Endocrine Parameters and Phenotypes of the Growth Hormone Receptor Gene Disrupted (GHR−/−) Mouse

The aim of this review is to summarize data on the GH receptor gene disrupted mouse. The absence of GH induced signaling results in dwarf mice with depressed serum IGF-1 and insulin along with increased GH levels. Among their most notable physiological phenotype is increased adiposity found preferentially in the subcutaneous depot. Despite being obese, these mice are extremely insulin sensitive, possess a decreased incidence of cancer, and have an extended life span.

Abstract

Disruption of the GH receptor (GHR) gene eliminates GH-induced intracellular signaling and, thus, its biological actions. Therefore, the GHR gene disrupted mouse (GHR−/−) has been and is a valuable tool for helping to define various parameters of GH physiology. Since its creation in 1995, this mouse strain has been used by our laboratory and others for numerous studies ranging from growth to aging. Some of the most notable discoveries are their extreme insulin sensitivity in the presence of obesity. Also, the animals have an extended lifespan, which has generated a large number of investigations into the roles of GH and IGF-I in the aging process. This review summarizes the many results derived from the GHR−/− mice. We have attempted to present the findings in the context of current knowledge regarding GH action and, where applicable, to discuss how these mice compare to GH insensitivity syndrome in humans.

1. Introduction

2. Growth

   1. Body and tissue weights

   2. Body composition

   3. Energy balance

   4. Response to altered diets

3. Blood Chemistry Profiles of the GHR−/− Mouse

   1. Glucose and insulin

   2. Glucagon

   3. Glucocorticoids

   4. Adipokines

   5. Lipid profiles

   6. Thyroid hormones

   7. Other biochemical profiles

4. Metabolic Changes in the GHR−/− Mouse

   1. Glucose metabolism

   2. Insulin signaling

   3. PPAR isoforms

   4. Effects of diets on glucose homeostasis and lipid profiles

5. Reproduction

   1. Effects of GH on female reproduction

   2. Effects of GH on male reproduction

6. Effects on Specific Tissues

   1. Adipose tissue

   2. Skeletal muscle

   3. Heart

   4. Bone

   5. Brain

7. Aging

   1. Influence of insulin/IGF-I/GH on lifespan

   2. Comparison of transgenic GHA mice and GHR−/− mice

   3. Caloric restriction and GHR−/− mice

   4. Insulin sensitivity in GHR−/− mice: chronological age and biological age comparison

   5. Disease

8. Concluding Remarks/Future Perspectives

I. Introduction

The GH receptor (GHR) gene disrupted (GHR−/−) mouse was generated in our laboratory nearly 15 yr ago (1) and has been used in a variety of GH-related studies. Over the years, the mouse has been referred to by several names, including the Laron mouse, GHR knockout, GHR-KO, GHR gene-disrupted, and the GHR−/− mouse. More correctly, this mouse is also called the GHR/GH binding protein (GHBP) gene-disrupted (GHR/BP−/−) mouse because the GHBP gene is embedded in the GHR gene. By targeting exon 4 of the GHR/BP gene, both the GHR- and GHBP-encoded proteins were eliminated (1). Throughout this review, we will refer to these mice as GHR−/−, realizing that GHBP is also removed in these animals.

Currently, numerous publications have resulted from research on these mice. Without functional GHRs, the mice are insensitive (or resistant) to the action of GH. As will be discussed, these mice are dwarf (Fig. 1), with severely depressed levels of serum IGF-I and elevated levels of GH (1). GHR−/− mice are fertile; however, at times breeding is a challenge (1). GHR−/− mice are strongly hypoinsulinemic and insulin sensitive and transition from hypo- to normoglycemic with age (2–11). Additionally, these mice are extremely long-lived (7, 12), which is somewhat unexpected because they are also obese (13).

Fig. 1.

Size comparison of the three genotypes of the GHR−/− mice; WT (left), heterozygous (center), and homozygous (right) GHR−/− mice.

The GHR−/− mice have proven useful to study clinical conditions associated with GH action as well as the basic physiological function of GH. Regarding clinical impact, these mice mimic many phenotypic aspects of human GH-insensitive [Laron syndrome (LS)] patients (14, 15). In this regard, we will cite similarities and differences between individuals with LS and the GHR−/− mice throughout this review. Furthermore, these mice continue to be used to study disease states in which GH has been implicated including diabetes and cancer.

GH-induced intracellular signaling has been studied using this mouse. For example, the generation of GHR−/− animals that express truncated analogs of the GHR has defined areas of the GHR that are important for in vivo GH-induced signaling (16).

Studies employing GHR−/− mice have also helped to determine the basic role of GH in growth, reproduction, longevity, obesity, insulin and glucose metabolism, and the GH/IGF-I feedback loops of the pituitary. Thus, the use of these mice as a research “tool” has been widespread.

In 2001, we reported a 3-yr update on the GHR−/− mice (15). At that time, there were nine publications. Now, 10 yr later, there are over 100 publications resulting from research worldwide. Thus, we felt it appropriate to provide an update and summary of the published work on the GHR−/− mice. These animals, along with others that display alterations in GH-induced signaling, have been important “in vivo reagents” for defining specific effects of GH, many of which intersect the action of other hormones.

II. Growth

For many years, the multiple actions of GH were thought to be mediated by IGF-I. This concept was referred to as the somatomedin hypothesis. This hypothesis suggests that GH acts mainly on the liver to generate IGF-I, and IGF-I in turn stimulates proportional body growth (17, 18). The somatomedin hypothesis has since been modified with the recognition that GH has direct and independent effects (19). Specifically, Green et al. (20) found that GH stimulates differentiation of preadipocytes, and, as a result of differentiation, these cells become responsive to IGF-I. This theory of GH action has also been applied to other tissues such as the epiphyseal growth plate in bone (21). This modified form of the somatomedin hypothesis is known as the dual effector theory of GH action. The discovery that a number of nonhepatic tissues (22–25) produce IGF-I supports the possibility that not only hepatic IGF-I but also IGF-I secreted from many different tissues upon GH stimulation might act locally by autocrine or paracrine mechanisms (21, 26, 27). Indeed, hepatic IGF-I is not required for normal growth because liver-specific IGF-I−/− mice have a 75% reduction in serum IGF-I and surprisingly possess normal bone and overall body length (28). Although IGF-I transgenic mice have 30% increased weight, they do not grow longer than their nontransgenic littermates (29). In contrast, GH transgenic mice are significantly larger (weight and length) than wild-type (WT) controls (30). The GHR−/− mice offer additional insight into the impact of GH on growth and tissue development that is described below.

A. Body and tissue weights

Both male and female GHR−/− mice are dwarf, with a body weight consistently reported to be about half (41–56%) of littermate controls depending on the specific study, age, and background strain of the mice (1, 12, 31, 32). However, at birth there is no difference in body size or weight of mice of the following genotypes: GHR+/+, GHR+/−, and GHR−/− (1). The difference in weight becomes apparent at approximately 3 wk of age, with male and female GHR−/− mice being significantly smaller than WT counterparts (1, 12, 31). Overall, heterozygotes are not significantly different in size than control mice for both genders at most ages (1, 12), although the trend is for GHR+/− mice, especially females, to be slightly smaller than WT mice at older ages (12). Measurements of body length (nose-to-anus) follow the same trend with GHR−/− mice having shorter body lengths than heterozygotes and WT controls, as well as female heterozygotes being slightly shorter than WT mice (1). Studies reporting body weight over the lifespan of the GHR−/− mice reveal that both males and females have a notable decline in body weight that occurs at approximately 90 wk of age (31, 32). The decline in body weight is not unique to these mice and probably does not account for their extended longevity because WT mice show a comparable decline at a similar age.

As might be expected, most organs are proportionally smaller (allometrically scaled) in GHR−/− mice. Specifically, heart, bone, stomach, gastrocnemius muscle, spleen, and testis are smaller in GHR−/− mice but are not different than WT animals when normalized to body weight (7, 33, 34). However, there are exceptions. For example, even after normalizing to body weight, kidney and liver are smaller in GHR−/− mice than controls (1, 3, 5, 7, 32, 33). In contrast, brain (3, 7, 33), pituitary (35–37), and specific adipose depots (3, 5, 13, 32, 38) are larger in GHR−/− mice relative to body weight. Several findings related to tissue weights will be more thoroughly discussed later in this review.

B. Body composition

Adipose, bone, and muscle tissues are well-recognized targets of GH action, making GH highly influential on overall body composition. Measurement of body composition in GHR−/− mice of varying ages and in both genders has been performed using dual-energy x-ray absorptiometry and nuclear magnetic resonance. In fact, body composition has been evaluated longitudinally in both male and female GHR−/− mice over the course of their life span (Fig. 2). Results consistently show that male and female GHR−/− mice have a marked reduction in absolute lean mass; however, lean mass normalized to body weight (% lean mass) is not significantly different between GHR−/− and WT mice (32, 39). This indicates that the reduction in total lean mass is proportional to the decreased size of the GHR−/− mice. Two studies (32, 39) also report no significant decline in lean mass in older animals (∼2 yr of age) as might be expected with advancing age.

Fig. 2.

Absolute fat mass and lean mass for male (top) and female (bottom) GHR−/− mice (right) and WT controls (left). Data are expressed as mean ± sem; n = 7 male GHR−/−, 6 male WT, 8 female GHR−/−, and 8 female WT mice. Using the same cohort of mice, weight and body composition measurements were taken periodically up to 112 wk of age in duplicate using the Bruker Minispec. [Reproduced with permission from D. E. Berryman et al., Two-year body composition analyses of long-lived GHR null mice. J Gerontol A Biol Sci Med Sci 65:31–40, 2010 (32).]

Although lean mass is proportional to body size, differences in body composition persist in GHR−/− mice largely due to the changes in the abundance of adipose tissue. Male GHR−/− mice are relatively obese in comparison to littermate controls (3, 4, 13, 32, 39) with percentage body fat ranging from 21–42% vs. 13–22%, respectively, depending on the specific study. The only exception to this trend appears in one report in which fat mass percentage of “very young” male mice (6 to 7 wk of age) did not show a statistically significant difference from controls (39). Female GHR−/− mice showed a similar trend of increased percentage body fat at several ages, but the difference from controls is not as profound as that seen in males; also, a difference is not observed at all ages in every study. Specifically, one report showed no difference in percentage body fat in “very young” (6 to 7 wk of age) or “adult” (7 to 10 months) female GHR−/− mice compared with WT females (39), whereas another showed a statistically significant increase between female GHR−/− mice and controls at all younger ages that dissipated by 22 months of age (32). We have reported a decline in body weight in older GHR−/− mice, and because lean mass in those older mice is preserved, the decline in body weight reflects a reduction in body fat (32). Strikingly, despite the significantly reduced body weight of the GHR−/− mice, the absolute weight of their total fat mass is comparable to that of littermate controls (3, 13, 32); thus, adipose tissue appears to be one of the few tissues not reduced in size in these dwarf mice. Overall, these results demonstrate a gender- and age-dependent difference in body composition for GHR−/− mice, primarily due to changes in adiposity, which highlights the need to consider these variables in study design.

Individuals with LS also have increased percentage fat mass (40, 41). Furthermore, the increase in percentage body fat for LS is initiated in early life (42) as shown for the GHR−/− mice. However, unlike the GHR−/− mice, females with LS are reported to have a greater percentage fat mass than the males (41).

C. Energy balance

Accumulation of excess adipose tissue, as seen in GHR−/− mice, often results from a positive energy imbalance in which energy intake exceeds energy expenditure. GHR−/− mice consume less total energy, which would be expected given their dwarf size (3, 4, 7, 13, 44). However, despite a few exceptions (7, 13), many studies show that GHR−/− mice consume significantly more energy when values are normalized to body mass (3, 4, 7, 44).

Energy expenditure was determined using adult males (6–12 months of age) (3, 45) and older females (17 months of age) (44). Both male and female GHR−/− mice displayed increased oxygen consumption as well as lower respiratory quotient values (44, 45). Lower values indicate a preference for fat oxidation, suggesting GHR−/− preferentially oxidize lipids. Moreover, Longo et al. (44) report differences in the light and dark phases with a shift in energy oxidation and storage in female GHR−/− mice. Collectively, the energy balance studies do not fully explain the increased adiposity observed in these mice because increased energy intake is somewhat offset by a concomitant increase in energy expenditure.

D. Response to altered diets

Several studies have reported growth or body composition in GHR−/− mice fed altered diets. In certain mouse strains (such as C57BL/6J), high-fat (HF) feeding results in diet-induced obesity and the onset of type 2 diabetes (46–49). The effects of HF feeding on male GHR−/− mice have been analyzed by two separate groups (3, 50). Both studies started the dietary manipulation at a relatively young age (2.5 or 3.5 months) and continued the diet for either 12 or 17 wk. Both studies show significant increases in body weight for GHR−/− mice with calorically dense diets and report a greater susceptibility to diet-induced obesity in GHR−/− mice compared with control mice. The one study that assessed body composition (3) further showed that the excessive weight gain due to HF feeding in GHR−/− mice is due almost exclusively to gains in adipose tissue mass. Overall, all fat pads measured are increased in GHR−/− mice with HF diets. Interestingly and contrary to what is expected in obesity, the increase in body fat gained with the calorically dense diet does not lead to significant alterations in glucose homeostasis in GHR−/− mice (see Section IV.D).

Caloric restriction (CR) has been used in a number of studies related to aging in these mice (see Section VII.C). Two studies that used GHR−/− mice report the impact of CR on body weight. Using a 20% CR regimen, there is no significant effect of the diet on final body weights of GHR−/− mice (31). However, intermittent fasting resulted in no effect on body weight in WT females or GHR−/− males, but reduced growth of WT males and female GHR−/− mice (51). Neither of these studies assessed body composition changes with CR.

III. Blood Chemistry Profiles of the GHR−/− Mouse

The circulating levels of several hormones and metabolites have been measured in GHR−/− mice and will be described in this section.

A. Glucose and insulin

Fasting and nonfasting glucose levels are significantly lower in young male and female (<10 months old) GHR−/− mice compared with WT controls (2–9). However, glucose levels increase in GHR−/− males at older ages, becoming similar to those of WT controls (2, 7, 10, 11). Unfortunately, glucose levels of older GHR−/− females have not been reported. Individuals with LS show a similar pattern of circulating glucose, with very low levels in childhood and an increase later in life, but remaining below normal values (52). However, cases of hyperglycemia, glucose intolerance, and type 2 diabetes are also observed in older individuals with LS (53).

Insulin levels are extremely low in GHR−/− mice and, unlike glucose, remain significantly lower than WT controls for their entire life span (2–5, 7–11, 54). Therefore, GHR−/− have increased insulin sensitivity (2, 5, 10, 54–56), which is consistent with their extended longevity (see Section VII). On the contrary, individuals with LS show increased insulin levels and insulin resistance (52). However, despite increased circulating insulin, levels are lower than would be expected for individuals with such a degree of obesity (57).

B. Glucagon

In a similar age-dependent pattern as glucose, circulating glucagon levels in GHR−/− mice are lower than WT at 2 months of age (5) and not different than WT by 21 months (11). In addition, after a glucose load, the decrease in glucagon levels is more marked in GHR−/− compared with WT mice at 2 to 4 months of age (54). In the case of LS, glucagon response is normal after an arginine infusion test (Z. Laron, personal communication).

C. Glucocorticoids

Glucocorticoids are elevated in young and old male GHR−/− mice but not in young females (4, 8, 11); unfortunately, no measurements of corticosterone have been reported for older females. Consistent with higher corticosterone levels, expression of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) is higher in livers of young male GHR−/− mice compared with WT mice. Together with low insulin, increased corticosterone levels are consistent with the activation of gluconeogenesis in the liver. In fact, increased levels of several gluconeogenic enzymes have been found in the livers of GHR−/− mice (11) (see Section IV.B).

D. Adipokines

The adipokines, leptin and adiponectin, have been assessed in GHR−/− mice. Specifically, leptin levels are elevated in male mice at young (4) and older ages (11), consistent with their increased adiposity. However, the increase does not consistently reach statistical significance (13) and may depend on the age, genetic background, or fasting method used. Total adiponectin levels are also elevated in male (11, 13, 58) and female GHR−/− mice (58). Unlike leptin, adiponectin is typically correlated negatively with obesity and positively with insulin sensitivity. Interestingly, GHR−/− mice represent a counterintuitive situation in which obesity is accompanied by an improvement in insulin sensitivity. Thus, the high adiponectin levels in the obese GHR−/− mice may relate more to adiponectin's positive correlation to insulin sensitivity rather than its negative correlation to obesity. Alternatively, the fraction of higher molecular weight species of adiponectin, which is considered to have the predominant bioactivity, may be less indicative of adiposity than glucose homeostasis. In fact, our laboratory has data suggesting that not only total adiponectin but also high molecular weight adiponectin is elevated in GHR−/− mice (our unpublished results). Like GHR−/− mice, individuals with LS also have elevated leptin as well as total and high molecular weight adiponectin (59, 60).

E. Lipid profiles

Regarding lipid profiles, circulating cholesterol levels are generally lower in GHR−/− than WT mice. In males, total cholesterol levels are lower than WT at young and old age (4, 6, 56). However, when male and female GHR−/− mice are analyzed together, total cholesterol levels are not different between genotypes (5). Unfortunately, no data are available on cholesterol levels of females alone. In young males, high-density lipoprotein (HDL)-cholesterol is also lower in GHR−/− than WT mice (4), and grouped male and female GHR−/− mice show a trend toward lower HDL-cholesterol than WT animals (5). Low-density lipoprotein (LDL)-cholesterol is decreased as well in young male GHR−/− mice when compared with WT mice (4). Similarly, individuals with LS usually show low to normal total and LDL-cholesterol in early childhood (61). However, in later life individuals with LS have increased total and LDL-cholesterol levels, probably due to their increased obesity (52, 61, 62). Triglyceride levels in adult individuals with LS remain normal (63). In GHR−/− mice, triglyceride levels are normal to low at a young age (4–6, 56) and apolipoprotein (Apo) B levels are also low (4). Regarding free fatty acids, normal levels were reported for older GHR−/− mice (56), whereas individuals with LS usually display high free fatty acid levels (61). This agrees with the fact that individuals with LS tend to develop fatty livers (63). Fat accumulation in the liver is also altered in GHR−/− mice. Compared with WT, GHR−/− mice show lower free and higher esterified cholesterol, together with higher triglyceride content in the liver (6, 32). When fat accumulation was measured in skeletal muscle, however, no change from WT was observed in triglyceride or fatty acid contents in older male GHR−/− mice (56).

F. Thyroid hormones

Thyroid hormone levels reported for GHR−/− mice suggest hypothyroidism (at least in females) (8). Levels of T₃ and T₄ are lower in young female GHR−/− mice than controls, but the T₃/T₄ ratio is not different from WT mice (8). In contrast, individuals with LS display normal T₃ and free T₄ levels (64).

G. Other biochemical profiles

Serum ghrelin levels have been assessed in GHR−/− mice by two separate groups. Nass et al. (65) report no changes in fed or fasted serum concentrations of ghrelin in young male GHR−/− mice compared with WT controls as well as no significant difference in stomach ghrelin mRNA expression. Likewise, a study by Egecioglu et al. (4) shows no change in serum concentrations in 4- to 5-month-old male mice. However, this same study does show that GHR−/− mice have a blunted feeding response to ghrelin when centrally injected.

A variety of other metabolic parameters have been measured in GHR−/− mice. Circulating levels of calcium, bilirubin, uric acid, creatinine, γ-glutamyltransferase (GGT), alkaline phosphatase (ALP), and total protein are normal, whereas chloride, creatine kinase (CPK), alanine aminotransferase (ALT), urea, and albumin are increased in GHR−/− compared with WT mice (5). In addition, potassium levels are normal or increased, and sodium levels are normal or decreased (5, 66). Decreased plasma renin and normal aldosterone levels have also been reported for GHR−/− mice (66) (see Section VI.C). Similarly, individuals with LS display normal sodium and potassium levels, normal ALP (Z. Laron, personal communication), and normal GGT (the latter mainly in women) (63). Conversely, male individuals with LS generally show increased GGT levels, and both genders have normal ALT (63); CPK and chloride levels are also normal (Z. Laron, personal communication). Additionally, in early childhood individuals with LS have low ALP and creatinine levels (61).

In summary, compared with WT, GHR−/− mice display constantly low insulin levels, whereas glucose and glucagon values start low and normalize as the mice age. Corticosteroids are high (at least in males), as are leptin and adiponectin. Lipid profiles generally show decreased cholesterol (total, LDL, and HDL) and triglycerides and normal free fatty acids. Also, GHR−/− mice show lower thyroid hormone levels, normal ghrelin, and mostly normal levels of ions and liver enzymes, with some exceptions (chloride, CPK, ALT, etc.).

IV. Metabolic Changes in the GHR−/− Mouse

A. Glucose metabolism

As discussed in the previous sections, GHR−/− mice show low to normal glucose levels and very low circulating insulin throughout their life span. The increased insulin sensitivity observed in GHR−/− mice is likely due to the removal of the antiinsulin activity of GH. Given the marked insulin sensitivity and increased longevity of GHR−/− mice, numerous studies have investigated the regulation of metabolism in these animals.

As already mentioned, very low circulating IGF-I and high GH levels are characteristic in GHR−/− mice and result from GH insensitivity due to the lack of GHR and less negative feedback of IGF-I on the pituitary gland (1, 67). We have already explained how, consistent with a lack of GH lipolytic action, GHR−/− mice display enhanced obesity. The coexistence of obesity and improved insulin sensitivity is a hallmark of GHR−/− mice and raises interesting questions about the regulation of metabolism in these animals. The activation of the insulin signaling cascade has been evaluated in liver and skeletal and cardiac muscle of GHR−/− mice, and interesting tissue and age-specific variations have been observed (see Section IV.B).

GHR−/− mice also exhibit specific alterations in pancreas structure. Compared with controls, GHR−/− mice display smaller islet and islet cell size (even after normalizing for body weight) (5, 54). Within cell types, β-cell mass is decreased in GHR−/− mice, together with insulin content and insulin mRNA. These results are consistent with lower circulating insulin levels and suggest lower insulin production capabilities in GHR−/− mice compared with WT. In fact, lower insulin secretion, compared with controls, is observed in GHR−/− mice after a glucose load (54), suggesting glucose intolerance in these animals. In this regard, it is worth noting that GHR−/− mice are not normally exposed to such high amounts of circulating glucose, and therefore their lower insulin secretion is usually sufficient for proper glucose clearance in physiological conditions; thus, designating these mice as glucose intolerant is somewhat misleading, and it is important to remember that these mice are extremely insulin sensitive and have low to low normal levels of glucose.

B. Insulin signaling

As stated above, increased insulin sensitivity is consistent with the lack of GH's antiinsulin activity in GHR−/− mice. However, the mechanisms by which GH antagonizes insulin's actions are not entirely clear. One hypothesis involves the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), named p85. This protein has several isoforms (p85α, p85β, p55α, p50α) that interact with the catalytic subunit of PI3K, named p110. Interestingly, only the regulatory subunit of PI3K, p85α, has been linked to insulin resistance when expressed in excess of p110; in mice, enhanced GH action has been associated with an up-regulation of p85α in adipose tissue and skeletal muscle (68, 69). This results in a high proportion of free (inactive) p85α monomers, which can blunt insulin-induced signal transduction by competing with (active) p85-p110 heterodimers for association to tyrosine-phosphorylated insulin receptor substrate 1 (pY-IRS-1). Low p85α levels in mice result in increased insulin sensitivity, even when p85β levels are normal (68, 69). Unfortunately, p85α levels have not been measured in GHR−/− mice. Nevertheless, in humans, a short-term GH infusion has no effect on the insulin-stimulated increase in IRS-1-associated PI3K activity in skeletal muscle (70), demonstrating that more information is necessary to unveil the different mechanisms by which GH might exert its insulin antagonistic actions. Numerous studies have investigated the activation of the insulin-signaling cascade in liver, skeletal muscle, and heart of GHR−/− mice. The current understanding of tissue-specific insulin signaling in these mice is reviewed below and summarized in Table 1.

Table 1.

Levels of proteins in the insulin-signaling cascade in liver, skeletal muscle, and heart of GHR−/− mice as compared to WT

Age (months)	Liver	Skeletal muscle	Heart
3–6	↑/↔ IR, ↑ pY-IR, ↓ pY-IR/total IR, ↔ rate of IR Y-phosphorylation	↓ rate of IR Y-phosphorylation	↔ mRNA IR
	↔ IRS-1, ↔ pY-IRS-1, ↔ rate of IRS-1 Y-phosphorylation	↓ rate of IRS-1 Y-phosphorylation	↑ mRNA IRS-1
	↔ p85, ↔ p85 associated to IRS-1, ↔ p85 associated to IRS-1/total IRS-1	↔ p85 associated to IRS-1/total IRS-1	↑ mRNA IRS-2
	↔ PI3K activity	Robertson et al., 2006 (50)	↔ mRNA GLUT4
	Dominici et al., 2000 (9); Robertson et al., 2006 (50)		Masternak et al., 2006 (76)
9–15	↑ mRNA IR, ↑ IR, ↑ pY-IR	↑ IR, ↔ pY-IR	↑ IR, ↑ pY-IR, ↔ pY-IR/total IR
	↑ mRNA IRS-1	↔ IRS-1, ↓ pS-IRS-1	↑ IRS-1, ↑ pY-IRS-1, ↔ pY-IRS-1/total
	↑/↔ mRNA IRS-2	↑ p85 associated to IRS-1	↔ p85, ↑ p85 associated to IRS-1
	↓ p85, ↓ p55α, ↓ p50α, ↔ p85 associated to IRS-1	↑ Akt1 and Akt2, ↑ p-Akt1 and p-Akt2	↑ Akt, ↑ p-Akt, ↔ p-Akt/total Akt
	↑ mRNA Akt, ↔ Akt1, ↔ p-Akt1	↓ mTOR, ↓ p-mTOR	↓ GLUT4, ↔ GLUT1
	↑/↔ mRNA Akt2, ↔ Akt2, ↔ p-Akt2	↑ GLUT4	Giani et al., 2008 (75)
	↑ mRNA Foxo1	Bonkowski et al., 2009 (10)
	↑/↔ mRNA PEPCK
	↑ mRNA PGC-1α
	↑/↔ mRNA GLUT2
	Bonkowski et al., 2009 (10); Panici et al., 2009 (2)
21	↑ mRNA IR	↔ mRNA IR	↔ mRNA IR
	↑ mRNA IRS-1	↔ mRNA IRS-1	↔ mRNA IRS-1
	↑/↔ mRNA IRS-2	↔ mRNA IRS-2	↔ mRNA IRS-2
	↑ mRNA Akt1, ↑ mRNA Akt2, ↔ Akt, ↓ p-Akt	↔ mRNA Akt2, ↑ Akt2, ↔ p-Akt,	↔ mRNA GLUT4, ↓ GLUT4
	↑ mRNA Foxo1, ↑ Foxo1	↔ mRNA PKCζ, ↓ p-PKCλ/ζ	Masternak et al., 2006 (76)
	↑ AMPK, ↑ p-AMPK	↓ p-JNK1, ↔ p-JNK2
	↑ mRNA PEPCK	↔ mRNA PGC-1α, ↔ PGC-1α
	↑ mRNA G6Pase	↔ mRNA Foxo1, ↓ Foxo1
	↑ mRNA PGC-1α, ↑ PGC-1α	↔ mRNA Foxo3, ↓ Foxo3
	↑ p-CREB	↔ p-AMPK
	↑ mRNA GLUT2	↔ mRNA GLUT4
	Masternak et al., 2005 (72); Panici et al., 2009 (2); Al-Regaiey et al., 2005 (11)	Masternak et al., 2005 (72); Al-Regaiey et al., 2007 (73)

Arrows indicate the direction of change (increase, ↑; decrease, ↓; no change, ↔) for the levels measured in GHR−/− mice as compared to WT. Unless specified, changes displayed represent protein levels. AMPK, 5′-AMP-activated protein kinase; Akt, protein kinase B; p, phosphorylated; pS, serine-phosphorylated; pY, tyrosine-phosphorylated.

1. Liver

Young and young adult (3 to 14 months old) GHR−/− mice show increased levels of insulin receptor (IR) and normal or increased pY-IR (tyrosine-phosphorylated IR) (9, 10, 50). However, all intracellular intermediates quantified downstream of the IR in the insulin-signaling cascade display normal levels of activation. These include the rate of tyrosine phosphorylation of the IR and IRS-1 after an insulin stimulus (50), the levels of pY-IRS-1 (9), phosphorylated Akt (p-Akt) (10), and the activity of PI3K (9). Although no changes were observed in PI3K activity, a decrease in the levels of p85 (total) and its isoforms p55α and p50α (10) was reported. These findings are difficult to interpret, given that not all isoforms of p85 have the same effect on insulin signaling. As mentioned above, monomeric (inactive) p85α vs. heteromeric (active) p85-p110 would provide a better idea of insulin responsiveness in the liver. On the other hand, the activation of the MAPK pathway was also analyzed in the liver of 3- to 5-month-old GHR−/− mice. Levels of Shc (Src homology 2 domain-containing-transforming protein C1) in these mice are not different from WT animals. Therefore, similar to the PI3K/Akt pathway, there seems to be no alteration in insulin signaling via MAPK in young GHR−/− compared with WT mice.

Despite the enhanced whole body insulin sensitivity observed in GHR−/− mice at all ages tested, the livers of older animals (21 months old) display a decrease in insulin signaling when compared with controls. Levels of p-Akt are reduced (11), and there is an increase in forkhead box protein O1 (Foxo1) and peroxisome proliferator-activated receptor (PPAR)γ coactivator 1α (PGC-1α) (11). Foxo1 is a transcription factor that binds PGC-1α and promotes the expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Consistent with this finding, PEPCK and G6Pase are higher in livers of 21-month-old GHR−/− than WT mice (11). Increased levels of 5′-AMP-activated protein kinase (p-AMPK) and cAMP response element-binding protein (p-CREB) are further indications of low insulin signaling and consequent activation of gluconeogenesis and fatty acid oxidation in the liver of older GHR−/− mice (11). CREB is subject to insulin inhibition, and p-CREB is thought to activate gluconeogenesis and fatty acid oxidation through PGC-1α (71). Thus, at older ages, the liver of GHR−/− mice displays a shift toward glucose production and lipid oxidation. A predominance of lipid utilization is also consistent with the data on energy expenditure discussed above (see Section II.C). Given their increased longevity (see Section VII), GHR−/− mice reach half of their median life span at approximately 21 months of age, whereas WT mice do so at approximately 15 months (2). Interestingly, the differences found between GHR−/− and WT mice at 21 months of age persist even when 21-month-old GHR−/− mice are compared with 15-month-old WT mice (2). Therefore, the differences between GHR−/− and WT mice exist even when comparing animals of similar biological ages (2).

2. Skeletal muscle

Early reports indicated that insulin signaling was diminished in skeletal muscle of GHR−/− mice compared with WT. This decrease was based on the delayed activation of the IR and IRS-1 after an insulin stimulus measured in GHR−/− mice at 6 months of age (50) and on the unchanged mRNA levels of IR, IRS-1, and glucose transporter 4 (GLUT4) in 21-month-old GHR−/− mice compared with controls (72). However, more recent studies point toward increased insulin sensitivity in this tissue. At 14 months, GHR−/− mice display unchanged pY-IR levels, but decreased serine-phosphorylation of IRS-1 and increased p-Akt and GLUT4 levels when compared with WT mice (10). Inhibition of IRS-1 activation via serine phosphorylation is performed by a number of enzymes, such as mammalian target of rapamycin (mTOR), c-Jun N-terminal kinase 1 (JNK1), and protein kinase Cζ (PKCζ). Consistent with increased insulin signaling, levels of p-mTOR at 14 months and p-JNK1 and p-PKCζ at 21 months were lower in skeletal muscle of GHR−/− than WT mice (73). Also, levels of (total) p85 associated to pY-IRS-1 were higher in 14-month-old GHR−/− mice than controls (10). However, active p85-p110 dimers and inactive p85α monomers were not distinguished in this study. In addition, levels of Foxo1 and Foxo3 were lower in GHR−/− than WT mice at 21 months, whereas p-Akt and PGC-1α remained unaltered (73). Although the function of Foxo transcription factors is not entirely clear in skeletal muscle, a negative role in muscle mass maintenance and glucose metabolism has been proposed (74). Therefore, a decrease in Foxo1 levels would be consistent with diminished muscle wasting and increased insulin sensitivity in skeletal muscle of GHR−/− mice. The general physiology of skeletal muscle in these mice is addressed in Section VI.B.

3. Heart

Similar to skeletal muscle, cardiac muscle in GHR−/− mice displays enhanced insulin sensitivity. One study by Giani et al. (75) reported increased pY-IR, pY-IRS-1, and p-Akt in 21-month-old GHR−/− mice relative to WT mice. Levels of (total) p85 associated to pY-IRS-1 were also increased (75), but individual levels of active p85-p110 dimers or inactive p85α monomers were not evaluated. On the other hand, GLUT4 levels were decreased at 14 and 21 months of age in hearts of GHR−/− mice when compared with WT mice (75, 76). However, GLUT1 levels were not different from WT in 14-month-old GHR−/− mice (75). Decreased GLUT4 levels contradict the increased activation of the insulin signaling cascade in GHR−/− hearts. A possible explanation for this decrease could involve a compensatory mechanism against excessive glucose uptake in this organ (75). Consistent with enhanced insulin signaling, molecules in the MAPK pathway (ERK1 and ERK2) are also increased in the heart of GHR−/− mice when compared with controls (75). As with skeletal muscle, the general physiology of heart in GHR−/− mice is described in Section VI.C.

In summary, when analyzing the activation of the insulin signaling cascade in various organs of GHR−/− mice, it becomes apparent that their high insulin sensitivity is not reflected uniformly across all insulin-responsive organs. In fact, as just described, molecules in the insulin signaling cascade show variable levels of activation in the liver, heart, and skeletal muscle of GHR−/− mice. Interestingly, at young ages, the liver shows normal activation of the signaling cascade (9) but data from older animals suggest decreased insulin signaling in GHR−/− mice, compared with WT levels (11). In contrast, heart and skeletal muscle of GHR−/− mice display enhanced insulin responsiveness as suggested by increased levels of activation of signaling molecules (10, 73, 75). Given that insulin acts through numerous and varied mechanisms, further research is necessary to thoroughly characterize the insulin sensitivity in each of these organs and other targets of insulin action, such as adipose tissue.

C. PPAR isoforms

PPAR isoforms are transcription factors that heterodimerize with retinoid X receptor (RXR) isoforms and bind to responsive DNA sequences to transcriptionally regulate processes such as lipid and glucose metabolism. Three isoforms of PPAR are differentially expressed in various tissues of mice and humans. Levels of PPARγ are high in adipose tissue, where it promotes differentiation of adipocytes, lipid accumulation, and secretion of adipokines, enhancing whole body insulin sensitivity (77). PPARα is expressed mainly in the liver and promotes oxidation of fatty acids, decreasing triglyceride content (77, 78). Whereas in mice PPARα decreases HDL, ApoA-I, and ApoA-II production, the effect is opposite in humans, resulting in higher ApoA-I, ApoA-II, and HDL-cholesterol (78, 79). Finally, PPARβ (or δ) has a less clear role. It appears to be involved in wound healing and possibly in lipid metabolism (80). The available data on PPAR and RXR isoform levels in different tissues of GHR−/− mice are summarized below.

1. Liver

At 21 months of age, GHR−/− mice display liver PPARγ and PPARα levels that are higher and PPARβ that are lower than WT mice (81). In this organ, GH negatively regulates PPARα and PPARα-regulated genes through signal transducer and activator of transcription 5b (82–84); therefore, it is not surprising that PPARα levels are elevated in the liver of GHR−/− mice. Increased PPARα levels are consistent with the activation of fatty acid oxidation, which agrees with increased Foxo1, p-CREB, and p-AMPK levels and low respiratory quotient values as mentioned above (see Sections IV.B and II.C). Furthermore, the low levels of circulating HDL-cholesterol found in GHR−/− mice (see Section III.E) may be a consequence of high PPARα action in the liver. Interestingly, high liver PPARα in humans would lead to increased HDL-cholesterol, which might contribute to the high total cholesterol observed in individuals with LS (52, 61, 62).

In the case of PPARγ, its action in the liver is not well characterized. However, given its effects on whole body insulin sensitivity, high PPARγ levels in the liver may serve to counteract the diminished insulin signaling detected in GHR−/− mice at older ages. Conversely, decreased PPARβ in the liver might reduce energy uncoupling and increase lipid accumulation, consistent with the increased liver triglyceride content found in these mice (see Section III.E). However, the exacerbated action of PPARβ might be counteracted by the increased PPARα levels (81). All isoforms of RXR show increased mRNA levels in GHR−/− mice, which may serve to enhance the action of PPAR isoforms (81). In addition, gene targets of PPARα were measured in the liver of 3- to 4-month-old GHR−/− mice (85). Consistent with PPARα activation and related to fatty acid catabolism, cytochrome P450 4a (Cyp4a) and thiolase mRNA levels were increased, although other related genes remained unchanged in GHR−/− mice compared with WT animals (85). Stress resistance genes were mainly unaltered [T-complex protein 1-α and 1-β, protein disulfide-isomerase A4 (ERp72), heat shock protein 25 (Hsp25), Hsp60, Hsp65, Hsp84], except for an increase in T-complex protein 1-ε (85). Consistent with the negative regulation of PPARα on inflammatory genes, expression of β-fibrinogen was decreased, suggesting protection against cardiovascular disease in GHR−/− mice (85).

2. Skeletal muscle

In skeletal muscle of 21-month-old GHR−/− mice, the protein levels of all three isoforms of PPAR are decreased when compared with WT animals (56). Given the enhanced activation of the insulin signaling cascade measured in skeletal muscle (see Section IV.B), low levels of PPARγ suggest that the insulin-sensitizing actions of this isoform might not be important in skeletal muscle (56). Decreased PPARα levels point toward lower lipid oxidation in this tissue in GHR−/− compared with WT mice (56). Similar to the liver, lower PPARβ levels in skeletal muscle suggest decreased energy uncoupling and increased lipid accumulation (56), although triglyceride and fatty acid content in this tissue are not increased (see Section III.E). Furthermore, unchanged mRNA levels of RXR isoforms in GHR−/− mice negate the possibility of any compensation for decreased PPAR isoform levels (56).

3. Heart

PPAR and RXR isoforms have been measured at the mRNA level in 3-month-old GHR−/− mice and at the protein and mRNA level in 21-month-old GHR−/− mice (76). Contrary to findings in skeletal muscle, cardiac muscle shows no change in PPARγ levels at both ages (76). However, PPARα protein levels are decreased in hearts of older GHR−/− mice compared with controls, suggesting lower fatty acid oxidation (76). This is consistent with the increased insulin sensitivity observed in this organ (see Section IV.B). Compared with WT animals, PPARβ mRNA levels are increased at both ages, but protein levels are decreased at 21 months in GHR−/− mice (76). Unfortunately, the function of PPARβ in this organ is not clear. Regarding RXR isoforms, mRNA levels of all isoforms are increased at both ages (except for unchanged levels of RXRγ at 3 months) (76). As in the liver, increased RXR expression in the heart might enhance the action of PPAR isoforms in this organ in GHR−/− mice (76).

Clearly, more information on the specific actions of PPAR isoforms is necessary to thoroughly interpret the variations in protein and mRNA levels measured in GHR−/− mice compared with WT mice in the tissues described above.

D. Effects of diets on glucose homeostasis and lipid profiles

GHR−/− mice have been fed various diets, mostly to study effects on longevity and glucose homeostasis. CR has been the most commonly used dietary manipulation. As described below (see Section VII.C), several reports have analyzed the effects of CR on longevity, insulin sensitivity, components of the insulin signaling cascade, and PPAR and RXR isoforms in liver, skeletal muscle, and heart (10, 56, 72, 73, 75, 76, 81).

The effects of HF feeding on body fat of GHR−/− mice have also been discussed above (see Section II.D). Relative to glucose homeostasis, a HF diet does not affect fasting glucose levels on 5-month-old GHR−/− mice (3). Although there is an increase in circulating insulin after HF feeding, the levels of insulin still remain significantly lower than those of low fat-fed WT mice (3). Similarly, no changes in insulin sensitivity or nonfasted insulin levels are observed in HF vs. low fat-fed GHR−/− mice at 3 months of age (50). In these mice, pancreatic β-cell mass increases in a similar manner as that of HF-fed WT animals (50). An enhanced need for insulin production after HF feeding is probably responsible for this increase (50). Therefore, despite the weight gain, GHR−/− mice remain insulin sensitive when fed a HF diet.

GHR−/− mice have been subjected to soy-derived diets. A high isoflavone-soy protein diet results in increased glucose clearance and cholesterol levels that rise to match those of normal WT mice (6). No changes in fasting glucose or triglyceride levels are detected in either GHR−/− or WT animals fed this diet (6). In contrast, when fed a similar soy-derived diet but with low isoflavone content, fasting glucose and triglyceride levels decrease, whereas cholesterol levels and glucose tolerance remain unchanged in both GHR−/− and WT mice (6). Hence, for soy-derived diets, differences in insulin sensitivity between GHR−/− and control mice are only observed when the isoflavone content is high.

V. Reproduction

As mentioned earlier, both male and female GHR−/− mice can reproduce; however, the lack of GH action results in significant defects in reproductive function. This section will summarize the observations regarding the suboptimal reproductive capabilities and provide information regarding several tissue-specific changes in GHR−/− mice that may explain these deficiencies.

A. Effects of GH on female reproduction

Observations that females with LS exhibit delayed sexual maturation but eventually prove to be fertile suggested that GH plays a role in female reproductive maturation (86). The GHR−/− mouse has provided a model allowing for the characterization of numerous GH-dependent effects on female development and fertility (1, 87). In female GHR−/− mice, the average age of the first pregnancy is approximately 10 wk, whereas that for WT and GHR+/− mice is approximately 6 wk. This delay in the age of first pregnancy suggests a delay in sexual maturation. Further evidence of this delay is seen in the age of vaginal opening. The average age of vaginal opening for GHR−/− mice is delayed by approximately 7 d compared with WT females (38 vs. 31 d) (87). Furthermore, the delay in vaginal opening can be reduced by approximately 3 d via the administration of recombinant human IGF-I (87). In contrast, female mice that express the GH transgene have accelerated sexual maturation but still exhibit poor overall reproductive performance (88).

Standard mating practices utilize heterozygous matings (GHR+/− males bred to GHR+/− females) to generate GHR−/−, GHR+/−, and WT controls and all three genotype offspring from these matings are normal size and length at birth (see Section II.A). It is not until approximately 3 wk of age that the GHR−/− offspring begin to demonstrate a dwarf phenotype (1, 87). In contrast, pups born from GHR−/− females have reduced body weight and length (1, 87), whereas their placental weights are increased.

GHR−/− females are subfertile, producing fewer pups per litter than WT or GHR+/− mice (1, 87). Inbreeding of WT and GHR+/− mice generated, on average, 6.75 pups per litter. However, inbreeding of GHR−/− mice on average produces only 2.7 pups per litter. GHR−/− females also carry fewer fetuses during their pregnancy than WT mice but do not have increased fetal mortality. These observations suggest a reduced ovulatory rate in GHR−/− mice (87). In contrast, female GH transgenic mice that become pregnant have increased litter sizes, which reflect an increased ovulation rate (88).

After birth, offspring of GHR−/− females experience increased perinatal mortality. Pups born from GHR+/+ and GHR+/− inbreeding experience a perinatal mortality rate of 5–6%, whereas the mortality rate of pups born from GHR−/− mice is approximately 26% (1). The observation that pups born to GHR−/− females appear to have decreased milk consumption may in part explain this increased mortality (1). Along this line, Gallego et al. (89) have demonstrated that GH preferentially activates signal transducer and activator of transcription 5 in the stromal compartment of mammary tissue and results in significantly retarded ductal outgrowth and branching in the developing mammary gland of GHR−/− females. However, this deficiency is overcome during pregnancy, and GHR−/− females are able to lactate (89).

To further study the smaller and larger litter sizes in GHR−/− and GH transgenic mice compared with WT mice, numerous studies have been performed to investigate the role of the GH/IGF-I axis on follicular growth and development and its effect on the overall ovulation rate. The fact that both GH and IGF-I receptors are expressed throughout the ovary as well as IGF-I itself suggests a role for the GH/IGF-I axis in normal ovulation (90–92). Because IGF-I gene-disrupted mice are sterile and fail to ovulate, there is a requirement for IGF-I in reproduction (93). Ovaries isolated from GHR−/− mice contain all developmental categories of follicles. However, GHR−/− ovaries contain a significantly higher number of primordial follicles and a concomitant decrease in the number of healthy follicles in the antral and preovulatory stages (92, 94, 95). In addition, a higher level of follicular atresia is observed in GHR−/− ovaries, indicating increased levels of follicular degeneration and resorption. Importantly, the treatment of GHR−/− mice with IGF-I results in decreased numbers of primordial follicles and increased numbers of healthy antral follicles. The altered follicular development in the GHR−/− mice leads to a decrease in the number of preimplanted eggs observed soon after fertilization as well as subsequent uterine implantation (94, 95). Finally, the number of corpora lutea observed in GHR−/− ovary sections is decreased compared with WT mice and, whereas superovulation of GHR−/− females results in an increased number of eggs, their numbers are still significantly lower than those produced by WT females (95). In contrast to GHR−/− mice, GH transgenic mice have a reduced percentage of ovarian follicles containing apoptotic cells compared with WT mice, which leads to an increased number of healthy preovulatory follicles (96). These results suggest that the GH/IGF-I axis plays a role in the development of follicles to the ovulatory stage. This may occur via primordial follicle recruitment and growth and a reduction in follicular atresia.

B. Effects of GH on male reproduction

Similar to females, males with LS exhibit delayed sexual maturation but eventually prove to be fertile (97–99). Delayed sexual maturation has also been documented for GHR−/− mice, as well as Snell dwarf mice and GH-deprived rats (achieved by passive immunization against rat GH-releasing factor) (1, 100, 101). Similar to females, there is an absolute requirement for IGF-I for male fertility in that IGF-I gene-disrupted mice are sterile (93). In addition to a delayed developmental maturation, male GHR−/− mice also exhibit several behavioral differences that contribute to an impaired reproductive rate. For example, their responses to females are suppressed and lead to a decrease in copulatory behavior and an increased interval from mating to conception (1, 102). A similar change in behavior occurs in humans with LS in that they exhibit a reduced level of sexual desire and erection (103).

A physical example of delayed maturation due to the lack of GH action is in penile development. In humans, although individuals with LS have normal androgen levels, they often suffer from a condition termed micropenis (104). In rodents, the effect of a lack of GH action is manifested in the age of balano-preputial separation. Androgen-dependent balano-preputial separation is used as an external index for the onset of puberty and refers to the separation of the prepuce from the glans penis. Keene et al. (105) have demonstrated that balano-preputial separation is delayed by approximately 5 d in GHR−/− mice compared with WT mice. A lack of GH action is also associated with reduced testicular, seminal vesicle, and epididymal weights in GHR−/− mice compared with WT mice (105). In addition, GHR−/− mice exhibit a delay in sexual maturation with respect to the level of spermatogenesis within the testes. Elongated spermatids can be detected in the testes of some WT mice at 25 d of age and in all animals examined at 35 d (105). In contrast, elongated spermatids are absent from testes of GHR−/− mice at 25 d and detected in only 29% of the animals at 35 d. However, elongated spermatids are detected in all GHR−/− animals beginning at 40 d. This delay in appearance of elongated spermatids may be explained by the reduced intratesticular testosterone levels observed in GHR−/− mice compared with WT animals (105).

The delay in sexual maturation in male GHR−/− mice described above may arise from changes in the secretion of hormones necessary for reproductive development due to the lack of GH action. LH is secreted from the gonadotroph cells of the anterior pituitary gland in response to the release of GnRH from the hypothalamus. In males, LH acts on the Leydig cells located within the testicle to produce testosterone. Testosterone then acts upon Sertoli cells of the testis to stimulate spermatogenesis by facilitating the conversion of round to elongated spermatids (106, 107). Although the level of basal LH secretion in GHR−/− mice is similar to that of WT mice, the increase in plasma LH levels in response to GnRH treatment is attenuated (102, 108, 109). The decreased response in GHR−/− mice to GnRH is believed to be due to the lack of circulating IGF-I. The LH-stimulated testosterone release from Leydig cells is diminished in GHR−/− mice compared with WT mice, although circulating plasma testosterone levels are normal (109). The reduced intratesticular testosterone level observed in GHR−/− mice implies a lack of GH action on Leydig cells. Leydig and Sertoli cells express GHR, and GH has been shown to stimulate testosterone secretion from Leydig cells (110, 111). Therefore, the lack of GHR on these cells in GHR−/− mice probably results in lower testosterone secretion. In addition, GHR−/− mice have a reduced volume of Leydig cells per testis (109, 111). Prolactin (PRL) may also play a role in the maturation process. GHR−/− mice have elevated circulating levels of PRL, and PRL has been shown to increase LH receptor numbers and increase LH action on the testes (112, 113). However, GHR−/− mice have significantly reduced numbers of testicular LH and PRL receptors compared with WT mice (109, 114). The reduced PRL receptor number results in lower LH receptor numbers, although the GHR−/− mice are hyperprolactinemic.

One final potential contributor to the delayed maturation observed in GHR−/− mice involves the action of FSH. Hypothalamic release of GnRH results in the secretion of FSH from the gonadotrophs of the anterior pituitary. This hormone acts on the Sertoli cells of the testis to increase the production of androgen binding protein (109). Overall, FSH prevents the premature death of spermatogonial cells and spermatocytes and supports the conversion of spermatocytes to spermatids (meiosis) (106, 115, 116). GHR−/− mice also have decreased levels of circulating FSH (109). In summary, the lack of circulating IGF-I due to a lack of GH action may result in reduced FSH and testosterone secretions in the GHR−/− mice. This causes a delay in sexual maturation but does not inhibit final maturation.

Finally, reproduction studies involving the GHR−/− mouse have revealed several roles of the GH/IGF-I axis in reproduction. They have confirmed earlier observations in humans such as the delayed onset of puberty and various morphological differences. Nevertheless, animals that are GH insensitive exhibit these reproductive delays but eventually attain sexual maturity and become capable of reproducing.

VI. Effects on Specific Tissues

A. Adipose tissue

Adipose tissue is now understood to be a more complex organ than previously realized, having a major intrinsic endocrine function, complex cellular composition, and clear depot-specific differences in metabolism (117–119). As described earlier (see Section II.B), GHR−/− mice have a disproportionate amount of adipose tissue, remain relatively obese throughout much of their lives, yet still exhibit improved insulin sensitivity and longevity. This is an intriguing characteristic that challenges much of what is considered dogma, where increases in adiposity are often associated with decreases in life span and impaired insulin sensitivity. Thus, these mice have fueled interest in understanding the unique features of their adipose tissue.

1. Disproportionate accumulation in white adipose depots of GHR−/− mice

As discussed previously, the absolute mass of adipose tissue is not different between WT and GHR−/− animals. Thus, it is tempting to assume that adipose tissue is unscathed by the lack of GH action in GHR−/− mice. However, this is not the case because the accumulation of excess total body fat in GHR−/− mice is not uniform, with certain depots being disproportionately enlarged (Fig. 3). Specifically, a profound increase in the mass of the depot has been reported in both young and older GHR−/− mice (3, 5, 13, 32, 38, 120). This preferential deposition in sc fat is maintained throughout life span. The mass of the sc depot in these dwarf mice is quite impressive, with an absolute sc fat mass that is the same size or larger than that of the much larger WT mice.

Fig. 3.

Regional body fat distribution of male control mice (top) and male GHR−/− mice (bottom) using magnetic resonance images. The serial images are acquired using a Bruker 4T small animal magnetic resonance scanner and a T1-weighted multi-echo spin echo acquisition (matrix, 128 × 512 × 128; field of view, 2.5 cm × 10 cm × 2.5 cm; resolution, 0.1953 × 0.1953 × 0.1953 mm³; T1-weighted, 15 echoes; exposure time, 15, 30, 4…225 msec). The mouse is positioned such that the anterior part of the mouse is at the bottom of the image. Image voxels were classified using a multichannel classification algorithm into three classes: 1) fat, 2) lean muscle, and 3) bone/air (156). An interactive segmentation method is used to identify the peritoneum and to label the fat into two groups (intraabdominal and sc). Subcutaneous fat is noted by the color yellow and intraabdominal by the color blue. Male mice were 5 months of age. Images were provided by Kevin Behar and Xenios Papedemtrius at the Yale Mouse Metabolic Phenotyping Center.

Data are less consistent for the intraabdominal white adipose depots routinely assessed. For example, several studies have indicated that, relative to body weight, the fat pad next to the kidney (retroperitoneal) is increased in male GHR−/− mice when compared with controls (4, 13). However, other studies suggest no difference in normalized depot weight between genotypes (3, 5). These discrepancies may be influenced by the age or genetic background of the mice or the greater difficulty in defining the boundaries of the retroperitoneal depot. Other depots are repeatedly shown to be normal or disproportionately smaller in the dwarf GHR−/− mice. For example, the epididymal fat pad is a male perigonadal depot commonly studied because of its ease of dissection and its relative abundance. When normalized to body weight, this depot is of similar size (3, 4, 13, 32) or smaller (7, 38) than the same depot in male WT mice, indicating that there is no preferential accumulation of fat in this region in GHR−/− mice. Interestingly, early reports that used the mass of only the epididymal fat pad to extrapolate whole body adiposity levels (7, 15) suggested that GHR−/− mice were not obese compared with control mice. Collectively, these data reveal the importance of studying multiple adipose depots because distinct white adipose tissue (WAT) depots would likely yield differing conclusions.

Individuals with LS also have marked increases in sc adipose tissue, although other depots may be enlarged as well (41). Long-term IGF-I treatment decreases the sc depot significantly in these individuals (121). Unfortunately, a systematic characterization of the distribution of adipose tissue in individuals with LS has not been reported.

2. Additional depot-specific differences

The cause of the differences in adipose depot accumulation in GHR−/− mice remains unresolved, yet additional differences have been observed. There are variations in adipocyte sizes and numbers among depots in GHR−/− mice relative to WT (120, 122). Subcutaneous and retroperitoneal depots display increased mean cross-sectional area of adipocytes in GHR−/− mice compared with controls, exhibiting a 33 and 11% increase, respectively (122). In contrast, there is no significant increase in mean adipocyte size in the epididymal fat pads of GHR−/− mice. Thus, an increase in adipocyte size appears to contribute to the enlargement of the sc, and possibly retroperitoneal, depot in GHR−/− mice. The trend in female mice may be different because an increase in adipocyte number has been suggested to be the main cause for the increased sc adipose mass in GHR−/− females (120).

In addition to differences in the size of mature adipocytes, there are also differences in the intrinsic properties of the preadipocytes isolated from distinct depots. Preadipocytes isolated from a sc depot of GHR−/− mice do not differ in their ability to proliferate, differentiate, and respond to insulin and isoproterenol when compared with preadipocytes isolated from control animals (120). In contrast, preadipocytes isolated from the parametrial fat pad (a perigonadal fat pad in female mice) cannot proliferate or undergo differentiation like those isolated from control animals from the same depot. Thus, the effects of GH action on preadipocyte proliferation and differentiation are markedly different in distinct depots and provide an example of the inherent depot-specific effects of GHR absence in GHR−/− mice.

3. Brown adipose tissue enlargement

The changes in adipose tissue of GHR−/− mice are not exclusive to WAT; brown adipose tissue is altered in GHR−/− mice as well. When normalized to body weight, the interscapular brown adipose depot is enlarged in male GHR−/− mice (4, 38). The increased mass of brown adipose tissue is accompanied by increased expression of uncoupling protein-1 in young and older mice (10 and 52 wk, respectively) (38). In addition, there is some indication that there may be differences in the brown adipocyte pool found within the white adipose depots of GHR−/− mice (123). Recent data from other animal models show that a distinct population of brown adipocytes is interspersed within white adipose depots (124), where these brown adipocytes play important roles (125, 126).

In summary, the specific enlargement of the brown adipose depot as well as at least the sc white adipose depots of GHR−/− mice warrants further investigation. Although the underlying mechanism for the depot differences is unresolved, our laboratory and others are actively studying distinct depot differences in GHR−/− mice including changes in cell composition, innervation, macrophage infiltration, and cell senescence, as well as protein/gene expression that may explain the functional differences in these depots.

B. Skeletal muscle

GH has an anabolic effect on skeletal muscle. In fact, recombinant human GH is shown to increase lean mass in GH-deficient adult patients (127). Individuals with LS have decreased muscle mass (41) and reduced muscle force and endurance, which can be partially rescued by IGF-I treatment (128). Also, individuals with LS exhibit limited exercise capacity, partly due to peripheral muscle dysfunction (129). Similarly, GHR−/− mice have decreased muscle mass (1, 7, 32). Skeletal muscle results using GHR−/− mice will be discussed below.

1. IGF-I expression in skeletal muscle of GHR−/− mice

As mentioned earlier, GHR−/− mice have markedly reduced circulating IGF-I levels. In mouse skeletal muscle, two IGF-I mRNA isoforms resulting from alternative splicing exist: IGF-IEa (the form that is also expressed by liver), and a muscle-specific form called mechano growth factor (130). Regarding muscle expression of IGF-I in GHR−/− mice, controversial results have been reported. At the mRNA level, total IGF-I is down-regulated in 10-wk-old male GHR−/− mice (67). Moreover, both IGF-I isoforms are down-regulated in 3-month-old male GHR−/− mice (131), suggesting that muscle IGF-I mRNA expression relies to a large extent on intact GH signaling. However, protein levels of total IGF-I in skeletal muscle of 4-month-old female GHR−/− mice are not different from WT mice (132), suggesting that muscle IGF-I protein expression does not strongly rely on GH, at least in female mice. In humans, recombinant human GH treatment (0.075 IU/kg·d) for 2 wk does not change muscle IGF-I mRNA expression, although circulating IGF-I increased 3-fold in healthy young male human subjects (133). This supports the observation that muscle IGF-I protein expression in female mice is less dependent on GH. Future studies are needed to clarify any gender- and age-related differences in the skeletal muscle of GHR−/− mice.

2. Skeletal muscle fiber size, number, and type

Skeletal muscle is composed of multinucleated myofibers or myocytes. Muscle hypertrophy occurs in two ways: cell volume expansion and cell fusion with mononucleated satellite cells, which upon stimulation, proliferate, differentiate, and fuse with existing multinucleated myofibers to increase myofiber size. GHR−/− mice have decreased muscle mass (1, 7, 32), apparently resulting from smaller-sized myofibers, but not lower cell number (134, 135). In vitro experiments have revealed that in limb muscle cells derived from 4-wk-old WT mice, GH (but not IGF-I) increases myofiber size by promoting fusion of satellite-like myoblasts to nascent myotubes (134). Due to the lack of GHR, no such effect is observed in muscle cells from GHR−/− mice (134). In addition, GH shows no effect on the size, proliferation, or differentiation of myoblast precursor cells from WT mice (134). This finding suggests that GH acts on muscle at the level of cell fusion, but not through hypertrophy or hyperplasia of myoblast precursor cells.

There are two major types of skeletal muscle fiber: type I and II fibers. Type I (or slow twitch) fibers produce low force but have higher endurance, using energy generated primarily from oxidative phosphorylation. In contrast, type II (or fast twitch) fibers produce higher force over a short time using energy generated primarily by glycolysis. Hind limb muscle fiber type has been assessed in GHR−/− mice in two separate studies. Sotiropoulos et al. (134) found that 2-month-old GHR−/− mice (mixed gender) had a fiber type switch from type I (slow) to type II (fast) in soleus and tibialis anterior. However, Schuenke et al. (135) found unchanged proportions of fiber types in plantaris, soleus, and gastrocnemius using 4-month-old female mice. These results may be due to differences in strain, age, and gender of the animals used.

3. Interaction of GH and androgen on skeletal muscle hypertrophy

Androgens are essential for muscle mass increase during puberty and subsequent maintenance in male adulthood. Therefore, it is of interest to evaluate whether there is an interaction of androgens with the GH/IGF-I axis on muscle size. To address this issue, GHR−/− and WT mice have been orchidectomized and treated with dihydrotestosterone or testosterone during late puberty. Both treatments stimulate muscle mass equally in GHR−/− and WT mice, without changing muscle IGF-I mRNA expression (67). Because testosterone treatment does not produce a different effect in muscle mass stimulation in GHR−/− or WT mice, it appears that androgens and GH/IGF-I stimulate muscle growth via independent mechanisms.

4. Interaction of GH and glucocorticoids on skeletal muscle atrophy

Glucocorticoids are known to induce muscle loss, causing steroid myopathy (136). This process can be prevented by GH and/or IGF-I treatment in rats (137). The enzyme 11β-HSD1 catalyzes the conversion of inert cortisone and 11-dehydrocorticosterone into the active glucocorticoids (cortisol and corticosterone, respectively), and it is down-regulated by GH replacement therapy in adult hypopituitary patients (138). Interestingly, no difference in the mRNA levels of 11β-HSD1 and glutamine synthetase (a glucocorticoid-inducible enzyme) is found in skeletal muscle of 3-month-old GHR−/− mice when compared with WT mice (139). These results suggest that muscle glucocorticoids do not contribute significantly to the reduced muscle size in GHR−/− mice. Further studies on the protein levels are needed to confirm this observation.

In summary, skeletal muscle in GHR−/− mice is composed of a conserved number of muscle fibers. However, the smaller size of these fibers results in reduced muscle mass. Conflicting results suggest that the fiber type proportion depends on mouse age, strain, gender, and perhaps different muscle groups used. In addition, GHR−/− skeletal muscle expresses lower levels of IGF-I mRNA but contains the same protein level as control mice. This suggests that IGF-I protein expression in the muscle is not entirely dependent on GH, and also that autocrine action of IGF-I is not sufficient to restore the muscle mass of GHR−/− mice. Finally, the activation of muscle anabolism by GH/IGF-I is regulated independently of androgens and glucocorticoids in WT mice. Future studies are needed to address the gender- and age-related differences on the muscle phenotype of GHR−/− mice. It is interesting that GH, but not IGF-I, increases myofiber size by promoting fusion of satellite-like myoblasts to nascent myotubes (134). Also, IGF-I replacement therapy only partially rescues the reduced muscle force and endurance in individuals with LS. These data suggest that mechanisms other than via IGF-I for GH-induced muscle hypertrophy should be considered for further studies.

C. Heart

Acromegalic patients present a vast array of cardiovascular abnormalities characterized by ventricular hypertrophy, interstitial fibrosis, extracellular collagen deposition, and areas of monocyte necrosis in the heart (140). In addition, these patients have an increased atherogenic lipid profile (141). An increased prevalence of cardiovascular abnormalities is also observed with GH deficiency. Alterations in lipid profiles, coagulopathy, atherosclerosis, endothelial dysfunction, and reduced cardiac dimensions are among the negative factors associated with GH deficiency (140, 142, 143).

The association between GH hypersecretion/deficiency and cardiovascular health has been studied employing genetic mouse models of acromegaly (144, 145) and GH deficiency (146); however, the impact of an intact GH/IGF-I axis in cardiac morphology and function is still not well established. The GHR−/− mouse has provided additional insight into the role of the GH/IGF-I axis in cardiac structure and function.

Heart structure and physiology

Studies have shown that GHR−/− mice present reduced cardiac weight and volume (7, 66, 147). These observations are consistent with the heart morphology associated in humans with LS (148). Echocardiography (ECG) measurements show that GHR−/− mice produce a normal cardiac output. In contrast, individuals with LS show a significant lower cardiac output than control subjects (148). Additional measurements also show that GHR−/− mice exhibit a 25% decrease in left ventricular posterior wall thickness compared with WT mice (66). This finding is associated with a significant reduction in left ventricular diameter at diastole; however, no differences in the relative wall thickness are found between GHR−/− mice and controls (66). In addition, no significant difference in the average early diastole/late atrial contraction ratio is present in GHR−/− mice, which indicates that the diastolic function is not different between genotypes (66). Accordingly, no major differences in early diastole/late atrial contraction ratio between individuals with LS and control subjects are found (148). On the other hand, GHR−/− mice exhibit significant reductions in left ventricular diameter at systole, fractional shortening, and velocity of circumferential shortening (66), suggesting that GHR−/− mice have a decreased systolic cardiac function. ECG data from individuals with LS show that left ventricular septum, posterior wall, and end-diastolic diameter are reduced compared with the control group. However, these alterations do not significantly impact the overall cardiac function in LS (148). Interestingly, no major differences in the systolic and diastolic function are found in LS when compared with controls (148).

The reduced systolic cardiac function observed in the ECG studies of GHR−/− mice correlates with the decreased systolic blood pressure observed in GHR−/− females at 4 months of age (66) and in 9-month-old GHR−/− males (147). Female GHR−/− mice (4 months) exhibit a 25% reduction in systolic blood pressure compared with WT mice (66). The decreased systolic blood pressure is consistent with the low levels of renin and increased levels of K⁺ in plasma of GHR−/− mice, although total aldosterone levels are not altered (66). Thus, the mechanisms underlying the reduced systolic blood pressure still need to be studied. One hypothesis is that increased expression of endothelial nitric oxide (NO) synthase (eNOS) in large vessels may contribute to this effect (66). NO is released in response to stress and contributes to maintain vasodilator tone pressure (149). Thus, augmentation in NO production leads to increased vasodilation (149). RT-PCR studies show that female GHR−/− mice have increased aortic eNOS mRNA expression compared with WT mice (66). Increased eNOS mRNA may result in systemic elevation in NO production, explaining the reduced systolic blood pressure observed in GHR−/− mice. However, endothelial cells of GHR−/− mice exhibit a normal contractile function in response to stimulation with norepinephrine and acetylcholine (66, 147). Thus, despite the increase in eNOS mRNA expression, endothelial cell function is not compromised in GHR−/− mice (66, 147). Additional studies are required to further explain these contrasting results.

In summary, whereas GHR−/− mice exhibit functional and morphological changes in heart and vasculature, the health and life span of these mice are not compromised, suggesting the existence of compensatory mechanisms to overcome the negative physiological effects of a disrupted GH/IGF-I axis on the cardiovascular system.

D. Bone

Longitudinal bone growth is regulated by GH in a dose-dependent manner (150). In fact, the preferred method to measure GH activity in vivo evaluates the rate of increase in the width of the tibial growth plate in hypophysectomized rats (151). During bone growth, germinal cells located between two areas of calcified tissue differentiate into chondrocytes, which enter an actively proliferating layer where they undergo limited clonal expansion (20, 21, 152). Ossification occurs where cells move away from the germinal layer after cell division and enter the hypertrophic layer. In humans, normal growth of the longitudinal bone ends with epiphyseal closure induced by sexual maturity (21). However, in rodents, the closure is not completed in some growth plates including proximal tibia, where longitudinal growth continues beyond sexual maturity.

1. Longitudinal bone growth and bone morphology in the GHR−/− mouse

In GHR−/− mice, the width of the epiphyseal plate is reduced by 20% (at 20 d) to 30% (at 10 wk) with a 65% reduction in linear growth rate measured by tibia length compared with WT littermates (153, 154). However, the overall bone structure is similar to that of WT mice. Proliferation, number, and hypertrophy of chondrocytes in the germinal zone are all reduced and hypoplastic in GHR−/− mice. In comparison, the growth plate of IGF-I−/− mice shows significant enlargement of the germinal zone, whereas the proliferation and amount of chondrocytes remain normal with reduced overall hypertrophy (153). These data are consistent with the phenotype differences observed between GHR−/− and IGF-I−/− mice, in that GHR−/− mice have greater linear growth deficiency compared with IGF-I−/− mice. Thus, GH seems to exert a dual action (i.e., IGF-I-dependent and -independent effects) on chondrocytes (153). However, conflicting data were reported by Lupu et al. (155), who found a nearly equal deficit in linear growth in IGF-I−/− and GHR−/− mice, and only an additional 5% reduction in double deletion (GHR−/− IGF-I−/−) mutant mice. These data indicate that the effects of IGF-I and GH are rather independent in chondrocytes. The inconsistencies reported might be due to the fact that each study used IGF-I−/− mouse lines that were independently generated in different genetic backgrounds. However, both studies clearly demonstrated that the absence of IGF-I causes the expansion of the germinal zone.

There are also conflicting reports as to whether the timing of ossification is delayed in GHR−/− mice relative to WT (33, 155). However, these discrepancies may be derived from the technical difficulty to align tissue sections when comparing dwarf and WT mice (153, 155).

Multiple studies have assessed bone mineral density (BMD) in GHR−/− mice. For example, reduction of the ratio of total bone mineral content to crown-rump length by 45% and the ratio of total bone area to crown-rump length by 23% is observed in 3.5-month-old GHR−/− mice compared with controls (4). Also, a reduction in BMD is observed in whole body, spine, femur, tibia, and cranium by 11, 20, 32, 29, and 29%, respectively, in 3-month-old GHR−/− mice (33). The reduction in mineral content of the middiaphyseal section of the femur and tibia is approximately 50% in GHR−/− mice, whereas serum osteocalcin level remains similar to controls (33). Figure 4 shows images of femora of GHR−/− and WT mice at 2 yr of age measured in our laboratory (32). Femora from GHR−/− and WT females are significantly longer than GHR−/− and WT males, respectively. More importantly, BMD is reduced in both male and female GHR−/− mice when compared with controls (32).

Fig. 4.

Right femora images of GHR−/− and WT mice. Right femora of 2-yr-old female (A and B) and male (C and D) GHR−/− (A and C) and WT (B and D) were mounted in foam and scanned using GE eXplore Locus Small Animal MicroCT Scanner at 20 μm voxel, 80 kV, 450 μA, and 2000 msec exposure time. Femora were significantly longer in females. BMD was significantly lower both in male and female GHR−/− compared with WT; however, there were no gender differences (32). [Reproduced with permission from D. E. Berryman et al., Two-year body composition analyses of long-lived GHR null mice. J Gerontol A Biol Sci Med Sci 65:31–40, 2010 (32).]

2. Sex steroids and bone

Stimulation of longitudinal growth by estrogens involves the GH/IGF-I axis, and estrogens can promote GH secretion and induce hepatic synthesis of IGF-I (157). Treatment of orchidectomized GHR−/− male mice with 17β-estradiol restores the growth rate of longitudinal bone, cross-sectional area, cortical thickness, as well as periosteal perimeter in bone by a net increase in osteoblast number (157). These changes are accompanied by the up-regulation of hepatic IGF-I gene expression in GHR−/− mice, thus restoring serum IGF-I levels to approximately 25% of normal levels. This indicates that radial growth of cortical bone is mainly determined by circulating IGF-I levels via increased chondrocyte proliferation and growth plate thickness, independent of GH action (67). In contrast, trabecular bone modeling is not affected by low serum IGF-I levels in GHR−/− mice because trabecular BMD, bone volume, number, width, and bone turnover are not affected (67). Dihydrotestosterone and testosterone administration increase periosteal bone growth and cortical thickness without affecting serum or skeletal IGF-I levels. These androgens also restore the trabecular bone volume that is significantly reduced in WT and GHR−/− mice by orchidectomy to the same extent, indicating that androgens stimulate trabecular and cortical bone modeling independently of local or systemic IGF-I production (67).

Overall, studies in GHR−/− mice demonstrate that the development and maintenance of trabecular bone does not require GHR signaling or circulating IGF-I, although locally expressed IGF-I may regulate trabecular bone modeling independent of GH. However, serum IGF-I levels stimulated by GH determine the growth of periosteal bone and cortical bone mass. GH signaling, serum IGF-I levels, and androgen receptor signaling all stimulate periosteal growth and cortical bone mass, but these factors act independently. Because the stimulatory effects of androgens on cortical bone mass during puberty parallel the effects of androgens on muscle, these effects may be, at least in part, indirectly mediated through an increase in muscle mass, resulting in increased mechanical loading (67).

3. Effect of GH on craniofacial complex and the development of tooth

GH influences craniofacial bone growth by accelerating cartilage formation in both humans and animals (158–161). In contrast, facial bone growth and fontanelle closure are delayed in individuals with LS (162). Disproportional growth of craniofacial bone causes frontal bossing, saddle nose, shallow orbits, small chin, and sunset look (162). GH particularly influences the posterior height of the face determined by the length of the mandibular ramus (159, 163). Similar to humans, GH overexpression in mice increases craniofacial length, upper face height, and the length of the mandibular corpus. In fact, GHR−/− and GH antagonist (GHA) transgenic mice show a significant reduction in these measurements (158) (for a more detailed description of GHA mice, see Section VII). The effect of GH is different in different regions of the skull as demonstrated by the comparison of craniofacial length of 45-d-old bovine GH transgenic (164), GHA (165), and GHR−/− (164) mice. As with the upper face height, the posterior face height determined by the mandibular ramus is significantly reduced in GHA and GHR−/− mice (158).

Tooth development is delayed in individuals with LS and often shows defects and crowding because of the relatively small mandible (162). Hypodontia has been reported as well for individuals with LS (166). In contrast, teeth of patients with gigantism or acromegaly are normal (167). In mice, the cross-sectional area of the cementum is reduced by 10-fold in GHR−/− and 3-fold in GHA mice, whereas it is increased by 2-fold in bovine GH mice (168). Crown dimensions are significantly reduced in GHR−/− and GHA mice when compared with controls (168). On the other hand, GH overexpression results in the increase of the length of incisors, whereas GHR−/− mice show the opposite effect (158). Thus, GH seems to influence dentin size and shape by affecting appositional dentin growth as well as tooth crown and root development before dentinogenesis (168).

E. Brain

GH, GHR, and IGF-I are expressed in neural tissues at various stages of development and in adulthood, suggesting an important role for the GH/IGF-I axis in brain development and activity (169–171). However, much of the research investigating the role of the GH/IGF-I axis in the brain has yielded contradictory results. Depending on the parameters examined, studies have shown improvements in learning and memory under certain conditions for animals with GH deficiency/resistance (172–174) and for animals with elevated GH (175, 176). Studies of human subjects that are GH deficient or resistant have also produced contradictory data, with some showing cognitive defects, and others showing high or normal cognitive ability [reviewed by Forshee (177)].

1. Effects of GH on brain growth and development

Tissue weights of various organs have been examined in 3-month-old GHR−/− mice. The brain is one of few organs that is increased in size relative to other organs in GHR−/− animals. In proportion to total body weight, brain mass in GHR−/− mice is 151.5% of WT, although absolute brain weights in GHR−/− mice are decreased (33). This finding was confirmed in a later analysis that found GHR−/− brains were only 20% lower in absolute weight compared with WT controls (178), whereas total body weight of GHR−/− mice is approximately half that of littermate controls (178). In addition, several parameters of brain size have been measured, including cortical, subcortical, and striatal area of striatum and hippocampus sections, as well as cortex length and thickness. By most of these measures, the dwarf GHR−/− brain is smaller in absolute terms, with the exception of cortical thickness (178). The disproportionate size of the GHR−/− brain may be reflective of the fact that much of brain growth occurs early in life at a time when somatic growth is largely independent of GH action, even though GHR is expressed in prenatal brain tissue of WT animals (169, 179). While the function of GHR in cells of the developing brain is not known, it does not appear to play a predominant role in stimulating growth. These data suggest that GHR signaling does not play a critical role in promoting brain growth in the mouse.

2. Neuron populations of the central nervous system (CNS)

To date, there has been one in-depth analysis of cell populations in the CNS of GHR−/− mice to elucidate a potential role of GH in CNS development (178). Analysis of brain sections has revealed interesting parameters of cell size, total cell number, and cell density of various cell populations of the CNS. In the somatosensory cortex, GHR−/− mice have 23–26% greater neuron density compared with WT. Regarding cell populations, there is some layer-to-layer variability, but the total cell number in GHR−/− mice is not different from controls (178). Staining for neuronal nuclei protein revealed that there is a greater proportion of neural cells in the GHR−/− motosensory cortex compared with controls.

Further studies have been performed to determine whether ablation of GH signaling has differential effects on specific subpopulations of cortical neurons. Both calretinin- and calbindin-positive neurons are found at increased density in GHR−/− cortex, whereas parvalbumin-positive neurons are not different from WT (178). Regarding noncortical neurons, cholinergic neurons in the striatum, defined as choline acetyl-transferase-positive cells, are found at a higher density in GHR−/− mice, whereas the total number of cholinergic neurons per section is not different from WT. Overall, these data indicate that the brain of the GHR−/− mouse has the same number of neurons but at increased density compared with WT. It appears that GH signaling does not significantly influence the level of neurogenesis, largely a prenatal event. Gliogenesis, a process that mostly occurs after birth, has also been examined. GHR−/− astrocytes are smaller and less abundant, thus resulting in a greater neuron-to-glia ratio in GHR−/− mice. Measurement of cell soma size shows a decrease in cell size in GHR−/− samples for calretinin- and calbindin-positive neurons in the cortex and for choline acetyl-transferase-positive neurons in the striatum (178). Although not all neuronal cell types have been studied, these data clearly illustrate that the absence of GHR has differential effects on various cell populations of the CNS.

3. Prenatal motoneuron development

In 2003, a set of experiments (180) were designed to investigate a potential prenatal role for GH in CNS development. Citing previous research showing antiapoptotic action of GH and a transient increase in GH and GHR expression in the rodent CNS during a period of remodeling via neuronal programmed cell death, Parsons et al. (180) hypothesized that GH is a neurotrophic factor for motoneurons during programmed cell death. As with pre- and perinatal body weight, no significant difference is found in spinal cord volume at embryonic day (E) 13.5 or 18.5 or postnatal day 2 between GHR−/− and WT pups. Brachial motoneurons and lumbar lateral motor columns are indistinguishable in appearance and position between GHR−/− and WT at E13.5, E18.5, and postnatal d 2. The number of brachial and lumbar lateral motor column motoneurons in GHR−/− mice is the same as WT at these ages, whereas motoneuron cell size is smaller (measured indirectly via nuclear area), consistent with findings of neural populations in the brain cortex and striatum of adult mice (178, 180). Motoneuron number in cranial motor nuclei at E18.5 is the same between GHR−/− and WT. Thus, although various lines of in vitro and in vivo evidence suggest a conserved role for GH during CNS development, GH does not appear to play a significant role in motoneuron survival in mice (180).

4. Memory

As early as 1979, GH was shown to have an effect on memory (176). A more recent report found that GH mRNA expression is induced in the hippocampus after trace conditioning, a process of memory formation that requires the hippocampus (181). As discussed elsewhere in this review, GHR−/− mice live dramatically longer than their WT littermates. Two studies have analyzed cognitive function in GHR−/− mice as they age (172, 174). In 2001, Kinney et al. (172) performed an inhibitory avoidance test to analyze learning and memory in 17- to 20-month-old GHR−/− mice. In their first experiment, all mice received equivalent shocks. Memory retention was measured by latency to enter shock compartment 24 h, 7 d, and 28 d after training. GHR−/− mice were found to have greater retention at 28 d after training compared with controls. In a follow-up experiment, GHR−/− mice were trained with a shock adjusted for their dwarf size. Both young and old WT and GHR−/− mice were compared. Old WT animals showed a decline in performance at 7 and 28 d after training compared with young WT mice. This decline in performance with age in WT mice was not observed in GHR−/− animals (172). Further research has been conducted to characterize behavioral and cognitive aging in GHR−/− mice (174). A water maze test compared both young and old WT and GHR−/− mice. In this test, mice were placed in an opaque water reservoir with a hidden platform, and the time to reach the platform was recorded. Old WT mice showed reduced performance, whereas old GHR−/− mice showed the same performance as young WT and GHR−/− mice, suggesting that the long-lived GHR−/− mice are protected from age-related decline in memory (174).

5. The pituitary and hypothalamus

The pituitary and hypothalamus closely coordinate the production of several endocrine hormones, including GH, PRL, LH, FSH, ACTH, and TSH. Production of GH in the pituitary is subject to a complex feedback system that includes ultrashort, short, and long feedback loops, all thought to rely on intact GHR signaling (as reviewed in Refs. 182–184). Disruption of these feedback loops in GHR−/− mice results in a 30-fold increase in serum GH (1). Whole body weights were approximately 45% of WT in adult GHR−/− mice, whereas GHR−/− pituitaries weighed 53, 58 and 61% of WT pituitaries in three different studies (35–37); thus, as with brain, pituitary size in GHR−/− mice is relatively larger in mass when normalized to body weight. Morphological analysis of the pituitaries in GHR−/− mice has revealed structural abnormalities. The reticulin-fiber network is disrupted with acini expansion and loss of reticulin (35). Immunocytochemical staining revealed an increase in the proportion of GH-positive cells in GHR−/− pituitaries, ranging from 76–79% in GHR−/− and 61–67% in WT (35). An increased proportion of somatotrophs is also observed (35, 37, 185), with 64% of pituitary cells being GH positive (37). These somatotrophs are abnormal, showing reduced cytoplasmic GH staining accompanied by prominent juxtanuclear golgi GH staining (35). Instead of typical somatotrophs, a hyperactive population of sparsely granulated cells is present showing numerous mitochondria, expanded endoplasmic reticulum membranes, and large golgi complexes. GHR−/− pituitaries also display evidence of mild hyperplasia (35). These results are similar to morphological changes found in stimulated lactotrophs and in human somatotroph adenomas, suggesting that these changes are the result of the increase in GH production and secretion in the pituitary of GHR−/− animals. The number and percentage of pituitary cells that stain positive for PRL are reduced (185). Even with a reduction in lactotrophs in GHR−/− pituitaries, serum PRL levels are either elevated or the same as WT controls (36, 185). Examination of other cell types showed no difference in the percentage of TSH- and ACTH-immunopositive cells between GHR−/− and WT animals (185), indicating that development of these cell types is largely independent of GH signaling.

The hypothalami of GHR−/− mice have increased expression of hypothalamic GHRH and decreased expression of somatostatin and neuropeptide Y (37). Additionally, there is increased expression of GHRH receptor and GH secretagogue receptor in the pituitary (37).

In summary, the negative feedback loops in GHR−/− mice are disrupted due to a complete absence of GHR signaling, which the regulatory mechanisms rely upon to sense the appropriate amount of GH to produce. Pituitaries of GHR−/− mice produce high levels of GH in a futile attempt to satisfy negative feedback loops that have been perturbed.

VII. Aging

Aging is associated with a progressive decline in physiological function and an increase in disease occurrence. Evolutionarily, the process of aging has likely been shaped by the natural selection of genes that are favorable in early life but unfavorable once fertility has diminished. These particular genes would in essence escape natural selection (186, 187).

Several theories are proposed for aging. The oxidative stress theory involves susceptibility of biological molecules to oxidative damage. Molecules such as proteins, lipids, and nucleic acids slowly accumulate oxidative damage over time, resulting in age-related functional decline (188–190). Damage to mitochondrial DNA via oxidative stress is also thought to have an important role in aging (191–193). The insulin exposure theory suggests that decreased exposure to insulin and insulin signaling results in increased longevity (194, 195). Furthermore, conditions that decrease insulin can also result in a global decrease in other growth factors such as IGF-I. More recently, a hybrid theory called the epigenetic oxidative redox shift theory has been proposed to account for both oxidative stress and insulin exposure (196). Although many other theories of aging exist, reduced insulin level is a common factor in two of the most constant interventions that routinely extend life span across multiple species: CR and inhibition of the GH/IGF-I axis. Both of these have been studied extensively using the GHR−/− mice and will be discussed below.

A. Influence of insulin/IGF-I/GH on life span

GHR−/− mice have an extremely extended life span and are considered one of the longest lived laboratory mouse models. In fact, one GHR−/− mouse survived 1819 d and is recognized as the longest lived laboratory mouse. At the time this review was prepared, the GHR−/− mouse was still the holder of the “Methuselah Mouse Prize” for the world's longest lived mouse, an award bestowed by the Methuselah Foundation (http://www.methuselahfoundation.org/). It is important to note that the life span extension can vary by background strain. In 2000, when the GHR−/− mice were first described as long lived, the mice were maintained as a mixed genetic background and had an increase in mean life span of 55% in males to 38% in females (12). When GHR−/− mice are maintained in the C57BL/6J strain, life span is still significantly increased but to a lesser degree, with average increases of 26% in males and 16% in females (7).

Several other mouse models that exhibit extended longevity have defects in insulin, GH, or IGF-I pathways. For example, Ames and Snell dwarf mice have severe decreases in the GH/IGF-I axis and exhibit increased longevity. However, it is important to recall that these mice also carry homozygous recessive mutations at the Prop-1 (Ames) and Pit-1 (Snell) loci. These mutations result in impaired development of somatotrophs, lactotrophs, and thyrotrophs within the anterior pituitary resulting in deficiencies in GH, PRL, and TSH, respectively. Although decreases in GH and IGF-I are thought to be responsible for extended longevity in these dwarf mice (197, 198), the fact that multiple hormones are affected limits the utility of these mice for elucidating the specific impact of the GH/IGF-I axis on aging. To this end, the GHR−/− mouse offers a more precise tool for dissecting out the role of GH in aging.

Additional mouse models with disruptions to the GH/IGF-I axis also show increased life span. Although homozygous disruption of the IGF-I receptor gene (IGF-IR−/−) is lethal, heterozygous mice with one functioning allele for the IGF-IR (IGF-IR+/−) survive. These mice have approximately half the levels of IGF-IR, and extended life spans (199). Mice that have disruptions to the IGF-IR substrate p66shc (p66shc−/−) also have extended life spans and are more tolerant to oxidative stress induced by paraquat (200). Mice with a homozygous mutation in the GHRH receptor gene (GHRHR−/−), commonly known as “little” mice or “lit/lit” mice, have severely decreased GH and are also long lived (201). Transgenic mice that overexpress Klotho have an extended life span (202). Klotho is a transmembrane protein primarily expressed in the kidney and brain. Among other functions, Klotho fragments are found in the bloodstream and are suggested to bind to and repress insulin and IGF-I intracellular signaling molecules (202). Targeted disruption of IRS-1 and IRS-2, which serve as signaling molecules for insulin and IGF-I, may also extend life span. However, results are not clear-cut. Only female IRS-1−/− mice (and not males) have extended longevity despite mild insulin resistance (203). While homozygous IRS-2−/− mice are short lived, heterozygous IRS-2+/− mice have been reported to have an extended life span in one study (204) but a normal life span in another (203). Furthermore, tissue-specific reduction of IRS-2 in brain (as seen in heterozygous brain-specific IRS-2+/− disrupted mice) increases life span (204). Tissue-specific disruption of the insulin receptor in adipose tissue, which occurs in the FIRKO mouse (fat-specific insulin receptor knockout), leads to a 50–70% reduction in adipose tissue mass (205). These mice are more glucose tolerant at older ages and have an extended longevity. Thus, many mice models with repressed activity of GH, IGF-I, or insulin display extended longevity.

B. Comparison of transgenic GHA mice and GHR−/− mice

GH-deficient Ames and Snell dwarf mice have extended longevity (201, 206–208); however, as stated above, questions remain about the exact role of GH in these models due to the lack of other pituitary hormones (PRL and TSH). Therefore, to specifically determine GH's role in aging, we have assessed life span in two dwarf mouse lines (generated in our laboratory) that are specifically deficient in GH signaling: the GHA mouse (209) and the GHR−/− mouse (1). Because GHA competes with endogenous GH for the GHR, GH signaling is inhibited in GHA transgenic mice resulting in low serum IGF-I and a dwarf phenotype (165, 209–212). Although GHA and GHR−/− dwarf mice share many similarities, several key differences exist. GHA mice have similar fasting blood glucose and insulin levels as WT controls, whereas the GHR−/− mice have significantly low or normal fasting blood glucose and significantly decreased insulin. More importantly, the GHA mice have normal life span compared with controls, whereas GHR−/− mice have significantly increased life span (7). Thus, dwarfism in and of itself does not appear to increase life span in mice.

C. Caloric restriction and GHR−/− mice

CR, usually achieved by specific reduction in daily caloric intake or by every other day feeding, is a potent intervention that extends life span in a wide range of species (213).

The specific mechanisms by which CR extends life span are not completely understood; however, they are likely to involve the collective effects of multiple systems including those that modulate glucose, insulin, and IGF-I levels as well as accumulation of reactive oxygen species and oxidative damage (214, 215). In 2001, Bartke et al. (198) established that CR in long-lived Ames dwarf mice further increased mean and maximal life span. Because both GH deficiency and CR increased life span in an additive manner, it appears that separate and distinct mechanisms are responsible. However, the fact that multiple hormones are affected in this mouse line (GH, PRL, and TSH are all deficient in the Ames mice) complicates interpretation of the results. Yet, in 2006 when CR was applied to the GHR−/− mice, no further life span extension was observed (55) (Fig. 5). Since Ames dwarfs have additional deficiencies beyond GH, it is likely that the differences between these two mouse lines involve hormones other than GH (PRL and TSH). The authors concluded that the failure of CR to increase life span in GHR−/− mice is related to its failure to further increase insulin sensitivity (55).

Fig. 5.

Comparison of life span in male (left) and female (right) WT mice fed ad libitum (AL), WT mice with CR, GHR−/− mice fed AL, and GHR−/− mice with CR. [This figure was modified from M. S. Bonkowski et al., Targeted disruption of growth hormone receptor interferes with the beneficial actions of calorie restriction. Proc Natl Acad Sci USA 103:7901–7905, 2006 (55), with permission. Copyright 2009 National Academy of Sciences.]

The overlap between CR and GHR gene deletion suggests that similar genes are altered under these conditions. To test this, expression levels of 2352 genes in livers of GHR−/− mice and WT controls subjected to CR have been compared (216). In control mice, 352 of the genes are significantly altered by CR. In stark contrast, not one of the 2352 genes is significantly altered in the GHR−/− mice when subjected to CR. Thus, the effect of CR is quite different in GHR−/− mice as opposed to WT control mice. The authors conclude that the effect of CR on gene expression is stronger in the liver of WT mice and that conversely the GHR−/− genotype blunts the effects of CR (216).

Meta-analysis of hepatic gene expression has been performed on several longevity-associated studies mentioned above (217). Included in the analysis were long-live dwarf mice (GHR−/−, Ames, Snell, and lit/lit mice) as well as the effect of several diet regimens associated with increased longevity. When the four dwarf mouse strains are compared, 13 genes are found to be differentially expressed in all dwarf models, with three up-regulated (Hao3, Sult2a2, and Spink3) and 10 down-regulated (Socs2, Mup4, Es31, Keg1, Hsd3d5, Igfals, Lifr, Mup3, Igf1, and Egfr). When all four dwarf mouse lines are compared with at least one CR in the meta-analysis, only three of the 13 genes mentioned above are differentially expressed (Igf1, Igfals, and Lifr) (217).

Studies focusing on specific proteins and mRNAs have also been performed on GHR−/− and WT mice subjected to CR. Protein and mRNA levels of PPARγ, PPARα, and RXR are higher in livers of GHR−/− mice than in WT mice and do not change with CR in either genotype, indicating that CR may increase insulin sensitivity via different mechanisms (81). In addition, liver Akt phosphorylation is decreased by CR in WT mice but not in GHR−/− mice, suggesting an important role for Akt as a common regulator of longevity (11). In skeletal muscle, Akt2 and PGC-1α were increased, whereas phosphorylated JNK1 was reduced in response to CR in WT mice but not in GHR−/− mice (73). These results again suggest an overlap between CR and GHR gene disruption.

Studies in GHR−/− mice subjected to every other day feeding (which results in mild ∼15% CR) have also been performed (10). In WT mice, CR increases insulin-stimulated activation of insulin-signaling molecules in liver and muscle. However, GHR−/− mice respond differently with increased activation in only the early steps of insulin signaling in liver, whereas muscle has increased activation of only downstream signaling events. Chronic CR in GHR−/− mice does not result in further changes in insulin signaling compared with WT mice, which may explain why CR does not increase longevity in the GHR−/− mice. Furthermore, muscle of GHR−/− mice exhibits severely reduced serine phosphorylation of IRS-1. Because serine phosphorylation of IRS-1 is inhibitory to insulin signaling and is reduced in the muscle of GHR−/− mice, it is possible that reduced serine phosphorylation of IRS-1 accounts for the heightened insulin sensitivity found in GHR−/− mice (10) (see Section IV.B).

The degree and type of CR have been modified in efforts to determine whether the particular dietary regimen accounted for the lack of further extension in life span in GHR−/− mice. It appears that 30% CR (55) as well as CR accomplished by every other day feeding (51) were unable to extend life span in GHR−/− mice and point to an overlap in the effects of GHR-gene disruption and CR on insulin signaling.

D. Insulin sensitivity in GHR−/− mice: chronological and biological age comparison

Based on all the data compiled in GHR−/− mice, it is evident that insulin plays a role in the extended longevity of these mice. However, the studies that show a difference in insulin sensitivity have compared GHR−/− mice and WT mice at the same chronological age (see Section IV.B). Given their extended life span and delayed sexual maturation, chronological age in GHR−/− mice does not necessarily reflect the same biological age as in WT mice. To address this, the relative expression levels of 14 hepatic genes that are associated with glucose metabolism have been analyzed in male mice at three ages (9.5, 15, and 21 months) (2). The latter two ages were selected such that 15-month-old WT mice and 21-month-old GHR−/− mice represented a comparable biological age (at ∼50% of median life span). Results show that regardless of chronological or biological age, GHR−/− mice have lower circulating insulin and lower homeostasis model analysis. Furthermore, regardless of chronological or biological age, all 14 of the hepatic genes analyzed show significant differences between GHR−/− and WT littermates. Thus, the changes to these insulin-related genes are due to the suppression of GH signaling rather than differences in biological ages.

E. Disease

GHR−/− mice are apparently protected from several diseases. Specifically, GHR−/− mice do not appear to have cardiac abnormalities (66), do not develop nephropathy when type 1 diabetes is induced (218), and are resistant to the development of certain types of cancers (219–221), which may contribute to their increased longevity.

Regarding cardiovascular health, no major cardiac abnormalities have been detected in GHR−/− mice by ECG and vascular function measurements (66). Also, consistent with the negative regulation of PPARα on inflammatory genes, expression of β-fibrinogen is decreased in GHR−/− mice, suggesting protection against cardiovascular disease (85). Therefore, the overall cardiovascular risk seems to be diminished in GHR−/− mice, despite their obesity, gender, and age.

GHR−/− mice are also protected against diabetic kidney disease. When type 1 diabetes is induced using streptozotocin, WT mice develop glomerulosclerosis; however, the glomeruli of diabetic GHR−/− mice remain unchanged, suggesting a role of GH and/or IGF-I in the development of diabetic kidney disease (218).

Pathological assessment of GHR−/− mice after normal aging reveals a 49% lower incidence of fatal neoplasms, which includes significant reductions in lymphomas and adenocarcinomas (219). GHR−/− mice are also resistant to simian virus 40 large T-antigen-induced breast (221) and prostate (220) cancers. Furthermore, the decreased incidence and delayed occurrence of neoplastic lesions in GHR−/− mice have been described as similar to the effects of CR, suggesting that resistance to cancer is a contributing factor to extended longevity in the GHR−/− mice and yet another point of overlap with CR (219). Thus, the increase in longevity may be related not only to alterations in insulin sensitivity, but also to how these or other factors alter susceptibility to disease states.

Comparison to individuals with LS becomes difficult due to very low numbers of individuals with this disease; however, according to Dr. Zvi Laron (personal communication), not one of the individuals with LS he has studied over 50 yr has had cancer. Furthermore, a 2007 report by Shevah and Laron (222) provides similar data. In the study, 222 patients with congenital IGF-I deficiency (this group included LS, GH gene deletion, GHRH receptor defects, and IGF-I resistance) were compared with 338 relatives (first and second degree). Although 9–24% of the relatives had a history of cancer, not one of the congenital IGF-I-deficient patients did.

VIII. Concluding Remarks/Future Perspectives

Numerous studies in GHR−/− mice have been able to help define the key roles of GH in relation to growth, metabolism, and aging. GHR−/− mice are roughly half the body weight of WT littermates. Likewise, most tissues and organs in GHR−/− mice are smaller in absolute terms, but still remain proportional to body size. However, the exceptions, such as a smaller liver and a larger brain, provide evidence of a tissue-specific effect that has not yet been fully explored.

One of the most striking differences is in adipose tissue, which is not different in absolute mass despite having a body size that is half that of WT littermates. Thus, GHR−/− mice are markedly obese. The accumulation of fat mass in GHR−/− mice is dramatically different among adipose depots with preferential enlargement of sc WAT. Because these mice have disproportionally enlarged sc WAT and display improved insulin sensitivity with an extended life span, it is tempting to speculate that this particular depot confers health benefits over the other WAT depots. Although some differences among these depots in GHR−/− mice have been reported, there is a need to more fully understand how these depots respond distinctly to the absence of GH action. For example, perhaps sc WAT is more sensitive to the action of insulin than other WAT depots. Furthermore, these relatively long-lived, healthy mice demonstrate that obesity itself can be separated from the unfavorable metabolic consequences that are typically associated with increased adiposity. Consequently, GHR−/− mice could not only provide important information regarding how GH function influences adipose tissue but could also help establish the features of excess adipose tissue that commonly result in the metabolic dysregulation that accompanies obesity.

Overall, one of the most interesting results obtained from the GHR−/− mice is their extended life span that is typically 30–50% longer than control littermates. Strikingly, one mouse has lived to nearly 5 yr (1819 d) and is the longest lived mouse on record (223) (http://www.methuselahfoundation.org/). Although other dwarf mouse models with altered GH or IGF signaling have implicated this axis in aging, the GHR−/− mice have the distinct advantage of being specific for disrupting GH signaling while leaving other pituitary hormone actions intact.

Due to the effects of GHR disruption on improving glucose homeostasis (improved insulin sensitivity with low insulin levels and low to normal glucose), these mice provide evidence that lack of GH signaling promotes longevity via improved insulin sensitivity. Interestingly, whereas CR has been proven to extend life span in other long-lived mouse models, CR has not been shown to be effective in extending the life span in GHR−/− mice (55), pointing toward an overlap between the effects of GHR-gene disruption and CR. The role of GHR disruption in controlling certain disease states such as cancer may also aid in extending longevity.

To further dissect the role of GH in insulin sensitivity and aging, our laboratory and others are currently generating and using tissue-specific GHR−/− mice. Much of the initial effort has been to delete GHR in insulin-sensitive tissues, such as liver, muscle, and adipose tissue. Because these tissues are major sites of both GH and insulin action, removal of GHR in these tissues will establish their individual contribution to the high insulin sensitivity and improved life span observed in the GHR−/− mice. Although many studies are currently under way, one recent report has provided phenotypic characteristics of liver-specific GHR−/− mice (224). These data imply that tissues other than liver are most likely responsible for improving life span and insulin sensitivity in GHR−/− mice. Beyond the major insulin-sensitive tissues, groups are also disrupting the GHR gene in other tissues such as macrophages (43), where a novel role for GH in adipogenesis has been revealed.

Over the last 15 yr, the GHR−/− mouse has revealed much about GH and its many physiological roles. We believe that the GHR−/− mouse and derivatives thereof will continue to provide new insights and exciting directions into the various actions of GH.