Abstract
The aim of the study is to find possible explanations for vanishing juvenile hypoglycemia in growth hormone receptor deficiency (GHRD) in human patients and animal models. We reviewed parameters of glucose metabolism in distinct age groups into two human cohorts (Israeli and Ecuadorian) of Laron syndrome (LS) patients, a mouse model (Ghr-KO mouse) and provided additional data for a porcine model (GHR-KO pig). Juvenile hypoglycemia is a common symptom of GHRD and vanishes in adulthood. In the Israeli cohort, developing metabolic syndrome is associated with decreasing insulin sensitivity, insulinopenia and glucose intolerance, and increasing glucose levels with age. In the Ecuadorian patients and both animal models, insulin sensitivity is preserved or even enhanced. Alterations in food intake and energy consumption do not explain the differences in glucose levels; neither is the accumulation of body fat associated with negative effects in the Ecuadorian cohort nor in the animal models. A reduced beta-cell mass and resulting insulin secretory capacity is common and leads to glucose intolerance in Ghr-KO mice, while glucose tolerance is preserved in Ecuadorian patients and the GHR-KO pig. In human patients and the GHR-KO pig, a simultaneous occurrence of normoglycemia with the onset of puberty is reported. Reduced gluconeogenesis in GHRD is discussed to cause juvenile hypoglycemia and a counter-regulatory stimulation of gluconeogenesis can be hypothesized. A coherent study assessing endogenous glucose production and beta-cell capacity in the hypoglycemic and normoglycemic age group is needed. This can be performed in GHR-KO pigs, including castrated animals.
Introduction
Growth hormone receptor deficiency (GHRD) – the human Laron syndrome (LS) – is a hereditary disorder leading to diminished growth hormone (GH) binding and intracellular signal transduction. LS patients have low serum levels of insulin-like growth factor 1 (IGF1) and elevated levels of GH. Phenotypically they have a small stature and are somewhat obese (1). In the Israelicohort of LS patients (2), several inactivating mutations of the GHR gene have been reported (1), while a cohort in Ecuador is more uniform with only one inactivating GHR mutation (3). In total, just 350 LS patients are estimated worldwide, of which the Ecuadorian cohort is the largest in numbers, with approximately 100 affected individuals (4, 5). A mouse model for LS – the Ghr-KO mouse – was produced by disruption of Ghr exon 4 (6). A porcine LS model has been established by inducing a frameshift mutation in GHR exon 3 (7). Similar to human LS patients, these animal models show the characteristic phenotype of postnatal growth retardation and increased accumulation of adipose tissue.
Juvenile hypoglycemia is commonly observed as a key symptom of LS but vanishes when the patients get adult (1, 8, 9). Nevertheless, differences in glucose homeostasis between various cohorts of LS patients have been described – ranging from insulin resistance (10) to a metabolically healthy phenotype with increased insulin sensitivity even in obese LS patients (9). This review summarizes the alterations of glucose homeostasis in different cohorts of LS patients and in rodent and pig models with GHRD. Mechanisms potentially involved in the phenomenon of transient juvenile hypoglycemia are discussed.
Glucose homeostasis in growth hormone receptor deficiency
Laron syndrome patients
Juvenile hypoglycemia is a major symptom in LS patients from both cohorts (8, 11) and has clinical implications in infants and children after longer periods of fasting (Table 1). Hypoglycemic glucose levels of 3040 mg/dL were reported (8), leading to heavy sweating, pallor, headache, seizures, and loss of consciousness (12, 13). In the Israeli cohort, glucose values were found to vary widely but were always lower than in controls during childhood, although statistical significance disappeared at age 5 years (1, 10). Fasting glucose levels tend to normalize at puberty (10), and even occasional cases of hyperglycemia have been observed in patients aged 40 years and older (8). Young adultEcuadorian LS patients (age < 20 years) revealed normal fasting glucose levels (9, 14). In contrast to the Israeli cohort, hyperglycemic values were not reported, and a lifelong increased insulin sensitivity was assumed (9).
Parameters of glucose metabolism in distinct age groups. Age groups are divided according to glycemic status. In human LS patients (Israeli and Ecuadorian cohort), prepubertal, hypoglycemic patients are referred to as young, after puberty, reaching normoglycemia as adult. In Ghr-KO mice, animals up to an age of 9.5 months, exhibiting hypoglycemia are referred to as young according to (36), starting from 11 months of age, reaching normoglycemia according to (37), animals are referred to as adults. GHR-KO pigs are referred as young at the age of 3 months and adults at puberty with 6 months of age.
| Israeli cohort | Ecuadorian cohort | Ghr-KO mouse | GHR-KO pig (7) | |||||
|---|---|---|---|---|---|---|---|---|
| Young | Adult | Young | Adult | Young | Adult | Young | Adult | |
| Glucose | ↓ (1) | ↓↔↑ (8) | ↓ (11) | ↔ (9) | ↓ (35, 36) | ↔ (37, 38); ↑ (36) | ↓ | ↔ |
| Insulin | ↑↑ (10) | ↑↔ (8, 10, 16) | ↓ (9, 14) | ↓ (36, 37); ↔ (35) | ↓ (35, 36, 37) | ↔ ↓ | ↔ ↓ | |
| GTT | ||||||||
| Glucose response | EN – NO (10, 17) | EN (10, 17) | NO (9) | EN (39, 40) | EN (41) | NO | NO | |
| Insulin response | IM (10, 18) | IM (10); NO – EN (17, 19) | IM (9) | IM (39) | IM | IM | ||
| Caloric intake | ↔ (8) | ↔ (53) | ↑ (37, 56) | ↑ (37, 57) | ||||
| Energy expenditure | ↑ (53) | ↑ (55) | ↑ (57) | |||||
| HOMA-IR | ↑(8) | ↑ ↔ (8) | ↓ (9) | ↓ (36) | ↓ (36) | ↔ ↓ | ↔ ↑ | |
| ITT | ||||||||
| Glucose response | IM (10, 19) | NO (10, 19) | IM (35, 64) | IM (41, 74) | ||||
| Beta-cell mass | ↓ (10) | ↓ (9) | ↓ (35, 39) | |||||
EN, enhanced; IM, impaired; NO, normal.
Under physiological conditions, insulin levels increase at puberty, earlier in females than in males, and decrease in adulthood (15). A similar age-dependent peak was observed in LS patients of the Israeli cohort, and their insulin levels were always higher than in age-matched control subjects. The difference appeared earlier in females than males and was more pronounced from age 6 to 10 years than from age 11 to 22 years. Thus, the degree of hyperinsulinemia decreased with age, and in adult patients, a trend of relative insulinopenia was observed (8, 10, 16). Studies of insulin levels in the Ecuadorian LS cohort did not focus on a comparison of different ages; in adult LS patients, insulin levels in the range of one-third of those of control siblings and relatives were reported (9, 14).
In Israeli LS patients, a high incidence of glucose intolerance was revealed by oral (17) and intravenous (18) glucose tolerance tests (GTTs) (10). Glucose intolerance was first observed at early puberty, and the incidence increased with age (19). The amplitude of insulin response following glucose load was commonly lower in LS patients compared to controls (10, 18) and decreased with age, that is, was lower in young adults (23–30 years) than in pubertal patients (11–22 years) (8). However, cases of hyperinsulinemic peaks (17) and rising insulin levels with age in LS patients presenting with insulin-resistant diabetes were reported (19), highlighting the broad variability among patients in this cohort (8). Comparing Ecuadorian LS patients to BMI- and age-matched control relatives, no sign of glucose intolerance was observed. In oral GTTs, the area under the curve values for glucose was not significantly different, but those for insulin were significantly lower in the LS patients compared to healthy controls, arguing for increased insulin sensitivity (9).
While environmental circumstances, especially differences in diet between the cohorts have been discussed to cause the contrary phenotype in glucose homeostasis (20), genetic causes appear just as likely. Several mutations of the GHR gene are described in the Israeli cohort (1), whereas patients from Ecuador have the E180 precursor mRNA splice mutation in common (3). The E180 mutation seems to lower the risk for diabetes, as one Israeli patient of Moroccan heritage under Zvi Laron`s supervision sharing this mutation (21) did not develop insulin resistance at the age of 36 years (22). Insulin sensitivity – in the context of type 2 diabetes (T2D) – has a genetic component with more than 400 identified T2D-associated gene variants affecting the disease risk (23, 24). In view of the high degree of consanguinity in LS patients (21), it is likely that the differences in glucose homeostasis between the various LS cohorts are related to the respective genetic background.
Treatment of Laron syndrome patients and effects on glucose homeostasis
Recombinant human (rh) IGF1 is available for more than 30 years and can be used as a therapeutic in LS patients (25). Long-term treatment starting at infancy accelerates – but not fully restores – linear growth velocity and modifies the craniofacies (26). Treatment of children decreases fat mass and hyperlipidemia in the first months to years (2) but over time adipogenic effects of rhIGF1 lead to an increase in fat mass and hyperlipidemia (27). The most prevalent adverse effect of treatment is IGF1-induced hypoglycemia after overdosage or administration in a fasting state (2). Adult LS patients from the Israeli cohort treated with rhIGF1 for a limited time showed a reduction of fat mass and hyperlipidemia, which reversed upon discontinuation of treatment (28). Long-term rhIGF1 replacement therapy reversed insulin resistance and improved glucose tolerance in the Israeli LS cohort (10). Glucose levels were reported to stabilize as patients were more likely able to tolerate fasting episodes without hypoglycemic episodes and insulin levels persistently decreased (29). In the Ecuadorian cohort of LS patients, rhIGF1 replacement therapy has been used in clinical trials and showed that a reduced dosage could decrease fat mass accumulation and osseous maturation, increasing adult height potential (30). Administration to adults for 7 days successfully suppressed GH levels and decreased serum insulin levels without hypoglycemic events (31). Administration of rhIGF1 over 12 months to prepubertal Ecuadorian LS patients showed a promotion of growth velocity without a significant increase of hypoglycemic incidents (32). In conclusion, the treatment with rhIGF1 appears desirable for all LS patients as the phenotype of short stature normalizes and the metabolic phenotype of patients from the Israeli cohort improves. Nevertheless, the therapy is not available for all LS patients due to the high cost of rhIGF1 and the costs for obtaining approval by authorities (33).
The Ghr-KO mouse
Low serum levels of glucose were reported for Ghr-KO mice up to an age of 9.5 months (referred to as 'young'; (34, 35, 36)) but became similar to controls at age 11 months (37). In contrast to human LS patients, normalization of glucose levels at puberty has not been observed in the Ghr-KO mouse model. Inconsistent results have been obtained in 'old' Ghr-KO mice (21 months), which showed glucose levels similar to controls (38) or even significantly higher levels (36).
Insulin levels were reported to be low in young Ghr-KO mice and always remained lower than in age-matched controls (35, 36, 37). Only one study observed normal levels in very young animals (3 days; 35).
During GTTs, the secretion of insulin in ~2-month-old Ghr-KO mice was not sufficient to clear the glucose, revealing glucose intolerance (39, 40, 41).
In addition to global Ghr-KO mice, multiple lines of conditional mutants lacking GHR in specific tissues have been generated (reviewed in 42, 43). Many of these lines show alterations in glucose homeostasis and insulin secretion/action. For instance, mice lacking GHR in the liver have elevated blood glucose and insulin levels, impaired glucose tolerance, and reduced insulin sensitivity (44, 45), whereas inactivation of Ghr in adipose tissue resulted in reduced serum insulin levels but increased insulin sensitivity (42). Male mice with muscle-specific deletion of GHR revealed reduced circulating levels of glucose and insulin, and improved glucose tolerance (46), and in another line, elimination of GHR in skeletal muscle increased insulin sensitivity of mice on a high-fat diet (47). The consequences of beta-cell-specific inactivation of Ghr (48) will be discussed below.
Conditional deletion mutants of Ghr provided important insights into the tissue-specific roles of GHR, but for dissecting the mechanisms involved in transient juvenile hypoglycemia in GHRD the global Ghr-KO mouse appears to be the more relevant model.
The GHR-KO pig
Consistent with the human phenotype, we observed cases of clinical hypoglycemia in young GHR-KO piglets in the early morning after long periods of not being fed by the sow, reaching glucose levels <15 mg/dL (measured as previously described 7). It has to be noted that the clinical symptoms in GHR-KO piglets were not as severe as reported in human infants; slight dizziness could easily be treated by oral administration of milk or glucose solution. At the age of 3 months, fasting glucose levels were significantly lower in GHR-KO pigs compared to age-matched controls (41.5 ± 3.8 mg/dL vs 63.1 ± 3.1 mg/dL; P = 0.0001) and normalized at age 6 months (57.9 ± 3.1 mg/dL vs 58.2 ± 2.8 mg/dL; P = 0.9429) (Fig. 1A; 7). Sexual maturity at that age was confirmed during necropsy by the presence of corpora lutea or disrupted Graafian follicles in the ovaries or mature seminiferous tubules in the testis and sperm in the epididymis (reviewed in 49). Therefore, normalization of glucose levels in GHR-KO pigs was associated with sexual maturation.
Fasting glucose levels, fasting insulin levels and HOMA-IR score in GHR-KO and control animals at the age of 3 months (young) and 6 months (adult) (modified from 7).
Citation: European Journal of Endocrinology 185, 2; 10.1530/EJE-21-0013
Fasting glucose levels, fasting insulin levels and HOMA-IR score in GHR-KO and control animals at the age of 3 months (young) and 6 months (adult) (modified from 7).
Citation: European Journal of Endocrinology 185, 2; 10.1530/EJE-21-0013
Fasting glucose levels, fasting insulin levels and HOMA-IR score in GHR-KO and control animals at the age of 3 months (young) and 6 months (adult) (modified from 7).
Citation: European Journal of Endocrinology 185, 2; 10.1530/EJE-21-0013
In young GHR-KO pigs, insulin levels were slightly lower than in controls (3.37 ± 1.3 µU/dL vs 4.48 ± 1.4 µU/dL; P = 0.5668). During puberty, insulin levels increased similarly in both groups but remained as a tendency lower in 6-month-old GHR-KO vs control pigs (4.86 ± 1.3 µU/dL vs 6.0 ± 0.9 µU/dL; P = 0.4917) (Fig. 1B; 7).
Intravenous GTTs confirmed the low glucose levels in young GHR-KO pigs before glucose infusion (Fig. 2A), but AUC analysis of glucose levels did not reveal significant differences between 3- and 6-month-old GHR-KO pigs or controls of both ages (Fig. 2A and B). The AUC insulin in both age groups of GHR-KO pigs was significantly smaller than in controls (Fig. 2C and D), thus mirroring observations in the Ecuadorian cohort of LS patients (9).
Intravenous glucose tolerance in GHR-KO and control pigs at age 3 and 6 months. Intravenous glucose tolerance tests were performed as described previously (78). Plots A and C show the mean ± s.e.m. for glucose and insulin. Area under the curve analyses presenting LSMean ± s.e. for glucose (B) and insulin (D), calculated applying the PROC GLM procedure (SAS 8.2) taking the effects of age and genotype into account. ***P < 0.001; ns, not significant.
Citation: European Journal of Endocrinology 185, 2; 10.1530/EJE-21-0013
Intravenous glucose tolerance in GHR-KO and control pigs at age 3 and 6 months. Intravenous glucose tolerance tests were performed as described previously (78). Plots A and C show the mean ± s.e.m. for glucose and insulin. Area under the curve analyses presenting LSMean ± s.e. for glucose (B) and insulin (D), calculated applying the PROC GLM procedure (SAS 8.2) taking the effects of age and genotype into account. ***P < 0.001; ns, not significant.
Citation: European Journal of Endocrinology 185, 2; 10.1530/EJE-21-0013
Intravenous glucose tolerance in GHR-KO and control pigs at age 3 and 6 months. Intravenous glucose tolerance tests were performed as described previously (78). Plots A and C show the mean ± s.e.m. for glucose and insulin. Area under the curve analyses presenting LSMean ± s.e. for glucose (B) and insulin (D), calculated applying the PROC GLM procedure (SAS 8.2) taking the effects of age and genotype into account. ***P < 0.001; ns, not significant.
Citation: European Journal of Endocrinology 185, 2; 10.1530/EJE-21-0013
Similarities and differences between LS patient cohorts and GHRD animal models
LS patients from the Israeliand Ecuadorian cohorts have juvenile hypoglycemia in common, but with age the phenotype develops in a different manner: patients from the Israeli cohort tend to develop the metabolic syndrome, while patients from Ecuador appear metabolically healthy. The Ghr-KO mouse resembles juvenile hypoglycemia; however, glucose levels do not normalize at puberty but at an older age. Low insulin levels are similar to findings in the Ecuadorian LS cohort, whereas glucose intolerance reflects observations in the Israeli cohort of LS patients.
The porcine GHR-KO model closely resembles the phenotype of the Ecuadorian LS cohort in terms of glucose and insulin levels, as well as glucose and insulin responses during intravenous GTTs. Therefore, the GHR-KO pig appears as a suitable model for investigating mechanisms underlying transient juvenile hypoglycemia in GHRD.
Potential mechanisms causing juvenile hypoglycemia and normalization with age
Commonly, the etiology of the juvenile hypoglycemia in GHRD is explained by the lack of diabetogenic GH action (50). Under physiological conditions, the acute administration of GH increases hepatic glucose output and stimulates glucagon release (51). Insulin antagonizing effects of GH are mediated by alterations of insulin-related intracellular signaling molecules resulting in insulin resistance, as shown in rodent models overexpressing GH (52). In the following, we outline potential mechanisms leading to the normalization of glucose levels in GHRD with age (summarized in Fig. 3).
Overview of mechanisms potentially involved in the normalization of blood glucose levels toward adulthood in cohorts of LS patients and in animal models of GHRD. '' indicates that there is evidence for the relevance of this mechanism, while '' indicates that the mechanism is probably not relevant. '?' indicates that there is currently not enough clinical or experimental evidence to draw a conclusion.
Citation: European Journal of Endocrinology 185, 2; 10.1530/EJE-21-0013
Overview of mechanisms potentially involved in the normalization of blood glucose levels toward adulthood in cohorts of LS patients and in animal models of GHRD. '' indicates that there is evidence for the relevance of this mechanism, while '' indicates that the mechanism is probably not relevant. '?' indicates that there is currently not enough clinical or experimental evidence to draw a conclusion.
Citation: European Journal of Endocrinology 185, 2; 10.1530/EJE-21-0013
Overview of mechanisms potentially involved in the normalization of blood glucose levels toward adulthood in cohorts of LS patients and in animal models of GHRD. '' indicates that there is evidence for the relevance of this mechanism, while '' indicates that the mechanism is probably not relevant. '?' indicates that there is currently not enough clinical or experimental evidence to draw a conclusion.
Citation: European Journal of Endocrinology 185, 2; 10.1530/EJE-21-0013
Changes in food intake and energy expenditure
Increased food intake and/or reduced energy expenditure are potential causes for increasing blood glucose levels.
Food intake and energy expenditure have been assessed in the Israeli LS cohort, showing that – when corrected for body weight – patients do not consume significantly more energy than normal subjects, but their resting energy expenditure is increased (8, 53). It has to be noticed, that for the Ecuadorian cohort, a traditional diet can be assumed for the past, while a transition toward a western diet comparable to the Israeli cohort is ongoing (4). Nevertheless, the effect of a high caloric diet on the Ecuadorian cohort cannot be foreseen, as the increased insulin sensitivity in Ghr‑KO mice persists after feeding a high fat diet (54). Increased food consumption of Ghr-KO mice, observed after normalization for their smaller size, was interpreted as compensation of an increased energy expenditure (55). Caloric intake was found to be significantly increased in hypoglycemic Ghr-KO mice animals at age 2 (37) and 3 months (56) and remained elevated at age 9 (37) and 17 months (57). A significant increase in energy expenditure – measured by oxygen consumption – was seen in hypoglycemic Ghr-KO mice at age 21 weeks (55), 7–12 months (58) and 17 months (57).
Therefore, it can be concluded that adiposity in GHRD is not conclusively due to a positive energy balance (8). Neither do alterations in energy supply correlate with the changes in blood glucose levels. It has been discussed that alterations of body composition may affect energy expenditure, and increased energy consumption can be seen as an adaptation to increased energy utilization due to smaller body size (8, 58).
A recent systematic review of 12 studies investigating changes in energy expenditure and intake during puberty in healthy nonobese adolescents concluded that the absolute basal or resting metabolic rate (BMR/RMR) and total daily energy expenditure (TDEE) were higher in pubertal than in prepubertal adolescents (59). However, several studies noted a decrease in lean or fat-free mass-adjusted BMR/RMR with increasing pubertal maturation. As the proportion of body fat increases and the proportion of lean mass decreases toward puberty both in Laron patients and in GHR-KO pigs, a relative decrease in BMR/RMR during sexual maturation can be expected.
Increasing obesity
The lack of lipolytic GH action in GHRD leads to progressive obesity (8, 20, 54), and the effects on metabolism – especially on glucose homeostasis – have been addressed in several publications.
Differences in the distribution of fat tissue accumulation, that is, a preferential enlargement of subcutaneous vs visceral fat depots, have been observed in Ghr-KO mice (60, 61) and were also suggested for human LS patients (62, 63). The site of fat accumulation plays a major role in metabolism, as visceral fat accumulation promotes insulin resistance while a preferential accumulation of subcutaneous fat decreases the risk of developing type 2 diabetes mellitus (64, 65). Therefore, the enlargement of subcutaneous fat depots in Ghr-KO mice can be seen as a 'healthy obesity' (63). Furthermore, visceral fat depots in Ghr-KO mice show an altered secretory activity, even enhancing insulin sensitivity (64). For instance, leptin and adiponectin (both low and high molecular weight adiponectin) levels were found to be elevated in Ghr-KO mice (66), as in LS patients (67). Elevated adiponectin levels can result in increased insulin sensitivity in the presence of increased adipose tissue, especially in the subcutaneous depot (68). A similar situation can be expected for GHR-KO pigs, which have significantly increased subcutaneous fat tissue and elevated serum leptin levels without obvious negative effects on metabolism (7).
Based on the findings in the Ecuadorian cohort of LS patients, in Ghr-KO mice and GHR-KO pigs, GHRD results in an enlargement of preferentially subcutaneous fat depots leading to 'healthy obesity'. Consequently, no common obesity-associated comorbidities have been observed (68). Progressive accumulation of adipose tissue is thus unlikely to play a major role for the normalization of blood glucose levels following juvenile hypoglycemia. The preferential use of circulating triglycerides for storage lipid synthesis in adipocytes leads to low serum levels in young Ghr-KO mice (54) and GHR-KO pigs (7) which normalize with age. In LS patients from the Israeli cohort, a progressive increase of serum lipids was reported (1), while low levels of triglycerides were described for adult patients from the Ecuadorian cohort (9). Enhanced insulin action accompanied by diminished insulin resistance and decreased lipolysis in the Ecuadorian cohort and potentially in the Ghr-KO mouse and the GHR-KO pig can lead to a decrease of free fatty acids and glycerol as a substrate for gluconeogenesis in young individuals (69). On the other hand, normalization of circulating lipid levels after assembling storage lipids can increase the substrate pool for gluconeogenesis and contribute to the normalization of glucose levels in adults.
Changes in insulin sensitivity
Insulin sensitivity is the major difference between the Israeli LS cohort on the one hand and the Ecuadorian LS cohort (9), the Ghr-KO mouse (54), and the GHR-KO pig (7) on the other hand. Hyperinsulinemia, developing hyperglycemia, and homeostasis model assessment for insulin resistance (HOMA-IR) values above 2.0 in Israeli LS patients indicate insulin resistance up to an age of 40 years (8). In addition, cases of insulin-dependent diabetes mellitus were reported (16).
In contrast, LS patients from the Ecuadorian cohort displayed low HOMA-IR scores compared to age-, gender- and BMI-matched healthy controls (9). While the HOMA-IR is not validated for animals (70), similar results have been reported for the Ghr-KO mouse (36), with lower scores than in age-matched controls at 9.5, 15, and 21 months. In GHR-KO pigs, HOMA-IR scores corresponded to 0.25 ± 0.20 and 0.70 ± 0.20 in 3- and 6-month-old animals (7) and were thus clearly below the threshold value (2.0) for insulin resistance in humans. The insignificant increase in HOMA-IR from age 3 to 6 months reflects the normalization of glucose levels. Future studies need to determine insulin sensitivity in 3- and 6-month-old GHR-KO pigs and corresponding controls using hyperinsulinemic-normoglycemic clamp experiments that are routinely established in pig models (71).
The outcome of insulin tolerance tests (ITTs; leading to insulin-induced hypoglycemia) in LS patients from the Israeli cohort was age-dependent (8, 10, 19). While children up to the age of 5.5 years did not recover from hypoglycemia, older children (6–8 years) showed a slight recovery of glucose levels, and in children at early puberty, normoglycemia was completely restored. These outcomes suggest age-dependent differences in counter-regulatory mechanisms to hypoglycemia. The insulin-induced hypoglycemia test is frequently used as a diagnostic tool in endocrinology to assess pituitary–adrenal function (72, 73). As GH and the adrenocorticotropic hormone (ACTH) are secreted in response to hypoglycemia, the secretion patterns of glucocorticoids, epinephrine and norepinephrine are of special interest in the context of GHRD-associated transient juvenile hypoglycemia (discussed below).
In Ghr-KO mice, ITTs were performed at age 5.5 months (64), 7 months (35), and 1 year (74). All studies observed a more pronounced decrease and a delayed recovery of glucose levels in Ghr-KO mice compared to the respective controls.
In conclusion, decreasing insulin sensitivity with age can indeed contribute to the normalization of blood glucose in Israeli LS patients, while this is not the case in the Ecuadorian LS cohort and in animal models for LS.
Changes in beta-cell mass and secretory capacity
GH stimulates beta-cell proliferation, insulin gene transcription and insulin secretion (35, 75). In patients with LS there is only indirect evidence that beta-cell mass may be affected. In Israeli LS patients, the relative insulinopenia seen after a period of hyperinsulinemia was discussed as a sign of beta-cell exhaustion due to insulin resistance (10). In insulin-sensitive Ecuadorian LS patients, a significantly reduced homeostasis assessment model for beta-cell function (HOMA-β) score was described (9), which may imply a reduction in beta-cell mass.
While detailed morphological and functional analyses of the endocrine pancreas in LS patients are lacking, the consequences of GHRD for the size and composition of the islets of Langerhans have been extensively studied in the Ghr-KO mouse model (35). The average size of the pancreatic islets was found 28% and 68% reduced in 10-day-old and 2-month-old Ghr-KO mice compared to age-matched controls. Moreover, a significantly decreased proportion of islet cells staining positively for the proliferation marker Ki67 was observed in 3-day-old Ghr-KO mouse pups. A detailed analysis of islet cell populations was performed in 10- to 11-month-old animals. The beta-cell mass of Ghr-KO mice was markedly reduced (−80% compared to controls), even when corrected for body weight (−50% compared to controls). The alpha-cell mass was not significantly different between Ghr-KO and control mice, and thus significantly (100%) increased the Ghr-KO group after correction for body weight. The reduced islet size in Ghr-KO mice could be rescued by beta-cell-specific expression of an Igf1 transgene (39).
In contrast to mice with a global Ghr-KO, mice lacking GHR specifically in the beta cells did not show a reduced beta-cell mass when maintained on a chow diet; nevertheless, their glucose-stimulated insulin secretion was impaired (48). When challenged with a high-fat diet, beta cells lacking GHR showed reduced proliferation, resulting in an overall reduced beta-cell mass, blunted glucose-stimulated insulin secretion, and further deterioration of glucose homeostasis (48). The authors concluded that GHR in beta cells is important for glucose-stimulated insulin secretion and beta-cell proliferation in response to a high-fat diet. The results from this study need to be interpreted with caution, as the used mouse line expressing Cre recombinase under the control of a rat insulin gene promoter (RIP-Cre transgenic mouse line) also harbors a human growth hormone (hGH) minigene. Locally expressed hGH signals via the prolactin receptor in pancreatic islets and induces gene expression augmenting beta-cell mass and insulin content (76). Moreover, the question whether age-related changes in the abundance, composition and function of islet cells underly the phenomenon of transient juvenile hypoglycemia in GHRD was not addressed in the mouse model with the beta-cell-specific Ghr-KO (48).
It will thus be interesting to perform quantitative sterological investigations of beta cells and other endocrine cell populations in pancreas samples from 3- to 6-month-old GHR-KO and control pigs. Random systematic sampling is routinely performed to ensure the recovery of representative samples (77). Our previous studies showed that the proliferation rate of beta cells in pigs is markedly higher in 11-week-old compared to 5-month-old animals (78). Thus, the lack of a growth stimulus is expected to have more pronounced effects on cell proliferation in the younger age group, as previously observed in a pig model with impaired glucose-dependent insulinotropic polypeptide receptor (GIPR) function (78).
Metabolic changes during puberty
A link between sexual development and glucose metabolism has already been proposed by Laron, Avitzur and Klinger (10) as the hypoglycemia vanishes with the onset of puberty. Notably, LS patients show hypogenitalism, hypogonadism and a delay in puberty but preserved fertility (1). In the Ghr-KO mouse model, normalization of glucose levels was reported at age 11 months (37) while sexual maturity – although delayed – already occurred at 38 days (investigated in female mice) and deficits in reproduction such as delayed maturation of males and females and a reduced litter size were reported (79).
In line with observations in LS patients, no obvious deficits in reproduction have been observed in GHR-KO pigs and the normalization of glucose levels occurred with sexual maturity at the age of 6 months (7).
Puberty is physiologically associated with a decrease in insulin sensitivity and a compensatory increase in insulin secretion (15, 80, 81), with clinical implications for diabetic patients at puberty (82). Interestingly, the changes in insulin sensitivity are not mediated by sexual hormones but by the GH/IGF1 axis. IGF1 levels are significantly related to the level of insulin resistance and show a similar pattern of rise and fall during the Tanner stages of puberty (83). In contrast, while changing dramatically during puberty, neither testosterone nor estradiol levels are associated with the level of insulin resistance during puberty and remain higher in adulthood, while insulin sensitivity returns to prepubertal values (84). Furthermore, sexual hormones protect against the development of insulin resistance, as testosterone improves insulin sensitivity and reduces body fat in men and estrogen protects females against insulin resistance and increases hepatic insulin sensitivity (85, 86, 87).
Another potential factor contributing to the normalization of glucose levels during puberty is the relative use of fatty acids vs glucose as fuels for cellular respiration and energy generation. In normal human subjects, the pubertal increase in GH/IGF1 contributes to insulin resistance. Thus, the effect of insulin to suppress fatty acid oxidation is reduced and – due to the competition of fatty acids and glucose for substrates – glucose oxidation is decreased, resulting in a glucose sparing effect (reviewed in 80). In GHRD this mechanism is at least partially disrupted. Normoglycemic 6-month-old GHR-KO pigs had significantly reduced serum glycerol concentrations and, as a tendency, reduced free fatty acid levels. Moreover, a decreased ratio of long-chain acylcarnitines to free carnitine suggested reduced activity of carnitine palmitoyltransferase 1A and thus reduced mitochondrial import of fatty acids for beta-oxidation (88). However, a preference for fat oxidation was suggested in older Ghr-KO mice, even in the absence of lipolytic GH action (54).
Although there is a clear temporal association between normalization of glucose levels and puberty in both LS patients and in the GHR-KO pig model, the underlying mechanisms remain unclear. Systematic studies of glucose vs fatty acid oxidation in 3- and 6-month-old GHR-KO pigs and age-matched controls may provide new insights into the complex relationships between GHRD, puberty and energy homeostasis.
Altered endogenous glucose production
Endogenous glucose production is regulated by multiple hormones modulating the rates of gluconeogenesis and glycogenolysis (reviewed in 89).
Postprandial insulin peaks promote hepatic glucose uptake and glycogen synthesis, while basal insulin inhibits gluconeogenesis and glycogenolysis through the PI3K/AKT pathway (reviewed in 90). A multi-omics analysis of liver samples from a pig model for mutant INS gene-induced diabetes of youth (MIDY) (91) revealed increased levels of retinol dehydrogenase 16 (RDH16) and retinoic acid as mechanistic link between insulin deficiency and stimulated gluconeogenesis (92).
In contrast to insulin, glucagon promotes hepatic glucose output by increasing gluconeogenesis and glycogenolysis and by decreasing glycogenesis and glycolysis (reviewed in 93). Similarly, GH stimulates gluconeogenesis, partly through the STAT5 pathway, and can also stimulate glycogenolysis (reviewed in 89).
Reduced hepatic glucose production/gluconeogenesis was discussed as a potential cause for juvenile hypoglycemia in LS patients (10, 69).
In 2-month-old hypoglycemic Ghr-KO mice, glucagon levels were lower than in control mice (35), potentially resulting in reduced gluconeogenesis. In contrast, normal or even elevated levels of glucagon were measured in 21-month-old Ghr-KO mice (38). In addition, the abundance of the gluconeogenic transcription factor forkhead box protein O1 (FOXO1) and the mRNA levels for key enzymes of gluconeogenesis such as phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC) were increased in liver tissue from Ghr-KO vs control mice. These observations suggested that counter-regulatory activation of gluconeogenesis can normalize blood glucose levels in aged Ghr-KO mice (8).
Proteomic and metabolomic investigations of liver and serum samples derived from 6-month-old GHR-KO pigs showed normal abundances of enzymes involved in gluconeogenesis but a decreased abundance of pyruvate kinase (PKLR, liver and red blood cell isoform), which catalyzes a rate-limiting step of glycolysis (88). A reduction in PKLR levels can result in the preservation of substrates for gluconeogenesis and therefore contribute to the normalization of glucose levels in the absence of GH action. In addition, multiple enzymes involved in amino acid metabolism were found to be upregulated, providing substrates for gluconeogenesis. Future studies need to perform similar analyses of liver tissue from 3-month-old GHR-KO pigs and controls to see if proteome and metabolome changes provide hints for reduced gluconeogenesis. Importantly, in the pig, the rate of gluconeogenesis can be determined by the 2H2O-method in vivo (94, 95).
Additional hormones potentially involved in the normalization of glucose levels in GHRD upon puberty include catecholamines (especially epinephrine) and glucocorticoids. Epinephrine levels are known to increase in a state of hypoglycemia, promoting gluconeogenesis by the mobilization of gluconeogenic substrates from peripheral tissues and thereby intensifying the effect of glucagon (96). While epinephrine levels were not investigated in GHRD so far, elevated levels of glucocorticoids were reported in Ghr-KO mice at a normoglycemic age (8). Accompanied by low levels of insulin, the promotion of hepatic glucose production by gluconeogenesis has been discussed (54).
Taken together, impaired gluconeogenesis has been assumed as potential cause for juvenile hypoglycemia in LS patients. This concept and the normalization of glucose levels by an age-related increase in gluconeogenesis are supported by observations in the Ghr-KO mouse model. The GHR-KO pig model, which is hypoglycemic at age 3 months but normoglycemic at age 6 months, provides a unique model for testing this hypothesis by quantifying the rate of gluconeogenesis in the two age groups using the 2H2O-method and by quantifying the abundances of gluconeogenic enzymes and substrates by multi-omics profiling of corresponding liver and serum samples.
Conclusions and perspectives
Juvenile hypoglycemia is a major consequence of GHR deficiency in both cohorts of LS patients and in both animal models. Decreasing insulin sensitivity and relative insulinopenia in the context of metabolic syndrome explains the normalization of glucose levels or may even lead to hyperglycemia in the Israeli cohort of patients. In the Ecuadorian LS cohort as well as in both animal models, insulin sensitivity is preserved or increased; therefore, other factors normalizing glucose levels need to be considered. While differences in food consumption and energy expenditure have been assessed, their influence on the normalization can be considered as minor. An increased accumulation of body fat is a common hallmark of GHR deficiency, but a preferred accumulation of healthy subcutaneous fat does not lead to a decreasing insulin sensitivity in Ecuadorian patients and both animal models. A reduced beta-cell mass in the absence of proliferative GH action can lead to a decreased secretory capacity for insulin, which results in glucose intolerance in Ghr-KO mice but not in Ecuadorian LS patients nor in the GHR-KO pig model. While there is a clear temporal association between puberty and the normalization of glucose levels in LS patients and GHR-KO pigs, the underlying counter-regulatory mechanisms and metabolic changes deserve further investigation. An increase in endogenous glucose production – especially by gluconeogenesis – appears most likely and can be mediated by several hormones, including glucagon and catecholamines.
By now, no coherent study assessed these parameters in different age groups, wherefore a study in a suitable animal model is required. The GHR-KO pig resembles the phenotype of the Ecuadorian cohort more closely than the Ghr-KO mouse regarding glucose tolerance, potentially secretory capacity for insulin and the association of normoglycemia and puberty. Therefore, a study in GHR-KO pigs needs to compare hypoglycemic animals at the age of 3 months with normoglycemic animals aged at least 6 months. The in vivo quantification of gluconeogenesis by applying the 2H2O-method in combination with a stereological examination of beta-cell mass in both age groups can clarify (a) to which extent insufficient gluconeogenesis contributes to juvenile hypoglycemia; (b) if stimulation of gluconeogenesis serves as a counter-regulatory mechanism normalizing glucose with age; and (c) whether differences in the secretory capacity of beta cells modulate glucose levels in an age-dependent manner. Furthermore, the inclusion of castrated GHR-KO pigs can clarify if the recovery of normoglycemia at pubertal age depends on the presence of sex hormones and, more generally, help to dissect the roles of sex steroids and of the GH/IGF1 axis in the metabolic changes associated with puberty.
Another interesting question is if brain glucose consumption plays a role in transient juvenile hypoglycemia in Laron syndrome. The mammalian brain critically depends on glucose as a fuel for ATP production, oxidative stress management, and synthesis of neurotransmitters, neuromodulators, and structural components (reviewed in (97)). Due to its large size, the human brain requires particularly high amounts of glucose. Kuzawa and colleagues (98) calculated the glucose consumption of the human brain from birth to adulthood using positron emission tomography(PET) and MRI data. This study revealed the peak of brain glucose consumption not at birth (when the brain to body ratio is highest) but around age 5 years, reflecting the high energetic costs associated with extensive development of neuronal processes and synapses before activity-dependent pruning in late childhood and adolescence. Interestingly, the peak in brain glucose uptake corresponds closely with the age of slowest body weight gain, thus reducing energy expenditure on somatic growth. In adult humans, the daily glucose use of the brain is just 50% of the peak glucose uptake during childhood (98). Thus, the increased brain glucose consumption in young children could contribute to transient juvenile hypoglycemia in LS patients. Conversely, juvenile hypoglycemia could affect normal brain development. Studies of intelligence in humans with GHRD revealed mixed results from normal intelligence (99) and normal brain appearance to varying degrees of parenchymal loss and below-average intelligence (100), probably due to effects of specific GHR mutations, genetic background, or other factors unrelated to GHRD (discussed in 101). The latter study investigated young adult LS patients from the Ecuadorian cohort, revealing enhanced cognitive performance and greater task-related activation in frontal, parietal, and hippocampal regions compared with their GHR-intact relatives (101). This was explained by the increased insulin sensitivity in this LS cohort, ensuring proper glucose supply, enhanced cognitive function and a protective effect delaying cognitive aging (4). In line with the increased relative brain weight of rodent (102) and porcine models (7) of GHRD, young adult Ecuadorian LS patients had larger surface areas in several frontal and cingulate regions and showed trends toward larger dentate gyrus and CA1 regions of the hippocampus (101). Final clarification if functional and structural changes of the brain in LS patients are related to juvenile hypoglycemia requires systematic studies of brain glucose consumption in different age cohorts (98).
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by the Deutsche Forschungsgemeinschaft (HI 2206/2-1 to A H and TRR 127 to E W) and the DZD (to E W).
Author contribution statement
A H wrote the original manuscript, S R, M B, J J K and E W commented on the manuscript and revised the draft.
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