The THs are essential for development, growth, and metabolism 
. The present study shows that transient neonatal hypothyroidism in male rats gave rise to endocrine alterations that not only affected postnatal growth but also influenced hepatic physiology and responsiveness to THs replacement in adulthood.
Growth-inhibiting conditions exist during development in association with malnutrition, glucocorticoid excess, systemic diseases, GH-IGF-I deficiency, or hypothyroidism 
. In this work, several biomarkers of neonatal hypothyroidism (i.e., decreased circulating THs and hepatic mRNA expression levels of ME and Spot14) and high expression of IGFBP-2 were associated with decreased circulating IGF-I and a delayed somatic growth rate on PND30. Furthermore, when the growth-inhibiting condition (i.e., MMI) was removed, somatic growth rate (weight and tail lenght gain) and food efficiency increased in CH rats, which is a phenomenon known as catch-up growth 
. By PND80, however, this effect had subsided, and somatic growth rate (see body weight gain and tail length gain in Supplementary File S3
) in CH animals was similar to age-matched INTACT controls. Alternatively, higher food efficiency remained in euthyroid CH group on PND80 along with significance differences in total body weight and size in comparison with INTACT group, which suggests, in agreement with previously reported data 
, a higher but less efficient rate of metabolism (i.e., a reduced ability to transform calories consumed into total body weight and size) in rats previously exposed to CH. The CH rats on PND80 also showed increased mRNA levels of several GH target genes (i.e., IGF-I, SOCS-2, CIS, CYP2C11, CYP2C13) suggesting that the increased hepatic GH activity observed in these animals was possibly associated with catch-up growth. In contrast, other well-known GH target genes in female rats such as CYP2C7 and CD36 were downregulated in CH group. This apparent paradox could be explained by sexually dimorphic pattern of gene expression in rat liver 
. The downregulation of female-predominant genes (e.g., CYP2C7 and CD36) concomitant with the induction of male-predominant genes (e.g., CYP2C11 and CYP2C13) suggests that a male pattern of gene expression was enhanced in CH rat liver.
In the current study, we show that transient CH is associated with changes in SOCS-2 and CIS expression, which are key negative regulators of GH-dependent somatic growth in vivo
. GH resistance can be shown in rat models of sepsis and uremia and in small rats for gestational age (SGA) without catch-up growth. This was associated with an increased expression of SOCS-2 and CIS and impaired JAK/STAT signaling 
. In our model, however, catch-up growth was associated with the overexpression of SOCS-2 and CIS in adult CH rats. Whether the overexpression of SOCS and CIS is associated with delayed growth development and catch-up growth in CH rats requires further research.
Growth-inhibiting conditions during fetal-neonatal period of life may influence lipid metabolism in adulthood 
. Human and rats, who do show catch-up of somatic growth and increased feed efficiency after withdrawal of growth-inhibiting condition (e.g., SGA or caloric restriction), have higher risk of fat in the liver and increased adiposity in adulthood 
. Now, we show that somatic growth inhibition by neonatal hypothyroidism influences hepatic lipid metabolism in adulthood. CH rats showed a concomitant upregulation of PPARα and CPT1, a gene related to fatty acid catabolism.
Furthermore, adult CH rats showed a downregulation of CD36, which is involved in fatty acid uptake and a well-known PPARα target gene, along with the reduced transcription of genes involved in cholesterol uptake (LDLR), cellular sterol efflux (ABCA), triglyceride assembly (MTTP), bile acid synthesis (CYP8B1, CYP7A1 and CYP27A1), and lipogenesis (SREBP1c) .
These data indicate that CH significantly influenced lipid metabolism in adulthood and, most likely, contributed to the diminished hepatic levels of triglycerides, cholesteryl esters, and FFA. Conversely, because expression levels of several lipid genes in CH adult group were altered in similar direction to that detected in WP group, our data could be explained, in part, as a consequence of delayed growth 
. Furthermore, a reduced content of hepatic lipids in CH group could be caused by prolonged catch-up growth which might cause an increased lipid catabolism in growing animals (i.e., CH group) in comparison with those that have completed their body growth (i.e., INTACT). To determine whether some of these changes caused by CH are life-long adaptations, similar analysis would need to be performed in older animals 
. Furthermore, the reduced content of hepatic lipids in CH group could be caused by prolonged catch-up growth which might cause increased lipid catabolism in growing animals (i.e., CH group) in comparison with those that have completed their body growth (i.e., INTACT). However, several reports have suggested that catch-up growth is likely associated with increased level of hepatic lipids and adiposity 
, which would not be in agreement with this explanation. Additionally, despite all of the changes observed in liver, the levels of circulating lipids (triglycerides and cholesterol) and lipoproteins (data not shown) were similar to those in the INTACT aged-matched littermates, which suggest that CH rats were able to maintain lipid homeostasis and support the increased energy demands imposed by an accelerated growth rate. This is apparently achieved by redistributing lipids from the liver towards peripheral tissues rather than through active hepatic lipogenesis, which is an energy-consuming process that would compete with peripheral energy needs.
In this study, we show that transient neonatal hypothyroidism influences transcriptional program in adult liver. Despite being euthyroid, adult CH animal showed a modified transcriptional profile in liver in comparison with age-matched INTACT rats, which might be explained by altered tissue responsiveness to T3 and/or GH, two hormones that are drastically reduced by hypothyroidism 
. However, independent of CH status, several of the responses to hypothyroidism and hormone replacement were similar. As expected, development of TX increased circulating cholesterol levels and decreased the serum triglyceride levels, while T3 hormone replacement restored circulating cholesterol level. Additionally, T3 and GH treatments increased the body weight gain and hepatic levels of IGF-I mRNA. At first glance, these results suggested that the CH rats, after suffering a biological insult (i.e., a second burst of hypothyroidism in adulthood), showed a biological response similar to age-matched INTACT rats. However, several of the responses to TX or hormone replacement suggested an altered lipid metabolism in the CH rats. First, a significant reduction of circulating FFAs by TX in TX/+CH group but not in TX/−CH group, an effect that was restored by T3 replacement. Second, GH-increased hepatic cholesterol esterification occurred in the TH/−CH animals but not in the TH/+CH animals. Third, GH treatment reduced serum VLDL fraction in the TX/−CH but not in the TX/+CH rats (data not shown). We did not observe major alterations in hepatic reactivity to GH in terms of lipid changes which suggests that the capacity of GH treatment to reduce serum triglyceride levels in TX/−CH rats, but not in TX/+CH rats, is most likely due to altered GH activity in extrahepatic tissues, such as fat and muscle 
. Finally, hepatic concentrations of lipids in T3-treated TX/+CH rats did not differ significantly from TX/−CH group, which suggested that the homeostatic capacity of CH tissue in response to T3 was not dramatically affected. However, this is in contrast to the enhanced ME expression in the TX/+CH group in response to the T3 treatment. Increased ME mRNA expression did not seem to be a general response to T3 replacement because SREBP1c and 2 showed a less pronounced change (data not shown). ME is directly regulated by the binding of TR to a TRE in the promoter of the ME gene 
. Our measurement of mRNA levels for TR receptors in liver showed unaltered expression of the major isoform TRβ and reduced levels of TRα, making it unlikely that changes in ME expression can be attributed to altered TR content. Noticeably, the ME regulatory region also contains binding sites for PBX1 and 2 
, CEBPα 
, and an E-box 
that can modulate the response to T3. At this point, we cannot exclude the possibility that CH effects on these transcription factors and other nuclear co-regulators influence ME expression but additional experiments are needed to test this hypothesis. Likewise, a clearer mechanistic explanation for the metabolic changes observed in the CH rats would require, among others measures, the analysis of fat and muscle metabolism.
Androgens may influence the hepatic response to CH. It is well known that neonatal hypothyroidism results in increased circulating levels of testosterone in male rats, which is secondary to increased testis size 
. We also observed two-fold higher serum testosterone levels in the CH rats compared to the age-matched adult INTACT rats (data not shown). Although the liver is not considered to be a primary target of testosterone action, androgens maintain specific male pattern of pituitary GH secretion and actions on liver 
, and it has been shown that specific deletion of androgen receptor (AR) in liver of male animals causes hepatic insulin resistance with decreased fatty acid β-oxidation and steatosis 
, which implicates the hepatic AR as a positive factor in maintaining physiological control of glucose and lipid homeostasis. Alternatively, in prostate, a well-known target tissue of androgen action, testosterone has lipogenic effects, such as inducing the expression of FAS 
. Therefore, we cannot exclude the possibility that some of the transcriptional effects detected in the liver of the CH rats (e.g., increased CYP2C11, CYP2C13, and PPARα) were secondary to increased levels of circulating testosterone.
In summary, our findings support the hypothesis that TH deprivation during neonatal period of life causes long-lasting influence on the liver transcriptome and provokes an altered responsiveness to biological insult in adulthood. Several findings that cannot be explained by the lower body weight in CH rats, compared to WP, include genes regulated by GH (e.g., IGF-I, CIS, CYP2C11, and CYP2C13) and genes involved in hepatic lipid metabolism (e.g., PPARα, CPT1, ME, ACC1, FAS, CYP4F1, AOX, L-FABP, LXR, HMGCoA-S, and HMGCoA-R). Being clinically relevant, the changes observed in the transcriptional responses to T3 highlight the possibility that CH influences tissue reactivity to thyromimetic drugs in adulthood 
. Interestingly, thyroid-disrupting compounds, which can cause neonatal hypothyroidism, include a wide range of chemicals from naturally occurring compounds, pharmaceuticals, and a number of xenobiotics 
. The long-lasting influence of growth-inhibiting conditions on hepatic metabolism is intriguing and warrants further study to explore whether the alterations observed in this study cause metabolic disruptions or chronic diseases.