- Research Paper
- Open Access
Gene expression profile in bone of diabetes-prone BB/OK rats fed a high-fat diet
Genes & Nutrition volume 8, pages99–104(2013)
A high-fat diet (HFD) has been recognized as a risk factor for diseases such as dyslipidemia, atherosclerosis, obesity, and osteoporosis. However, studies analyzing gene expression after HFD in bone are rare. That prompted us to analyze the expression of selected genes in bone of 4-week-old diabetes-prone B(io)B(reeding) rats. Two breeding pairs were fed a HFD (+10 % tallow) or were fed a normal diet (ND; Ssniff R-Z) before mating and afterward during pregnancy. After the birth of progeny, parents continued to be given HFD or ND until the progeny was weaned (3 weeks). Thereafter, offspring were weaned and were fed the same food as their parents up to an age of 4 weeks. Body weight was measured at an age of 4 weeks, and subsequently 13 HFD rats and 13 ND rats were killed and the tibial bone was harvested to analyze the expression of 53 genes in bone. All rats fed HFD were significantly heavier than rats fed ND after 3 and 4 weeks. The diet also influenced the expression of genes in bone. There were significant differences in 20 out of 53 genes studied between rats fed HFD compared with rats fed ND. Four out of 20 had a lower and 17 out of 20 genes a higher expression in HFD rats, but differences in gene expression showed obvious differences between males and females. There were only two genes that were similarly different between males and females: Bmp4 and Atf4. Two genes, Foxg1 and Npy, were inversely expressed in males and females. It seems that the gene expression is differently regulated by diet during pregnancy and later in life between males and females. Nevertheless, it cannot be excluded that HFD also acts as an epigenetic factor in the development of offspring in utero.
A high-fat diet (HFD) has been recognized as a risk factor for diseases such as dyslipidemia, atherosclerosis, obesity, and osteoporosis (Hou et al. 1990; Steinberg 1991; Yang et al. 2008; Salem et al.1992; Kopelman 2000; Parhami et al.2000; Chen et al. 2010; Cao 2011). In growing animal models, a HFD or high-energy diet could deleteriously affect bone mineral content, structure, and mechanical properties (Li et al. 1990; Zernicke et al. 1995; Ward et al. 2003). In addition, significant correlations were observed between body composition, adiponectin, and bone parameters in growing mice and rats fed a HFD (Lac et al. 2008; Devlin et al. 2010). Furthermore, it was shown that an atherogenic diet inhibits bone formation by blocking differentiation of osteoblasts in growing mice, possibly resulting from lipid oxidation products (Parhami et al. 2001). A recent study has suggested that a HFD may induce an increase in bone resorption in mice (Cao et al. 2009, 2010). However, HFD can also have positive effects as shown in the diabetes-prone BioBreeding/OttawaKarlsburg (BB/OK) rat that is an excellent animal model for type 1 diabetes (Yang and Santamaria 2006; Bahr et al. 2011). HFD protects BB/OK rats from developing type 1 diabetes in a sex-specific manner. Furthermore, the expression of lipid-related genes was especially influenced in subcutaneous adipose tissue by a high-fat diet (Bahr et al. 2011). Studies analyzing gene expression after a HFD in bone are rare and mostly done in mice (Xiao et al. 2010, 2011). That prompted us to analyze the expression of selected genes in bone of 4-week-old BB/OK rats, which were fed a HFD (n = 13) or a normal diet (ND; n = 13) during pregnancy of their mothers and up to an age of 4 weeks. The expression of 53 genes playing a role in bone and lipid metabolism as well as in immunologic reactions was studied in bone (cf. Table 1 in Supplementary Material and M&M).
Materials and methods
All rats were bred and housed in our own animal facility. They were kept under strict hygienic conditions and had free access to food and acidulated water. All experiments were performed in accordance with the regulations for animal care of the Ministry of Nutrition, Agriculture, and Forestry of the Government of Mecklenburg-Vorpommern (Germany).
Two breeding pairs were fed a high-fat diet (HFD; Ssniff R-Z + 10 % tallow, Soest, Germany) or a normal diet (ND; Ssniff R-Z) 1 week before mating and afterward during pregnancy. After the birth of progeny, parents continued to be given a HFD or ND until the progeny was weaned (3 weeks). At 3 weeks postpartum, offspring were weaned, and the sexes caged separately and fed the same food as their parents up to an age of 4 weeks. Body weight of one complete litter was measured at an age of 4 weeks, and then 13 rats (6M:7F) fed HFD and 13 rats (7M:6F) fed ND were killed with an overdose of anesthesia (Sevofluran, Abbott, Germany) to analyze the gene expression in bone.
At the time of euthanasia, the tibial bone was harvested from the proximal metaphysis to the tibiofibular junction, excluding all cartilaginous and soft tissue. The tibias were snap frozen in liquid nitrogen and pulverized. Total RNA was extracted with Trizol (Qiagen, Hilden, Germany). Residual DNA was removed by DNase treatment (RNase-Free DNase Set; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A defined amount of purified RNA (1.5 μg) from bone samples was transcribed into cDNA and stored at −20 °C until use, as detailed before (Klöting et al. 2005a, b).
Real-time polymerase chain reaction (qRT-PCR)
Real-time polymerase chain reaction (qRT-PCR) was performed using the ABI PRISM Sequence Detection System 7000 (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions, using ABI PRISM 7000 SDS Software v1.1. described in detail elsewhere (Klöting et al. 2005a). Each quantitative PCR was performed in duplicate.
Target cDNAs were amplified by primer sets as summarized in Table 1 in Supplementary Material and described earlier (Klöting and Klöting 2004; Klöting et al. 2005a, 2006). The rat 18SrRNA gene (eukaryotic 18SrRNA endogenous control; FAM Dye/MGB Probe, Applied Biosystems) served as the endogenous reference gene. The melting curve was done to ensure specific amplification.
The standard-curve method was used for relative quantification. For each experimental sample, the amounts of targets and endogenous reference, 18SrRNA, were determined from the calibration curve. The target amount was then divided by the endogenous reference amount to obtain a normalized target value. The relative gene target expression was also normalized to the normal diet-group tissue sample (calibrator). Each of the normalized target values was divided by the calibrator-normalized target value to generate the final relative expression. Final results are expressed as N-fold differences in selected gene expression relative to the 18srRNA gene and the calibrator.
Data are given as mean ± SEM, and differences were assessed by two-way analysis of variance using the statistical analysis system SPSS (SPSS Inc., Chicago, IL, USA). Significant differences are given as follows: *p < 0.05; **p < 0.01; ***p < 0.001.
At first, all rats fed a HFD were significantly heavier than rats fed a ND after weaning and 1 week later, as shown in Fig. 1. The diet also influenced the expression of genes in bone as summarized in Fig. 2. There were significant differences in 20 out of 53 genes studied between rats fed a HFD compared with rats fed a ND. One can differentiate between genes that were significantly more highly expressed in ND-fed rats (4/20) or more highly expressed in HFD rats (17/20), but gene expression also showed obvious differences between males and females. Contrary expression patterns between males and females were observed in two genes (Npy and Foxg1) differing between HFD and ND. In males, the gene expression was significantly increased, whereas in females, the gene expression was decreased in HFD. Another expression behavior was found in Bmp4 and Aft4: both genes were significantly increased in male and female rats fed a HFD. Comparing HFD males and females, there were eight out of 20 genes in males (Lpr, Repin1, Dlk1, Ccl2, Col11a2, Mmp9, RT1Da, and Bmp3) and eight out of 20 genes in females (Lep, Slc2a4, Rnd3, Nfkb, Ppp3a, Vegf, Cd1, and Ibsp), which were significantly more highly expressed in only one sex. They were uniquely increased in males and females, respectively. In addition, three genes in males (Foxg1, Mmp9, and Bmp3) and two genes in females (Rnd3 and Ibsp) particularly stand out, because they are several times more highly expressed in HFD than ND rats (p < 0.001) and than all the other genes shown in Fig. 2.
Contrary to expectations, no significant differences were found in expression of genes, which are markers of osteoclasts (Tcirg), of osteocytes (Sost), or of genes regulating osteoclast differentiation like Tnfsf11 (Rankl). Also the expression of osteocalcin (Bglap) and insulin receptor (Insr) showed no significant differences in bone between BN, DA, and WOKW.
Nutrition, including dietary fat, is related to molecular markers of bone remodeling and may contribute to the risk of bone-related diseases, which is supported by our findings (Watkins et al. 2000; Seibel 2002). Several genes studied were significantly different between HFD and ND rats, a result supporting the findings of Xiao et al. (2010, 2011) analyzing the gene expression between male mice fed HFD or ND. They used a gene microarray system that contained over 22,000 probe sets for 12,960 different genes. Therefore, they found 89 genes that were changed in the HFD group compared with the control group. There were three genes that were also different in our study: Col1a1, Mmp9, and Bmp4. However, the difference between HFD versus ND was not comparable. In addition, they studied only males, so that sex differences could not be found, a phenomenon that was observed in our study. There were only two out of 20 genes that were comparable between males and females: Bmp4 and Atf4. All other genes were differently expressed between males and females.
In contrast to Bmp4, Atf4 belongs to a large family of transcription factors. Its protein binds DNA via their basic regions and dimerize via their leucine zipper domain to form a variety of homo- and heterodimers to regulate gene transcription (De Angelis et al. 2003). It was demonstrated that Atf4 plays a direct role in regulating osteoclast (OCLs) differentiation and findings suggest that it may be a therapeutic target for treating bone diseases associated with increased OCL activity (Cao et al. 2010). It is also known that Aft4 influenced the diet-induced obesity. From knockout (KO) mouse studies, it is known that Atf4 null mice are lean, and they are resistant to age-related and diet-induced obesity. Atf4 null mice are also hypoglycemic, indicating that Atf4 regulates mammalian carbohydrate metabolism (Seo et al. 2009; Yoshizawa et al. 2009).
It is noteworthy that Foxg1 and Npy are inversely expressed in males and females. They are more highly expressed in HFD males and lower in HFD females. Foxg1 encodes a developmental transcription factor with repressor activities (Murphy et al. 1994). The specific function of this gene has not yet been determined. However, it plays a role in the development of the brain and telencephalon and is therefore mainly expressed in the brain, but also in the testes. Therefore, ours is the first study to show that this gene is also expressed in bone in a sex-dependent manner. Mutations in Foxg1 gene cause the so-called Rett syndrome, which is a severe neurodevelopmental disorder affecting almost exclusively girls (Jacob et al. 2009). In vitro studies have shown a strong expression of Foxg1 in androgen receptor (AR)-abundant areas of the adult brain, which suggests possible involvement in neuroendocrine regulation. Furthermore, because of the repression of transcription by direct binding to DNA, Foxg1 may interact with AR in vivo, thereby targeting its repressor function specifically to sex hormone signaling (Obendorf et al. 2007). Therefore, one could speculate that the sex-specific expression of Foxg1 in bone of HFD BB/OK rats may be caused by the sex-specific action of this gene.
In contrast to Foxg1, Npy is a neuropeptide that is widely expressed in the central nervous system and influences many physiological processes, such as stress response, food intake, circadian rhythms, and cardiovascular function (Abe et al. 2010). Studies with KO mice have shown that the Npy system in the hypothalamus is also implicated in bone remodeling, since Npy KO mice have increased bone mineral density (BMD) (Cawley and Yanik 2010). This might be interpreted as a physiological interplay between Npy in energy metabolism and bone formation. That is true for our findings in bone; however, this study found sex dependence in bone under a HFD. Edelsbrunner et al. (2009) have shown that female Npy null mice are characterized by a significant decrease in water and food intake, but this was not true in males. The difference between males and females found in our study may indicate that the regulation of Npy is sex-influenced under a HFD.
Now it is well-accepted that the skeleton is an endocrine organ that, through the secreted molecule osteocalcin (Bgla), favors insulin secretion by insulin-producing β cells and insulin sensitivity in liver, muscle, and adipocytes (Ferron et al. 2008; Fukumoto and Martin 2009; Hinoi et al. 2008; Lee et al. 2007; Lee 2010; Lieben et al. 2009; Schwetz et al. 2012). The protein osteocalcin is produced by osteoblasts and odontoblasts and has been known as a marker of bone turnover (Brown et al. 1984; Ducy 2011). But, we were not able to observe significant differences in Bgla expression in bone. Also other genes that are markers of osteoclasts (Tcirg), of osteocytes (Sost), or of genes regulating osteoclast differentiation like Tnfsf11 (Rankl) or Insr showed no significant differences. Therefore, HFD seems not to influence endocrine function of bone in BB/OK rats developing insulin-dependent type 1 diabetes. But, we have not measured serum osteocalcin or have studied the morphology of bone, so that this assumption remains speculative.
Further important findings were the sex dependence of gene expression in bone of males and females. Nearly 50 % of genes demonstrated significantly higher expression under a HFD, but the genes were not comparable between males and females. That could mean that genes of males and females react differently upon fat consumption, or in other words, the digestion of fat is controlled differently depending on sex. This is a plausible consideration, because in mice, it was shown that different protein diets (high vs. normal protein diet) during pregnancy and later in the life of offspring using a HFD versus ND can cause different gene expression between males and females. Sellayah et al. (2008) showed that the mRNA expression for Lepr and for Npy genes was significantly lower in male than female offspring. Comparable results were also found in this study for the expression of Lepr. It was only significantly increased in HFD males compared to ND males. The values in females were comparable between ND and HFD. Therefore, the gene expression can be differently regulated by diet during pregnancy and later in life in males versus females. However, it cannot be excluded that HFD may also act as an epigenetic factor in the development of offspring in utero. To answer this question, further studies are needed (Gallou-Kabani et al. 2010; Tamashiro and Moran 2010).
Abe K, Kuo L, Zukowska Z (2010) Neuropeptide Y is a mediator of chronic vascular and metabolic maladaptations to stress and hypernutrition. Exp Biol Med 235:1179–1184
Bahr J, Klöting N, Wilke B, Klöting I, Follak N (2011) High fat diet protects BB/OK rats from developing type 1 diabetes. Diabetes Metab Res Rev 27:552–556
Brown JP, Delmas PD, Malaval L, Edouard C, Chapuy MC et al (1984) Serum bone Gla-protein: a specific marker for bone formation in postmenopausal osteoporosis. Lancet 1:1091–1093
Cao JJ (2011) Effects of obesity on bone metabolism. Orthop Surg Res 6:30–37
Cao JJ, Gregoire BR, Gao H (2009) High-fat diet decrease cancellous bone mass but has no effect on cortical bone mass in the tibia in mice. Bone 44:1097–1104
Cao H, Yu S, Yao Z, Galson DL, Jiang Y, Zhang X et al (2010) Activating transcription factor 4 regulates osteoclast differentiation in mice. J Clin Invest 120:2755–2766
Cawley NX, Yanik T, Woronowicz A, Chang W, Marini JC et al (2010) Obese carboxypeptidase E knockout mice exhibit multiple defects in peptide hormone processing contributing to low bone mineral density. Am J Physiol Endocrinol Metab 299:E189–E197
Chen JR, Lazarenko OP, Wu X, Tong Y, Blackburn ML et al (2010) Obesity reduces bone density associated with activation of PPARγ and suppression of Wnt/β-catenin in rapidly growing male rats. PLoS ONE 5:e13704
De Angelis R, Iezzi S, Bruno T, Corbi N, Di Padova M et al (2003) Functional interaction of the subunit 3 of RNA polymerase II (RPB3) with transcription factor-4 (ATF4). FEBS Lett 547:15–19
Devlin MJ, Cloutier AM, Thomas NA, Panus DA, Lotinun S et al (2010) Caloric restriction leads to high marrow adiposity and low bone mass in growing mice. J Bone Miner Res 25:2078–2088
Ducy P (2011) The role of osteocalcin in the endocrine cross-talk between bone remodelling and energy metabolism. Diabetologia 54:1291–1297
Edelsbrunner ME, Herzog H, Holzer P (2009) Evidence from knockout mice that peptide YY and neuropeptide Y enforce murine locomotion, exploration and ingestive behaviour in a circadian cycle- and gender-dependent manner. Behav Brain Res 203:97–107
Ferron M, Hinoi E, Karsenty G, Ducy P (2008) Osteocalcin differentially regulates beta cell and adipocyte gene expression and affects the development of metabolic diseases in wild-type mice. Proc Natl Acad Sci USA 105:5266–5270
Fukumoto S, Martin TJ (2009) Bone as an endocrine organ. Trends Endocrinol Metab 20:230–236
Gallou-Kabani C, Gabory A, Tost J, Karimi M, Mayeur S et al (2010) Sex- and diet-specific changes of imprinted gene expression and DNA methylation in mouse placenta under a high-fat diet. PLoS ONE 21:e14398
Hinoi E, Gao N, Jung DY, Yadav V, Yoshizawa T et al (2008) The sympathetic tone mediates leptin’s inhibition of insulin secretion by modulating osteocalcin bioactivity. J Cell Biol 183:1235–1242
Hou JC-H, Zernicke RF, Barnard RJ (1990) High-fat sucrose diet effects on femoral neck geometry and biomechanics. Clin Biomech 5:162–168
Jacob FD, Ramaswamy V, Andersen J, Bolduc FV (2009) Atypical Rett syndrome with selective FOXG1 deletion detected by comparative genomic hybridization: case report and review of literature. Eur J Hum Genet 17:1577–1581
Klöting N, Klöting I (2004) Congenic mapping of type 1 diabetes-protective gene(s) in an interval of 4 Mb on rat chromosome 6q32. Biochem Biophys Res Commun 323:388–394
Klöting N, Follak F, Klöting I (2005a) Diabetes per se and metabolic state influence gene expression in tissue-dependent manner of BB/OK rats. Diabetes Metab Res Rev 21:281–287
Klöting N, Follak F, Klöting I (2005b) Is there an autoimmune process in bone? Gene expression studies in diabetic and nondiabetic BB rats as well as BB rat-related and -unrelated rat strains. Physiol Genomics 24:59–64
Klöting N, Blüher M, Klöting I (2006) The polygenetically inherited metabolic syndrome of WOKW rats is associated with insulin resistance and altered gene expression in adipose tissue. Diabetes Metab Res Rev 22:146–154
Kopelman PG (2000) Obesity as a medical problem. Nature 404:635–643
Lac G, Gavalie H, Ebal E, Michaux O (2008) Effects of a high fat diet on bone of growing rat. Correlations between visceral fat, adiponectin and bone mass density. Lipids Health Dis 7:16–20
Lee NK (2010) An evolving integrative physiology: skeleton and energy metabolism Cell 130:456–469
Lee NK, Sowa H, Hinoi E, Ferron M, Ahn JD et al (2007) Endocrine regulation of energy metabolism by the skeleton. Cell 130:456–469
Li KC, Zernicke RF, Barnard RJ, Li AF (1990) Effects of a high fat-sucrose diet on cortical bone morphology and biomechanics. Calcif Tissue Int 47:308–313
Lieben L, Callewaert F, Bouillon R (2009) Bone and metabolism: a complex crosstalk. Horm Res 71:134–138
Murphy DB, Wiese S, Burfeind P, Schmundt D, Mattei MG (1994) Human brain factor 1, a new member of the fork head gene family. Genomics 21:551–557
Obendorf M, Meyer R, Henning K, Mitev YA, Schröder J (2007) FoxG1, a member of the forkhead family, is a corepressor of the androgen receptor. J Steroid Biochem Mol Biol 104:195–207
Parhami F, Garfinkel A, Demer LL (2000) Role of lipids in osteoporosis. Arterioscler Thromb Vasc Biol 20:2346–2348
Parhami F, Tintut Y, Beamer WG, Gharavi N, Goodman W et al (2001) Atherogenic high-fat diet reduces bone mineralization in mice. J Bone Miner Res 16:182–188
Salem GJ, Zernicke RF, Barnard RJ (1992) Diet-related changes in mechanical properties of rat vertebrae. Am J Physiol 262:R318–R321
Schwetz V, Pieber TR, Obermayer-Pietsch BR (2012) The endocrine role of the skeleton—background and clinical evidence. Eur J Endocrinol. doi:10.1530/EJE-12-0030
Seibel MJ (2002) Nutrition and molecular markers of bone remodelling. Curr Opin Clin Nutr Metab Care 5:525–531
Sellayah D, Sek K, Anthony FW, Watkins AJ, Osmond C et al (2008) Appetite regulatory mechanisms and food intake in mice are sensitive to mismatch in diets between pregnancy and postnatal periods. Brain Res 27:146–152
Seo J, Fortuno ES 3rd, Suh JM, Stenesen D, Tang W et al (2009) Atf4 regulates obesity, glucose homeostasis, and energy expenditure. Diabetes 58:2565–2573
Steinberg D (1991) Antioxidants and atherosclerosis. A current assessment. Circulation 84:1420–1425
Tamashiro KL, Moran TH (2010) Perinatal environment and its influences on metabolic programming of offspring. Physiol Behav 14:560–566
Ward WE, Kim S, Robert Bruce W (2003) A western-style diet reduces bone mass and biomechanical bone strength to a greater extent in male compared with female rats during development. Br J Nutr 90:589–595
Watkins BA, Li Y, Allen KG, Hoffmann WE, Seifert MF (2000) Dietary ratio of (n-6)/(n-3) polyunsaturated fatty acids alters the fatty acid composition of bone compartments and biomarkers of bone formation in rats. J Nutr 130:2274–2284
Xiao Y, Cui J, Li YX, Shi YH, Le GW (2010) Expression of genes associated with bone resorption is increased and bone formation is decreased in mice fed a high-fat diet. Lipids 45:345–355
Xiao Y, Cui J, Shi Y, Le G (2011) Lipoic acid increases the expression of genes involved in bone formation in mice fed a high-fat diet. Nutr Res 31:309–317
Yang Y, Santamaria P (2006) Lessons on autoimmune diabetes from animal models. Clin Sci 110:627–639
Yang RL, Li W, Shi YH, Le GW (2008) Lipoic acid prevents high-fat diet-induced dyslipidemia and oxidative stress: a microarray analysis. Nutrition 24:582–588
Yoshizawa T, Hinoi E, Jung DY, Kajimura D, Ferron M et al (2009) The transcription factor ATF4 regulates glucose metabolism in mice through its expression in osteoblasts. Clin Invest 119:2807–2817
Zernicke RF, Salem GJ, Barnard RJ, Schramm E (1995) Longterm, high-fat-sucrose diet alters rat femoral neck and vertebral morphology, bone mineral content, and mechanical properties. Bone 16:25–31
We thank Silvia Sadewasser, Susanne Schuldt, Edeltraut Lübke, and Kathrin Stabenow for expert technical assistance. This research is partially supported by Grant No. KL 771/11-2 of Deutsche Forschungsgemeinschaft.
Conflict of interest
The authors report no conflicts of interest.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
Lange, J., Barz, T., Ekkernkamp, A. et al. Gene expression profile in bone of diabetes-prone BB/OK rats fed a high-fat diet. Genes Nutr 8, 99–104 (2013) doi:10.1007/s12263-012-0299-1
- Weight gain
- Gene expression