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. 2015 Feb 8;9(2):143–150. doi: 10.1007/s12079-015-0264-z

Gender-specific effects on food intake but no inhibition of age-related fat accretion in transgenic mice overexpressing human IGFBP-2 lacking the Cardin-Weintraub sequence motif

Petra Wiedmer 1, Franziska Schwarz 1, Birgit Große 1, Nancy Schindler 2, Armin Tuchscherer 3, Vincenzo C Russo 4, Matthias H Tschöp 5, Andreas Hoeflich 2,✉
PMCID: PMC4458247  PMID: 25663268

Abstract

IGFBP-2 affects growth and metabolism and is thought to impact on energy homeostasis and the accretion of body fat via its heparin binding domains (HBD). In order to assess the function of the HBD present in the linker domain (HBD1) we have generated transgenic mice overexpressing mutant human IGFBP-2 lacking the PKKLRP sequence and carrying a PNNLAP sequence instead. Transgenic mice expressed high amounts of human IGFBP-2, while endogenous IGFBP-2 or IGF-I serum concentrations were not affected. In both genders we performed a longitudinal analysis of growth and metabolism including at least 4 separate time points between the age of 10 and 52 weeks. Body composition was assessed by nuclear magnetic resonance (NMR) analysis. Food intake was recorded by an automated online-monitoring. We describe negative effects of mutant human IGFBP-2 on body weight, longitudinal growth and lean body mass (p < 0.05). Very clearly, negative effects of mutant IGFBP-2 were not observed for fat mass accretion throughout life. Instead, relative fat mass was increased in transgenic mice of both genders (p < 0.05). In male mice transgene expression significantly increased absolute mass of total body fat over all age groups (p < 0.05). Food intake was increased in female but decreased in male transgenic mice at an age of 11 weeks. Thus our study clearly provides gender- and time-specific effects of HBD1-deficient hIGFBP-2 (H1d-BP-2) on fat mass accretion and food intake. While our data are in principal agreement with current knowledge on the role of HB-domains for fat accretion we now may also speculate on a role of HBD1 for the control of eating behavior.

Keywords: IGFBP-2, Age, Gender, Growth, Metabolism, Energy, Obesity, Transgenic, Mouse, Heparin, Food intake

Introduction

Forced expression of IGFBP-2 in transgenic mice (Hoeflich et al. 1999, 2001) was used to simulate the effects of elevated levels of IGFBP-2 in vivo but also to study potential modes of IGFBP-2 action. IGFBP-2 acts by negative interference with IGF-I or -II, which is particular relevant for growth and muscle accretion. In addition, IGFBP-2 has been demonstrated to block age- and diet-induced obesity in mice (Wheatcroft et al. 2007). IGFBP-2 was thus discussed in the context of metabolic homeostasis (Wheatcroft and Kearney 2009). Interestingly, IGFBP-2 expression is regulated by leptin (Hedbacker et al. 2010; Munoz-Gutierrez et al. 2005) and it was recently demonstrated that IGFBP-2 mediates leptin effects on glucose metabolism and insulin-sensitivity in sheep (Yau et al. 2014).

IGFBP-2 binds to heparin, extracellular matrix (Arai et al. 1996) and to proteoglycans (Russo et al. 1997, 2004). This is achieved by the presence of two independent heparin binding domains (HBD) present in the linker domain (HBD1: PKKLRP) (Russo et al. 2005) and in the carboxy-terminal region (HBD2: KHGLYNLK…H) (Kuang et al. 2006). Both HB-domains have been shown to be required for its negative effects on preadipocyte differentiation (Xi et al. 2013). It further has been demonstrated that IGFBP-2 via HBD1 present in the linker domain binds to the receptor protein tyrosine phosphatase β (RPTPß) and thereby regulates IGF-I-dependent cell signaling (Shen et al. 2012). In vitro, mutation of HBD1 (PKKLRP to PNNLAP) has revealed a role for growth and metastatic potential of neuroblastoma cells (Russo et al. 2005).

According to our results at younger ages overexpression of mouse IGFBP-2 has positive effects on fat mass (Hoeflich et al. 2001; Rehfeldt et al. 2010) contrasting the inhibitory effects of human IGFBP-2 on fat accretion at advanced ages (Wheatcroft et al. 2007). Thus, longitudinal studies of IGFBP-2 excess are required to better understand the in vivo actions of IGFBP-2. As demonstrated recently, IGFBP-2 stimulates expression of estrogen receptor α (Foulstone et al. 2013) and expression of IGFBP-2 itself is regulated by estrogen in reproductive tissues but also in the brain (Hoeflich et al. 2014). Therefore it is clear that IGFBP-2 also possesses gender-related effects, as demonstrated already e.g., in bones (DeMambro et al. 2008), which further have to be considered by the longitudinal approach.

In order to investigate the role of the HB-domain present in the linker domain of IGFBP-2, we established a novel mouse model overexpressing the HBD1-deficient (H1d) mutant form of human IGFBP-2 lacking the PKKLRP-sequence motif as we have previously described (Russo et al. 2005). In the present study we characterized the phenotype of this novel H1d-BP-2-mouse model and provide age- and gender-specific phenotypes concerning energy metabolism and food intake.

Materials and methods

Generation and verification of the H1d-BP2-mouse model

Transgenic mice were generated by microinjection of an expression vector as described before (Hoeflich et al. 1999) with the exception of using an expression vector containing complementary DNA coding for human IGFBP-2 lacking the heparin-binding domain (HBD1) within the linker domain of IGFBP-2 (Russo et al. 2005). By this mutation the positively charged Cardin-Weintraub sequence (PKKLRP) becomes neutralized by means of three amino acid exchanges resulting in PNNLAP (Russo et al. 2005). The expression vector was injected into pronuclei of fertilized eggs from FVB mice. Mice were genotyped by gene-specific PCR (forward: CGG AGC AGG TTG CCA GAC AA; reverse: GTG ACC TTC TCC CGG AAC AC) in lysates from tail-tip biopsies. Four independent transgenic mouse lines were established, all were characterized by elevated expression of HBD1-deficient IGFBP-2 (H1d-BP2) in different tissues (not shown). Animal experiments were performed according to the guidelines of the Ministry of Agriculture, Brandenburg. All animals were kept under standard conditions and received tab water and standard chow ad libitum (sniff RIM, V1534-000, Soest, Germany). Male hemizygous H1d-BP2-mice of FVB genetic background were mated with female non-transgenic NMRI outbred mice. The F1 generation of this breeding was used for phenotypic characterization of male and female transgenic mice expressing human H1d-BP2. Transgene expression of human H1d-BP2 was verified by ELISA (R&D Systems, Minneapolis, USA). Endogenous expression of mouse IGFBP-2 and IGF-I in serum from H1d-BP2-mice and controls also was investigated by specific ELISA kits (R&D Systems, Minneapolis, USA). The analysis of serum mouse IGF-1 levels required an acidic extraction in advance to applying the ELISA according to Kratzsch et al. (Kratzsch et al. 1993).

Analysis of physiological parameters

Body weights of male and female wildtype (wt) and transgenic (tg) offspring were recorded weekly starting at an age of 3 weeks (Fig. 1). From 8 weeks of age body composition was analyzed using nuclear magnetic resonance (NMR, minispec mq 10, Fa. Bruker Optik GmbH, Ettlingen, Germany) monthly. We further performed a longitudinal analysis of body composition and body length at an age of 5, 10, 18, 29 and 51 weeks. Fat mass analyzed with NMR served to calculate lean body mass as the difference from body weight. Body length was analyzed by measuring the distance between nose tip and anus under isoflurane anesthesia in an outstretched position. In all mice food consumption was quantified at an age of 11, 19, 29 and 51 weeks. Glucose tolerance was assessed after overnight fasting in mice as described (Sartorius et al. 2014). D-glucose was injected intraperitoneally at a dose of 2 g/kg body weight. Glucose concentrations in the blood (tail vein) were quantified before (time point 0) as well as 15, 30, 60, and 120 min after glucose injection using a commercial glucometer (Elite, Bayer, USA) at the age of 12, 20, 29 and 51 weeks after birth.

Fig. 1.

Fig. 1

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Schematic presentation of the longitudinal animal study performed in male and female mice. Body lengths were recorded at an age of 5, 10, 18, 29 and 51 weeks. Body weights and body composition were recorded every 4 weeks and in the course of body lengths assessment. Glucose tolerance tests were performed at an age of 12, 20, 29 and 51 weeks after birth (FDM: assessment of food intake, GTT: glucose tolerance test)

Statistical analysis

The data analysis was generated using SAS software, Version 9.4 for Windows. Copyright, SAS Institute Inc., Cary, NC, USA. Descriptive statistics and tests for normality were calculated with the UNIVARIATE procedure of Base SAS software (SAS Institute Inc 2013a). Data that could be considered as approximately normal were analyzed by ANOVA / repeated measurement ANOVA using the MIXED procedure of SAS/STAT software (SAS Institute Inc 2013b). The repeated measurement ANOVA model for the variables body length, body weight, body composition (fat mass, lean body mass), food consumption (absolute food, relative food), relative fat mass contained the fixed effects sex (levels: male, female), group (levels: wt, tg) and week and all interactions between these fixed effects. The levels of the repeated variable week were 5, 10, 18, 29 and 51 for body length and body composition and 11, 19, 29 and 51 for food consumption and relative fat mass. For body weight the levels of week were 4 to 53 by 1. Repeated measurements on the same animal were taken into account by the “repeated statement” of the MIXED procedure using an unstructured type for the blockdiagonal residual covariance matrix for body length, body composition, food consumption and relative fat mass. For body weight an AR(1) type for the blockdiagonal residual covariance matrix was used. For the variables blood glucose ANOVA model was used with the fixed effects sex (levels: male, female), group (levels: wt, tg), week and time all interactions between these fixed effects. The levels of the repeated variables week and time were week 11, 20 29, 51 and time with hour 1 to 22 for the time before and 15, 30, 60, 120 minutes after glucose infusion for blood glucose. The ANOVA model for the variables mouse-IGFBP-2 and IGF-I contained the fixed effects sex (levels: male, female), group (levels: wt, tg) and the interaction sex and group. For human-IGFBP-2 a one-way model with the fixed effect sex (levels: male, female) was used for tg-animals. Least-squares means (LSM) and their standard errors (SE) were computed for each fixed effect in the models, and all pairwise differences of LS-means were tested by the Tukey-Kramer procedure. Effects and differences were considered significant if p < 0.05.

Results

Detectable amounts of human IGFBP-2 were present in plasma of H1d-BP2-mice but not in non-transgenic littermates (Fig. 2a). While in H1d-BP2-mice human IGFBP-2 concentrations ranged around 800 ng/ml in non-transgenic littermates human IGFBP-2 was undetectable. At an age of 71 weeks concentrations of mouse IGFBP-2 ranged around 50–70 ng/ml and were similar in transgenic and non-transgenic animals (Fig. 2b). Also plasma IGF-I concentrations were similar in transgenic and non-transgenic mice (at an age of 71 weeks: ≈350–400 ng/ml) (Fig. 2c). Transgene expression reduced body weight after birth at all times assessed between 3 and 52 weeks in males tg and with the exception of weeks 4, 30, 32, 44 and 46 also in females tg compared to wt controls (Fig. 3). Body weight reductions in transgenic mice amounted up to 10 and 14 % in males and females, respectively. Mild reductions of body lengths were found in all age groups assessed but did not reach statistical significance if compared within separate weeks (Fig. 4). As an effect over all age groups the transgenic mice had significantly reduced body lengths within males and females (p < 0.05). Transgene expression significantly reduced fat-free mass (Fig. 5) at all time-points assessed (p < 0.001). The reductions of lean body mass amounted almost up to 20 % in both tg genders (Fig. 6). In male mice absolute fat mass was increased by transgene expression over all age groups and specifically at the age of 18 weeks (p < 0.01). In tg female mice slight increases of absolute fat mass were statistically not significant. Relative fat mass (Fig. 7) was increased in transgenic animals of both genders (male 20-week mice: 230 % of controls; female 20-week mice: 130 % of controls; p < 0.05). Food consumption was significantly reduced in male transgenic mice (p < 0.05) at the age of 11 and 19 weeks (Fig. 8). Also, if compared over all age groups the negative effect of transgene expression was statistically significant for male mice (p < 0.05). By contrast, in female transgenic mice food consumption was either increased at the age of 11 weeks or not different compared with wildtype females at the other investigated age groups. Oral glucose tolerance tests revealed no significant effects of transgene expression on glucose clearance (Fig. 9).

Fig. 2.

Fig. 2

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Serological parameters of transgenic mice overexpressing mutated human IGFBP-2. Serum concentrations of human (panel A) and mouse (panel B) IGFBP-2 and mouse IGF-I (panel C) were assessed in male and female mice at an age of 71 weeks. Data are presented as means ± SEM (N ≥ 5; h: human; m: mouse)

Fig. 3.

Fig. 3

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Longitudinal analysis of body weights in male and female H1d-BP2 transgenic mice and non-transgenic littermates from week 3 through week 52. Data are presented as means ± SEM. P < 0.05. Statistical within-group effects of transgene expression are indicated for male and female mice (N = 10)

Fig. 4.

Fig. 4

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Longitudinal analysis of body lengths in male and female H1d-BP2 transgenic mice and non-transgenic littermates from week 5 through week 51. Data are presented as means ± SEM. p < 0.05. Statistical within-group effects of transgene expression are indicated for male and female mice (N = 10)

Fig. 5.

Fig. 5

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Longitudinal analysis of lean body mass in male and female H1d-BP2 transgenic mice and non-transgenic littermates from week 5 through week 51. Data are presented as means ± SEM. ***: p < 0.0001 (N = 10)

Fig. 6.

Fig. 6

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Longitudinal analysis of absolute body fat mass in male and female H1d-BP2 transgenic mice and non-transgenic littermates from week 5 through week 51. Data are presented as means ± SEM. **: p < 0.001. Statistical effects of transgene expression within group are indicated for male mice (N = 10)

Fig. 7.

Fig. 7

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Longitudinal analysis of relative body fat in male and female H1d-BP2 transgenic mice and non-transgenic littermates from week 8 through week 52. Data are presented as means ± SEM. *: p < 0.05. Statistical within-group effects of transgene expression are indicated for male and female mice (transgenic male at week 20: N = 6; all other groups: N = 10)

Fig. 8.

Fig. 8

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Longitudinal analysis of food intake in male and female H1d-BP2 transgenic mice and non-transgenic littermates from week 11 to week 51. Data are presented as means ± SEM. *: p < 0.05; **: p < 0.01. Statistical within-group effects of transgene expression are indicated for male mice (N ≥ 8)

Fig. 9.

Fig. 9

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Longitudinal analysis of glucose tolerance in male and female H1d-BP2 transgenic mice and non-transgenic littermates from week 11 to week 51. Data are presented as means ± SEM (N ≥ 5)

Discussion

Here we present a novel transgenic model overexpressing mutant IGFBP-2 which has a non- functional Cardin-Weintraub sequence (Russo et al. 2005) allowing for studying the influence of the HB-domain present in the linker domain (HBD1) of IGFBP-2 (Russo et al. 2005) on somatic growth and energy metabolism. The transgenic line was originally established on an FVB genetic background. For the study presented here we crossed male hemizygous FVB mice with female non-transgenic NMRI out-bred mice generating a mixed genetic background of 50 % FVB and 50 % NMRI. The phenotypes described herein thus are influenced by a broader genetic background compared to the inbred background from FVB mice. The effects of mutant IGFBP-2 were studied in a longitudinal approach in both genders.

Increased expression of human IGFBP-2 in mutant FVB/NMRI mice did not compromise endogenous IGFBP2 expression. Also, higher IGFBP-2 plasma levels did not affect the concentrations of plasma IGF-I.

Throughout life postnatal growth was significantly reduced in IGFBP-2 transgenic mice. This finding is in agreement with studies in transgenic mice overexpressing normal mouse IGFBP-2, which also had growth deficits starting around week 3 after birth (Hoeflich et al. 1999). We may thus neither assume a difference of human or mouse IGFBP-2 (mIFGBP-2) for inhibition of somatic growth nor postulate a major role of the HB present in the linker domain of IGFBP-2 for the control of somatic growth. In addition, lean body mass was significantly reduced at all ages assessed in both sexes and the extent of lean mass reduction exceeded negative effects on total body weight in H1d-BP2-mice. Thus, also for lean body mass differential effects of mouse IGFBP-2 or human H1d-BP2 cannot be observed. In absolute terms muscle and bone mass greatly contribute to fat free mass. Notably both, muscle (Hoeflich et al. 1999) and bone mass (Eckstein et al. 2002) have been demonstrated as sensitive tissues for the actions of IGFBP-2. Although we do not have data to further speculate in the present study we would like to mention that IGFBP-2 by its HB-domain stimulates osteoblast differentiation and thus bone mass acquisition in mice (DeMambro et al. 2012; Xi et al. 2014). Since also gender has been demonstrated to affect bone effects of IGFBP-2 (DeMambro et al. 2008; Khosla 2008) the analysis of H1d-BP2 effects on bone growth appears to represent an important issue although for future longitudinal assessment.

It was further shown previously in tg mice that mIGFBP-2 overexpression increased fat mass in male mice at an age of 10 weeks (Rehfeldt et al. 2010). Also this phenotype was reproduced in male H1d-BP2-mice, which had higher absolute fat mass over all age groups, with prominent increases particularly at an age of 18 weeks. However and very clearly, our mouse model did not reproduce the negative effect of wildtype hIGFBP-2 on fat mass accretion observed in mice at advanced ages (Wheatcroft et al. 2007). Instead, H1d-BP2-mice consistently had increased levels of relative body fat and a protection from age-related increases of fat mass observed by Wheatcroft et al. (Wheatcroft et al. 2007) definitely was absent in our mouse model. In fact an involvement of both HB-domains present in IGFBP-2 has been demonstrated to mediate inhibition of preadipocyte differentiation and fat accretion in mice (Wheatcroft et al. 2007). In addition evidence has been provided to postulate a major effect of the second HB-domain present in the C-terminal region of IGFBP-2 for the protective effect on fat accretion over the HB-domain present in the linker domain (Xi et al. 2013). However, as we observed in H1d-BP2-mice a block of age-related fat mass accretion is completely absent in the presence of HBD2. One potential reason for that discrepancy may be related to the fact that Xi et al. applied HB-domain peptides (Xi et al. 2013) while in our mouse model both domains are coupled via the IGFBP-2 backbone, which may imply mechanistic aspects. In transgenic mice expression of H1d-BP2 is permanent, while by Xi et al. (Xi et al. 2013) HBD-peptides were given temporally. As we discussed in a separate review on that topic this may make the difference (Hoeflich 2008). Therefore, results derived from permanent versus temporary exposure of IGFBP-2 have to be interpreted carefully.

In order to explore additional mechanisms potentially behind the higher relative fat mass in transgenic animals of both genders we have also studied food intake and glucose metabolism in the present study. We found that food intake was sexually dimorphic in transgenic mice, revealing rather higher food intake in females at the age of 11 weeks but rather decreased food intake in male mice at the age of 11 and 19 weeks. Food intake and growth performance are functionally related e.g., in pigs (Boudry et al. 2010) and the reason for the disruption of that relationship in female H1d-BP2-mice is unclear. Concerning gender effects it is intriguing that estrogen signaling has been identified as a novel target of IGFBP-2 (Foulstone et al. 2013). It is known that food intake is affected by estrogens (Asarian and Geary 2013; Davis et al. 2014) or by other steroids like corticosterone (Choleris et al. 2013). Our H1d-BP2-mouse model may therefore represent an attractive model for further analysis of IGFBP-2 dependent control of food intake in female versus male mice.

To summarize, overexpression of human IGFBP-2 lacking the HBD1 from the linker domain resulted in moderate reductions of body weight, body lengths and lean body mass in both genders. Interestingly, an age-related block of fat mass accretion is not observed in our mouse model. Instead, relative fat mass is increased throughout life in both genders, while in male tg mice at an age of 18 weeks even an increase of absolute fat mass was found. In absolute terms, young female H1d-BP2-mice eat more food while young male H1d-BP2-mice ate rather less food than non-transgenic littermates. Based on our data we demonstrate that the phenotype of elevated fat mass is associated with higher food intake in females but not in male H1d-BP-2-mice. All in all we identify age- and gender-specific effects of H1d-BP2 in our model and have to assume functions for food behavior and fat accretion for mutant IGFBP-2. While further studies are required to investigate the physiological mechanisms behind differential food intake and fat mass accretion in male and female H1d-BP2-mice, the present study particularly provides evidence for the physiological relevance of HBD1 from IGFBP-2 for energy metabolism in mammals.

Acknowledgments

We are grateful to Tamara Holy and Eckhard Wolf (Gene Center, Munich, Germany) for microinjection of the transgenic expression vector and development of the H1d-BP2 mouse model. VCR was supported by the NHMRC of Australia (Project Grant, # 1008062). AH was supported by the Deutsche Forschungsgemeinschaft (DFG HO2003/1-6).

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