Common and Uncommon Mouse Models of Growth Hormone Deficiency

Abstract

Mouse models of growth hormone deficiency (GHD) have provided important tools for uncovering the various actions of GH. Nearly 100 years of research using these mouse lines has greatly enhanced our knowledge of the GH/IGF-1 axis. Some of the shared phenotypes of the 5 “common” mouse models of GHD include reduced body size, delayed sexual maturation, decreased fertility, reduced muscle mass, increased adiposity, and enhanced insulin sensitivity. Since these common mouse lines outlive their normal-sized littermates—and have protection from age-associated disease—they have become important fixtures in the aging field. On the other hand, the 12 “uncommon” mouse models of GHD described herein have tremendously divergent health outcomes ranging from beneficial aging phenotypes (similar to those described for the common models) to extremely detrimental features (such as improper development of the central nervous system, numerous sensory organ defects, and embryonic lethality). Moreover, advancements in next-generation sequencing technologies have led to the identification of an expanding array of genes that are recognized as causative agents to numerous rare syndromes with concomitant GHD. Accordingly, this review provides researchers with a comprehensive up-to-date collection of the common and uncommon mouse models of GHD that have been used to study various aspects of physiology and metabolism associated with multiple forms of GHD. For each mouse line presented, the closest comparable human syndromes are discussed providing important parallels to the clinic.

Graphical Abstract

Graphical Abstract.

Essential Points.

• Mouse models with mutations that result in GHD have been used for 95 years to help uncover the many actions of GH

• Most syndromes with GHD have a corresponding mouse model (sometimes multiple models) to help medical researchers better understand molecular and cellular mechanisms of these diseases

• When mutations result in defects beyond GH or pituitary hormone production, health effects are diverse, often severe, and usually deleterious

• When mutations result in defects that are limited to the production of GH, unfavorable (obesity, decreased lean mass, low bone mineral density (BMD), poor fertility) as well as favorable (enhanced insulin sensitivity, slower aging, and protection from age-associated diseases including cancer) health effects occur

Growth hormone (GH) is a principal vertebrate hormone that affects many cell types and plays a substantial role in numerous physiological processes including growth. For more than a century, countless endocrine researchers have studied conditions resulting from aberrant production of GH (1). While clinical studies provide the best insight as to how GH acts in humans, there are obvious ethical limits to the molecular, cellular, and physiological depth of studies that can be conducted in humans. Because of this, animals are widely used in endocrine research. Mice are by far the most common mammal used since large colonies are easily housed, they can be genetically engineered to produced transgenic and gene-disrupted lines, they have physiologies and organ systems similar to humans, and they share most of their protein-coding genes with humans (2). According to The Jackson Laboratory (www.jax.org/about-us/why-mice), the effect of mouse-based research on biological discovery and medical progress over the past century has been so profound that 26 Nobel Prizes in Physiology or Medicine can be directly tied to Jackson Laboratory's mice alone. As such, numerous GHD mouse lines have also been generated and used in the GH field to elucidate the many actions of GH.

In this paper, we review 17 different mouse models that are segregated into “common” and “uncommon” models of GHD. In the first section, we provide a comprehensive review of 5 commonly used models with mutations in the Pou1f1, Prop1, Ghrhr, Ghrh, and Gh genes. These common mouse models have disruptions that are limited to the production of GH itself or GH and other pituitary hormones. The clinical consequences of mutations in the corresponding human gene are also discussed, and tables describing the mutations provided (Tables 1-4). Collectively, the phenotypes of the common mouse models of GHD have produced novel insights into our understanding of GH in growth, metabolism, reproduction, immunity, cancer, and aging.

Table 1.

Pathogenic mutations of the POU1F1 gene (chromosomal location 3p11.2) in humans

Mutation type	Mutation detail	Diagnosis	Year(s)	References
Homozygous nonsense	R172X	CPHD	1992	(3)
Heterozygous missense	R271W	CPHD	1992, 1994, 1995, 1997	(4-7)
Heterozygous missense	R271W	CPHD	1992, 2005	(8, 9)
Heterozygous missense	P24L	CPHD	1992	(9)
Homozygous missense	R143Q	CPHD	1992	(9)
Homozygous missense	A158P	CPHD	1992	(10)
Homozygous nonsense	E250X	CPHD	1995	(11)
Homozygous missense	F135C, W193R	CPHD	1996	(12)
Heterozygous missense	E174G	CPHD	1998	(13)
Homozygous missense	P239S	CPHD	1998	(14)
Heterozygous missense	P14L	CPHD	1998	(15)
Heterozygous missense	K216E	CPHD	1999	(16)
Homozygous nonsense	K145X	CPHD	2003	(17)
Heterozygous missense	Q167K	CPHD	2003	(18)
Homozygous deletion	Q242R (−2 bp)	CPHD	2005	(19)
Heterozygous missense	W193X, F262L	CPHD	2005	(19)
Homozygous missense	S179R	CPHD	2006	(20)
Homozygous deletion	4.11 kb deletion of exon	CPHD	2017	(21)
Heterozygous intron 1 of PIT-1α (exon 2 skipping in PIT-1α leaving PIT-1β intact)	c.143-83A > G	CPHD	2017	(22)
Heterozygous missense	S50A, S50S, I51I, I51S, L52W, S53A	CPHD	2021	(23)
Homozygous frameshift	c.634_638delGAAAG	CPHD	2021	(24)
Homozygous splice site	c.665 + 1G > T	CPHD	2021	(24)
Homozygous intronic	c.215-3C > G	CPHD	2021	(24)
Heterozygous frameshift	c.343delA	CPHD	2021	(24)
Heterozygous missense	c.97G > A	CPHD	2021	(24)
Homozygous deletion	c.605delC	CPHD	2016, 2019	(25, 26)
Compound heterozygous missense	R217Y (c.649C > T) and I221T (c.662T > C)	CPHD	2021	(27)
Heterozygous intron 1 of PIT-1α or heterozygous of PIT-1β (PIT-1β overexpression)	c.143-69T > G or c.152T > G	CPHD	2021	(28)
Homozygous splice-site deletion	c.744-5_749del	CPHD	2022	(29)

Abbreviation: CPHD, combined pituitary hormone deficiency.

Table 4.

Pathogenic mutations of the GH1 gene (chromosomal location 17q23.3) in humans

Mutation type	Mutation detail	Diagnosis	Effect on gene	Year(s)	References
Heterozygous gene deletion	6.7-kb deletion in GH1 cluster	IGHD1A	GH1 gene deletion	1981	(66, 67)
Homozygous gene deletion	7.5-kb deletion in GH1 cluster	IGHD1A	GH1 gene deletion	1985	(68)
Homozygous gene deletion	7.6-kb deletion in GH1 cluster	IGHD1A	GH1 gene deletion	1986	(69)
Homozygous gene deletion	40-kb double deletion in GH1 cluster	IGHD1A	GH1 gene deletion	1986	(70)
Homozygous gene deletion	6.7–7.6-kb deletion in GH1 cluster	IGHD1A	GH1 gene deletion	1988	(71)
Heterozygous gene deletion	7.1-kb deletion in GH1 cluster	IGHD1A	GH1 gene deletion	1990	(72)
Homozygous gene deletion	45-kb deletion in GH1 cluster	IGHD1A	GH1 gene deletion	1992	(73)
Heterozygous gene deletion	6.7-7.6-kb deletion in GH1 cluster	IGHD1A	GH1 gene deletion	1992	(74, 75)
Compound heterozygous deletion/frameshift	6.7-kb deletion in GH1 cluster + c.50del	IGHD1A	GH1 gene deletion	1990, 1994	(76)
Compound heterozygous deletion/frameshift	6.7-kb deletion in GH1 cluster + c.243-244delAG	IGHD1A	GH1 gene deletion	1994	(76, 77)
Homozygous insertion	c.64-65ins26	IGHD1A	Frameshift inserts 86 amino acids at exon-2	2008	(78)
Homozygous nonsense	W7X	IGHD1A	No mature GH	1993, 1994	(76, 79)
Homozygous splice site	IVS4 + 1G > T	IGHD1B	Loss of 24 amino acids in exon-4, frameshift in exon-5	1994	(76)
Homozygous missense	C182X	IGHD1B	Truncated protein, disrupted disulfide bond	2009	(80)
Homozygous missense	G120V	IGHD1B	Reduced bioactivity	2009	(80)
Heterozygous missense	C77R	IGHD2	Partial GH resistance	1996	(81)
Heterozygous splice site	IVS4-1G > A	IGHD2	Splicing affected	2006	(82)
Heterozygous splice site	IVS3 + 28G > A	IGHD2	Abnormal splicing, 17.5-kD isoform	1997, 2003	(83, 84)
Heterozygous splice site	IVS3 + 28-45del	IGHD2	18-bp deletion, exon-3 skipped, 17.5-kD isoform	1997, 2003	(83, 84)
Heterozygous splice site	IVS3 + 56-77del	IGHD2	Removal of branching point site at intron-3, exon-3 skipped	2006	(85)
Heterozygous splice site (at exon splice enhancer)	E33G, E3 + 5A > G	IGHD2	Exon-3 skipped (part/full), 17.5-kD isoform	2002, 2003	(84, 86)
Heterozygous splice site (at exon splice enhancer)	E32X, E3 + 1G > T	IGHD2	Exon-3 skipped	2002	(87)
Heterozygous splice site (at exon splice enhancer)	E32K, E3 + 1G > A	IGHD2	Exon-3 skipped	2008	(88)
Heterozygous splice site (at exon splice enhancer)	E32A, E3 + 2A > C	IGHD2	Exon-3 skipped (part/full), 17.5-kD isoform	2007	(89)
Heterozygous splice site	c.291 + 2T > G	IGHD2	Exon-3 skipped	2019	(90)
Heterozygous splice site	IVS2 -1G > A	IGHD2	3′-Acceptor splice site	2003	(91)
Heterozygous splice site	IVS2 -2A > T	IGHD2	3′-Acceptor splice site	2003	(91)
Heterozygous splice site	IVS3 + 1G > A	IGHD2	Exon-3 skipped	1995, 2003	(92, 93)
Heterozygous splice site	IVS3 + 6T > C	IGHD2	Exon-3 skipped	1994	(76, 94)
Heterozygous splice site	IVS3 + 2T > C	IGHD2	Exon-3 skipped	2003	(93)
Heterozygous splice site	IVS3 + 1G > C	IGHD2	Exon-3 skipped	1995	(95)
Heterozygous splice site	IVS3 + 5G > A	IGHD2	Exon-3 skipped	1999	(96)
Heterozygous splice site	IVS3 + 5G > C	IGHD2	Exon-3 skipped	1999	(97)
Heterozygous splice site	IVS3 + 6T > G	IGHD2	Exon-3 skipped	2001	(98)
Heterozygous missense	R178H	IGHD2	Disrupted GH secretion, binding, signaling	2009, 2010	(80, 99)
Heterozygous missense	R183H	IGHD2	Disrupted GH secretion	1997, 2001	(100, 101)
Heterozygous missense	K41R	IGHD2	Exon-3 skipped partly	2003	(91)
Heterozygous missense	C53S	IGHD2	Bioinactive GH	2005	(102)
Heterozygous missense	P89L	IGHD2	Disrupted GH secretion	2005	(103)
Heterozygous missense	S108C	IGHD2	Disrupted GH secretion	2003	(91)
Heterozygous missense	S108R	IGHD2	Reduced GH signaling	2003	(91)
Heterozygous missense	D112G	IGHD2	Bioinactive GH	1997	(104)
Heterozygous missense	T175A	IGHD2	Reduced GH signaling	2003	(91)

Abbreviations: GH, growth hormone; GH1, growth hormone 1.

In contrast, the uncommon GHD mouse lines include mice that have documented GHD and/or have mutations in genes involved in pituitary development. The uncommon models also include GHD mice due to nongenetic manipulation (ie, hypophysectomy and the use of diphtheria toxins for somatotroph destruction). As will be described, some of the uncommon GHD mice involve larger neural and sensory organ defects well beyond GH and/or pituitary hormone production, often resulting in severe abnormalities and/or embryonic lethality. Therefore, many of these mouse lines are not often the first choice for studies addressing the specific actions of GH and are more tailored for their associated rare genetic diseases. In this second section, we provide a brief phenotypic summary of the 12 uncommon mouse models of GHD. For all, we also provide a summary of the syndromes/phenotypes caused by human mutations of the same genes, and when available, the GH status of these individuals (accounting for the limitations of currently available tests in the clinical setting to unequivocally diagnose GHD). For convenience, a table that consolidates the information for all mouse lines discussed in this paper is included (Table 5). A timeline of when each mouse line was first generated is provided in Fig. 1, and Fig. 2 shows a graphic representation of genes involved in pituitary development or GH production that—when disrupted in mice—generate mouse models of GHD. We expect that this review will be helpful in choosing appropriate mice to address specific research questions that recapitulate the molecular, cellular, and genomic events of GHD in humans.

Table 5.

Common and uncommon mouse models of GHD

Mouse line	Human disease	Mouse phenotype	References
Common mouse models of GHD
Pou1f1 null (PIT1 or Snell)	CPHD1	Multiple pituitary hormone deficiencies (GH, PRL, TSH); resulting in ↓ IGF-1 and dwarfism, ↑ adiposity, ↑ leptin, ↑ adiponectin, ↓ insulin, enhanced insulin sensitivity, impaired glucose tolerance, infertility, hearing impairment, immune dysfunction, ↓ mTORC1 activity, ↑ mTORC2 activity, ↓ DNA methylation, cancer resistance, ↓ signs of aging, ↑ lifespan	(105-130)
Prop1 null (Ames)	CPHD1 and 2	Multiple pituitary hormone deficiencies (GH, PRL, TSH, low levels of LH and FSH) resulting in ↓ IGF-1 and dwarfism, ↑ adiposity, ↑ adiponectin, ↓ insulin, enhanced insulin sensitivity, impaired glucose tolerance, ↓ fertility, hearing impairment, immune dysfunction, ↓ inflammatory cytokines, ↓ body temperature, cancer resistance, ↓ signs of aging, ↑ lifespan	(124, 129, 131-150)
Ghrhr null (little or lit/lit)	IGHD4	Reduced pituitary production of GH and low PRL resulting in ↓ IGF-1 and dwarfism, ↑ adiposity, ↑ leptin, enhanced insulin sensitivity, impaired glucose tolerance, ↓ fertility, ↓ milk production, normal immune function, ↑ expression of xenobiotic detox genes, cancer resistance, ↑ lifespan	(109, 115, 121, 151-166)
Ghrh null	No cases (but suspected)	Reduced pituitary production of GH resulting in ↓ IGF-1 and dwarfism, ↑ adiposity, ↓ adiponectin mRNA in adipose tissue, enhanced insulin sensitivity, ↓ fertility, ↓ immune function, ↑ lifespan	(167-176)
Gh null	IGHD1A	Complete absence of GH (pituitary and extrapituitary) due to knockout of GH gene itself resulting in ↓ IGF-1 and dwarfism, ↑ adiposity, ↓ insulin, enhanced insulin sensitivity, impaired glucose tolerance, ↓ pancreatic islet size, ↓ fertility, ↓ maturity of gut microbiome, ↑ serum glycine, cancer resistance, ↑ lifespan	(177-180)
Uncommon mouse models of GHD
D2dr null	addiction disorders	Chronic hyperprolactinemia with ↑ lactotroph and ↓ somatotroph populations in the anterior pituitary, ↓ GH release, ↓ GH, ↓ IGF-1, ↓ IGFBP-3, impaired growth	(181-184)
Egr2 null	CMTD, DSS, CHN1	Lethality in early life with half dying within 2 d, and others dying within 2 wk after birth, ↓ rhombomeres 3 and 5 of developing hindbrain, ↓ orobuccal reflex, ↓ respiratory and jaw opening rhythms, ↓ somatotrophs, ↓ body size	(185-191)
Ghsr null	GHDP	↓ Pituitary expression of Pit-1, ↓ circulating GH and PRL, ↓ IGF-1, ↓ trabecular bone mass and ↑ rate of bone resorption, mild ↓ in body weight, nonsignificant trend for ↓ body length. Overall mild growth phenotype	(170, 192-195)
Hesx1 null	CPHD5, SOD	Many neurological defects, ↓ prospective forebrain tissue, abnormal morphogenesis of Rathke pouch, forebrain midline defects with pituitary dysplasia; 2 types of pups observed (class 1 and 2). Class 1: severe ↓ head size, short nose, eyes absent, display mortality shortly after birth. Class 2: less severe phenotype with less craniofacial dysplasia, only 1 eye is affected, ∼25% survive weaning and can produce viable offspring	(196-199)
Lhx3 null	CPHD3	Lack of anterior pituitary, mice are stillborn or die within 24 h of birth	(200)
Lhx4 null	CPHD1 and 4	Die shortly after birth, with substantial lung and neural defects, lack anterior pituitary	(201)
Otx2 null	MCOPS5, CPHD6	Embryonic lethality with no midbrain or forebrain; disruption to pituitary gland, pineal gland, inner ear, optic nerve, and eye	(202-205)
Snord116null	PWS	Early GHD with ↓ GH and IGF-1 levels, ↓ body length, ↓ processing of proinsulin, proGHRH and proghrelin, ↓ motor learning, ↑ ghrelin, ↑ Arp and Npy ↑ hyperphagia, normal fertility	(206-208)
Sox2 null	MCOPS3	Homozygous mice die shortly after blastocyte stages. Heterozygous mice show abnormal anterior pituitary development with ↓ LH, ↓ TSH and ↓ GH, some with ↓ body weight and ↓ fertility	(198)
Sox3 null	PHPX	Craniofacial abnormalities, midline CNS defects, and hypopituitarism, low to normal body weight, ↓ GH, LH, FSH, TSH, abnormal Rathke pouch, usually normal growth but stunted growth occasionally observed, ↓ fertility, ↑ growth of upper and/or lower teeth	(198, 199, 209)
Nongenetic models of GHD (somatotroph destruction)
Hypophysectomy	Hypophysectomy	Complete loss of pituitary, including all pituitary-derived hormones; phenotype varies based on procedure, stain, age, etc, but generally similar to Ames and Snell dwarf mice including ↑ lifespan when performed at age 1 and 9 mo	(210-214)
Diphtheria toxin	No human equivalent	Somatotroph toxicity due to DT-A expressed in somatotrophs. Phenotype varies based on age of onset. rGH-DT: ↓ IGF-1, dwarfism, ↓ fertility, lactotrophs toxicity detected, progressive lack of coordination. AOiGHD: ↓ GH, ↓ IGF-1, no difference in body weight, ↑ adiposity, enhanced insulin sensitivity, cancer resistance	(215-219)

Abbreviations: AOiGHD, adult-onset, inducible growth hormone deficiency; CHN, congenital hypomyelinating neuropathy; CMTD, Charcot-Marie-Tooth disease; CNS, central nervous system; CPHD, combined pituitary hormone deficiency; DSS, Dejerine-Sottas syndrome; DT-A, diphtheria toxin A; FSH, follicle-stimulating hormone; GH, growth hormone; GHDP, isolated partial growth hormone deficiency; IGF-1, insulin-like growth factor-1; IGHD, isolated growth hormone deficiency; LH, luteinizing hormone; MCOPS, microphthalmia; mRNA, messenger RNA; PHPX, X-linked hypopituitarism; PRL, prolactin; PWS, Prader-Willi syndrome; SOD, septo-optic dysplasia; TSH, thyrotropin.

Figure 1.

Timeline of when each mouse model of growth hormone deficiency was first generated. Orange text indicates genetically engineered mouse lines, blue text indicates mouse lines discovered with spontaneous mutations, and red text indicates physical disruption of the pituitary.

Figure 2.

Targeting genes involved in pituitary development or growth hormone (GH) production to generate mouse models of GH deficiency (GHD). Targeting genes involved in pituitary development or GH production to generate mouse models of GHD. Most of the mouse models of GHD result from disruptions to genes with known roles in pituitary development or GH production and are listed in black.

Common Mouse Models of Growth Hormone Deficiency

Pou1f1 Null Mice (Gene for PIT1 Protein; Commonly Known as Snell Dwarf Mice)

Snell dwarf mice—the first known GHD mouse line—was described by Snell and colleagues at Harvard University in 1929 (105). Dwarfism in these mice results from a W261C spontaneous point mutation in the Pou1f1 gene, which encodes a POU family transcription factor, PIT1 (106). This mutation interferes with the interaction between PIT1 and its target enhancer sequences. Since PIT1 plays an important role in the differentiation and expansion of lactotrophs, somatotrophs, and thyrotrophs in the anterior pituitary (107), this mutation results in GH, prolactin (PRL), and thyrotropin (TSH) deficiencies (108-110) with a subsequent severe reduction in plasma insulin-like growth factor-1 (IGF-1) (111-113).

Snell dwarf mice grow normally until approximately age 2 weeks, when dwarfism becomes apparent. Young adult Snell mice are only one-third to one-fourth the weight of age-matched controls (105, 114, 115). Injections of GH and thyroxine (T4) for 11 weeks (4- to 15-week-old) increases body weight of young Snell mice by 45% (116). In addition to pronounced dwarfism, Snell mice have several physiological and metabolic differences such as increased adiposity with elevated leptin levels (115), decreased circulating insulin, increased adiponectin, and a decreased rate of gluconeogenesis and glycogenolysis (resulting in a 60% reduction of plasma glucose utilization) (117).

Snell dwarf mice exhibit dysregulations in reproduction, hearing, and immune parameters. Male and female Snell mice are infertile (105) with reduced plasma gonadotropins (118); thus, heterozygous mice must be used for breeding. Male Snell mice have poorly differentiated Sertoli and Leydig cells and decreased production of primary spermatocytes and spermatids (118). Also, these mice suffer from congenital deafness likely due to TSH deficiency (119) as hypothyroidism has been linked with deafness (220, 221). Altered immune parameters in Snell dwarf mice include lower splenic T- and B-cell populations at age 4 months (120, 121). T4 administration for 10 days increases splenic CD4+ and CD8+ T-cell numbers, suggesting this effect is due to TSH deficiency as opposed to GHD in these mice (122).

Snell dwarf mice have a 42% longer lifespan compared to wild-type (WT) controls, with males and females living 50% and 29% longer, respectively (115, 123, 124). Following the discovery that these mice are long-lived, numerous studies have identified aging pathways affected in these mice. For example, Snell dwarf mice have reduced mTORC1 activity (125) and exhibit slower aging as per a blood-based age prediction assessment that evaluates DNA methylation levels (126). mTORC2 activity is higher in the liver, muscle, heart, and kidney of fasted Snell dwarf mice, while feeding suppresses activity of mTORC2 and increases mTORC1 activity (125). Additionally, skin fibroblasts from Snell mice are resistant to multiple stressors, such as peroxide, UV light, paraquat, heavy metals (Cd), and heat (127, 128), which are thought to be due to increased rates of autophagy (129, 222). Snell dwarf mice also are resistant to age-related diseases such as cancer (116), cataract development (116), collagen denaturation (115), glomerular basement membrane damage (116), and neuropathological decline (130). Furthermore, reduced adipose tissue senescence in Snell mice compared to age-matched controls has been reported (223).

In humans, POU1F1 (MIM 173110) loss-of-function mutations were first reported in 1992 (3) and comprise, along with PROP1 mutations, the majority of patients with nonsyndromic combined pituitary hormone deficiency (CPHD) of a genetic origin. These patients mainly have a combined deficiency of GH, PRL, and TSH (named type 1 CPHD, MIM 613038) and a variable degree of anterior pituitary hypoplasia with a normal posterior pituitary and no associated midline abnormalities (224). Patients with POU1F1 mutations tend to present initially with GH and PRL deficiency, with a later onset of TSH deficiency (225). There are more than 46 different human mutations (>31 are pathogenic; see Table 1) for POU1F1, and depending on the patient cohort tested, POU1F1 mutations can account for 2% to 26% of CPHD cases (24). The position of the mutation often determines either autosomal dominant (19%) or recessive (67%) inheritance in patients (24, 226). Table 1 summarizes the most common pathogenic POU1F1 mutations reported in human patients, wherein more than 75% are missense/nonsense mutations (recently reviewed (24)). A gross deletion of the POU1F1 locus (homozygous for exon-1 and exon-2 deletion) leading to severe CPHD phenotype has also been described in a patient from a consanguineous Turkish pedigree (21). Furthermore, while most POU1F1 mutations affect the α isoform (which is the main transcript), recently, pathogenic variants in the coding region of the β isoform (larger transcript that contains an extra 26-amino acid sequence called the β domain) and the intron near the exon-intron boundary for the β domain have been identified (22, 28).

Morphologic and physiologic phenotypes of POU1F1 mutations display extensive variation from patient to patient, differing additionally on nature and scale of pituitary hormone deficiencies, times of onset, and responses to hormone therapy. Profound hypothyroidism, along with complete lack of GH and often PRL, is present in patients affected by POU1F1 mutations (8). The patients usually respond to thyroid hormone therapy and partially to GH administration (227). Among the POU1F1 mutants, heterozygous groups have a higher peak GH level compared to homozygotes and compound heterozygotes (24). Typically, patients with POU1F1 variants share the clinical features of GHD including proportionate short stature, “doll-like” face, protruding forehead, frontal bossing, abdominal and thigh adipose tissue accumulation and, on the presence of early and severe GHD, neonatal hypoglycemia and hypogenitalism in male patients. If severe TSH deficiency is present from an early age, cretinism—including myxedema, hoarse voice, macroglossia, and psychomotor delay—could be observed (4, 5, 228) along with additional early symptoms like prolonged jaundice, low appetite, poor muscle tone, and constipation (27). However, although TSH deficiency can present shortly after birth, it usually occurs with or after the onset of GHD, and hypothyroidism is usually mild (229), thus classic signs and symptoms of congenital hypothyroidism are possible, but not common in POU1F1-related CPHD (27) (also considering that central hypothyroidism is usually milder than primary hypothyroidism). Gonadotropin or adrenocorticotropin (ACTH) deficiency are not considered within the typical clinical spectrum of POU1F1-related CPHD, though isolated cases of delayed pubertal development, infertility, or hypocortisolemia have been reported (24, 230). No information on systemic pathophysiology and lifespan of POU1F1 mutation in humans has been reported.

Prop1 Null Mice (Commonly Known as Ames Dwarf Mice)

Ames dwarf (df/df; Prop1 null) mice were described by Schaible and Gowen from Iowa State University in Ames, Iowa, in 1961 (131). These mice have a pathogenic (T83C) homozygous mutation, located within the homeodomain of the transcription factor prophet of Pit-1 (Prop1). For normal pituitary development, PROP1 induces a ZEB2-mediated epithelial-to-mesenchymal transition in pituitary stem cells (132) as well as represses and activates gene expression of pituitary transcription factors HESX1 and POU1F1, respectively (132, 231-236). Since Prop1 gene expression is required for development of somatotrophs, lactotrophs, thyrotrophs, and gonadotrophs in the anterior pituitary gland, the Prop1 deficient mutants have poorly vascularized and dysmorphic pituitaries (114), lack pituitary-derived GH, PRL, and TSH, and have greatly reduced levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and IGF-1 (133). This mouse line is a model for human CPHD type 2.

Ames dwarf mice exhibit severe growth retardation after approximately age 1 week (134). By the time they reach age 2 months, their weight is roughly half of WT littermates, and as adults, their body size is merely one-third of littermate controls. Daily injections of GH, T4, GH and TSH, or GH and T4 for 40 days significantly increases body weight and organ size (133). Ames dwarf mice have severely reduced plasma gonadotropin levels (135) and are mostly infertile (136). Female Ames mice have ovaries that are small and lack large follicles and corpora lutea (136), while males have poorly developed testes. Ames mice also exhibit abnormalities in immune parameters with decreased weights of and lower lymphocyte numbers in their thymus and spleen as well as reduced splenic natural killer cell activity (137). Increased adiposity with increased adiponectin, enhanced insulin sensitivity, and reduced levels of circulating insulin and blood glucose are seen in these mice (138, 237). Despite enhanced insulin sensitivity, Ames mice are glucose intolerant, likely due to decreased islet size and subsequent reduction in insulin production (138). Ames mice exhibit decreases in lipid synthesis, plasma free fatty acids and triglycerides, core body temperature, and inflammatory cytokines interleukin-6 and tumor necrosis factor-α (129, 139, 222).

These mice are long-lived compared to WT littermates (140). Male Ames mice live an average of 350 days more (49%) and females live an average of 470 days more (68%) than sex-matched controls (124, 134). Importantly, the enhanced lifespan in Ames dwarf mice is reduced to normal levels by GH treatment during postnatal weeks 1 to 7 (238). Ames dwarf mice have delayed onset in a number of age-associated physiological declines, including cognition (124), cancer (141), and neuromusculoskeletal frailty (142). Multiples studies indicate that Ames dwarf mice are also resistant to oxidative stress (143-148, 239). Additionally, Ames dwarf mice have elevated metallothionein-induced coenzyme Q10 synthesis, which protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson disease (149), and despite severe suppression of hippocampal GH, adult Ames dwarf mice have similar or increased levels of hippocampal IGF-1 RNA and protein levels vs controls (240). This finding is accompanied by an enhanced rate of neurogenesis in the dentate gyrus of the brain (240). Interestingly, calorie restriction (CR), which is a known intervention to extend lifespan in yeast, worms, flies, rodents, and monkeys, has been assessed in Ames mice. The long-lived Ames mice experience even greater improvements in longevity with CR and exhibit significantly delayed occurrence of neoplasms and improved cognitive performance compared to ad libitum–fed Ames mice (241, 242), suggesting overlapping yet distinct pathways being affected.

In humans, the first documented case of a PROP1-related CPHD was identified in 1967 (32). Inactivating mutations of PROP1 (MIM 601538) are the most frequent causes found in nonsyndromic (mainly familial) CPHD of a genetic origin (224). Patients are characterized by a severe deficiency in postnatal pituitary ontogenesis, resulting in GH (and as a result, IGF-1), gonadotropin (LH and FSH), TSH, and PRL deficiencies (named type 2 CPHD, MIM 262600), that vary in their severity and time of onset, developing throughout an individual's lifetime. ACTH (and consequently cortisol) deficiency is less common and, when present, usually occurs in adolescence or adulthood (43, 229). Mutations in PROP1 show an autosomal recessive inheritance, and their description as the underlying factor for CPHD type 2 was first reported in 1998 (30). Several different pathogenic mutations in the gene have been identified to date (see Table 2) using molecular genetic testing. Some of the mutational hot spots identified are the 2bp-A301-G302-deletion and the 1bp-A150-deletion, both resulting in frameshift leading to premature translation termination (33) and a homozygous codon mutation generating an R120C amino acid substitution with a greater than 85% reduced PROP1 function (30, 31). Several cases of gross deletions of the entire PROP1 locus, resulting in severe CPHD phenotypes, have been reported in cohorts from Brazil (243), patients with Kurdish origin in the United Kingdom (244), Turkey (21, 245), China (246), and India (247). Similar to the Ames mice, the diagnosis of GHD in these patients usually precedes that of TSH with almost complete loss of pituitary production of both hormones over time (39); consequently, neonatal hypoglycemia and prolonged jaundice are much less frequently reported in patients with PROP1 mutations than in those with POU1F1 ones (39). Growth retardation with proportionate short stature and GHD (with its typical phenotype including central adiposity) are usually observed in the first years of life. TSH and gonadotropin deficiencies can be present at birth (with neonatal hypothyroidism features and undescended testes and micropenis in males) or show up later in life, as hypothyroidism of variable severity and delayed puberty or infertility, respectively) (39). Isolated cases of GH sufficiency and spontaneous pubertal development even on the presence of PROP1 mutations have been reported (224). Patients with pituitary hormone deficiencies due to PROP1 mutations usually show a good response to GH and L-thyroxine treatments. Unlike in Prop1 null mice that display marked pituitary hypoplasia (248), several reports of anterior pituitary enlargements (resembling macroadenoma or craniopharyngioma or a Rathke pouch cyst, which can be transient) have been reported in patients with PROP1 deficiency (39, 47). However, although the anterior pituitary can also be normal or show some degree of hypoplasia, whereas posterior pituitary and stalk usually show no abnormalities.

Table 2.

Pathogenic mutations of the PROP1 gene (chromosomal location 5q35.3) in humans

Mutation type	Mutation detail	Diagnosis	Year(s)	References
Homozygous missense	R120C	CPHD	1998	(30, 31)
Compound heterozygous (deletion/frameshift + missense)	2bp-A301G302-deletion + F117I	CPHD	1998	(30)
Homozygous/heterozygous deletion-frameshift	2bp-A301G302-deletion L102C-frameshift-Ter8	CPHD	1967, 1998, 1999, 2000, 2004	(32-36)
Heterozygous deletion/frameshift	1bp-A150-deletion R53D-frameshift-Ter112	CPHD	1999, 2001, 2016	(37, 38)
Homozygous, heterozygous missense	R73C, R73H, F88S, F117I, R120C, R120H, R99X	CPHD	2000, 2001, 2002, 2004	(39-42)
Homozygous, heterozygous missense	C217T, T349A, C358T	CPHD	1999	(43)
Homozygous splice site	Nucleotide 343-2 (A-to-T)	CPHD	1999	(43)
Heterozygous frameshift	149-del-GA, 296-del-GA	CPHD	1999	(43)
Homozygous deletion	13-bp-(112-124-exon2)-deletion S38P-frameshift-Ter123	CPHD	1999	(44)
Homozygous nonsense	W194X	CPHD	2004	(45)
Homozygous nonsense	R112X	CPHD	2016	(46)
Homozygous insertion	c.113_114ins28 bp in exon 2	CPHD	2019	(47)
Homozygous missense	R71C	CPHD	2023	(48)

Abbreviation: CPHD, combined pituitary hormone deficiency.

One of the best studied groups with PROP1 deficiency–related CPHD are the patients from the island of Krk in the Adriatic Sea, harboring homozygosity for the 1bp-A150-deletion. Similar to the Ames mice, these patients have severe deficiency of GH (with low serum IGF-1), TSH, and PRL but normal ACTH levels (249). However, unlike the Ames mice, the Krk cohort as well as patients of PROP1-related CPHD are also severely gonadotropin deficient. The Krk patients display characteristic short stature, multiple reproductive defects, coarse wrinkled skin, soft nails, high-pitched voice, and hypersensitivity to cold (249). Interestingly, in these individuals there is no incidence of diabetes despite their high prevalence of obesity, no reports of any malignancies, and no reports of heart disease despite hypercholesterolemia (249). Importantly, a 20-year (1988-2007) follow-up study on 25 consanguineous patients in the Krk cohort reports that they live a relatively long-life (up to 91 years) despite hypopituitarism, indicating beneficial effects on longevity due to congenital lack of GH action, similar to Ames mice (249).

Ghrhr Null Mice (Commonly Known as lit/lit or Little Mice)

Lit/lit mice (Ghrhr null) were first described in 1976 by Eicher and Beamer at The Jackson Laboratory (151). This mouse line was named “little” due to its small size, and the homozygous mice are thus referred to as “lit/lit.” The genetic basis for dwarfism in lit/lit mice is an A-to-G mutation in codon 60 of the GH-releasing hormone receptor (GHRHR) gene (152). This mutation produces an Asp-to-Gly change in the N-terminal ligand-binding domain of the GHRHR, which prevents binding of GHRH and results in a marked decrease in pituitary GH, and some decrease in PRL (153, 250, 251). Since GHRHR is required for the release of GH from the somatotrophs of the pituitary, there is a significant (∼92%) decrease in circulating serum GH (154, 155, 251) and a concomitant reduction (>77%) in serum IGF-1 in lit/lit mice (154, 156, 251). While it is not known why there is also a reduction in PRL, Lin and colleagues point out that in addition to somatotrophs, lactotrophs, thyrotrophs, gonadotrophs, and corticotrophs, a small percentage of anterior pituitary cells, referred to as somatomammotrophs, coexpress GH and PRL. Immunohistochemistry analysis by Lin et al reveal very few cells coexpressed GH and PRL in lit/lit pituitaries (251); thus, a reduced somatomammotroph population may possibly account for PRL reduction in this mouse line.

Growth retardation becomes evident starting at age 15 days (151), and adult body weight of lit/lit mice is approximately two-thirds of littermate controls (115, 154). Femoral length and BMD are reduced by 8% to 10% in 23-day-old lit/lit mice and by 24% to 32% in 56-day-old mice (157). Male and female lit/lit mice are both subfertile, and females typically lose their first litters due to delayed mammary gland development/failed milk production (151). Lit/lit mice have reduced insulin-like growth factor binding protein 3 (IGFBP-3) with normal levels of IGFBP-1, -2, and -4 (156). The histological appearance of somatotrophs and lactotrophs are normal at birth in these mice; however, somatotrophs become difficult to locate and have fewer and smaller secretory granules between ages 14 and 24 days (109, 158). Lit/lit mice have increased fat mass with decreased lean mass (156) along with reduced serum leptin (115). GH treatment in these mice restores body composition (decreases fat mass and increases lean mass) and several bone parameters (increased bone mineral content, density, and surface area) (157). Lit/lit mice have enhanced insulin sensitivity but impaired glucose tolerance (159, 160). In contrast to previously discovered GHD dwarf mouse lines with multiple pituitary hormone deficiencies (ie, Snell and Ames dwarf lines), the lit/lit mice appear to have normal immune function (thymopoiesis, myelopoiesis, hematopoietic stem cell production, progenitor cell production, and B-cell development) (121). Furthermore, common myeloid progenitor cells isolated from lit/lit mice perform as well as controls in colony-forming assays (121). Challenges with T-independent and T-dependent antigens and with Listeria monocytogenes result in normal humoral and cell-mediated immune responses (161).

Lit/lit mice are long-lived with a 23% increase in male mean lifespan and a 25% increase in female mean lifespan compared to littermate controls (115). These long-lived mice exhibit cancer resistance in xenograft/transplant studies including reduced growth in sarcoma (162), breast (163), and prostate (164) cancers. Lit/lit mice have increased expression of xenobiotic detoxification genes (165), which are associated with an increased resistance against a variety of compounds including zoxazolamine, acetaminophen, and bromobenzene.

In humans, inactivating mutations of the GHRHR gene (MIM 139191) cause autosomal recessive isolated GHD (IGHD), classically included into the IGHD type 1B group in an early classification based exclusively on the model of inheritance; however, it is currently excluded from this group and named type IV IGHD (MIM 618157) in a new classification that accounts for the underlying gene defect. Unlike IGHD1A, these mutations result in decreased but detectable spontaneous and stimulated GH secretion, with subsequent low serum IGF-1 levels, recapitulating several features of lit/lit mice. More than 20 different pathological mutations of the GHRHR gene have been reported (Table 3), wherein several missense, nonsense, and splice mutations of the gene are found in patients from Sri Lanka (E72X), India (E72X), Pakistan (E72X, A222E), Spain (L144H), the United States (E72X, K329E, A176V), Israel (R357C), Turkey (S317T, K264E, S330L, G369V, T257A), and Brazil (c.57 + 1G > A). Common characteristics among these patients, comparable to the lit/lit mice, are pituitary hypoplasia, severe growth retardation with proportionate short stature, reduced muscle mass, central adiposity, delayed puberty, high-pitched voice, along with very low serum IGF-1 and IGFBP-3 levels. Most patients show a positive response to GH replacement. As a whole, the GHD phenotype in these patients is usually less severe than that in patients with type 1A GHD (with absolute lack of GHD) in which some features such as neonatal hypoglycemia are more frequently observed.

Table 3.

Pathogenic mutations of the GHRHR gene (chromosomal location 7p14.3) in humans

Mutation type	Mutation detail	Diagnosis	Year(s)	References
Homozygous nonsense	E72X	IGHD4	1996, 1997, 1998, 1998, 2000, 2010	(49-54)
Homozygous/heterozygous splice site	IVS1DS, + 1G > A	IGHD4	1999, 2003	(55, 56)
Homozygous missense	L144H	IGHD4	2001	(57)
Homozygous missense	F242C	IGHD4	2001	(57)
Homozygous missense	A222E	IGHD4	2001	(57)
Heterozygous missense	K329E	IGHD4	2002	(57)
Compound heterozygous missense	K329E and -124A-C, promoter	IGHD4	2002	(58)
Homozygous missense	A176V	IGHD4	2003, 2012	(59, 60)
Homozygous splice site	IVS12 + 2T > A	IGHD4	2004	(61)
Homozygous missense	R357C	IGHD4	2006	(62)
Homozygous splice site	IVS7-1G > A	IGHD4	2012	(60)
Homozygous frameshift	c.340delG	IGHD4	2012	(63)
Heterozygous missense	S317T	IGHD4	2014	(64)
Homozygous missense	K264E, S330L, G369V, T257A, c.380inserC	IGHD4	2014	(64)
Homozygous nonsense	E214X	IGHD4	2023	(65)

However, the phenotype among patients with GHRHR mutations also vary significantly, with some endogamic populations showing phenotypic singularities. For instance, the highly consanguineous Sindh (Pakistan) cohort of patients with an E72X mutation show severe growth impairment, with adult height around 130 cm for men and 113.5 cm for women. Additionally, these patients have significantly lower blood pressure but no facial dysplasia, microphallus, truncal obesity, or hypoglycemia (49). Dual-energy x-ray absorptiometry scans in male patients of the Sindh cohort show suppressed bone elongation and overall bone size, but no difference in BMD (252).

Aguiar-Oliveria and colleagues have studied GHRHR^–/– individuals from Itabaianinha, Brazil, for more than 30 years (105 patients studied for >8 generations) (253-255). These individuals have IGHD due to a homozygous 5′ precursor messenger RNA (mRNA) splice-site G-A mutation at position +1 of intron 1 of the GHRHR gene and display several clinical features similar to those found in lit/lit mice (55, 256-261). For example, all patients display marked anterior pituitary hypoplasia and proportionate short stature of postnatal onset (after normal birth length) with severely reduced—but detectable—GH, IGF-1, IGF-2, IGFBP-3, and acid-labile subunit (ALS) throughout life (256), causing a (256) decrease in IGF-1/IGFBP-3/ALS ternary complexes next to an increase in IGF-1/IGFBP-2 complexes in serum (256). The demonstration of GH secretion in these patients following treatment with a GH secretagogue indicates that GH production in somatotrophs remains intact (257).

Additionally, the Itabaianinha cohort display increased truncal obesity but improved insulin sensitivity with higher serum adiponectin (259, 261) and lower incidence of diabetes, postulated as a consequence of enhanced insulin sensitivity (261). The incidence of nonalcoholic fatty liver disease is higher than normal in the Itabaianinha cohort but is less severe than that found in normal individuals and does not progress to advanced forms of hepatitis (261). However, despite higher total and low-density lipoprotein cholesterol and C-reactive protein, no increase in carotid wall thickness or evidence of premature atherosclerosis is found in these patients (258). Interestingly, 6 months of GH treatment in these patients results in a progressive increase in the number of atherosclerotic carotid plaques and intima-media thickness in these patients despite improving body composition and metabolic profile in adult patients in this cohort (260), which is reversible after stopping treatment (262). The positive effect of GH treatment on lipid profile is also observed in children and adolescents (257, 263). Despite the lack of GH signaling, the Itabaianinha cohort reports smaller but strong bones, normal to improved muscle strength, no substantial reproductive defects, delayed pigmentation at youth, and virtual absence of graying of hair in old age (261). Moreover, adult patients with lifetime IGHD present with fewer problems in performing upper-extremity activities with fewer tendinous injuries (264) although hip-joint problems have been reported (265). Recently, a single case of cancer-related death has been reported in this cohort as well as some cases of skin tumors (255, 261). Although there is a higher frequency of death prior to age 20 years (266), the Itabaianinha cohort of patients with IGHD due to GHRHR mutations have relatively long lives and can live to more than 100 years (261). They also appear to have a “prolonged healthspan,” with better attention and executive function in patients older than 50 years compared to age-matched, GH-sufficient controls (267). Interestingly, a recent report indicates that these patients had a similar anti-SARS-CoV-2 antibody production as their GH-sufficient relatives but have lower frequency of confirmed cases or evolution to symptomatic stages of COVID (268).

Ghrh Null Mice

The Ghrh null mice were generated by Alba and Salvatori from John Hopkins University School of Medicine in 2004 (167). The GH-releasing hormone (GHRH) peptide is the activating ligand for the GHRHR mentioned earlier. In contrast to previously mentioned GHD mouse lines that resulted from naturally occurring mutations, Ghrh null mice were genetically engineered by replacing the Ghrh gene fragment spanning the last part of intron 2 and the front half of exon 3 with a bacterial neomycin resistance gene. The targeted Ghrh gene fragment encodes 3 amino acids of the N-terminal secretory signal peptide and 14 amino acids of the mature GHRH protein (167).

Ghrh null mice have significantly reduced levels of GH mRNA and protein from pituitary extracts with concomitant reductions in liver IGF-1 mRNA, serum IGF-1, and body weight (167-169). Reduced body weight becomes apparent in Ghrh null mice after age 3 weeks, and by age 8 weeks, body weight is reduced by 55% compared to littermate controls. As with other GHD lines, body composition is significantly altered with a reduction in percentage of lean mass and increased percentage of fat mass. Ghrh null mice exhibit enhanced insulin sensitivity (170) with decreased levels of adiponectin and visfatin mRNA in adipose tissue (171). Ghrh null mice also have increased locomotor activity, food consumption, body temperature, brown adipose tissue mass, and UCP-1 expression in brown adipose tissue (172, 173). These mice are subfertile with testes showing reduced levels of prostaglandin D2, cyclooxygenase-2, lipid peroxidation, macrophages, and apoptosis rate (174). Several measures of immune function are altered in Ghrh null mice. For example, while thymic weight and cellular function appear normal, these mice have an increased peripheral T-cell population, increased splenic atrophy, reduced B-cell population, and a decreased ability to clear a low S pneumoniae dose (175). Furthermore, a decreased immunoglobulin M response—which can be rescued with GH treatment—is observed following inoculations with pneumococcal polysaccharides (PPS) or protein-PPS conjugate (PCV13) (175). Curiously, Ghrh null mice are resistant to experimental autoimmune encephalomyelitis (EAE), and GH treatment restores susceptibility to EAE, strongly suggesting that GH supports the development of this disease (176).

Male and female Ghrh null mice both have extended longevity (50% and 43%, respectively) compared to normal-sized littermates (170). The addition of CR can further increase the lifespan in female Ghrh null mice by 21% but not in males (170). Despite the additive effect of CRs observed in at least one sex, microarray analysis performed on Ghrh null and CR mice indicates many similarities, suggesting substantial overlap in mechanisms responsible for lifespan extension (170).

In humans, inactivating mutations in the GHRH gene (MIM 139190) had been previously postulated, but not demonstrated, to underlie some cases of IGHD that respond to GHRH treatment (269, 270). It was not until 2024 that the first heterozygous mutation in the GHRH gene (frameshift: c.91dupC) was identified and functionally characterized (aberrant cytoplasm GHRH accumulation and inability to stimulate GHRHR expressing cells) in a boy of Chinese origin with IGHD (271). This patient was born full-term with normal birth weight and length (50 cm), showing at age 11 proportionate short stature (−3.27 SDS) more than 2 SDS away from modified mid-parental height, along with a 4-year delay in skeletal maturation, low serum IGF-1 level, and detectable but low GH after stimulation (peak < 5.0 ng/mL). No skeletal abnormalities, impairment of the rest of anterior pituitary hormones, or alteration in lipid profile were observed, and hypothalamic-pituitary magnetic resonance imaging examination showed normal pituitary structure. The patient responded to GH replacement treatment (even on irregular compliance). This novel etiology of human IGHD constitutes a new diagnostic entity (no MIM reference to date) and broadens the spectrum of genes to be considered and studied on the presence of IGHD.

Gh Null Mice

While numerous mutant mouse lines with inactivating mutations of various genes involved in the pituitary development or GH regulation have been created, surprisingly it was only recently that the GH gene (Gh) itself was targeted via homologous recombination to generate a GH null mouse. In 2019, Gh null mice were described by List and colleagues at Ohio University in Athens, Ohio (177). Gh null mice were generated by deleting the entire protein coding region of the Gh gene from the translational start codon located in exon 1 to the stop codon located in exon 5. Unlike the previously mentioned GHD mouse lines, the Gh gene itself was deleted. Thus, Gh null mice exhibit complete GHD and are a model of human IGHD type 1A.

Gh null mice have undetectable serum GH and a greater than 90% reduction in serum IGF-1 with a concomitant reduction to body length and weight. The difference in size becomes apparent after approximately age 3 weeks, with Gh null mice weighing approximately half that of normal littermates in adulthood (177). Similar to the previously mentioned GHD mouse lines, Gh null mice are relatively obese with a decrease in percentage of lean mass and an increase in percentage of fat mass. These differences in body composition increase with age (177). Treatment with exogenous GH reduces body fat and increases IGF-1, body length, body weight, and lean body mass in Gh null mice (177). Analysis of various white adipose tissue (WAT) depots indicates that the greatest enlargement (in dissected depot weight as well as adipocyte cell size) occurs in the inguinal subcutaneous inguinal depot. Furthermore, subcutaneous WAT from Gh null mice has reduced measures of collagen deposition, compared to other WAT depots, suggesting that GH's effect is more pronounced in subcutaneous WAT compared to other depots (177). Gh null mice have enhanced insulin sensitivity but are intolerant to exogenous glucose loads. Histological analysis of pancreatic islets suggests that reduced islet size may lead to glucose intolerance despite enhanced insulin sensitivity (177). Gut microbiome analysis in Gh null mice reveals an immature microbiome as well as decreased abundance in Actinobacteria, Proteobacteria, and Campylobacterota (178). Transcriptional profiling of liver, muscle, and WAT in Gh null mice with or without GH treatment has been reported (272) with 900 differentially expressed genes in liver, 723 in muscle, and 337 in adipose tissue. Fifty-five of these genes are common to all 3 tissues, including the canonical GH targets Igf-1, Igfals, and Cish. Enrichment analysis confirms the GH responses—such as fibrosis, metabolism, cell proliferation in select tissues, and as expected, the JAK/STAT pathway—is significantly altered in all 3 tissues. Gh null mice have also been used to compare 22-kDa human GH to that of 20-kDa human placental GH (273). In these mice, 22-kDa human GH and 20-kDa human placental GH both stimulate IGF-1 and increase bone growth, body length, and lean mass, while reducing fat mass. However, 20-kDa human placental GH has significantly reduced diabetogenic and lactogenic activity in these mice (273).

Gh null mice, although still a relatively new model, have other intriguing characteristics. In addition to enhanced insulin sensitivity and reduced fibrosis, Gh null mice have elevated levels of glycine (179), which have been shown to be important for healthy aging (274). Acute GH treatment of Gh null mice results in decreased serum glycine, which may be due to a reduction in Shmt1 and Shmt2 (glycine synthesis genes) mRNA expression in the liver (179). These mice also appear to be resistant to certain types of cancer—like other dwarf mouse lines (180). More specifically, when inoculated intradermally with fluc-B16-F10 melanoma cells, the resulting tumors in Gh null mice are about 50% that of WT controls (180). Since this line is relatively new, lifespan has not yet been reported in the literature for this mouse line. However, our laboratory has recently completed aging studies on a large cohort of these mice; thus, results indicating that male and female Gh null mice are both indeed long lived—like the 4 other common GHD mouse lines—should be made available in the near future.

In humans, several inactivating mutations of the GH1 gene (MIM 139250) have been identified, leading to a range of clinical outcomes with variable severity of GHD and associated phenotypic features. These outcomes include IGHD type 1A (MIM 262400, autosomal recessive, showing the most severe GHD phenotype); IGHD type 1B (MIM 612781, autosomal recessive); IGHD type 2 (MIM 173100, autosomal dominant), and Kowarski or bioinactive GH syndrome (MIM 262650, autosomal recessive). However, while IGHD is estimated to affect 1 of 4000 to 1 of 10 000 individuals (76), most cases have no known origin, and only a minority are proven to be caused by underlying genetic defects (66, 76). Some of the earliest cases of IGHD type 1 were described in 1970 in 3 Swiss children (67). However, mutations of the GH1 gene as an underlying cause of IGHD was not established until 1981, when a greater than 7.5-kb deletion—including the GH1 gene—was reported in this cohort (67). Since then, different lengths of deletions in the GH1 cluster, ranging from 7.5 to 45 kb, have been reported in patients affected with IGHD type 1A, postulated to arise from “unequal recombination and crossover events of the GH gene cluster during meiosis’ (275) (Table 4). In contrast, no deletions in GH1 but splice-site, frameshift, and missense mutations have been reported in IGHD type 1B patients (see Table 4). IGHD type 2 (the only IGHD type with an autosomal dominant mode of inheritance) is mainly caused by mutations affecting GH1 precursor RNA splicing either via missense mutations or splice-site mutations in intron 3 (or mutations in the exonic splice enhancer motif) that lead to exon 3 skipping (see Table 4). The latter is associated with the production of the 17.5-kDa isoform of GH that largely replaces the 22-kDa isoform production in a dominant-negative effect.

As previously stated, IGHD type 1A shows the most severe GHD phenotype, characterized by early-onset severe growth failure (often < −4 height SDS at age 6 months), undetectable GH concentrations, and a tendency to develop antibodies despite an initial good response to GH treatment (180). In contrast, patients affected with IGHD type 1B show detectable (but low) stimulated GH levels, usually less severe short stature, and lack of antibody production during GH treatment (66, 76, 276). In IGHD type 2, patients also show low but detectable stimulated GH levels, can vary considerably in height (< –4 SDS to normal), and can also present with a variable degree of pituitary hypoplasia. Albeit rarely, some etiologies of IGHD type 2 patients (R183H and E3 + 1G > A mutations) may show catch-up growth even without GH treatment (275).

Conflicting reports exist regarding insulin sensitivity in IGHD type 1 and IGHD type 2 patients ranging from normal to decreased (277), unlike the insulin sensitive Gh null mice. However, IGHD type 1 and IGHD type 2 patients both present with lifelong suppressed IGF-1, increased fat mass, and decreased lean mass, similar to the Gh null mice (177). No systematic report on cancer incidence or lifespan of GH1 gene mutation–related IGHD population is available.

Uncommon Mouse Models of Growth Hormone Deficiency

D2 Dopamine Receptor Null Mice

D2 dopamine receptor (Drd2) null mice were first generated by Baik et al at the Institut de Génétique et de Biologie Moléculaire et Cellulaire in Strasbourg, France, in 1995 (278) and later by Kelly et al from Oregon Health Sciences University in 1997 (181). DRD2 proteins are G protein–coupled receptors that are the predominant dopamine-receptor subtype in the anterior pituitary. The effect of Drd2 ablation on the GH/IGF-1 axis in mice has been characterized by Diaz-Torga et al in 2002 and Garcia-Tornadu et al in 2006 (182, 183). Drd2 null mice have a significant decrease in the somatotroph population in the anterior pituitary and decreased serum GH, IGF-1, and IGFBP-3 levels (182). More striking and better studied than GH alterations, Drd2 null mice have chronic hyperprolactinemia with marked pituitary lactotroph hyperplasia (182, 279). As might be expected, Drd2 null mice have impaired growth. Although body weights are similar at birth, somatic growth is decreased in Drd2 null mice by age 2 months. Additionally, femur length and weight of the liver and WAT are decreased in Drd2 null male mice (182). The growth phenotype is sexually dimorphic, with males having more dramatic growth retardation. Using whole tissue, pituitary cells from Drd2 null mice in vitro have a lower response rate to GHRH, although cells from Drd2 null mice respond to somatostatin and ghrelin at a similar rate as WT pituitary cells (183). The number of GHRH receptors are decreased in pituitary cells derived from Drd2 null mice to 46% of levels found in control cells. Moreover, while dopamine administration reduces PRL in WT cells, dopamine treatment has no effect on PRL in Drd2 null cells and an inconsistent effect on GH in Drd2 null cells and WT cells. These findings collectively suggest that DRD2—independent of dopamine—reduces the release of GHRH from hypophysiotropic neurons, leading to inadequate clonal expansion of the somatotroph population and decreased GH production (183).

In humans, genetic variants influencing DRD2 (MIM 126450) signaling have been extensively studied (especially the rs1800497—TaqIA polymorphism) with varying levels of frequency dependent on the population studied (280). As might be expected of a gene central to the dopaminergic system, DRD2 polymorphisms have been most strongly linked to addiction disorders (184), Alzheimer risk (281) and Parkinson disease (282) as well as brain (cortical) mass and cognitive function (283). As for growth, the data are mixed. Specific genetic variants of DRD2 have been correlated with idiopathic short stature (279), with significant differences in height and weight among different haplotypes (284). However, other studies have suggested that these same variants result in no alteration in height (285). Like height, there is evidence that some DRD2 polymorphisms show association with weight-related parameters (286), with rs2075654 and rs2587552 polymorphisms showing association with a better response to intervention in childhood obesity (287, 288), though no reference to patient's height is reported in these studies.

Early Response Factor 2 (Egr2) Null Mice

Several global and conditional Egr2 null mouse lines have been generated by the Charnay laboratory at Unite 368 de L’Institute National de la Sante et de la Recherche Medicale, with the first global Egr2 null mouse line (documented as either Krox20lacZ or Egr2lacZ) described by Schneider-Maunoury et al in 1993 (185). Early response factor 2 (EGR2, also known as Krox20) is a transcription factor that plays an important role in development, including that of the anterior pituitary. EGR2 is initially expressed in all differentiating pituitary cells, but after birth, is mostly restricted to somatotrophs (186). Egr2 null mice have anterior pituitary hypoplasia and die shortly after birth (within the first 2 weeks) (187, 188). Accordingly, several conditional (temporal and tissue-specific) knockout mouse lines have been created (186, 189) to assess the long-term effects on growth. Some of the tissue-specific mouse lines relevant to this review include pituitary-specific specific Egr2 gene disruption and mice with specific ablation of pituitary Egr2-expressing cells (primarily somatotrophs) (186).

Pituitary-specific Egr2 null mice are of normal size at birth with no obvious defects throughout life. However, mice with ablation of Egr2-positive cells (Pit1-Cre;Egr2GFP^(DT)/+ mice) have reduced body size and weight by postnatal day 10 (186) and stop growing by day 50. By age 6.5 months, Pit1-Cre;Egr2GFP^(DT)/+ mice have dramatic weight loss, weighing almost half of their littermate controls. These mice show a specific and progressive depletion of the somatotrophs and so represent a novel model of early-onset IGHD. Interestingly, these mice do not have altered body composition compared to controls. However, these Pit1-Cre;Egr2GFP^(DT)/+ mice do have increased insulin sensitivity and energy expenditure as seen with other mice lines with reduced GH action accompanied by reduced metabolic adaptability between glucose and lipid oxidation conditions.

In humans, EGR2 (MIM 129010) mutations are rare and cause substantially diverse phenotypes, varying in severity and age of onset (190). As EGR2 is best known as a transcription factor associated with transition of promyelinating to myelinating Schwann cells, EGR2 mutations in humans cause a variety of peripheral neuropathies. For example, humans with a mutation in this gene have been observed in Charcot-Marie-Tooth disease (MIM 607678) and Dejerine-Sottas syndrome (MIM 145900) (289) as well as congenital hypomyelinating neuropathy type 1 (MIM 605253). Other studies also suggest that mutations in EGR2 result in other defects in neural plasticity, learning/memory, as well as susceptibility to schizophrenia (290). However, few case studies report growth- or endocrine-associated defects. Of note, an upregulation of EGR2 in peripheral mononuclear cells after GH administration has been reported in children (291).

Growth Hormone Secretagogue Receptor (Ghsr) Null Mice

Two mouse lines disrupting the Ghsr gene were generated at almost the same time. The first mouse line was described by Sun et al at Baylor College of Medicine in 2004 (192), and the second mouse line was initially characterized by Zigman et al at Harvard Medical School in 2005 (193). GH secretagogue receptor (GHSR) is the receptor for ghrelin and is primarily located on somatotrophic cells. As such, GHSR has been implicated in linear growth and altering GH production.

Body weight is modestly decreased in Ghsr null mice compared to controls from ages 16 to 24 weeks (192), which becomes more pronounced—albeit still modest—by age 12 months (292). Ghsr null mice have decreased GH and IGF-1 levels, decreased Pit1 expression in the anterior pituitary, and reduced GH and PRL mRNA in the pituitary (194). Disruption of the Ghsr gene results in a modest difference—albeit not always statistically significant—in body length, BMD, and bone mineral content (193). Ghsr null mice do have lower trabecular bone mass and increased rate of bone resorption (osteoclastogenesis) (170). Of note, many studies have reported the phenotypes of Ghsr null mice using varying background strains, which influences the effect on body weight/size. For example, in a C57Bl/6J background, Zigman et al (193) report reduced body weight and adiposity in females but not males, yet Sun et al (192) report reduction in body weight both of male and female Ghsr null mice with no change in adiposity using a mixed genetic background. Regardless, these data suggest that Ghsr deletion modestly alters the growth phenotype in mice, which may indicate that ghrelin mainly regulates appetite. In a series of aging studies, the Sun laboratory has demonstrated that Ghsr null mice are protected against age-associated dysfunctions including insulin resistance (193, 293), increased adiposity (293), adipose tissue inflammation (294), thermogenic impairment (295), and metabolic decline in skeletal muscle (296). Curiously, no reports of whether these improvements result in increased lifespan in these mice have been published, which would be of great interest.

In humans, while genome-wide association studies and haplotype tagging, single-nucleotide variation (formerly known as single-nucleotide polymorphism) studies have not shown an association between GHSR (MIM 601898) variants and height (297-299), however, select rare mutations have been associated with idiopathic short stature and GHD. For example, a variant in GHSR (c.611C > A) (documented in 2 families) is associated with decreased receptor expression at the cell surface of HEK293 cells, albeit with normal binding of ghrelin. This mutation results in IGHD in the homozygous state but shows different phenotypes (ranging from normal to short stature) in the heterozygous state (195). Other mutations are reported to reduce ghrelin binding affinity and GH production (300). Recently, new GHSR variants have been reported to be associated with short stature: low IGF-1 levels but normal stimulated GH levels in the homozygous state (normal height and IGF-1 levels in the heterozygous state) in a familial pedigree (301), in a patient born small for gestational age without catch-up growth (302), and in a patient with diagnosed GHD and low IGF-1 levels (303). As ghrelin also plays a critical role in appetite through NPY/AGRP hypothalamic arcuate nucleus neuron stimulation, consequently, several studies have postulated a possible role for GHSR mutations in obesity (304).

Hesx1 Null Mice

Hesx1 null mice were described in 1998 by Dattani et al from London Centre for Paediatric Endocrinology and Metabolism, Institute of Child Health, United Kingdom (196). Hesx1 is a homeobox protein that acts as a transcriptional repressor needed for anterior visceral endoderm formation of the embryo, which is required for the establishment of the anterior-posterior body axis. HESX-1 is one of the earliest specific markers of the pituitary primordium. Hesx1 null mice have many neurological defects including a significant reduction in prospective forebrain tissue, abnormal morphogenesis of the Rathke pouch, forebrain midline defects associated with pituitary dysplasia, and GHD. At birth, 2 types of newborns are observed. One type has severe microphthalmia with a decreased head size, short nose, and an absence of eyes. These mutants display mortality shortly after birth. The second type has a less severe phenotype with relatively less craniofacial dysplasia, and only one eye (left or right) is affected. For the less severe phenotype of Hesx1 mice, approximately 25% survive weaning and can produce viable offspring (196, 197).

In humans, mutations in the HESX1 gene (MIM 601802) are associated with 3 clinical entities (MIM 182230): isolated GHD with pituitary hypoplasia (305); combined pituitary hormone deficiency (named CPHD type 5) (306); and septo-optic dysplasia (SOD, previously known as de Morsier syndrome). SOD is a rare syndrome with a wide and heterogeneous clinical spectrum including forebrain abnormalities, impairment of eye and optic nerve development, and midline defects such as partial or total absence of the septum pellucidum and pituitary hypoplasia (196, 307), with approximately 62% of these patients displaying hypopituitarism (summarized by Webb and Dattani, 2010 (307)). Most cases of SOD are sporadic and idiopathic; however, a minority of cases (mainly familial) have been described in association with mutations in developmental transcription factors (eg, HESX1, SOX2, SOX3, OTX2, and other genes are implicated in its etiology).

Lhx3 Null Mice

The Lhx3 null mouse was generated in 1996 by Sheng et al at the National Institute of Child Health and Human Development, National Institutes of Health in Bethesda, Maryland, United States (200). Lhx3 is a LIM homeobox gene (LIM for its initial discovery in the genes Lin-11, Isl-1, and Mec-3) expressed in the pituitary throughout development and is essential for the differentiation and proliferation of pituitary cell lineages. Lhx3 null mice lack the intermediate and anterior lobes of the pituitary, which results in the mice being stillborn or dying within 24 hours of birth (200). The posterior lobe of the pituitary appears normal. In contrast, heterozygous mutants are indistinguishable from their WT counterparts in size. It is important to note that a spontaneous recessive mutation in mice called “stubby” (stb), which produces mice with disproportionate dwarfism and male infertility, was previously suggested to be due to LHX3 (308) because of the dwarf phenotype and because it maps to the same area on chromosome 2 as the Lhx3 gene. However, Iwanaga and colleagues (309) have recently questioned the role of Lhx3 in the stubby mice and suggest that a nonsense mutation in Adamtsl2 is the culprit. More studies are needed to address this debate.

In humans, mutations in the LHX3 gene (MIM 600577) are associated with CPHD type 3 (MIM 221750), encompassing GH, PRL, FSH/LH, and TSH deficiency without ACTH impairment in the initial description, but with later cases reporting ACTH deficiency in up to 50% patients (224, 310), as well as neurosensorial deafness (310) and spine abnormalities in some patients (311). Magnetic resonance imaging findings can range from normal (infrequent) to aplasia or hypoplasia of the anterior pituitary, with enlargement anecdotally reported (224).

LIM Homeobox Gene-4 (LHX4) Null Mice (Also Called the GSH4 Knockout)

The LHX4 null mouse was developed by Hung Li and colleagues in 1994 at the Children's Hospital Research Foundation, Cincinnati, Ohio, United States (201). The LIM homeobox gene-4 (Lhx4) gene is also referred to as genomic screening homeobox-4 (Gsh4) in the mouse genome. The Lhx4 gene encodes a LIM-homeodomain transcription factor that is required for early pituitary development. Lhx4 null mice experience 100% mortality shortly after birth with underdeveloped lungs (201). Using Lhx3 and Lhx4 single- and double-null mutant mice, Sheng et al (1996) (200) demonstrated that both genes are needed for pituitary gland development. While the Rathke pouch is first formed as a rudiment and second as a definitive pouch, Sheng's studies indicate that Lhx3 and Lhx4 have redundant control over formation of the definitive pouch.

In humans, mutations in the LHX4 gene (MIM 602146) have been described in association with CPHD type 4 (MIM 262700). There are several known human mutations of the LHX4 gene, most heterozygous, that result in a hypoplastic anterior pituitary with congenital hypopituitarism (312) but with IGHD cases also reported (224). Additionally, a lethal phenotype associated with severe pituitary aplasia and hypopituitarism and several other abnormalities associated with a homozygous LHX4 mutation has been described (313). Due to its involvement in brain and spinal development, mutations in the LHX4 gene can result in cerebellar defects, Chiari malformation, and abnormalities of the sella turcica and corpus callosum (314).

Otx2 Null Mice

Otx2 null mice were generated by Acampora and Mazan and colleagues in a collaboration between 2 laboratories from Naples, Italy, and Paris, France, in 1995 (202). OTX2 is a homeobox family protein generally considered a head organizer in the primitive-streak stage of embryonic development and is expressed in the dorsal and ventral regions of the telencephalon, diencephalon, and mesencephalon of the developing brain (203). OTX2 functions in the regional patterning of the midbrain and forebrain and plays a role in the development of the pituitary gland, pineal gland, inner ear, optic nerve, and eye. Given its importance in head and sensory development, as expected, the Otx2 null mutation is embryonically lethal with the embryos growth retarded. Otx2 null mice are characterized by the absence of midbrain and forebrain region by 9.5 days post coitum (203) thus, no pituitary development occurs. Heterozygotes show highly variable phenotypes ranging from assorted craniofacial malformations to normal, depending on the genetic background of the mice (315).

In humans, loss-of-function mutations in the OTX2 (MIM 600037) have been described in association with CPHD type 6 (MIM 613986); syndromic microphthalmia or anophthalmia or early-onset retinal dystrophy with or without pituitary dysfunction (both MIM 610125). CPHD type 6 encompasses deficiencies of GH, TSH, LH, FSH, and ACTH (204, 205) with anterior pituitary hypoplasia, ectopic posterior pituitary, and possible Chiari malformation (205). Of note, Del Blanco et al point out that the majority of the OTX2 mutations found in patients with CPHD type 6 have been found in exon 5 and recommend starting mutational screening in this gene region (316).

Snord116 Null Mice (Also Called Snord116del Mice)

Snord116 null mice were first described by Ding et al from Stanford University in 2008 (206). Snord116 is a noncoding RNA that modulates expression of several genes, including IGFBP-7, which in turn can affect IGF-1 and GH (317). Accordingly, Snord116 null mice exhibit early-onset GHD with decreased GH and IGF-1 levels despite having normal pituitary structure and somatotroph population (206, 207). These mice exhibit hyperphagia, obesity, and have deficiencies in motor learning (206). Additional studies indicate that Snord116 null mice have increased ghrelin, hypothalamic agouti-related protein and neuropeptide Y (NPY), all of which may contribute to their hyperphagia (206, 208).

In humans, SNORD116 (MIM 605436) is related to Prader-Willi syndrome (PWS) (MIM 176270) as multiple copies of this gene are located within the introns of a large primary noncoding transcript originated from the PWS-critical region in chromosome 15q11.2. Children with PWS can show short stature in a widely variable clinical spectrum including hypotonia, impaired GH, ACTH and gonadotropin secretion, cognitive impairment, anxiety, behavior problems, hyperphagia, and obesity. PWS in humans is caused by lack of expression of paternally inherited imprinted genes in the chromosome 15q11 to q13 region (318). Within that region, SNORD116 is thought to play an important role (319). Similar to Snord116 null mice, the endocrine dysfunctions seen in patients with PWS lead to hypotonia and developmental delay, cognitive impairments, impaired motor development, and hyperphagia that leads to obesity (320). Of note, some symptoms of PWS overlap with those seen in the typical GHD phenotype, such as small hands and feet, short stature, changes in body composition (increased body fat and low muscle mass), and reduced muscle strength. Because of this (mainly intending to increase muscle), GH replacement has been successfully implemented to improve health outcomes of patients with PWS (321).

Sox2 Null Mice

Sox2 null mice were generated in 2003 by Ariel Avilion et al at the MRC National Institute for Medical Research, London, United Kingdom (198). SOX2 is a sox family (SRY-related high-mobility group box) transcription factor needed to maintain pluripotency of embryonic stem cells and to regulate neural development (322). Accordingly, Sox2 null mice die shortly after blastocyte stages. In a separate study, Kelberman et al (2006) investigated Sox2 heterozygous gene–disrupted mice and observed abnormal anterior pituitary development with reduced levels of GH, LH, and TSH, with reduced fertility in males (323).

In humans, mutation in the SOX2 gene (MIM 184429) cause optic nerve hypoplasia, central nervous syndrome abnormalities and syndromic microphthalmia-3 (MCOPS3) (MIM 206900) (324). MCOPS3 is characterized by anophthalmia or microphthalmia with or without defects of the optic nerve, optic tract, and optic chiasm. Extraocular abnormalities include sensorineural hearing loss, brain anomalies, seizures, esophageal atresia, motor disability, and neurocognitive delays. Hypoplasia of the anterior pituitary can be observed, with gonadotropin deficiency being the most consistent endocrinopathy described in individuals with SOX2 mutation; however, GHD can also be present (324).

Sox3 Null Mice

Sox3 null mice were first generated by Weiss et al in 2003 at Northwestern University (199) and soon after by Rizzoti et al at the MRC National Institute for Medical Research in London in 2004 (209). SOX3 is a transcription factor located on the X chromosome, expressed early on in central nervous system (CNS) development (325, 326). SOX3 plays an important role in normal development of the pituitary, brain, and face in mice and humans (327). Accordingly, Sox3 null mice have pleiotropic phenotypes including craniofacial abnormalities, midline CNS defects, and hypopituitarism. Likewise, body weight is highly variable but low body weight animals tend to be Sox3 null mice (209). Based on the data of Rizzoti et al, defects of pituitary function vary between individual mice and include reduced levels of GH, LH, FSH, and TSH. These defects are accompanied by abnormal development of the Rathke pouch and aberrant cells in the ventral diencephalon, which also varies in severity.

In humans, a range of phenotypes are associated with SOX3 (MIM 313430) mutations in males (due to its location in the X chromosome), including craniofacial abnormalities, intellectual disability, hearing impairment, and a variable spectrum of hypopituitarism ranging from IGHD (MIM 300123) to panhypopituitarism (MIM 312000) (198), which is usually present prior to puberty. As for pituitary defects, anterior pituitary and infundibular hypoplasia, absent pituitary stalk, persistence of the craniopharyngeal canal next to undescended or ectopic posterior pituitary, with or without abnormalities of the corpus callosum can be observed. Most SOX3-pathogenic variants reported are duplications and deletions of the whole or part of SOX3. Patients harboring duplication of SOX3 can present with IGHD (328), whereas gene deletions are usually associated with CPHD, including GH, TSH, ACTH, and gonadotropin deficiencies. Recently, Li et al (329) reported a SOX3 point frame-shift mutation in a patient showing GH and gonadotropin deficiency, with learning difficulty.

Growth Hormone Deficiency Due to Nongenetic Means

Growth hormone deficiency in mice due to hypophysectomy

Removal of the pituitary gland in mice (a process called hypophysectomy) was performed as early as 1933 by Selye et al (330). In 1946, studies by Korteweg and Thomas used hypophysectomized mice and hypophysis implants to evaluate mammary cancer and lifespan in female mice (210). Since then, hypophysectomies in mice have been performed to further understand the effect of anterior pituitary hormones on downstream endocrine organs (adrenal glands and ovaries), metabolism, growth, and various metabolic and growth disorders. This procedure has also provided a tool to study the replacement of one or more anterior pituitary hormones on rodent growth and metabolism. For instance, in 1958, Lostroh and Li from the University of California, Berkeley examined the separate effect of bovine GH and thyroxine on body weight in hypophysectomized C3H mice (211). Mice that received bovine GH over 7 days had increased linear growth, body weight, and organ weight (adrenals, ovaries, uterus, thymus, and spleen). Publications on hypophysectomies in mice increased from the 1950s (∼20 studies) to the 1960s (141 publications) and 1970s (178 publications). With the rise of transgenic and knockout mouse lines, the number of publications using hypophysectomized mice has declined.

Hypophysectomies are still used experimentally, albeit to a lesser degree, and provide a means to manipulate these pituitary hormones at any age. Of note, Powers et al from The Jackson Laboratory observed in 2006 that pituitary removal in mice at ages 1 and 9 months significantly extends lifespan (212). In 2017, Cernowicz et al from Jagiellonian University Medical College used hypophysectomies in mice to determine the effect of ghrelin through the GH/IGF-1 axis on acetic acid–induced colitis (213). Their findings led to the conclusion that ghrelin's protective effect on the intestines is mediated through GH and IGF-1. Several commercial production facilities provide hypophysectomized mice of various stocks or strains, and Hoff et al in 2006 provided an updated methodology for performing parapharyngeal hypophysectomy in mice (214). While hypophysectomized mice have been used to test GH in numerous studies as discussed earlier, the biological activity of human GH preparations by the pharmaceutical industry is commonly measured by evaluation of body weight gain or tibia growth in hypophysectomized rats although the use of lit/lit mice has been proposed as a better in vivo model (331).

In humans, hypophysectomy involves removal of part or most of the anterior pituitary to normalize or treat disease. Previously, hypophysectomy was used to slow the progression of vascular complications associated with severe diabetes (332, 333), to slow the progression of poorly responding cancers (334, 335), and is even currently being considered as a palliative treatment for pain reduction in refractory cancer pain (336). However, the vast majority of partial or total hypophysectomies performed in the clinic today are for the treatment of conditions associated with pituitary tumors, including Cushing disease, acromegaly, prolactinomas, and nonfunctioning adenomas (337, 338).

Growth hormone deficiency due to diphtheria toxin–disrupting somatotrophs

GHD mice can be produced by transgenic expression of diphtheria toxin A (DT-A) in somatotrophs of the pituitary, resulting in somatotroph cell death as described by Behringer et al at the University of Pennsylvania in 1988 (215). The rGH-DT-A transgenic mice have only 0.01% of the normal somatotroph population in the anterior pituitary (215, 216). Lactotrophs are also decreased in the anterior pituitary of rGH-DT-A mice, suggesting a shared common origin with somatotrophs and/or a “leaky” promoter/enhancer construct driving the DT-A transgene expression. As might be expected, these mice have undetectable circulating GH (215, 217), and IGF-1 levels are decreased by 8-fold compared to littermate controls (215). Preprosomatostatin mRNA in the periventricular hypothalamus is decreased to 60% of levels seen in controls (217), while GHRH mRNA in the hypothalamic arcuate nucleus is increased (339). The rGH-DT-A mice weigh significantly less than controls by age 5 days and cease growing by approximately 6 weeks (215). These mice are 50% smaller than controls, maintaining a weight between 10 and 15 g (215, 217). Pituitaries of the rGH-DT-A mice are also approximately 25% of the size of normal controls (215). The rGH-DT-A mice have shorter tibia and smaller visceral organs (liver, duodenum, and pancreas) and brains compared to controls (216). These mice exhibit subfertility and progressive lack of coordination and contracture of the front paws.

Adult-onset, inducible GHD (AOiGHD) mice have also been generated using diphtheria toxin. These mice were characterized by Luque et al in the Kineman Laboratory at the University of Illinois at Chicago in 2011 (218). As an inducible Cre system, destruction of the somatotroph population is induced by diphtheria treatment at approximately age 10 to 12 weeks. AOiGHD mice have a reduction (∼90%) in anterior pituitary size due to the significant decrease in the proportion of somatotrophs (218). Circulating GH levels are significantly decreased in AOiGHD mice compared to controls (218). It is important to note that the decline in GH in AOiGHD mice is not proportional to the decline in the number of somatotrophs, potentially due to increased GHRH levels and resultant increased GH production of the remaining somatotrophs (218). IGF-1 hepatic mRNA and circulating levels are both reduced in AOiGHD mice. Despite this reduction in the GH/IGF-1 axis, no difference in body weight is observed; however, these mice do exhibit improved insulin sensitivity, decreased lean mass, increased adiposity, and are less susceptible to DMBA-induced mammary tumors (218, 219). Aging studies in these mice demonstrate that female AOiGHD have increase lifespans while males have normal lifespans, although both sexes having increased osteoarthritis (340).

In humans, while no such mechanism has been described that parallels the diphtheria toxin–induced GHD in mice, the decline of GH secretion with age (a process called somatopause) is well documented. Pulsatile GH secretion in humans (mean secretion rate and mean duration of secretion) remains stable throughout childhood and peaks at puberty (341). After puberty and after achieving adult body height and sexual maturation, GH secretion declines by approximately 14% per decade (342). This decline is the reason why the substitution, therapeutic GH dose indicated in GH-deficient adults is 10-fold lower than the growth-promoting dose needed for growing children and adolescents. Importantly, this decline in GH levels with age is not consistent in all humans with the values for individuals of the same ages showing great variation. In fact, several studies have shown that approximately 40% of healthy individuals older than 50 years studied still secrete young adult levels of GH (341, 343, 344).

Concluding Remarks

The important role of the GH/IGF-1 axis in growth and disease is highlighted in this overview of GHD mouse lines (common and uncommon) along with a summary of comparable clinical correlates of these mice. Importantly, the phenotypes of the mice generally reflect, at least qualitatively, the findings in humans, suggesting these mice can serve as valuable tools in the study of GHD. That said, there are phenotypic differences that should be considered. The differences may, in part, be due to global loss of function—as would be seen in mice with gene ablation—vs the various single-nucleotide variation mutations reported for clinical conditions. For some of the uncommon forms of GHD, the affected genes also have broad effects on the pituitary, brain, or other organs, resulting in a phenotype that has greater implications than simply GHD. As such, these uncommon forms are often overlooked as growth disorders but provide additional insight as to the upstream or downstream molecules involved in GHD.

Abbreviations

ACTH

adrenocorticotropin

AOiGHD

adult-onset, inducible growth hormone deficiency

BMD

bone mineral density

CNS

central nervous system

CPHD

combined pituitary hormone deficiency

CR

calorie restriction

Drd2

D2 dopamine receptor

DT-A

diphtheria toxin A

EGR2

early response factor 2

FSH

follicle-stimulating hormone

GH

growth hormone

GHD

growth hormone deficiency

GHDP

partial growth hormone deficiency

GHRH

growth hormone–releasing hormone

GHRHR

GH-releasing hormone receptor

Ghsr

growth hormone secretagogue receptor

IGF-1

insulin-like growth factor-1

IGFBP-3

insulin-like growth factor binding protein 3

IGHD

isolated growth hormone deficiency

LH

luteinizing hormone

Lhx4

LIM homeobox gene-4

MCOPS

microphthalmia

mRNA

messenger RNA

NPY

neuropeptide Y

PHPX

X-linked hypopituitarism

PRL

prolactin

Prop1

prophet of Pit-1

PWS

Prader-Willi syndrome

SOD

septo-optic dysplasia

T4

thyroxine

TSH

thyrotropin

WAT

white adipose tissue

WT

wild-type

Funding

This work was supported by the National Institutes of Health (NIH R01-AG059779).

Disclosures

The authors have no conflicts of interest relevant to the subject matter or materials included in this work.