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. 2024 Jan 1;13(1):e230054.
doi: 10.1530/ETJ-23-0054. Online ahead of print.

The hypothalamic-pituitary-thyroid axis is intact in male insulin receptor substrate 4 knockout mice

Emilie Brûlé  1 , Xiang Zhou  2 , Ying Wang  3 , Evan R S Buddle  4 , Luisina Ongaro  5 , Mary Loka  6 , Anita Boelen  7 , Daniel Bernard  8
Affiliations

The hypothalamic-pituitary-thyroid axis is intact in male insulin receptor substrate 4 knockout mice

Emilie Brûlé et al. Eur Thyroid J. .

Abstract

Objective: Loss of function mutations in the insulin receptor substrate 4 (IRS4) gene cause a rare form of X-linked congenital central hypothyroidism in boys and men. Affected individuals show decreased thyroid-stimulation hormone (TSH) secretion. Members of the IRS family canonically act as scaffold proteins between tyrosine kinase receptors and downstream effectors. How loss of IRS4 affects TSH synthesis or secretion is unresolved. We therefore assessed IRS4's role in the hypothalamic-pituitary-thyroid axis of Irs4 knockout mice.

Methods: We generated two global Irs4 knockout mouse lines harboring either two or four base-pair deletions that result in frameshifts and loss of most of the IRS4 protein.

Results: Under normal laboratory conditions, Irs4 knockout males did not exhibit impairments in pituitary expression of TSH subunit genes (Tshb or Cga) or in the thyrotropin-releasing hormone (TRH) receptor. Additionally, their serum thyroid hormone, T3 (triiodothyronine) and T4 (thyroxine), and hypothalamic Trh expression levels were normal. When Irs4 knockouts were rendered hypothyroid with a low-iodine diet supplemented with propylthiouracil (PTU) for 3 weeks, their serum TSH increased similarly to wild-type males.

Conclusions: Overall, Irs4 knockout mice do not exhibit central hypothyroidism or otherwise appear to phenocopy IRS4 deficient patients. Compensation by another IRS protein may explain euthyroidism in these animals.

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Conflict of interest statement

The authors declare that they have no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported. Anita Boelen is on the editorial board of European Thyroid Journal. Anita was not involved in the review or editorial process for this paper, on which she is listed as an author.

Figures

Figure 1
Figure 1
IRS4/Irs4 mutations in humans and mice.(A) Schematic representation of IRS4 protein domain structure. Mutations identified in human IRS4 are shown, as is the location of the CRISPR-Cas9 introduced mutations in mice. PH, pleckstrin homology domain; PTB, phosphotyrosine binding domain; PI3K, PI3K binding motifs; Grb2, Grb2 binding site. (B) CRISPR-Cas9 introduced 2 and 4 bp deletions in Irs4 in the mouse models analyzed here. WT, wild type; – , deleted bp in the Δ2 and Δ4 strains. The PAM sequence is marked in green. Amino acids are numbered. (C) Pituitary Irs4 mRNA expression in euthyroid adult male WT and Irs4 Δ2/y mice as determined by RT-qPCR. ****, P < 0.0001. (D) Immunoblot (IB) of myc-tagged murine WT or Δ2 IRS4 and human IRS4. Black arrow indicates the location of the WT proteins. Blue arrow indicates the location of the Δ2 IRS4 protein. Tubulin was used as a loading control.
Figure 2
Figure 2
Irs4 knockout mice are euthyroid. Comparison of serum hormone levels and pituitary gene expression levels in adult male wild-type (Irs4+/y) and Irs4 knockout mice (Irs4Δ2/y). (A) Serum T3 and (B) T4 levels. Pituitary (C) Tshb, (D) Cga, (E) Trhr, and (F) Gh mRNA levels as measured by RT-qPCR. (G) Hypothalamic Trh mRNA levels in a subset of the animals (hypothalami were not available for all animals in this experiment). (H) Body weights. In all panels, bar heights show group averages. Individual data points are shown.
Figure 3
Figure 3
TSH production is normal in hypothyroid Irs4 knockout mice. Comparison of serum TSH levels and pituitary gene expression in adult male wild-type (Irs4+/y) and Irs4 knockout mice (Irs4Δ2/y) on a low iodine-PTU diet for 3 weeks. (A) Serum TSH before (pre) and 3 weeks after (post) mice were transferred from normal chow to LoI-PTU diet. Note that in five animals per genotype, TSH levels were below the limit of quantification (LOQ). These animals were assigned values of 153 pg/mL (the LOQ) for graphing and statistical purposes. ****, P < 0.0001, main effect of diet. Pituitary (B) Tshb, (C) Cga, (D) Trhr, (E) Irs4, and (F) Igsf1 mRNA levels as measured by RT-qPCR. ****, P < 0.0001. (G) Hypothalamic Trh mRNA levels in a subset of the animals (hypothalami were not available for all animals in this experiment). Bar heights show group averages. Individual data points are shown.
Figure 4
Figure 4
Expression of IRS/Irs subtypes in murine and human pituitary and brain. Dot plots showing expression of IRS subtypes in pituitary cell types in adult male (A) mice and (B) humans. Data were derived from snRNA-seq datasets (26, 33). Dot plots showing expression of IRS subtypes in (C) PVN cell types in male mice and (D) hypothalamic cell types in humans. Data were derived from snRNA-seq datasets (32, 36).
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References

    1. Lauffer P Zwaveling-Soonawala N Naafs JC Boelen A & van Trotsenburg ASP. Diagnosis and management of central congenital hypothyroidism. Frontiers in Endocrinology 202112686317. (10.3389/fendo.2021.686317) - DOI - PMC - PubMed
    1. Boelen A van Trotsenburg ASP & Fliers E. Congenital isolated central hypothyroidism: novel mutations and their functional implications. Handbook of Clinical Neurology 2021180161–169. (10.1016/B978-0-12-820107-7.00010-0) - DOI - PubMed
    1. Heinen CA, de Vries EM, Alders M, Bikker H, Zwaveling-Soonawala N, van den Akker ELT, Bakker B, Hoorweg-Nijman G, Roelfsema F, Hennekam RC, et al.Mutations in IRS4 are associated with central hypothyroidism. Journal of Medical Genetics 201855693–700. (10.1136/jmedgenet-2017-105113) - DOI - PMC - PubMed
    1. Patyra K Makkonen K Haanpaa M Karppinen S Viikari L Toppari J Reeve MP & Kero J. Screening for mutations in isolated central hypothyroidism reveals a novel mutation in insulin receptor Substrate 4. Frontiers in Endocrinology 202112658137. (10.3389/fendo.2021.658137) - DOI - PMC - PubMed
    1. Sun Y, Bak B, Schoenmakers N, van Trotsenburg AS, Oostdijk W, Voshol P, Cambridge E, White JK, le Tissier P, Gharavy SN, et al.Loss-of-function mutations in IGSF1 cause an X-linked syndrome of central hypothyroidism and testicular enlargement. Nature Genetics 2012441375–1381. (10.1038/ng.2453) - DOI - PMC - PubMed

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