Tertiary hypothyroidism and hyperglycemia in mice with targeted disruption of the thyrotropin-releasing hormone gene

Abstract

Thyrotropin-releasing hormone (TRH) is a brain hypothalamic hormone that regulates thyrotropin (TSH) secretion from the anterior pituitary and is ubiquitously distributed throughout the brain and other tissues including pancreas. To facilitate studies into the role of endogenous TRH, we have used homologous recombination to generate mice that lack TRH. These TRH-/- mice are viable, fertile, and exhibit normal development. However, they showed obvious hypothyroidism with characteristic elevation of serum TSH level and diminished TSH biological activity. Their anterior pituitaries exhibited an apparent decrease in TSH immunopositive cells that was not due to hypothyroidism. Furthermore, this decrease could be reversed by TRH, but not thyroid hormone replacement, suggesting a direct involvement of TRH in the regulation of thyrotrophs. The TRH-/- mice also exhibited hyperglycemia, which was accompanied by impaired insulin secretion in response to glucose. These findings indicate that TRH-/- mice provide a model of exploiting tertiary hypothyroidism, and that TRH gene abnormalities cause disturbance of insulin secretion resulting in marked hyperglycemia.

Figure 1

Targeted disruption of the pTRH gene. (a) Schematic representation of the mouse pTRH gene (Upper), targeting vector, expected targeted allele, and pTRH (Lower). Solid boxes represent exons. Allele-specific PCR primers for selection of homologous recombinants are indicated by arrows, and probes for Southern and Northern blot analyses are also indicated. Hatched bars represent TRH progenitor sequences in pTRH. (b) Southern blot analysis of DNA from ES cell clones and siblings generated by crossing heterozygous mice (F² mice). The wild-type (3.1 kb) and targeted (2.0 kb) HindIII and EcoRI fragments were detected by the 3′ external probe indicated in a. (c) Analysis of pTRH mRNA expression by Northern blot analysis of F² mice. Total RNAs (20 μg) from the hypothalamus of wild-type (+/+), heterozygous (+/−), and homozygous mice (−/−) were hybridized with pTRH cRNA. (d) Hypothalamic TRH contents of the wild-type (+/+, n = 9), heterozygous (+/−, n = 5), and homozygous (−/−, n = 10) mice. ∗∗∗, P < 0.001 vs. wild type.

Figure 2

Immunohistochemical analysis of the pituitary. When compared with those of the wild type (a), numbers of TSH positive cells in the TRH^−/− pituitary (b) were decreased. This decrease was restored by TRH replacement (d). No significant change was found in the pituitary of the hypothyroid mice induced by MMI treatment (c).

Figure 3

TRH stimulation test. After TRH administration, blood TSH (a) and T₃ levels (b) were determined in the wild-type (+/+, n = 6) and homozygous (−/−, n = 6) mice. The increment in T₃ over the basal level was reduced in the homozygotes.

Figure 4

Blood glucose and plasma insulin levels in TRH^−/− mice. (a) Fasting blood glucose levels in the wild-type (+/+, n = 11), heterozygous (+/−, n = 10), homozygous (−/−, n = 10) mice, and homozygous mice with T₄ replacement (−/− + T₄, n = 7). ∗∗, P < 0.01 vs. the wild-type and heterozygous mice. (b) Glucose tolerance tests. Blood glucose levels were determined after glucose challenge. Data indicate means ± SEM (n = 10–15). (▵), wild type; (▪), heterozygotes; (○), homozygotes. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 vs. wild type. (c) Plasma insulin levels before and 30 min after glucose challenge, and the ratio of fasting plasma insulin to glucose. +/+, wild type, n = 7; −/−, homozygotes, n = 7. ∗∗, P< 0.01 vs. wild type. (d) Insulin tolerance tests. Results are presented as the percentage of the fasting blood glucose and are means ± SEM. (○), wild type, n = 5; (▴), homozygotes, n = 5.

Figure 5

Characterization of TRH^−/− islets. Insulin secretion in response to glucose and arginine was determined in vitro. Values are expressed as means ± SEM (n = 10–15). Solid bars, wild type; hatched bars, homozygotes. ∗, P < 0.05; ∗∗, P < 0.01 vs. wild type.