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Impaired fetal growth resulting in low birth weight has long been associated with increased risk of developing abnormal metabolic functions in adulthood (1). Hence, low birth weight increases the risk of developing obesity and type 2 diabetes later in life in humans (2). A low birth weight in humans is associated with impaired insulin signaling in skeletal muscle in adult life (3–5), hepatic insulin resistance (6), and decreased insulin secretion (7). The exact mechanism whereby impaired fetal growth confers insulin resistance in the offspring is unknown, but mitochondrial oxidative stress was recently proposed as a possible mechanism (8).

Several animal models mimicking impaired fetal growth have been developed, all of which display dysregulated glucose metabolism and altered insulin sensitivity in adult life (9). One such model is gestational protein restriction, where dams are fed a low protein (LP) diet during pregnancy (9). Most studies on the effects of a maternal LP diet have focused on beta-cell dysfunction (9), although the influence of maternal LP diet on peripheral insulin sensitivity has also been studied, showing an increase in young animals (10) but a decrease in old animals (11,12).

Taurine is a sulfur-containing amino acid, which does not enter protein synthesis but has a number of other physiologic functions such as conjugation with bile acids, osmotic pressure regulation in brain, and antioxidant properties. Taurine is a chemical chaperone in conjugation with ursodeoxycholic acid relieving endoplasmatic reticulum stress, and it may be required for optimal mitochondrial protein synthesis through taurine modified tRNAs. It has also recently been suggested to be involved in skeletal muscle fatigue, most likely due to mitochondrial effects (13–15). Furthermore, taurine seems to have a positive effect on glucose homeostasis in type 2 diabetic patients (15,16) and has in rodents been shown to prevent or delay development of insulin resistance induced by fructose-overfeeding (17) as well as improving glucose homeostasis (18). However, the mechanism(s) whereby taurine exerts these effects is not known. Several studies have documented that taurine ameliorates some of the harmful effects that a maternal LP diet confers on the pancreas of the offspring by normalizing proliferation (19) and vascularization in pancreatic islets, and decreasing sensitivity of the pancreatic islets toward cytokines (20). Collectively, these studies suggest that taurine has a “reprogramming” or rescuing effect during fetal development, perhaps via epigenetic and/or organogenesis-related mechanisms. There is, however, no information on possible taurine effects on the development of peripheral organs like skeletal muscle and liver in fetal life.

In this study, we report that a maternal LP diet induces significant and qualitatively different changes in gene expression in liver and skeletal muscle and that taurine partially prevents the change in 30% of these expression changes in liver and 46% in skeletal muscle.

METHODS

Animals.

Virgin female (7–8 wk old, n = 3 per diet group) C57BL/6 mice (Taconic, Ejby, Denmark) were mated with C57BL/6 male mice. After observation of a vaginal plug (gestation day 0), the mice were randomized into four different diet groups: Normal protein (20% casein; NP; Hope Farms 4400.00, Woerden, NL) or LP (8% casein; LP; Hope Farms 4400.01) with or without 1% (wt/vol) taurine in the drinking water (tau, i.e. NP + tau or LP + tau) (synthetic taurine; Sigma Chemical Co.-Aldrich, St. Louis, MO).

The four different diet groups were considered largely isocaloric because the taurine supplementation contributed only marginally to the calorie intake: Mice drink approximately 7 mL of water per day and eat approximately 7 g of chow per day (21). For an NP diet, this corresponds to a total chow protein intake per day of 1.4 g protein (20% casein) or roughly 0.22 g N-equivalents per day (assuming 16% nitrogen). Similarly, total chow protein intake per day for the LP diet (8% casein) is 0.56 g protein or roughly 0.09 g N-equivalents. The taurine enriched water will supply 0.07 g taurine or 0.0078 g N-equivalents (taurine content is 11.2% nitrogen), i.e. less than a 10% increase of the nitrogen intake. Irrespective of its N-contribution, taurine cannot make up for the decreased intake of essential amino acids caused by the LP diet.

Mice were kept in a 12-h light/dark cycle. At day 19, the mice gave birth and newborn pups were weighed, killed by decapitation and liver and hind leg skeletal muscle quickly and quantitatively removed, weighed, quick frozen in liquid nitrogen and stored at −80°C for further analysis. A total of 25 pups from 12 different dams were analyzed, with 5 ≤ n ≤ 7 pups per group, all groups being a mixture of male and female pups. All experimental procedures were approved by The National Committee on Animal Experimentation, Denmark and by the local animal facility at the University of Copenhagen, Denmark.

RESULTS

Birthweight and enzyme activities in liver and muscle of newborn mice.

Mouse dams were subjected to four different isocaloric diet regimes from day 1 of pregnancy until giving birth as described in materials and methods. The LP diet caused a ∼40% decrease (p < 0.001) of the birth weight of the pups (Fig. 1 and Table 1). Supplementing the maternal LP diet with taurine reduced the birth weight loss by half (p < 0.01) (Fig. 1; Table 1). Both liver and skeletal muscle mass of newborn pups were decreased in proportion to body weight by the maternal LP diet (Table 1).

Figure 1
figure 1

Maternal LP diet confers changes in body mass; partial rescue by taurine. Newborn mice subjected to different diet regimes in utero as described in materials and methods were weighed at birth. NP (20% casein) or LP (8% casein) during pregnancy. Taurine, 1% taurine supplementation in the drinking water during pregnancy. ¶, p < 0.001 NP vs. LP; **, p < 0.01 LP vs. LP + tau; *, p < 0.05 NP vs. LP + tau. Bars depict means ± SEM 5 ≤ n ≤ 7 per diet group.

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Table 1 Maternal low protein diet confers changes in body mass, but not in liver and skeletal muscle citrate synthase specific activity in newborn mice
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As a measure of the mitochondrial fraction of tissue mass, citrate synthase (CS) specific enzyme activity and mRNA level (22) was determined in liver and skeletal muscle of the newborn pups. There was no difference between diet groups in either CS mRNA levels (Table S1, http://links.lww.com/PDR/A56) or CS enzyme specific activities (Table 1). Similarly, we detected no difference between diet groups with regard to creatine kinase specific activity in skeletal muscle. Lactate dehydrogenase specific activity increased in skeletal muscle with taurine supplementation (p < 0.05), whereas there was no such effect in liver (Table 1).

Effects of a maternal LP diet on gene expression levels in liver and skeletal muscle of newborn mice.

Gene expression profiles of three randomly chosen offspring samples from each diet group of both liver and skeletal muscle were analyzed using Affymetrix gene expression microarrays as described (Supplemental methods, http://links.lww.com/PDR/A56) (Table 2; Fig. 2A and C).

Table 2 General data from the microarray analyses
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Figure 2
figure 2

Liver and skeletal muscle gene expression profiling of newborn mice subjected to a maternal LP diet with or without taurine supplementation. Hierarchical clustering of samples and expression patterns of genes significantly different between newborn mice subjected to either a maternal LP diet or a maternal NP diet. (A) Liver (2012 genes), (B) liver, genes rescued by taurine (600 genes), (C) skeletal muscle (967 genes), (D) skeletal muscle, genes rescued by taurine (444 genes). NP (20% casein) or LP (8% casein) during pregnancy, tau, 1% taurine supplementation in the drinking water during pregnancy. Numbers indicate biologic replicates.

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In liver, the expression of a total of 2012 nonredundant transcripts, all encoding known genes, were significantly changed by the maternal LP diet (Table S5A, http://links.lww.com/PDR/A56), while a somewhat smaller number of 967 nonredundant transcripts, all encoding known genes, were changed in skeletal muscle (Table S5B, http://links.lww.com/PDR/A56) (pairwise false discovery rate <10%). There was an overlap of 210 known genes, for which changes in expression levels were seen in both liver and skeletal muscle (Table S5C, http://links.lww.com/PDR/A56). Of the 2012 known genes affected in the liver, 667 transcripts were up- and 1345 down-regulated. Similarly, in skeletal muscle, of the 967 known genes affected, 312 were up- and 655 transcripts were down-regulated compared with a maternal NP diet.

A number of significantly changed transcripts and one not significantly changed (CS) were randomly selected for RT-PCR validation of the microarray data. Positive validation was obtained for seven out of eight and six out of nine transcripts encoding known genes in liver and skeletal muscle, respectively (Table S1, http://links.lww.com/PDR/A56).

Taurine partially rescues the maternal LP diet offspring phenotype.

Taurine supplementation to the mother had no effect on the gene expression patterns in the offspring of the NP diet groups (Fig. 2; statistical analysis comparing NP with NP + tau, data not shown). In contrast, taurine supplementation prevented a major part of the gene expression changes seen with the maternal LP diet (Fig. 2; Table 2). Qualitatively, this is most clearly demonstrated when comparing all genes whose expression was changed by the LP diet (Fig. 2A and 2C) with those that were rescued (Fig. 2B and 2D) and noticing that in the latter, the LP + tau samples cluster together with NP and NP + tau in both liver and skeletal muscle. In all, taurine had a rescuing effect on 30% (600 out of 2012) of the changed transcripts of known genes in liver and on 46% (444 out of 967) in skeletal muscle (Table 2), rendering the taurine effect more pronounced on muscle than on liver (p < 10−16, Fishers exact test).

Among the changed genes, we found that mitochondrial genes (Tables S2 & S3, http://links.lww.com/PDR/A56) and among those in particular genes involved in oxidative phosphorylation (Table S4, http://links.lww.com/PDR/A56) were the most over-represented gene sets in both liver and skeletal muscle (Tables 3 and 4). Genes involved in tri carboxylic acid (TCA) cycle, pyruvate metabolism, and glycolysis were over represented only in skeletal muscle (Table 4). Over representation of genes involved in amino acid metabolism was observed both in liver and skeletal muscle (Tables 3 and 4). However, the key regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), was down-regulated by the LP diet in both tissues (Table S1, http://links.lww.com/PDR/A56).

Table 3 Analysis of gene set over-representation in liver
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Table 4 Analysis of gene set over-representation in skeletal muscle
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When genes were grouped according to function (Fig. 3A), a large difference was seen between up- and down-regulated genes in energy metabolism in both liver and skeletal muscle, albeit in a different direction in the two tissues. Similarly, when the largest significant gene set, mitochondrial genes (Fig. 3B), was grouped according to function, a clear difference between liver and skeletal muscle could be seen. No overt difference in the taurine rescue effect was seen between tissues when genes were grouped according to function.

Figure 3
figure 3

Classification of changed genes by biologic function. A, Genes significantly changed in newborn mice in response to an in utero LP diet in liver and skeletal muscle. For comparison, only genes that were changed in both liver and skeletal muscle are shown. B, Mitochondrial genes changed in newborn mice in response to an in utero LP diet in liver and skeletal muscle. Gray/black bars indicate down/up-regulated genes in newborn mice subjected to a LP diet in utero compared with a NP diet. Hatched bars indicate the number of genes rescued by taurine supplementation.

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The rescuing effect of taurine was significantly higher on genes involved in fatty acid metabolism in liver compared with other gene sets (p = 0.027, Fishers exact test) (Table 3), whereas in skeletal muscle this rescuing effect was present only for genes involved in oxidative phosphorylation and the TCA cycle (p = 0.010 and p = 0.029, respectively, Fishers exact test) (Table 4).

DISCUSSION

The novel findings of this study were 1) that a maternal LP diet induced substantial changes in gene expression patterns in newborn offspring in both liver and skeletal muscle, 2) that the changes in mitochondrial genes in general and genes of oxidative phosphorylation in particular were prominent in both liver and skeletal muscle, with predominantly up-regulation in liver and down-regulation in skeletal muscle, and finally 3) that maternal taurine supplementation in the drinking water during pregnancy to a surprisingly large extent prevented these changes in gene expression patterns in both liver and skeletal muscle (preventing 30% in liver and 46% in muscle). Importantly, taurine also partially prevented the decrease in birthweight caused by a maternal LP diet.

Birth weight.

We found a 40% decrease in birthweight of LP offspring compared with NP offspring. This is a somewhat larger decrease in birthweight than previously reported in rodents, i.e. 18–35% in rats (11,23) and 28% in C57BL/6 mice (24), whereas others have observed only a 7% weight loss in the same mouse strain (25), most likely due to differences in diet composition. We observed no difference in the ratio of organ weight:total body weight, which seems to disagree with a previous study in rats (23). Furthermore, it came as a surprise that taurine supplementation almost prevented the low birthweight; in particular because this effect was not observed in rats in a similar study (19). However, these disagreements likely reflect species differences.

Changes in global gene expression patterns in liver and skeletal muscle.

Despite the fact that the NP and LP diets are isocaloric, genes involved in energy metabolism, oxidative phosphorylation, and other mitochondrial genes were the ones most over represented on the list of changed gene sets (Tables 3 and 4). However, as CS activity can be considered a measure of mitochondrial mass (22), the results suggests a dysregulation of gene expression related to mitochondrial function rather than a change in mitochondrial mass. This may be in contrast to studies by Park et al. (26), who found a significant decrease in mtDNA:gDNA ratio in both liver and skeletal muscle in 5-wk-old offspring of maternal LP rats. Although genes of oxidative phosphorylation are over represented in the list of changed genes in both tissues, a striking difference between liver and skeletal muscle was observed (Fig. 3). In liver, a large fraction of genes involved in amino acid metabolism, protein synthesis, TCA cycle, and energy metabolism genes involved in both Complex I-IV and ATP synthesis showed increased expression, whereas in skeletal muscle the opposite effect was observed. One may speculate that these findings can be explained by the difference in the taurine requirement of the two tissues, as the concentration of taurine in skeletal muscle is ∼6-fold higher than in the liver in mice (27), when taking into consideration the decrease in available taurine in LP (28). Furthermore, taurine is a constituent of mitochondrial tRNA (29), and may thus be required for normal mitochondrial function.

In another model of intrauterine growth retardation in rats, uterine artery ligation, hepatic glucose production, the activity of phosphoenolpyruvate carboxykinase, and PGC-1α was found to be increased in liver (30), and the capacity of oxidative phosphorylation diminished (31). A similar observation was made in skeletal muscle (32). Gestational protein restriction does not suffer from the side effect of a decrease in available O2 as seen in uterine artery ligation and differences in observations may be credited to this.

Down regulation of oxidative phosphorylation in skeletal muscle has in several cases been linked to development of type 2 diabetes and insulin resistance (33–35). Although the exact mechanism is unknown, a decrease in the number of skeletal muscle mitochondria and/or in the activity of oxidative phosphorylation as well as mitochondrial dysfunction at birth may, if it is permanent, be associated with a decreased capacity for β-oxidation, which may lead to increased intracellular lipid content and subsequent disruption of insulin signaling (36). However, this hypothesis has recently been questioned (37).

Interestingly, in humans birthweight is positively associated with the expression of PGC-1α in skeletal muscle in adults (6), but despite PGC-1α controlling mitochondrial biogenesis (38), no concomitant difference in mitochondrial gene expression and oxidative phosphorylation was observed in adults with a low birthweight (6). However, the decrease in mitochondrial gene expression seen in this study, and not in adult humans, may reflect the difference between newborns and adults or species difference.

Partial phenotype rescue by taurine supplementation.

Taurine has previously been shown to rescue the detrimental effects of a maternal LP diet regarding pancreatic function in the offspring (19,20,39,40). However, such effects have not been examined in liver and skeletal muscle.

We find a large rescuing effect on gene expression profiles in both liver and muscle by taurine supplementation, with significantly more mitochondrial genes rescued in skeletal muscle than in liver, suggesting a preferential mitochondrial rescue effect by taurine in skeletal muscle compared with liver. Furthermore, we were surprised to find that taurine exhibited an even more specific rescue effect, as genes of fatty acid metabolism in the liver and genes involved in oxidative phosphorylation and TCA cycle in skeletal muscle were preferentially rescued compared with other genes (Tables 3–4; Table S4, http://links.lww.com/PDR/A56). A similar study examining the effect of maternal LP diet and taurine on islet gene expression also found decreased expression of respiration and TCA cycle genes, changes which were rescued fully by taurine supplementation (40). Combined with our results, this strongly suggests that the effect exerted by taurine is mediated by a mitochondrial mechanism.

Plasma taurine concentration has been suggested to be a marker of fetal well being (41), and it has been suggested that sufficient taurine plasma concentrations may be a prerequisite for normal fetal development (41). This is corroborated by the observation that lack of the taurine transporter (TAUT) confers a large decrease in exercise capacity (13), which may also be related to the observation that TAUT expression increases during myogenesis and that taurine is able to protect against dexamethasone induced atrophy (42). Thus, a picture of taurine as a necessary factor in myogenesis, and perhaps mitochondrial biogenesis, emerges.

In liver, a deficiency of taurine, as seen in the TAUT knockout mouse, causes an increase in hepatic apoptosis and inflammation, as well as a decrease in mitochondrial respiratory control ratio (43). Also, taurine has been shown to have an anti-apoptotic effect on the liver and to normalize tamoxifen induced mitochondrial dysfunction in rats (44). There are several reports on the effect of a maternal LP diet on offspring DNA methylation patterns (45,46), but there is no evidence of taurine involvement in this process. Interestingly, taurine has been shown to decrease N-methylation in rat heart (47).

Methodological considerations.

The C57BL/6 mice are hypertaurinuric (15), and it is important to consider whether the taurine effect could be caused by the extra nitrogen or sulfur supplied as taurine. The nitrogen supply by taurine is a minor fraction of the total nitrogen intake (see Materials and Methods), and it is unlikely that the taurine effects seen in this study are due to an increase in sulfur amino acid availability because there appears to be very little, if any, metabolic conversion of taurine into methionine or cysteine (48). Most other similar studies have used a higher concentration of taurine in the drinking water (2.0% or 2.5%) (19,20,39,40) than the 1% concentration used in this study.

In conclusion, a LP diet to mice during pregnancy caused major changes in both body mass and gene expression profiles of liver and skeletal muscle of the newborn mice. The expression changes were predominantly related to mitochondrial genes but were markedly different in liver and skeletal muscle. Maternal taurine supplementation in the drinking water partially prevented both the change in body mass and changes in gene expression. In particular, skeletal muscle genes involved in oxidative phosphorylation were almost completely normalized by the taurine supplementation. The mechanism of these taurine effects remains unknown.