weeks HFD feeding for the wt mice of both sexes, as is shown by a significantly increased area under the curve (AUC) (Figure 6A, B). For the Cebpb^ΔuORF male mice, the already significantly better glucose clearance on normal diet does not change on HFD (Figure 6A). The female Cebpb^ΔuORF mice on HFD show reduced glucose clearance in the IPGTT compared to ND but they perform significantly better than the HFD fed wt females (Figure 6B). At the time of 7 weeks on HFD, both the wt and Cebpb^ΔuORF mice of both sexes did not develop insulin insensitivity as measured by intraperitoneal insulin sensitivity test (IPIST) (Figure 6C, D). Both the Cebpb^ΔuORF males and females, however, generally performed better on IPIST than the wt mice.

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In conclusion, our data show that Cebpb^ΔuORF mice on HFD feeding perform better in a glucose tolerance test, are protected against steatosis and show a lower inflammatory status of WAT, although the latter is less evident in females. These metabolically favorable phenotypes of the Cebpb^ΔuORF mice correlate with hyperplastic fat storage and in male mice with more efficient fat accumulation in the subcutaneous depot.

C/EBPβ is a known transcriptional regulator of fat cell differentiation and function (Siersbæk and Mandrup, 2011). We have shown earlier that the truncated C/EBPβ isoform LIP inhibits adipocyte differentiation and that fibroblasts derived from Cebpb^ΔuORF mice have an increased adipogenic differentiation potential (Zidek et al., 2015). We therefore analyzed the expression in the visceral WAT of the adipogenic transcription factors C/EBPα and PPARγ, the sterol regulatory element-binding protein 1 c (SREBP1c) as a key transcription factor for lipogenesis, and fatty acid synthase (FAS) as a key lipogenic enzyme, by quantitative PCR. In wt male mice all four transcripts are significantly lower expressed on HFD compared to ND (Figure 7A). This generally corresponds to their protein levels as determined by immunoblotting, although the expression of PPARγ and SREBP1c varies considerably between the mice (Figure 7—figure supplement 1A). In the WAT of Cebpb^ΔuORF males on ND, expression of the four transcripts is similar (C/EBPα and PPARγ) or higher compared to WAT from wt males on ND (SREBP1 and FAS, also shown in Zidek et al., 2015), and its reduction under HFD occurs to a lesser extend compared to wt mice (Figure 7A). With the exception of C/EBPα, the transcript levels generally correspond to the protein levels, although here variations of in particular PPARγ and SREBP1 levels complicate interpretation (Figure 7—figure supplement 1A). For the wt female mice, only the transcript levels of FAS were downregulated upon HFD and for Cebpb^ΔuORF mice only expression of PPARγ and FAS was significantly higher on HFD compared to wt females on HFD (Figure 7B). The better maintained expression of FAS in HFD fed Cebpb^ΔuORF females in the qPCR analysis however could not be recapitulated with immunoblotting (Figure 7—figure supplement 1B).

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To examine whether HFD feeding is associated with changes in LAP/LIP expression, we analyzed extracts from WAT isolated from wt and Cebpb^ΔuORF mice of both sexes under ND and HFD conditions. In wt males, both LAP and LIP isoforms were upregulated upon HFD feeding as shown in the immunoblot (Figure 8A) and determined by quantification of blot signals from a cohort (Figure 8B, C). The quantification reveals a small but significant decrease in the LAP/LIP ratio (Figure 8B), indicating that the inhibitory function of LIP increases upon HFD in wt males. Due to the LIP-deficiency caused by the Cebpb^ΔuORF mutation, the LAP/LIP ratio is very high for the Cebpb^ΔuORF males and does not change upon HFD feeding (some residual LIP expression is usually visible in Cebpb^ΔuORF mice due to leaky scanning over the not-optimal AUG-start codons for the LAP proteins). For the females, a significant increase in both LAP and LIP expression in response to HFD feeding was only observed in the Cebpb^ΔuORF mice (Figure 8D, F). However, no significant changes were measured in LAP/LIP expression ratios, despite an overall trend towards a lower LAP/LIP ratio on HFD in wt females (Figure 8D, E). Taken together, LAP/LIP isoform ratios decline in response to HFD feeding due to higher increase of LIP expression which is significant for males but not for females.

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The Cebpb^ΔuORF mutation prevents expression of the inhibitory C/EBPβ protein isoform LIP which results in unconstrained function of the C/EBPβ transactivator isoform LAP. In two previous reports, we have shown that Cebpb^ΔuORF mice display metabolic improvements and a delay in the onset of age-related conditions, collectively resulting in an extended lifespan in females (Müller et al., 2018; Zidek et al., 2015). Here, we demonstrate that Cebpb^ΔuORF mice are protected against the development of metabolic disturbances in response to HFD feeding. In males, this improved metabolic phenotype occurs although the total fat mass in Cebpb^ΔuORF mice is increased in response to HFD to a greater extent than in wt mice. Our data indicate that two special features of the white adipose tissue (WAT) in Cebpb^ΔuORF males contribute to these metabolic improvements. Firstly, Cebpb^ΔuORF males on a HFD store the surplus of nutrients in fat depots that expand through hyperplasia; they increase the number of adipocytes and thus the individual cells have to store less fat. These smaller adipocytes are metabolically more active and less inflamed compared to the inflated wt adipocytes residing in a hypertrophic fat depot. Hypertrophic adipocytes are known to secrete inflammatory cytokines that promote insulin resistance and other metabolic disturbances (Reilly and Saltiel, 2017; Weisberg et al., 2003). Furthermore, since the number of adipocytes in hypertrophic fat tissue does not increase and the amount of fat that can be stored in an adipocyte is limited, fat starts to accumulate in ectopic tissues like liver or muscle, compromising metabolic health (Frasca et al., 2017). Accordingly, in wt males on HFD we observed pronounced inflammation and macrophage infiltration in the visceral WAT and severe steatosis. In contrast, Cebpb^ΔuORF males on HFD are protected against these metabolic disturbances, which also correlated with better maintenance of glucose tolerance. We have shown earlier that the expression of genes related to fatty acid oxidation is enhanced in the liver of Cebpb^ΔuORF mice accompanied by a significant increase in fatty acid oxidation (Zidek et al., 2015). This enhanced fat utilization likely contributes to the reduced lipid accumulation in the liver of Cebpb^ΔuORF mice on HFD, and the healthier metabolic phenotype of Cebpb^ΔuORF mice is presumably the result of the combination of an increase in WAT function and fat utilization. In addition, the Cebpb^ΔuORF males store relatively more fat in the subcutaneous compartment than wt mice, which relieves the fat storage pressure for the visceral depots. Fat storage in the subcutaneous fat depot is associated with a better metabolic health status in humans and mice, while fat storage in the visceral fat depot is associated with insulin resistance and inflammation (Carey et al., 1997; McLaughlin et al., 2011; Tran et al., 2008). In contrast to the males, female Cebpb^ΔuORF mice showed reduced fat accumulation in the subcutaneous fat depot upon HFD compared to wt mice, and the visceral fat depot showed a trend towards a reduced fat storage although this difference was not statistically significant (Figure 1F and G). However, similar to the Cebpb^ΔuORF males, the adipocyte cell size in the visceral fat from Cebpb^ΔuORF females was significantly reduced and the calculated number of adipocytes was higher compared to wt females revealing increased adipocyte hyperplasia also in HFD fed Cebpb^ΔuORF females. Accordingly, also the female mice showed an improved metabolic phenotype on HFD including reduced hepatic steatosis and better maintained glucose tolerance. The difference in inflammation between wt and Cebpb^ΔuORF females was less pronounced compared to males and only visible in antibody staining of the macrophage marker CD68 indicating reduced macrophage infiltration in the visceral fat of Cebpb^ΔuORF females. However, macrophage infiltration seemed to be less pronounced in wt females on HFD than in wt males based on the CD68 immunohistological staining (compare Figures 3B and 4B), which might be explained by the known anti-inflammatory function of β-estradiol (Camporez et al., 2019). The generally lower vulnerability for inflammation in females may mitigate the differences in inflammatory cytokines between the two genotypes.

The HFD induced adipocytic hyperplasia in Cebpb^ΔuORF mice indicates that unconstrained LAP functionality – through loss of inhibitory function of LIP – stimulates adipocyte differentiation and function. It may explain why Cebpb^ΔuORF male mice store more fat in WAT on a HFD than wt littermates assuming that efficient fat storage by adaptive increase of the number of adipocytes prevents relocation of fat to peripheral tissues. Although fat storage in female Cebpb^ΔuORF mice upon HFD was rather reduced compared to wt littermates, also their adipocyte numbers in the visceral fat depot were increased. These observations are in line with our previous experiments showing that mouse embryonic fibroblasts (MEFs) derived from Cebpb^ΔuORF mice are much more efficiently induced to undergo adipogenesis than wt MEFs, and differentiation of 3T3-L1 preadipocytes is strongly suppressed upon ectopic induction of LIP (see data in Expanded View Figure 3 B, C of Zidek et al., 2015). Furthermore, the Kirkland lab has shown that endogenous LIP levels increase upon ageing in pre-adipocytes and in isolated fat cells from rats which correlated with reduced C/EBPα expression and reduced differentiation potential of the pre-adipocytes and that overexpression of LIP in preadipocytes from young rats impaired adipogenesis (Karagiannides et al., 2001). Similarly, we observed a shift of the C/EBPβ isoform ratio towards more LIP expression in visceral fat from wt males on HFD (Figure 8) which probably inhibits adipogenesis and thereby might contribute to adipocyte hypertrophy observed in wt mice.

In the current model of the regulatory cascade of adipocyte differentiation C/EBPβ and C/EBPδ induce the expression of C/EBPα and PPARγ, which by positive feedback stimulate each other’s expression (Siersbæk and Mandrup, 2011). Pharmacological activation of PPARγ by thiazolidines similarly to the Cebpb^ΔuORF mutation stimulates adipocyte differentiation, results in fat storage in hyperplastic adipocytes and in a shift to fat storage in the subcutaneous compartment, resulting in improved metabolic health (Adams et al., 1997; Fujiwara et al., 1988; McLaughlin et al., 2010; Okuno et al., 1998). Our finding that the mRNA expression of adipogenic transcription factors PPARγ, SREBP1 and C/EBPα in the visceral fat of Cebpb^ΔuORF males is better maintained on HFD compared to wt mice also fits to these observations. However, in contrast to our qPCR data, C/EBPα protein levels were rather downregulated in Cebpb^ΔuORF males both at ND and HFD conditions upon HFD (Figure 7—figure supplement 1) suggesting counter-regulation at the level of translation. This might be an adaptive response to increased LAP function yet does not seem to affect the adipocyte hyperplasia phenotype.

In the visceral WAT of wt females, LIP levels and the LAP/LIP isoform ratios do not significantly change in response to HFD feeding, in contrast to males. Accordingly, the mRNA expression levels of the adipogenic transcription factors are maintained upon HFD feeding in wt females. Whether this sex-specific difference might be due to the less pronounced inflammation (macrophage infiltration) observed in females or to other sex-specific responses to HFD feeding has to be examined in future studies. Probably, the slight increase in PPARγ expression together with the increased LAP function might be sufficient for the observed adipocyte hyperplasia in female Cebpb^ΔuORF mice on HFD, which however, seems to be less pronounced compared to HFD fed Cebpb^ΔuORF males. The downregulation of fatty acid synthetase (FAS) mRNA levels that we observe in wt mice upon HFD seems to be independent from the expression of the adipogenic transcription factors tested because at least in females these regulatory events were uncoupled and might be due to other effects of HFD feeding. Furthermore, in Cebpb^ΔuORF females the protein expression of FAS on HFD does not correspond to the FAS mRNA levels, it is efficiently reduced despite almost completely maintained mRNA levels suggesting interfering, post-transcriptional effects. What these effects are and why they are only observed in female mice is so far not known.

Taken together, our data propose pharmacological reduction of LIP expression as an approach to switch the unhealthy metabolic phenotype of obese individuals into a healthy obese phenotype to prevent the development of metabolic disease (possibly together with pharmacologic PPARγ activation). We have shown that a search for such an intervention is feasible through the identification of drugs that inhibit LIP expression similar to mTORC1-inhibition (Zaini et al., 2017). One drug that we identified as an inhibitor of LIP expression, the antiviral drug adevovir dipivoxil (Zaini et al., 2017), was recently tested on female wt mice upon ND and HFD feeding (Bitto et al., 2021). This study showed that adevovir is effective in increasing the LAP/LIP isoform ratio also in vivo (although in the mice LIP expression was not affected but rather LAP expression was increased), which resulted in increased C/EBPβ target gene activation and increased expression of β-oxidation genes in the liver similar to what we observed in the Cebpb^ΔuORF mice (Zidek et al., 2015). Remarkably, adevovir treatment resulted in a significant reduction of body weight and fat content particularly in the HFD fed mice (Bitto et al., 2021) similar to what we found with the Cebpb^ΔuORF females on HFD. Whether this effect on body weight and fat accumulation is caused only by increasing C/EBPβ function or whether additional not yet identified effects of adevovir contribute in addition is not known so far. Furthermore, it will be interesting to examine whether adevovir treatment of males results in reduced fat accumulation like in females or in increased (subcutaneous) fat accumulation as we observed in the Cebpb^ΔuORF males. Since the increased C/EBPβ function in Cebpb^ΔuORF mice also has the potential to extend the healthspan (Müller et al., 2018), therapeutic interference with the C/EBPβ isoform ratio may represent a promising strategy to attenuate the metabolic disturbances not only associated with overnutrition but also with ageing.

Source data are included in the Source Data files.

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Decision letter after peer review:

Thank you for submitting your article "C/EBPβ regulates hyperplastic versus hypertrophic fat tissue growth" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Matt Kaeberlein as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

There is consensus among the reviewers that this study provides an interesting and important advance in understanding the role of CEBP/b LAP in metabolism and response to a high fat diet challenge. The major discoveries reported here are fairly well supported by the data, including that male uORF KO mice show an increase in fat cell number as opposed to fat cell size, less inflammation, and improved glucose tolerance/insulin sensitivity.

Essential revisions:

However, all of the reviewers shared the major concern that it appears that only male mice were studied here, and that this fact – or the rationale for using only male mice – was not clearly articulated within the manuscript. This makes interpretation quite challenging, especially given that the authors previously published that lifespan extension in the uORF KO mice is much more pronounced in female compared to male mice. There was consensus that this is a substantial weakness to the current manuscript which limits its overall impact. It's possible the authors already have this data, and we would need to see inclusion of data supporting similar outcomes for the key experiments in female mice to recommend publication in eLife. If the outcomes are different in males and females, this is likely quite interesting and would need to be developed further.

The other significant concern was related to the RT-qPCR data, which is indicative but not conclusive support for the authors' conclusions, especially since many of the changes are small in magnitude. It was noted that most of the relevant proteins have ELISAs available, and they all have antibodies which could be used to support the robustness and importance of the small but plausibly important differences observed. IHC against CD68 in the fat depots could be performed and the authors could strengthen their claims about adipose tissue inflammation by measuring the expression levels of inflammatory cytokines in adipose depots.

Essential revisions:

However, all of the reviewers shared the major concern that it appears that only male mice were studied here, and that this fact – or the rationale for using only male mice – was not clearly articulated within the manuscript. This makes interpretation quite challenging, especially given that the authors previously published that lifespan extension in the uORF KO mice is much more pronounced in female compared to male mice. There was consensus that this is a substantial weakness to the current manuscript which limits its overall impact. It's possible the authors already have this data, and we would need to see inclusion of data supporting similar outcomes for the key experiments in female mice to recommend publication in eLife. If the outcomes are different in males and females, this is likely quite interesting and would need to be developed further.

The other significant concern was related to the RT-qPCR data, which is indicative but not conclusive support for the authors' conclusions, especially since many of the changes are small in magnitude. It was noted that most of the relevant proteins have ELISAs available, and they all have antibodies which could be used to support the robustness and importance of the small but plausibly important differences observed.

IHC against CD68 in the fat depots could be performed and the authors could strengthen their claims about adipose tissue inflammation by measuring the expression levels of inflammatory cytokines in adipose depots.

We now included data of female mice complementary to most of the originally performed experiments for males.

Please find all changes and additions:

Title: we propose a more extended version of the title.

Bar graphs: in the various figures we now grouped genotypes instead of diet type for – in our opinion – easier assessment.

Figure 1: panels E-G now show new data from female mice.

The body weight of female mice was obtained at the end of the experiment upon termination of the mice (Figure 1E). Due to our move to a different institute, we could not perform body composition analysis of females by micro-CT as we did with males and therefore used the weights of visceral and subcutaneous fat obtained from isolated fat tissue from terminated mice at the end of the experiment (Figure 1F, G).

Figure supplement 1: no changes.

We could not include results of food intake and energy efficiency from female mice.

Figure 2: panel A shows male data from previous Figure 1E and panel B show new data from females.

Figure 3: Panel A was shown in previous Figure 1F. The panels B and C shows new data for males.

IHC against CD68 in the visceral fat depot were performed that strengthen the claim about macrophage infiltration in the visceral adipose tissue (B). In addition, we included qPCR analyses of the inflammatory cytokines TNFα, MCP1, IL-1β an IL-6 in visceral fat from males (C).

Figure 4: Shows new data from females complementary to the male data in Figure 3.

Also here, qPCR analysis of CD68 expression (C), IHC against CD68 (B) and qPCR analyses of the inflammatory cytokines TNFα, MCP1, IL-1β an IL-6 in the visceral fat depot were performed.

Figure 5: Panel A and C show male data previously shown in Figure 2A and figure 2 supplement 1A. Panels B and D show new data for females.

The lipid staining in livers from female was performed using Oil-Red-O (Figure 5B) instead of Sudan III that was used for males.

Figure 5 supplement 1: Panels A, B and C show male data previously shown in Figure 2 B, C and Figure 2 supplement 1 B. Panel D shows new data from females.

Figure 6: Panel A and C show data from males previously shown in Figure 3. Panels B and D show new data from females.

Figure 7: Panel A shows data from males previously shown in Figure 4. Panel B shows data from females.

Figure 7 supplement 1: shows new data for males (A) and females (B).

We show immunoblot analysis of genes whose expression was different between the diets as determined by qPCR analysis.

Figure 8: shows all new data for males and females. We show immunoblots of C/EBPβ isoform expression in extracts from visceral fat of males (A) and females (D) and quantifications of the LAP/LIP isoform ratio (B, E) and the LAP and LIP isoforms separately (C, F).

Discussion: The section has been extended based on added results and suggestions by the reviewers and parts of the discussion on the connection of our findings to the ageing process was removed to limit the extend of this section and to focus more on HFD feeding and obesity.

Material and methods: experimental details have been supplemented based on added data.

Author details

1. Christine Müller

   European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, Netherlands

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review and editing

   Contributed equally with

   Laura M Zidek

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1974-4053

2. Laura M Zidek

   Leibniz Institute on Aging - Fritz Lipmann Institute, Jena, Germany

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review and editing

   Contributed equally with

   Christine Müller

   Competing interests

   No competing interests declared

3. Sabrina Eichwald

   Leibniz Institute on Aging - Fritz Lipmann Institute, Jena, Germany

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Gertrud Kortman

   European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, Netherlands

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

5. Mirjam H Koster

   Division Molecular Genetics, Department of Pediatrics, University Medical Center Groningen, University of Groningen, Groningen, Netherlands

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Cornelis F Calkhoven

   1. European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
   2. Leibniz Institute on Aging - Fritz Lipmann Institute, Jena, Germany

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review and editing

   For correspondence

   c.f.calkhoven@umcg.nl

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6318-7210

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