Introduction

Adipose tissue is a key endocrine organ regulating energy metabolism. Secreted adipokines are known to act centrally to play an important role in the maintenance of energy homeostasis, in part by communicating with the hypothalamus to regulate feeding, energy expenditure and glycemia [1]. Obesity can promote dysfunctional adipose tissues that produces deleterious adipokines, stimulating inflammation and contributing negatively to global health [2,3,4]. An increased production of pro-inflammatory adipokines participates in the development of metabolic dysfunction, including insulin resistance and other features of the metabolic syndrome [5].

Angiopoietin-like 2 (Angptl2) is a pro-inflammatory adipokine connecting obesity to insulin resistance [6,7,8]. Increased circulating levels of Angptl2 have been reported in multiple metabolic disorders in both humans and animal models [9,10,11], making Angptl2 a potential therapeutic target for obesity and related metabolic diseases [12]. Accordingly, weight loss induced either by diet [13], physical exercise [14], diet combined with exercise [15] or bariatric surgery [16] significantly reduced circulating levels of Angptl2 in overweight subjects. Angptl2 in the circulation mainly comes from the visceral adipose tissue, with a contribution of adipocytes, endothelial cells, and infiltrated macrophages, particularly in overweight patients in whom augmented fat tissue mass associates with a matched microcirculation density and infiltrated inflammatory cells [11]. Angptl2 is expressed in many tissues, including adipose tissue, blood vessels, heart, liver, kidney and brain [6, 17, 18]. In a seminal study, Tabata and colleagues [6] reported that under a normal diet, Angptl2 knockout (KO, Angptl2-/-) male mice weighed less and exhibited a lower body fat mass than wild-type (WT) littermates. Moreover, when exposed to a HFD, Angptl2-/- mice were protected against weight gain and accumulated less white adipose tissue (WAT) [6]. A lower respiratory quotient (RER), i.e., a higher use of lipids rather than carbohydrates as an energy substrate, was speculated to contribute to the lower body weights of male Angptl2-/- mice [6]. We previously reported that the knockdown (KD) of Angptl2 similarly protected male mice against HFD [19]: Angptl2-KD mice gained less weight under HFD despite similar food intake, had a better lipid profile, reduced liver lipid accumulation, lower liver triglycerides, smaller adipocytes and reduced WAT inflammation. We also showed that the KD of Angptl2 reduced feeding efficiency and mimicked the beneficial effects of intermittent fasting on weight gain and insulin sensitivity in male mice [20]. As these studies were only performed in males, the possibility of a sexual dimorphism in Angptl2 regulation of energy balance warrants investigation. Moreover, it is unclear whether Angptl2 affects hypothalamic processes controlling feeding and energy expenditure and if altered energy states affect hypothalamic expression of Angptl2 or its receptor. Among them, integrin α5β1 (ITGA5) [6] myelin associated glycoprotein (MAG) [17] and immunoglobulin-like receptor B (PIRB, mouse ortholog of leukocyte immunoglobulin-like receptor B (LILRB2)) [21], are receptors for Angptl2 that are expressed in brain microglia, endothelial cells, oligodendrocytes and astrocytes [22, 23].

Within the hypothalamus, different neuronal populations have been characterized as key players in the central regulation of energy homeostasis. The arcuate nucleus of the medio-basal hypothalamus (MBH) comprises two antagonistic neuronal populations: Agouti-Related-Peptide (AgRP) and Neuropeptide-Y (NPY) expressing neurons and proopiomelanocortin (POMC) neurons [24]. The latter have anorexigenic effects, stimulating energy expenditure and catabolic processes via the sympathetic arm of the autonomic nervous system (ANS) [25]. POMC neurons are activated in states of energy sufficiency, by signals such as leptin or insulin, and release α-melanocytes-stimulating hormone (α-MSH) that activates MC4R and downstream catabolic cascades. The other subpopulation of neurons release AgRP and NPY and inhibits POMC neurons during states (e.g., fasting, hypoglycemia) or signals (e.g., ghrelin) of energy deprivation [26, 27]. Activation of these neurons stimulate food intake and anabolic processes via the parasympathetic arm of the ANS [28].

The aims of the present study were to investigate the impact of Angptl2 loss-of-function on weight gain, feeding and diet-induced obesity and the expression and nutritional regulation of Angptl2 and its receptors as well as the expression of hypothalamic neuropeptides critical to energy balance control in both male and female mice. We report sexually dimorphic effects of Angptl2-KD on energy metabolism and nutrient partitioning that may be explained by opposing regulation of MBH neurons expressing NPY, AgRP and MC4R.

Methods

Animal experiments

This work follows the ARRIVE guideline for reporting animal research [29] and all experiments were performed in accordance with the Guidelines for the Care and Use of Experimental Animals of the Canadian Council on Animal Care and were approved by the ethics committees of the Montreal Heart Institute and the Research center of CHUM. Three-month-old C57BL/6 Angptl2-KD male and female mice and their WT littermates from our colony were used, as previously described [19, 20, 30]. All animals were housed individually. A control, standard diet (STD; Teklad #08485, Harlan Laboratories, Madison, WI, US) or a high-fat diet (HFD, 45% kcal fat from lard; Teklad #88137) was given for 16 weeks ad libitum. The energetic value of the HFD is 4.5 kcal/g versus 3.6 kcal/g for the STD. Body weight and food intake were measured weekly.

Mice were sacrificed by exsanguination under anesthesia (100 mg/kg ketamine and 10 mg/kg xylazine i.p.) after overnight fasting at ZT4. Plasma, brain, liver and visceral adipose tissue were collected and kept at -80 °C until further analysis.

Insulin tolerance test

Insulin tolerance tests (ITT) were performed after a 4 h fast following an intra-peritoneal injection of insulin (0.6 U/kg); blood was collected in the tail vein after 0, 30, 60, 90 and 120 min to measure blood glucose levels and total AUC was calculated, as previously described [20].

Indirect calorimetry

Respiratory exchange ratio (RER), energy expenditure (EE) and locomotor activity were measured by indirect calorimetry using a Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments International, Columbus, OH, USA), as previously described [31]. Animals were single-housed in CLAMS cages at 21 ± 0.2 °C in a dark-light cycle during 24 h to acclimate before any measurements. Following acclimation, energy balance parameters were measured during 24 h. Mice were sacrificed by decapitation after isoflurane anesthesia at the end of the CLAMS. Brains were extracted, immediately flash-frozen at -80°C and stored for further tissue analysis.

Real-time quantitative PCR

MBH is defined as a region containing the arcuate nucleus, ventromedial hypothalamus and the dorsomedial hypothalamus. Using a precision brain slicer (Braintree Scientific, BS-2000C), bilateral 1 mm sagittal lateral to the midline were collected and the MBH micro-dissected using a scalpel and immediately frozen on dry ice. RNA was extracted using TRIzol as previously described [32]. RNA concentration was quantified and purity verified using a NanoDrop 2000 spectrophotometer. Total RNA (1μg) was reverse-transcribed by M-MuLV reverse transcriptase with random hexamers following the manufacturer’s conditions. Quantitative gene expression was measured from 1:10 cDNA dilutions. Real-time quantitative PCR (qPCR) was performed using SYBR Green PCR kit (Qiagen) on a Corbett Rotor-Gene 6000. Data were analyzed using the standard curve method and normalized to either actin, cyclophilin, and 18S mRNA expression levels or their geometric means, and were calculated by a ΔΔCT method, determined using NormFinder software (MDL, [33]). Primers sequences are provided in Table 1.

Table 1 Primer sequences.
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Statistical analyses

Sample sizes were estimated by performing power calculations (5% level of significance with 84% power) using preliminary results from our laboratory. The experimenter performed real-time qPCR analyses blind of the groups (sex and treatment); all other data acquisition were performed unblinded. Data are expressed as mean ± SEM. Data normality was analyzed using D’Agostino and Pearson omnibus normality test; variability was similar between groups. Two-way ANOVA with genotype (WT and KD mice) and diet (STD and HFD) as fixed factors and Sidak’s post hoc multiple comparison tests were performed with Prism 9 (La Jolla, CA, USA) to assess differences between groups. No randomization methods were used. Statistical significance was set at p ≤ 0.05.

Results

Angptl2-KD decreases body weight and food intake in male but not female mice

We first sought to measure alterations in energy balance in Angptl2-KD and control WT male and female mice submitted to either a STD or HFD for 16 weeks. As expected, Angptl2 mRNA expression was negligible in KD mice of both sexes (Fig. 1A, C). The HFD regimen increased body weight in both WT and KD mice of both sexes; however, KD males weighed less than controls on both the STD (27.3 ± 0.9 vs. 32.4 ± 0.9 g, p < 0.05) and HFD (37.3 ± 1.3 vs. 41.9 ± 1.6 g, p < 0.05) (Fig. 1B, C). In contrast, WT and KD females exhibited similar body weight on a STD (22.0 ± 0.8 vs. 23.9 ± 1.0 g, p > 0.05) or a HFD (29.3 ± 0.9 vs. 28.3 ± 1.2 g, p > 0.05) (Fig. 1H, I). Elevated body weights of male and female mice during HFD was associated with increased food consumption (Fig. 1D, J). In accordance with a lower body weight, male KD mice ate less than WT in both STD (1064.4 ± 16.0 vs. 1165.5 ± 20.0 kcal, p < 0.05) and HFD (1269.4 ± 29.3 vs. 1431.6 ± 37.5 kcal, p < 0.05) conditions (Fig. 1D). Females Angptl2-KD mice had lower food intake only in HFD conditions (1220.6 ± 27.7 kcal vs. 1377.0 ± 31.5 kcal, p < 0.05) (Fig. 1J) without changes in body weight (Fig. 1I).

Fig. 1: Body weight, food intake and adipose and liver mass in male and female Angptl2-KD and control mice on STD or HFD.
figure 1

Angptl2 mRNA expression in the MBH of WT and Angptl2-KD male (A) and female (G) mice exposed to STD or HFD. Weekly body weight (B, H) and final body after 16 weeks of diet (C, I), cumulative food intake (D, J), adipose tissue weight (E, K) and liver weight (F, L) in males and females. N = 12 mice per group; Two-way ANOVA (genotype X diet); Sidak’s post hoc test. *p < 0.05 Angptl2-KD vs. WT mice; ‡p < 0.05 HFD vs. STD.

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As anticipated, HFD induced a significant increase in visceral (epididymal) adipose tissue and liver mass in both sexes (Fig. 1E, F, K, L). Male Angptl2-KD mice exhibited decreased liver weight compared to WT under both diets (1.0 ± 0.1 vs. 1.3 ± 0.1 g on STD; 2.1 ± 0.2 vs. 2.6 ± 0.2 g on HFD; main effect p < 0.05), while adipose tissue showed a trend for lower values (1.0 ± 0.2 vs. 1.3 ± 0.1 g on STD; 2.5 ± 0.2 vs. 2.7 ± 0.2 g on HFD; p > 0.05) (Fig. 1E, F). In contrast, female Angptl2-KD mice accumulated more visceral adipose tissue relative to WT controls (1.3 ± 0.1 vs. 0.7 ± 0.1 g, p < 0.05) (Fig. 1K). Liver weights were slightly decreased in female Angptl2-KD mice under both diets (Fig. 1L). Together, these findings suggest that Angptl2 loss-of-function produces greater catabolic actions in male mice, while it largely fails to improve metabolic function in female mice.

Angptl2 knockdown improves insulin sensitivity in male but not female mice

To further explore the impact of Angptl2 on nutrient partitioning, we measured whole-body insulin sensitivity, fuel utilization and energy expenditure. In male mice, HFD exposure similarly increased fasting glycemia in WT and Angptl2-KD mice (12.7 ± 0.7 vs. 10.2 ± 0.7 mmol/L in WT; 12.2 ± 0.6 vs. 10.0 ± 0.4 mmol/L in KD; p < 0.05), (Fig. 2A). Corroborating our previous report [20], HFD lowered insulin responsiveness in both WT and Angptl2-KD male mice (Fig. 2B, C); however, this effect was attenuated in male KD mice under both STD and HFD diets (437 ± 39 vs. 587 ± 41 on STD; 653 ± 42 vs. 818 ± 37 on HFD; p < 0.05) (Fig. 2B, C). In contrast, HFD increased fasting glycemia in female Angptl2-KD (Fig. 2D). In addition, glucose levels tended to increase in female KD mice under HFD (p = 0.0753, Two-way ANOVA, Sidak’s post hoc test) (Fig. 2D). Similar results were observed after the ITT: female Angptl2-KD mice on the HFD had lower insulin sensitivity (607 ± 51 vs. 379 ± 47 on HFD; p < 0.05) (Fig. 2E, F). Consistent with the sex-specific results above, these data suggest that lack of Angptl2 in male mice protects against insulin resistance and is associated with improved metabolic function while it largely induces insulin resistance and metabolic dysfunction in female mice.

Fig. 2: Glucose homeostasis in male and female Angptl2-KD and control mice on STD or HFD.
figure 2

Glycemia in male (A–C) and female (D–F) Angptl2-KD mice (vs. WT controls) during an ITT after 16 weeks of STD vs. HFD. A, D Fasting glycemia, B, E glycemia during insulin tolerance test (ITT) and C, F total AUC of glycemia during ITT in WT and Angptl2-KD mice. N = 7–10 mice per group; Two-way ANOVA (genotype X diet); Sidak’s post hoc test. *p < 0.05 Angptl2-KD vs. WT mice; ‡p < 0.05 HFD vs. STD.

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Angptl2 knockdown blunts metabolic health in female mice

Respiratory exchange (RER), energy expenditure (EE) and locomotor activity were measured via indirect calorimetry in automated metabolic cages over 24 h (Supplementary Fig. 1). As expected, EE and locomotor activity increased during the dark cycle coinciding with nocturnal activity levels (Fig. 3B, C, E, F, H, I, K, L). Also as anticipated, HFD lowered RER in WT and KD mice of both sexes from ~1.0 to 0.9 (Fig. 3A, D, G, J). In males, but not in females, Angptl2 KD increased RER under both diets (1.06 ± 0.01 vs. 0.99 ± 0.02 on STD; 0.91 ± 0.03 vs. 0.87 ± 0.01 on HFD; p < 0.05 main effect; Supplementary Fig. 1A, D), suggesting that Angptl2 KD augments carbohydrate utilization in males over 24 h. In accordance with lower body weights, Angptl2 KD increased EE in males (0.33 ± 0.02 vs. 0.27 ± 0.01 on STD; p < 0.05; Fig. 3B, E and Supplementary Fig. 1B). This effect was not observed in females (Fig. 3H, K and Supplementary Fig. 1E) which is in accordance with similar body weights between female KD and WT mice. It is also important to note that in females, but not males, HFD diet decreased EE in both genotypes (0.31 ± 0.01 vs. 0.38 ± 0.03 kcal/g in WT; 0.32 ± 0.02 vs. 0.4 ± 0.03 kcal/g in KD; p < 0.05 main effect; Fig. 3K). Finally, locomotor activity was blunted by HFD in both sexes and genotypes (Fig. 3F, L). Decreased locomotion was also observed in female KD on STD (6100 ± 800 vs. 13,500 ± 2200 beam breaks; p < 0.05) (Fig. 3L and Supplementary Fig. 1F).

Fig. 3: Metabolic parameters in male and female Angptl2-KD and control mice on STD or HFD.
figure 3

Respiratory exchange ratio (A, G) and its quantification (D, J), energy expenditure (B, H, E, K) and locomotor activity (C, I and F, L) measured over 12 h, in WT (open bars) and Angptl2-KD (filled bars) males (in blue) and females (in red) mice exposed to STD or HFD for 16 weeks. Data are mean ± SEM of 5 mice/groups on STD for both sexes; and mean ± SEM of 4-5 male and 2-6 female WT and KD mice on HFD; Two-way ANOVA, Sidak’s post hoc test. *p < 0.05 Angptl2-KD vs. WT mice; ‡p < 0.05 HFD vs. STD.

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That glucose utilization was increased (RER) only in males following Angptl2-KD once again highlights how Angptl2 deletion ameliorates metabolic function in male mice. On the contrary and consistent with body weight and insulin sensitivity outcomes, loss of Angptl2 lowered locomotor activity thereby contributes to impaired metabolic health in female mice.

Sex divergence in Angptl2-related and metabolic neuropeptide gene expression profiles

The MBH comprises nuclei supporting nutrient and hormone sensing and orchestrating peripheral nutrient partitioning via neurons expressing POMC, NPY, AgRP and MC4R [34]. As a first step, we sought to determine MBH gene expression of Angptl2 along with its receptors Itga5, Mag and Pirb [9] under fed and fasted states in WT mice. While MBH Angptl2 mRNA expression was unchanged by fasting in male WT mice (Fig. 4A), fasting enhance Angptl2 expression in females (1.30 ± 0.11 vs. 1.00 ± 0.08, p < 0.05) (Fig. 4B). Fasting failed to affect Itga5 and Pirb mRNA levels in the MBH in male (Fig. 4C, E) and female mice (Fig. 4D, F). However, Mag expression was increased in MBH of both fasted males (Fig. 4G) and females (Fig. 4H). Overall, these data suggest that a state of acute energy deficiency may cause compensatory upregulation in the Angptl2 receptor MAG in the MBH of male mice, and in both Angptl2 and MAG in female mice.

Fig. 4: Gene expression in the medio-basal hypothalamus in male and female Angptl2-KD and control mice.
figure 4

Gene expression of Angptl2 (A, B) and its receptors: Itga5 (C, D), Pirb (E, F) and Mag (G, H), in MBH of WT male (in blue) and female (in red) mice, fed with a STD or fasted for 16 h. Data are mean ± SEM of n = 4-6 male and n = 8 female mice. *p < 0.05 vs. fed mice, unpaired-t test. POMC (I, J), NPY (K, L), AgRP (M, N) and Mc4r (O, P) mRNA expression in the MBH. Data are mean ± SEM of n = 4/6 male (in blue) WT (open bars) and 6/4 Angptl2-KD (filled bars) mice, on STD/HFD respectively; and mean ± SEM of n = 5/6 female (in red) WT and 6/6 Angptl2-KD mice, on STD/HFD respectively; Two-way ANOVA (genotype X diet); Sidak’s post hoc test. *p < 0.05 Angptl2-KD vs. WT; ‡p < 0.05 HFD vs. STD.

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As a final step, we measured MBH genes involved in the hypothalamic regulation of energy balance in KD and control mice used for behavioral and metabolic measures described above. As expected, HFD significantly increased Pomc expression [35], but only in males (1.83 ± 0.17 vs. 1.00 ± 0.19 in WT; 1.34 ± 0.44 vs. 0.75 ± 0.24 in KD; p < 0.05 main effect); however, Pomc expression was not impacted by lack of Angptl2 in either sex (Fig. 4I, J). Npy expression under STD was lower in Angptl2-KD male mice (Fig. 4K), whereas both Npy and Agrp mRNA levels were higher in KD female mice (Fig. 4L, N). These data are in line with catabolic actions in males and largely anabolic effects in females observed with lack of Angptl2. HFD lowered NPY expression in females (Fig. 4L; p = 0.055) and AgRP expression in both males and females as expected (Fig. 4M, N). We also measured the expression of the melanocortin receptor 4 (MC4R), a critical downstream effector of α-MSH (cleaved product of Pomc) and AgRP and component of the melanocortin pathway control of energy balance (Fig. 4O, P). Mc4r expression in male MBH was similar between KD and controls on both diets which is consistent with the lack of change in Pomc and Agrp expression in males. In females, Mc4r expression was elevated following HFD and increased by loss of Angptl2 in STD conditions (Fig. 4P). These results suggest that high-fat feeding and Angptl2 loss-of-function upregulate MC4R preferentially in female mice, an outcome that may be linked to increased Agrp expression observed.

Discussion

Although previous work suggested that Angptl2 contributes to obesity development and insulin resistance [6,7,8], the impact of Angptl2 on energy balance and associated hypothalamic neuropeptide expression in male and female mice remained to be elucidated. Here, we show that the absence of Angptl2 leads to reduced food consumption in both male and female mice, but that the impact of Angptl2-KD on body weight, energy metabolism and hypothalamic neuropeptides regulation varies by sex. In male mice, Angptl2-KD resulted in less fat accumulation in the liver, improved insulin sensitivity and lowered body weight. These observations were accompanied by a lower orexigenic NPY expression, which can be linked to the higher EE and increased carbohydrate utilization observed. In contrast, body weight was unchanged in female KD mice and associated with decreased locomotor activity, greater fat accumulation, moderate insulin resistance and increased AgRP and NPY expression. Together, these results suggest that the absence of Angptl2 sex-dependently modulates glucose metabolism and energy homeostasis.

Tabata and colleagues reported that under a normal and HFD diet, male Angptl2-/- mice exhibit reduced body weight and fat mass [6]. The authors speculated that a lower respiratory quotient, i.e. a higher use of lipids vs. carbohydrates as an energy substrate, may explain why Angptl2-/- male mice are leaner than WT mice [6]. In the present study however, we found that WT and Angptl2-KD mice of both sexes had a similar RER, close to 1.0, implying the use of carbohydrates as the principal energy source. Exposure to a HFD reduced similarly RER in both WT and KD male and female mice. However, Angptl2-KD was associated with a moderate increase in RER under both diets in males, and we consistently found that male Angptl2-KD have a higher EE under STD compared to male WT mice. In contrast, Angptl2-KD did not influence EE in female mice under STD. Moreover, female Angptl2-KD mice were not protected from weight gain under HFD conditions like male Angptl2-KD mice. Reduced locomotor activity combined with lower food intake could explain the lack of changes in body weight in females. The discrepancy between the results in males of Tabata and colleagues and ours could be explained by different genetic models employed and both the composition and duration of the HFD.

While Angptl2 is a circulating adipokine, Angptl2 mRNA is also expressed in the brain where it may contribute to the regulation of energy balance. To investigate this possibility, we evaluated Angptl2 expression in the MBH in response to energy deficit. Angptl2 is expressed in the MBH of both male and female WT mice and its expression is increased by fasting in female, but not in male mice. This specific modulation of Angptl2 in females by negative energy balance further defines the sexually dimorphic role of Angptl2 and may underly the lack of protection against diet-induced obesity in female mice. Nevertheless, we observed that exposure to HFD did not change hypothalamic Angptl2 gene expression in both sexes. In contrast to our observations in the MBH, transcriptome analysis of WAT from 48 h fasted male mice showed a reduction in Angptl2 expression when compared to the fasted state [36]. Together, these findings suggest that central and peripheral nutritional regulation of Angptl2 expression differs.

NPY is an important hypothalamic orexigenic peptide and central NPY-positive neurons stimulate feeding [37]. In accordance with lower body weight and reduced cumulative food consumption in male Angptl2-KD mice, hypothalamic NPY expression was reduced. In female Angptl2-KD mice however, both NPY and AgRP expression were elevated. These data suggest that the sexual dimorphism in Angptl2-dependent control of body weight and adipose tissue weight may be explained by divergent modulation of neurons expressing NPY and AgRP. Despite upregulation of orexigenic neuropeptides in female KD mice, this did not translate into higher food consumption or body weight. AgRP neurons are also known to affect peripheral nutrient storage or utilization [38] and rapidly shift whole-body metabolism towards lipid storage. Indeed, the fact that KD female mice display increased fat accumulation together with decreased feeding suggests an impairment in peripheral fat utilization. NPY/AgRP are also important for orchestrating meal patterns and its components such as meal size and duration [39]. Thus, even if the observed increase in NPY/AgRP expression does not correspond to greater total food intake, it may affect how often and when meals are consumed. In view of the effects of Angptl2-KD in the reduced high-fat intake in both male and female mice, further studies are warranted to investigate the role of angptl2 on meal structure. Of note, we observed that POMC expression was similar in both sexes and genotypes, suggesting that Angptl2 does not influence anorexigenic POMC neurons. Finally, Mc4r expression was increased in female angptl2-KD. MC4R activation by a-MSH (cleaved POMC), or its inhibition by AgRP, influences the regulation of energy homeostasis; activation of MC4R is associated with catabolic and anorexigenic effects and lead to weight loss, while inhibition promotes food intake and weight gain [40]. Increased MC4R expression is found in females, which could seem counterintuitive has they are not protected from HFD or present a phenotype following Angptl2-KD mostly associated with decreased activity of the MC4R. But it is known that AgRP has the effect of not only biasing the signaling of the MC4R to an inhibitory pathway, but also of inducing endocytosis of the MC4R, depleting the number MC4R to interact with melanocortins and thus favoring anabolic actions [41]. Thus, increased expression of MC4R expression in females is in agreement with increased AgRP serving as a feedback loop in response to MC4R protein internalization. However, additional research is required to better understand the central mechanisms mediating the actions of Angptl2 on the regulation of energy homeostasis and food intake in males and females.

The majority of changes in neuropeptide gene expression observed were in mice consuming the STD. That these changes were weaker in HFD conditions may be due to the well-described effects of HFD to modulate MBH circuits and neuropeptide expression, specially increasing POMC while decreasing AgRP and NPY [35], a pattern globally recapitulated by HFD in the present study in control mice.

Among the eight known members of the Angptl family, others are also suggested to participate in the regulation of energy homeostasis [42]. Angptl8, for example, is synthesized by the liver and adipose tissue [43] and, unlike Angptl2, displays anorexic properties in male mice, lowering food intake, body weight and adipose tissue in male mice [44]. Angptl6, known as angiopoietin-like growth factor (AGF) [12, 45], also displays anorectic effects and antagonizes weight gain in male mice [46]. Nonetheless, to the best of our knowledge, no study has explored central regulation of appetite and body weight by any Angptl protein in male and female mice. Our results demonstrate a sexual dimorphism in the regulation of energy metabolism by Angptl2. Such sex differences could apply to the other Angptl proteins.

Conclusion

The absence of Angptl2 revealed a sexual dimorphism on body weight, adipose tissue weight, energy expenditure, NPY expression and insulin sensitivity, favoring males compared to females. Our data suggest that Angptl2 expression is centrally regulated according to the nutritional state, and that Angptl2 contributes to the central nutritional regulation in a sex-dependent manner.