# Neonatal hypothyroidism in male mice is associated to developmental delay and metabolic dysregulations in the adult

Anouk Fontaine, Noémie Sallen, Karine Gauthier

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Anouk Fontaine, Noémie Sallen, Karine Gauthier. Neonatal hypothyroidism in male mice is associated to developmental delay and metabolic dysregulations in the adult. 2026. hal-05519057

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1 Neonatal hypothyroidism in male mice is associated to developmental delay and metabolic

2 dysregulations in the adult.

3 Authors: Anouk FONTAINE*, Noémie SALLEN*, and Karine GAUTHIER

4 Institut de Génomique Fonctionnelle de Lyon, UMR5242, École Normale Supérieure de Lyon, INRAE

5 USC1370, CNRS, Lyon 69007, France

6

7 *these authors equally contributed to this work

8 Correspondence : Karine Gauthier, PhD, Institut de Génomique Fonctionnelle de Lyon, UMR5242,

9 École Normale Supérieure de Lyon, INRAE USC1370, CNRS, 32-34 avenue Tony Garnier, Lyon 69007,

10 France. E-mail : karine.gauthier@ens-lyon.fr. ORCID : 0000-0003-2961-6119.

11 Disclosure summary : the authors have nothing to disclose

12

13 Abstract

14 The postnatal period of mouse development is critical for the maturation of many organs and fine
15 tuning of hormonal feedback loops. These developmental transformations are necessary to allow the
16 transition from a suckling pup to an independent life but they also determine the metabolic features
17 of the adult. Thyroid hormones are key hormones that control these postnatal events. However, the
18 impact of a short-term exposure to maternal hypothyroidism during the perinatal period on the
19 metabolic outcome of the descendants was not clearly established. We used a new protocol to obtain
20 animals exposed to maternal hypothyroidism only during gestation or only during their two first
21 postnatal weeks. The developmental and the long-term consequences of these two short perinatal
22 periods of hypothyroidism were assessed. Animals born from hypothyroid mothers did not show
23 developmental delays. As adults they were undisguisable from the controls and could adequately face
24 metabolic stresses such as exposure to a high fat diet or cold temperature. In contrast pups exposed
25 to maternal hypothyroidism during lactation presented delayed growth and remained leaner than the
26 controls. The expression of genes involved in different metabolic pathways in liver or brown adipose
27 tissues were affected both during development and in adults. The adults were also more sensitive to
28 diet induced obesity and to cold exposure. In conclusion in mice severe hypothyroidism in the pregnant
29 dam is not detrimental for postnatal development or metabolic programing of the pups. In contrast
30 postnatal hypothyroidism leads to developmental delays and long-lasting metabolic dysregulation in
31 otherwise euthyroid animals.

32
## 33 Introduction

In the context of a worldwide pandemics of obesity and diabetes, it is highly important to understand the causes of the increased sensitivity to these metabolic disorders. Life style, including diet and physical activity, is a major determinant but could not account for the extent of the pandemics. It is now well established that fetal and neonatal periods are critical to establish the metabolic capacities of the adult. Any perturbation from either nutritional or environmental origin during this time window can predispose the adults to chronic diseases including obesity. This concept is referred to as the developmental origin of adult diseases (1-2).

Thyroid hormones (TH) are key players during the development of many organs such as brain, bones, intestine and brown adipose tissue (BAT) (3-4-5-6). Pax8⁻/⁻ mice that lack a functional thyroid gland die before weaning and can be saved by daily postnatal injection of a TH (7). In mice the fetus begins to produce his own TH just before birth. Beforehand it relies on the maternal TH. The blood concentrations of the inactive T4 and its active derivative T3 are very low at birth, increase to peak around weaning time (PDN21) and thereafter decrease and stabilize at adult levels (8).

T3 signals *via* binding to its nuclear receptors TRα and TRβ (9). TR are T3 dependent transcription factors. Their targets genes differ from one tissue to the other (10). Many models of classical or tissue selective inactivation of *Thra* and/or *Thrb* encoding for these receptors in mice allowed to study the specific role of TH in orchestrating the different maturation events necessary to transform mother dependent pups to independent young mice (11-12). In humans, pregnancy is frequently associated with hypothyroidism. An immediate supplementation of TH can avoid developmental consequences in the newborn. However, if not corrected this gestational hypothyroidism correlates with higher frequency of metabolic diseases in the euthyroid adults (13). No causative role of hypothyroidism has been established yet. The goal of our study was to determine in mice whether short windows of maternal hypothyroidism either during gestation or in the two first weeks of lactation had an effect on the development of their pups and most importantly on their sensitivity to metabolic dysregulation later in life.

Different studies performed in rats in which hypothyroidism was chemically induced from gestational day 7 (GD7) to weaning, mainly focus on the consequence on brain development (14-17). Here we developed a new protocol based on cross adoption to study the impact of either gestational or postnatal (PDN5 to PDN23) hypothyroidism. Our results show that in mice gestational hypothyroidism (Pre-HT) had minimal impact on all the parameters measured. In contrast transient early postnatal hypothyroidism (Post-HT) strongly impairs growth and has life-long impacts on body weight, fasted glycemia and the expression of metabolic genes in liver and BAT. These dysregulations in Post-HT mice are associated with increased sensitivity to a diet induced obesity and difficulties to adapt to colder environment as compared to controls (CTRL).

## 69 Material and methods

### 70 *Animal procedures and preparation of tissue samples*

C57BL/6JR GD4 female mice were ordered from Janvier Labs (LE GENEST ST ISLE) and kept under the Altromin 1310 breeding diet (Genestil, Beauvais) (CHOW). At GD7 a third of the females were switched to a propylthiouracil (PTU)-containing diet (Harlan Teklad TD95125, Madison, WI) to induce hypothyroidism. One day after delivery (PDN1) pups born from hypothyroid mothers were given for adoption to euthyroid mothers (Pre-HT), and vice versa (Post-HT). Animals from the control (CTRL) groups were born and raised by euthyroid mothers. PDN15 onward all mice were fed *ad libitum* CHOW and housed under recommended conditions until juveniles were separated from their mothers at
PDN60 (Figure 1). For the time course analysis (PDN0 to PDN60), pups, irrespectively of their sex, were decapitated, blood sampled and the different tissues flash frozen in dry ice. Pups from at least 2 different litters were used for each time point to avoid litter effect.

For metabolic experiments male mice only were included. The number of males per dam was kept between 4 and 5 to limit the litter size bias. After PDN60 males were dispatched 2 or 3 per cage.

For Glucose tolerance test (GTT) mice starved for 6 hours were intraperitoneally injected with glucose (2mg/g of body weight). Glycemia was measured before and 15, 30, 60, 120min post injection using a glucometer (Accu-Chek). For insulin tolerance test (ITT) mice starved for 6 hours were intraperitoneally injected with insulin (0,75 U/Kg of body weight). Glycemia was measured at the same time points as for GTT.

To address the sensitivity to diet induced obesity, 3-month-old male mice were fed a High Fat Diet (HFD) (Research Diets D12331 58% Fat and Sucrose, New Brunswick, NJ) up to 4 months.

Cold response was assessed in mice implanted under isoflurane anesthesia with IPTT transponders (Plexx BV, Netherlands) exposed to 4° for 75 hrs. Body temperature and weight were measured 4 times per 24 hours. Daily food intake was inferred from the food left in the feeders after 24, 48 and 72 hours.

At the end of the experiments, mice were anesthetized (Xylazine/Ketamine, 0,66mg/3,3mg per mouse), blood drawn to retrieve plasma and tissue dissected, flash frozen for later RNA preparation or fixed in 100% ethanol for further bone analyses.

All experiments were carried out in accordance with the European Community Council Directive of September 22, 2010 (2010/63/EU) regarding the protection of animals used for experimental and other scientific purposes. The research project was approved by a local animal care and use committee (C2EA015) and subsequently authorized by the French Ministry of Research (APAFIS # 4511-2023081617562798v3).

### RNA extraction and expression analyses by relative quantitative RT-PCR (RT-qPCR):

RNAs were extracted using Trizol (Invitrogen). Total RNA was converted to cDNA using MMLV reverse transcriptase (Promega). Relative RT-qPCR were performed using SYBRGreen mix (BioRad iQ supermix). Duplicates were run for each sample. The results were analyzed according to the ΔΔCT method (18). *Hprt* was used as the reference gene, and the control group is as indicated in the figure legends. All the primer sequences are listed in Table 1.

### Bone analysis: bones were stained with alizarin as previously described (19). The size of both femurs and tibia were measured using a caliper.

### Biochemistry:

Plasmatic free T3 (FT3) and free T4 (FT4) were quantified on a Cobas 6000 automat with the Cobas e601module (Roche, ECL analysers).

### Statistics:

The data presented represent the average values for the different animals from the same genotype given the same treatment. The error bars represent SEM. Statistical significance was determined using the one variable Anova method. * stands for $p \le 0,05$, ** $0,005 \le p \le 0,05$, *** $p \le 0,001$

## Results

### A cross adoption strategy to obtain two windows of perinatal hypothyroidism:

In this study our goal was to determine the impact of perinatal maternal hypothyroidism limited to either the gestational (Pre-HT) or lactation (birth to PDN15) (Post-HT) period on the development but
also on the ability of their pups to face metabolic challenges later in life. To this end we designed a
new breeding strategy (Figure 1). Severe hypothyroidism could be observed in the dams under PTU
treatment, with no circulating FT4 and highly reduced FT3 as soon as GD14. These decreases were
even stronger at delivery (Figure 2A). The hypothyroid status of the mother clearly affected the thyroid
status of the pups. In all cases FT4 (Figure 2B) was much more affected than FT3 (Figure 2B). In Pre-HT
neonates and Post-HT PDN7 and PDN 15, FT4 was hardly detectable whereas FT3 concentrations were
never decreased below 30% of the normal concentration. After switching back to CHOW these
circulating levels were largely restored after 7 days and back to normal after 15 days both in the Pre-
HT and Post-HT groups. In contrast FT4 levels only normalized after a month in both groups. To
indirectly assess the local availability of T3, the expression of direct well characterized T3 target genes
was measured in 3 different tissues, *Hcn2* in the heart (Figure 2C), *Dio1* in the liver (Figure 2D) and
*Ptdgs* in the brown adipose tissue (BAT) (Figure 2E). Normal local T3 availability was restored faster
than circulating FT3 in the 3 analyzed tissues but the speed of recovery was faster in heart and liver
than in BAT. As early as PDN60 all animals were euthyroid with normal levels of circulating TH.

Altogether, this cross-adoption strategy allows to obtain a transient hypothyroidism in the pups, from
late gestion to around PDN7 in the Pre-HT group, from the first days of life to PDN23 in the Post-HT
group. In the later the hypothyroidism period corresponds to the naturally occurring peak of FT3
measured between PDN15 and PDN33 in the controls (CTRL) (Figure 2B).

139 *Post-HT but not Pre-HT impacts postnatal development*

One well described consequence of hypothyroidism is growth delay (20). Post-HT clearly affected body
weight (Figure 3A) and linear growth measured by the length of the femur and the tibia (Figure 3B)
well after normalization of FT3 concentration (PDN33) (Figure 2B). Post-HT animals never normalized
their weight, despite reaching a normal bone size by PDN60. In Pre-HT bone length was only slightly
reduced at PDN7. T3 is also involved in the maturation of BAT (6). As for bone length, the weight of
BAT was only decreased in Post-HT from PDN15 to PDN33 then normalized at PDN60 (Figure 3C). No
significant difference was seen in the Pre-HT conditions. These data suggest that Pre-HT does not
impact postnatal development for the parameters tested. In contrast Post-HT delays the growth of
bone and BAT.

149 *Pre-HT and Post-HT impact postnatal metabolism*

As lower weight in Post-HT did not only result from decreased growth we hypothesized that TH status
could impact metabolism during this postnatal period. Activity of metabolic pathways were indirectly
assessed by measuring the expression of genes encoding key enzymes and proteins of these pathways.
In the liver (Figure 4A) of Post-HT animals the expression of two of the genes necessary for *de novo*
fatty acid (FA) synthesis (*Acc* and *Fas*) was delayed in Post-HT animals. In contrast the FA β-oxidation
pathway seemed to be more active up to PDN23 as assessed *via Cpt1a* and *Cpt1b* expression. In heart
the regulation of these two genes went in opposite direction (Figure 4B) despite a similar T3 status in
the two organs. At PDN33 the expression of all these genes was normalized. Hepatic glycolytic gene
*Gk* was highly up-regulated in the post-HT group at PDN 33. There was little change from either of
those pathways in the Pre-HT group. The Post-HT group clearly shows a different gene expression
pattern for metabolic pathways and this alteration persists after FT3 normalization at least for *Gk*.

BAT thermogenesis is a crucial energy demanding process during the first weeks of life for the
thermoregulation of the pup. There are two major thermogenic mechanisms in the BAT: the
UCP1/PGC1α uncoupling- and the CKB/Phospho1 futile cycle- pathways. *Ucp1* and *Pgc1a* expression
were up-regulated in early hypothyroid conditions (PDN 15) (Figure 4C). In contrast at PDN33 when
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euthyroidism was restored the CKB/Phospho1 pathway was higher in the Post-HT group. This suggests a long-lasting need for higher energy production by the BAT.

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*Post-HT but not Pre-HT is associated with lower body weight and fasted glycemia in the adults.*

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The most important part of the study was to decipher whether a short window of hypothyroidism early in life could have long lasting effects in the euthyroid adults. Up to 5 month of age the weight of Post-HT animals never reached the one of CTRL (Figure 5A). Their food intake (Figure 5B) as well as their fed glycemia (Figure 5C) were similar to CTRL but their fasting glycemia was consistently lower (Figure 5D). They showed an increased sensitivity to glucose in GTT (Figure 5E) and normal sensitivity to insulin in ITT (Figure 5F). At 5 months of age Post-HT mice remained about 15% lighter than the CTRL (Figure 5G) despite displaying similar size (Figure 5H) and weight of several organs such as inguinal WAT, liver and BAT (Figure 5I). Only gastrocnemius and gonadal WAT were lighter. The same analyses/measures were performed for the Pre-HT animals that behaved exactly as CTRL (Supplemental Figure 1) (21).

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We hypothesized that in the Post-HT group the lower weight and decreased fasting glycemia could result from a persistent altered metabolism. In liver (Figure 6A) the expression of all genes involved in the FA synthesis and β-oxidation pathways were higher in the Post-HT group, in contrast the expression of the *Gk* gene, encoding Glucose Kinase (GK), was lower. In striking opposition in the BAT (Figure 6B) FA synthesis and β-oxidation pathways were decreased and the thermogenic machinery apparently not affected. Altogether these results suggest that Post-HT animals mainly used FA as fuel in the liver and might present decreased BAT thermogenesis in absence of local FA synthesis and β-oxidation necessary for this process. These two dysregulations would be associated with higher energy cost. Pre-HT animals behaved as CTRL (Supplemental Figure 2) (21).

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*Short-term Post-HT increases sensitivity to diet induced obesity and is associated to lower fasted glycemia*

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To test whether these metabolic alterations change the sensitivity of Post-HT animals to diet induced obesity, 3.5 month-old CTRL and Post-HT male mice were switched to a High Fat Diet (HFD) that was maintained for 3 months. They were weighted just before the switch (Figure 7A). As expected Post-HT animals were 15% lighter. The body weight gain of Post-HT on HFD was similar to the one of CTRL animals (Figure 7B). At the end of the experiment the Post-HT group was still 15% lighter than the CTRL (Figure 7A). As the initial weight was different, weight gain was also expressed as a percentage of the initial weight (Figure 7C). After correction the Post-HT group did gain significantly more weight than the CTRL for the first 2 months. Their food intake was also higher (Figure 7D). However fasted glycemia was still lower in the Post-HT group (Figure 7E). The sensitivity of these mice to both glucose (Figure 7F) and insulin (Figure 7G) were higher. Strikingly and in contrast to what was observed on Chow, there was no more significant difference in the expression of in any of the genes involved either in FA synthesis, β-oxidation and glucose metabolism in the liver or in FA synthesis, β-oxidation and thermogenic activity in the BAT (Supplemental Figure 3) (21). In the same set of analyzes Pre-HT animals were undistinguishable from CTRL (Supplemental Figure 4) (21).

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*Short-term Post-HT impacts response to cold but allows maintenance of body temperature at 4°C*

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BAT activity is critical during cold exposure. Local production and β-oxidation of FA (22-23) are necessary for efficient thermogenesis in BAT. As both were affected in Post-HT animals we tested whether these animals could response properly to cold exposure. 5-month-old CTRL and Post-HT male mice were challenged to a 4° temperature. Both groups managed to maintain their body temperature under this stress (Figure 8A). However, during the 24 first hours Post-HT animals did at the expense of a higher loss of weight (Figure 8B) despite an increase of food similar to CTRL (Figure 8C). The BAT
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activity seemed to be turned on normally in both groups with increased expression of all the thermogenic genes. However, the expression of genes involved in local FA synthesis was much higher in the Post-HT group (Figure 8D).

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## Discussion

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In human low birth weight and perturbations during the maturation of metabolic organs can lead to higher risk to develop metabolic disorders when exposed to high fat, high sucrose diet in the adults. Thyroid hormones are necessary for an appropriate development of many organs and transient perturbation of TH signaling during this period could predispose individuals to metabolic dysregulation later in life.

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In this study we used animals exposed to maternal hypothyroidism only during gestation or their two first weeks of life, to assess both the developmental and the long-term consequences of the two short perinatal periods of hypothyroidism.

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In the Pre-HT group all the developmental parameters (body weight and bone length) normalized within two weeks after birth. These animals became undistinguishable from the CTRL at adult stage. These results suggest that adult mice exposed *in utero* to maternal hypothyroidism have normal metabolic competences. This contrast with previously published data indicating that pups born from hyperthyroid dams present higher glucose tolerance and increase BAT activity (24). This indicates that hyperthyroidism and hypothyroidism are not mirror conditions. Moreover, metabolic changes associated with hypothyroidism during pregnancy are less detrimental for the developing fetus.

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In contrast, transient hypothyroidism, limited to the first three postnatal weeks has severe consequences on developmental processes. The developmental defects are similar to those we previously described in *Pax*-/- mice (7) deprived of TH. However, Post-HT as opposed to *Pax*-/- do survive to weaning and to some large extent recover from transient hypothyroidism. We did not address brain development in this study. As brain is really sensitive to thyroid hormone and postnatal hypothyroidism associated to irreversible brain damages (25), some brain dysfunction is likely present in Post-HT adults. However, they do not present any obvious behavioral abnormalities suggesting that brain might partly recover. At two month of age they reach the same size as CTRL but their weight remained 15% lower suggesting a metabolic mis-programming. Glucose is the universal fuel essential for neonatal life. During pregnancy, glucose is provided through the transplacental circulation. However at birth pups need to produce glucose from glycogen before suckling. Hereafter they use FFA from the milk both as a source of energy *via* FFA oxidation and a source of glucose *via* gluconeogenesis in the liver (26). At weaning time, they will switch back to a carbohydrate-based metabolism: glucose is provided by the food and used for *de novo* FFA synthesis. This period of intense metabolic transition, corresponds to the period of transient hypothyroidism in Post-HT animals. They present altered expression of genes encoding enzymes and proteins involved in metabolic and thermogenic pathways in liver, heart and BAT. The transition to FFA synthesis before weaning is the most affected with a marked delayed expression of *Fas* and *Acc*. Hepatic FFA oxidation is also up-regulated during the whole postnatal period which might indicate a higher energy demand. This is supported by the higher expression of *Gk* encoding the limiting enzyme for glucose usage after the switch back to carbohydrates. Altogether this shows that early postnatal hypothyroidism is associated to major metabolic dysregulations during this growing phase that is highly energy demanding.

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Transient postnatal hypothyroidism does not impact FT3 and FT4 concentrations in the adults in agreement to what was published for hypothyroid rats exposed to MMI from birth to PDN21 or from

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gestation to weaning (14-17). It allows to interpret all phenotypes as long-term consequences of early hypothyroidism. Strikingly postnatal hypothyroidism is associated to lower body weight despite normal food intake, bone size and most organ weights. Only gonadal WAT and muscles were significantly lighter. As these two tissues are crucial for storage of energy, respectively triglycerides in the WAT and glycogen in the muscle, we hypothesized that reduction of body weight might result from altered metabolism. Post-HT present a simultaneous up-regulation of all the players of *de novo* hepatic FA synthesis and β-oxidation with lower *Gk* expression suggesting that these animals use lipid rather than glucose. At room temperature mice are cold stressed and BAT is activated to maintain body temperature. BAT thermogenesis relies on local *de novo* FFA synthesis and β-oxidation to produce heat. These two pathways are decreased in Post-HT animals suggesting that BAT might not be fully functional. The recruitment of more costly processes might be necessary to maintain the body temperature. This is supported by the higher expression of genes of the FA synthesis pathways in BAT at 4°C. Increased energy expenditure sustained by FFA metabolism would be responsible for lower body weight and associated lower fasted glycemia in the Post-HT group.

In response to metabolic stresses specific mechanisms are activated as to maintain body balance under HFD or to maintain body temperature in cold environments. Transient postnatal hypothyroidism does not prevent these responses: Post-HT sense the changes and respond adequately. Under HFD there is no difference in the expression of genes involved in the metabolic signaling pathways between Post-HT animals and CTRL. Similarly, under cold exposure the usual response triggered in BAT by an increase of sympathetic output can be observed: *ie* a strong up-regulation of *Dio2* and of all the thermogenic genes. Their central hypothalamic control of energy metabolism seems intact. However, Post-HT animals are hypersensitive to diet induced obesity especially during the early weeks of HFD and lose more weight despite an increase in food intake during the first hours at 4°C. Under these two metabolic stresses, Post-HT animals seem to have a delayed response. In both conditions, after the "adaptation" time they behave as CTRL. It suggests that Postnatal hypothyroidism decreases metabolic flexibility but further studies are needed to clarify the underlying mechanisms.

In conclusion we exclude that severe hypothyroidism in the pregnant dam is either detrimental for postnatal development or metabolic programming. In contrast only three weeks of postnatal hypothyroidism leads to developmental delays and long-lasting metabolic dysregulation in otherwise euthyroid animals. It confirms that the peak of thyroid hormones that normally occurs around PDN21 is necessary for the growth and maturation of many organs. If most of the developmental effects can be reversed with the restoration of euthyroidism, the metabolic consequences of postnatal hypothyroidism are long lasting. Indeed, Post-HT is associated to lower body weight and dysregulation of the expression of metabolic pathways in different organs in the adult. At this point we cannot conclude whether T3 "programs" each tissue affected or participate to establish hormonal/neuronal circuits involved in metabolic programming.

Extrapolation to humans is difficult since the timing of development and thyroid hormone production both largely differ from mice. Moreover, if the notions of metabolic programming and the developmental origins of metabolic diseases are well accepted, the exact window and nature of this programming is still unclear. However, our data clearly establish that T3 does participate to this early programming at least in mice. Perturbation of T3 signaling during this early period *via* endocrine disruptors or other, could have long term consequences not only on brain function but also on sensitivity to metabolic diseases.
300 Limitations of the study

301 Only males were used whereas hormonal and metabolic regulations are recognized to be sexually dimorphic. Metabolic changes were inferred from altered expression of metabolic genes, direct measurement would reinforce our conclusions.

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305

306 **Author contribution statements:** AF and NS performed and analyzed the experiments, KG designed performed analyzed the experiments and write the paper

308 **Funding information:** ENS Lyon (HypoMetabo program; Projet-Emergent 2024), Agence Nationale de la Recherche (Hypothyro program; ANR-22-CE14-0026-01)

310 **Author disclosure** : the authors have nothing to disclose

311 **Acknowledgements:** the contribution of Frederic Flamant for his advices and critical reading of the manuscript, SFR Biosciences (Université Claude Bernard Lyon 1, CNRS UAR3444, Inserm US8, ENS de Lyon): Nadine Aguilera and the Plateau de Biologie Expérimentale de la Souris (ANIRA-PBES) for mouse breeding; and ANIRA-AGC for mouse genotyping.

315 **Data availability** : Original data generated and analyzed during this study are included in this published article or in the data repositories listed in References.

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318

319 **Figure legends**

320 Figure 1: *a new protocol to induce hypothyroidism in narrow windows during in utero and postnatal development.*

322 Scheme of the strategy used to obtain hypothyroidism limited either to gestation or the two first weeks of lactation for the dam. GD and PDN respectively stands for Gestational Day and Postnatal Day. At GD7 pregnant mice were either kept on a breeding chow diet (CHOW) in black or switch to a PTU containing diet (PTU) in green. At PDN1, litters from hypothyroid dams were given for adoption to euthyroid dams (Pre-HT group for Prenatal Hypothyroidism) (green pups), and *vice versa* (Post-HT group for Postnatal hypothyroidism) (purple pups). The same number of pups were exchanged. The control group (CTRL) (black pups) are pups born and raised by euthyroid mice on CHOW. At PDN15 all the dams were either kept/ or switch back to CHOW. Pups were sacrificed at birth, PDN7, PDN15, PDN23, PDN33 and PDN60 to sample tissues for the studies of the developmental effects, both sexes were used. For adult studies mice were separated from their mothers at PDN60 to let hypothyroid pups recover and only males were used.

333 Figure 2: *Characterization of time course recovery of circulating thyroid hormones concentration and their action in the peripheral organs*

335 Thyroid status was analyzed in both dams and pups at the different key time points of the protocol. A-FreeT4 (FT4) (left panel) and FreeT3 (FT3) (right panel) blood concentrations were measured in the dam at GD14, PDN0 and PDN23. CHOW and PTU fed dams were respectively in black and purple dots. As only 2 samples per time points were provided we did not include error bars but measurements were
very similar and the mean is reported. B- FT4 (left panel) and FT3 concentrations were measured in the pups at PDN0, PDN7, PDN15, PDN23, PDN33, PDN60. Given the small amount of blood drawn, samples from one given condition were pooled, at least two litters (3 animals per litter) for each up to PDN33. FT4 and FT3 were also measured in the adults obtained from the protocol. The control (CTRL), Pre-HT and Post-HT are respectively depicted in black, green and purple. C/D/E- Expression of T3 target genes were measured by relative RT-qPCR as an indirect measure of T3 availability in different tissues: *Hcn2* in the heart (C), *Dio1* in the liver (D) and *PTDGS* in the BAT (E). The reference group was CTRL. Stars indicated a significant difference between CTRL and one of the treatment group.

Figure 3: *Postnatal hypothyroidism delays growth.*

Different parameters were measured to assess the growth delay due to hypothyroidism. A- Body weight (gr) was followed during all the developmental pre-weaning period from PDN5 to PDN60 in the CTRL (n=13, black), Pre-HT (n=7, green) and Post-HT (n=7, purple). B- Bone growth and body length were indirectly estimated by measuring both tibia (upper left panel) (n≥5) and femur (lower left panel) (n≥5) at PDN7, PDN15, PDN33 and PDN60. A representative bone for each time point is pictured after alizarin staining (right panels). C- BAT weight (gr) was measured at different time points (n≥5). Stars indicated a significant difference between CTRL and one of the treatment group at a given developmental stage.

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Figure 4: *Postnatal hypothyroidism modify the expression of metabolic genes during development*

To assess the impact of hypothyroidism on metabolism during development, expression of genes involved in different metabolic pathways was measured in Liver (A), Heart (B) and BAT (C) at different time points by relative RT-qPCR. A-In liver different pathways were analyzed: fatty acid (FA) synthesis (*Acc* and *Fas*), FA β-oxidation (*Cpta1* and *Cpt1b*) and glucose metabolism (*Gk* an *L-Pk*). B- In heart only FA β-oxidation (*Cpta1* and *Cpt1b*) was checked. C-In BAT, the two main thermogenic mechanisms were analyzed: the UCP1 related uncoupling (*Ucp1* and *Pgc1α*) and the creatine futile cycle (*Ckb* and *Phospho1*). For each condition n was always ≥5. CTRL are in black, Pre-HT in green and Post-HT in purple. The reference group was always CTRL at the earliest time point the expression can be detected. Stars indicated a significant difference between CTRL and one of the treatment group at a given developmental stage.

Figure 5: *Short-term Postnatal hypothyroidism is associated with lower body weight and fasting glycemia despite normal bones' length in the adult.*

Different parameters were analyzed to assess the consequence of short-term postnatal hypothyroidism on the physiology of adult male mice either CTRL (n=13, in black) or Post-HT (n=6, in purple) CHOW fed. A- Body weight (gr) was followed from PDN20 to PDN200. B-Daily food intake as well as fed (C) and fasted (D) glycemia were measured around 3 months of age (PDN85 to PDN95). A GTT (E) was performed at PDN95 and an ITT (F) at PDN110. AUC is provided for each test on the right panel. At the end of the experiment at PDN200, mice were weighted (G), bone length (H) was measured using both tibia and femur and different tissues sampled and weighted as well (I): inguinal and gonadal WAT, BAT, liver and gastrocnemius muscle. Stars indicated a significant difference between CTRL and Post-HT.

Figure 6: *Short-term Postnatal hypothyroidism increases the expression of genes involved in FA synthesis and β-oxidation in adult liver and decreases them in the BAT.*
To assess the long-term impact of short-term postnatal hypothyroidism on metabolism in adult male mice, the expression of genes involved in different metabolic pathways was measured by relative RT-qPCR in liver (A) and BAT (B) two key metabolic organs. A-In liver different pathways were analyzed: FA synthesis (Fas, Acc, Me, Chrebp and Scd-1), FA β-oxidation (Cpt1a, Cpt1b and Pgc1β) and glucose metabolism (PEPCK, GK and L-PK). B- In BAT, FA synthesis (FAS, ACC, and ChREBP) and FA β-oxidation (Cpt1a, Cpt1b and Pgc1β) as well as markers of BAT activity (Ucp1, Phospho1, Ckb and Pgc1α) were measured. CTRL are in black (n=13) and Post-HT in purple (n=6). Stars indicated a significant difference between CTRL and Post-HT.

Figure 7: Short-term Postnatal hypothyroidism increases the sensitivity to diet induced obesity and is associated to lower fasted glycemia.

3.5 month-old CTRL (n=7 in black) and Post-HT (n=8 in purple) male mice were switched from CHOW to a High Fat Diet (HFD). We measured their body weight just before the switch and at the end of the protocol (A). We measured their body weight gain after the switch during all the period on HFD (B) and also expressed it as the percentage of their initial weight (C). Food intake (D) was calculated over the first 2 weeks on HFD. Measurement of fasted glycemia (E) and GTT (F) were performed after 10 weeks of HFD and ITT (G) after 13 weeks. Stars indicated significant difference between CTRL and Post-HT.

Figure 8: Short-term Postnatal hypothyroidism impacts early response to cold but allows maintenance of body temperature at 4°C.

5 month-old CTRL (n=6 black) and Post-HT (n=8 purple) male mice implanted with IPTT transponders were exposed to a 4° temperature. During the time of exposure their body temperature (A), their body weight (expressed as a percentage of their weight before exposure) (B) and their daily food intake (C) were followed. After 72h mice were killed. As BAT is the key organ involved in adaptive thermogenesis to maintain body temperature, the expression of genes involved in different metabolic pathways necessary for this process was measured by relative RT-qPCR (D): FA synthesis (Fas, Acc, and Chrebp) and FA β-oxidation (Cpt1a, Cpt1b and Pgc1β) as well as markers of BAT activity (Ucp1, Phospho1, Ckb, Dio2 and Pgc1α). CTRL (n=13) and Post-HT (n=6) mice maintained as room temperature (RT) were the same one as presented in Figure 6 and were the same age as cold exposed groups. The number indicated on top of the 4°C bars represent for each group the induction factor between the 4°C versus RT conditions. Stars indicated a significant difference between CTRL and Post-HT for a given temperature.

<table><caption>Table 1: RT-qPCR primer sequences</caption><thead><tr><td>Targeted cDNA</td><td>Reverse Primer sequence (5' to 3')</td><td>Forward Primer sequence (5' to 3')</td></tr></thead><tbody><tr><td>Hcn2</td><td>ATCCTTGGGGAACTCAACGGG</td><td>TTGTCAGCATGGCTGTGACG</td></tr><tr><td>Dio1</td><td>AGAGAGCCAGATTCCCTGTGC</td><td>GCTTGTAGGAACCATAGGCATTGG</td></tr><tr><td>Ptdgs</td><td>ACTTATCCGGTTGGGGCAGG</td><td>ATGGCCACCCTCTACAGCAG</td></tr><tr><td>Acc</td><td>TGGAGAGCCCCACACACA</td><td>TGACAGACTGATCGCAGAGAAAG</td></tr><tr><td>Cpt1a</td><td>ACC TCT GCT CTG CCG TTG TT</td><td>GGC TTC CAT GAC TCG GCT CT</td></tr><tr><td>Cpt1b</td><td>GCA ACG TGG TGT TTG GCT CA</td><td>TGG GCA CCT CTG GGA GTT TG</td></tr><tr><td>Gk</td><td>ACGGATGTGAGTGTTGAAGC</td><td>CCCTGAGTGGCTTACAGTTC</td></tr><tr><td>L-Pk</td><td>GGGCCGCATCTACATTGAC</td><td>GTCCCTCTGGGCCAATTTT</td></tr><tr><td>Fas</td><td>AGAGACGTGCACTCCTGGACTT</td><td>GCTGCGGAAACTTCAGGAAAT</td></tr><tr><td>Ucp1</td><td>AAGCCACAAACCCTTTGAAAA</td><td>AAGCTGTGCGATGTCCATGT</td></tr><tr><td>Pgc1α</td><td>TGAACGAGAGCGCATCCTT</td><td>GACTCAGTGTCACCACCGAAA</td></tr></tbody></table>
<table><tr><td>Pgc1β</td><td>CGTACTTGCTTTTCCCAGATGA</td><td>GAGGGCTCCGGACTTC</td></tr><tr><td>Ckb</td><td>AGGCTGGACAGAGCTTCTACTG</td><td>TGACCTGGACCCCAACTACG</td></tr><tr><td>Phospho1</td><td>GAGTCGTCGCTGTTCTCGTC</td><td>TGCCTATCTAGGGACGGCAG</td></tr><tr><td>Me</td><td>TTTGTATGCATCTTGCACAATCTTT</td><td>GCCGGCTCTATCCTCCTTTG</td></tr><tr><td>Pepck</td><td>CTTCAGCTTGCGGATGACA</td><td>AGGAGGAGTACGGGCAGTTG</td></tr><tr><td>Chrebp</td><td>CAGGGTGTCGAATCCTAGCTTAA</td><td>AGAACCGACGTATCACACACATCT</td></tr><tr><td>Dio2</td><td>CCTAGATGCCTACAAACAGGTTAAA</td><td>TGTTACCTGATTCAGGATTGGA</td></tr></table>

413

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479
<figure><img src="image_2.png" /></figure>
A Dams

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B Pups

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C

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A

<figure><img src="image_18.png" /><figcaption>FA β-oxidation</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>Glucose metabolism</figcaption></figure>

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B

<figure><img src="image_21.png" /><figcaption>Cpt1b</figcaption></figure>

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C

<figure><img src="image_24.png" /><figcaption>Cpt1a</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>Ckb</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>Relative mRNA expression to *Hprt*</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>Phospho1</figcaption></figure>
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A

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B

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F

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G

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<figure><img src="image_78.png" /><figcaption>Glucose metabolism</figcaption></figure>

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H

<figure><img src="image_80.png" /><figcaption>FA oxidation</figcaption></figure>

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<figure><img src="image_82.png" /></figure>
## Supplemental figure legends:

Supp Figure 1: Short-term Prenatal hypothyroidism does not impact body weight and glycemia in the adult.

Different parameters were analyzed to assess the consequence of short-term prenatal hypothyroidism on the physiology of CHOW fed adult male mice either CTRL (n=13, in black) or Pre-HT (n=7, in green). A- Body weight was followed from PDN20 to PDN200. Daily food intake (B) as well as fed (C) and fasted (D) glycemia were measured around 3 months of age (PDN85 to PDN95). A GTT (E) was performed at PDN95 and an ITT (F) at PDN110. At the end of the experiment (PDN200), mice were weighted (G), tibia and femur length (H) were measured and different tissues sampled and weighted: inguinal (I) and gonadal (J) WAT and BAT (K). Stars indicated a significant difference between CTRL and Pre-HT.

Supp Figure 2: Short-term Prenatal hypothyroidism barely impacts the expression of metabolic genes in liver and BAT.

To assess the long-term impact of short-term prenatal hypothyroidism on metabolism in adult male mice, the expression of genes involved in different metabolic pathways was measured in liver (A) and BAT (B) two key metabolic organs by relative RT-qPCR. A- In liver different pathways were analyzed: FA synthesis (Fas, Acc, Me, Chrebp and Scd-1), FA β-oxidation (Cpt1a, Cpt1b and Pgc1b) and glucose metabolism (Pepck, Gk and L-Pk). B- In BAT, FA synthesis (Fas, Acc, and Chrebp) and FA β-oxidation (Cpt1a, Cpt1b and Pgc1b) as well as markers of BAT activity (Ucp1, Phospho1, Ckb and Pgc1a) were measured. CTRL are in black (n=13) and Pre-HT in green (n=7). Stars indicated a significant difference between CTRL and Pre-HT.

Supp Figure 3: Short-term postnatal hypothyroidism hardly modifies the expression of metabolic genes under HFD.

3.5 month-old CTRL (n=7 in black) and Post-HT (n=8 in purple) male mice were switched from CHOW to a High Fat Diet (HFD). Tissues were sampled after 80 days. The expression of genes involved in different metabolic pathways was measured in liver (A) and BAT (B) two key metabolic organs by relative RT-qPCR. CTRL (n=13) and Post-HT (n=6) CHOW mice were the same one as those presented in Figure 6 and were the same age as HFD fed animals at the time of tissue sampling. Stars indicated significant difference between CTRL and Post-HT on a given diet. The number indicated on top of the HFD bars represent for each group the induction factor between the HFD versus CHOW conditions. (A) in liver different pathways were analyzed: FA synthesis (Fas, Acc, Me, Chrebp and Scd-1), FA β-oxidation (Cpt1a, Cpt1b and Pgc1b) and glucose metabolism (Pepck, Gk and L-Pk). (I) In BAT, FA synthesis (Fas, Acc, and Chrebp) and FA β-oxidation (Cpt1a, Cpt1b and Pgc1b) as well as markers of BAT activity (Ucp1, Phospho1, Ckb and Pgc1a) were measured. Stars indicated a significant difference between CTRL and Post-HT for a given diet.

Supp Figure 4: Short-term Prenatal hypothyroidism does not predispose to diet induced obesity

3.5 month-old CTRL (n=7 in black) and Pre-HT (n=10 in green) male mice were switched from CHOW to High Fat Diet (HFD). We measured their body weight gain after the switch during all the period on HFD (A) and also expressed it as the percentage of their initial weight (B). Food intake (C) was calculated over the first 2 weeks on HFD. Measurement of fasted glycemia (D) and GTT (E) were performed after 10 weeks of HFD and ITT (F) after 13 weeks. To assess the long-term impact of short-term prenatal hypothyroidism on the response to diet induced obesity the expression of genes involved in different metabolic pathways was measured organs by relative RT-qPCR in liver (G) and BAT (H) two key metabolic. (G) In liver different pathways were analyzed: FA synthesis (Fas, Acc, Me, Chrebp and Scd-
1), FA β-oxidation (Cpt1a, Cpt1b and Pgc1b) and glucose metabolism (Pepck, Gk and L-Pk). (H) In BAT, FA synthesis (Fas, Acc, and Chrebp) and FA β-oxidation (Cpt1a, Cpt1b and Pgc1b) as well as markers of BAT activity (Ucp1, Phospho1, Ckb and Pgc1a) were measured. Stars indicated a significant difference between CTRL and Pre-HT.