Roles of the Lactogens and Somatogens in Perinatal and Postnatal Metabolism and Growth: Studies of a Novel Mouse Model Combining Lactogen Re
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内分泌学杂志 2005年第1期
Division of Pediatric Endocrinology and Diabetes (D.F., J.O., S.W., J.K., M.F.), Duke University Medical Center, Durham North Carolina 27710; Institut National de la Santé et de la Recherche Médicale Unité 584-Hormone Targets (P.A.K., S.A., M.P.), Faculte de Medecine Necker, Paris, France 75015; and Musculoskeletal Diseases Center (S.M.), Jerry L. Pettis Veterans Affairs Medical Center, Loma Linda, California 92357
Address all correspondence and requests for reprints to: Dr. Michael Freemark, Division of Pediatric Endocrinology and Diabetes, Box 3080, Duke University Medical Center, Durham, North Carolina 27710. E-mail: freem001@mc.duke.edu.
Abstract
To delineate the roles of the lactogens and GH in the control of perinatal and postnatal growth, fat deposition, insulin production, and insulin action, we generated a novel mouse model that combines resistance to all lactogenic hormones with a severe deficiency of pituitary GH. The model was created by breeding PRL receptor (PRLR)-deficient (knockout) males with GH-deficient (little) females. In contrast to mice with isolated GH or PRLR deficiencies, double-mutant (lactogen-resistant and GH-deficient) mice on d 7 of life had growth failure and hypoglycemia. These findings suggest that lactogens and GH act in concert to facilitate weight gain and glucose homeostasis during the perinatal period. Plasma insulin and IGF-I and IGF-II concentrations were decreased in both GH-deficient and double-mutant neonates but were normal in PRLR-deficient mice. Body weights of the double mutants were reduced markedly during the first 3–4 months of age, and adults had striking reductions in femur length, plasma IGF-I and IGF binding protein-3 concentrations, and femoral bone mineral density. By age 6–12 months, however, the double-mutant mice developed obesity, hyperleptinemia, fasting hyperglycemia, relative hypoinsulinemia, insulin resistance, and glucose intolerance; males were affected to a greater degree than females. The combination of perinatal growth failure and late-onset obesity and insulin resistance suggests that the lactogen-resistant/GH-deficient mouse may serve as a model for the development of the metabolic syndrome.
Introduction
PLACENTAL LACTOGEN (PL), prolactin (PRL), and GH constitute a somatolactogen superfamily of polypeptides that have similarities in structure and biological activity. Through binding to cognate PRL and GH receptors in target tissues, the lactogens (PL and PRL) and somatogens (GH) regulate reproductive behavior and function, mammary growth and milk protein production, carbohydrate, lipid, protein, and mineral metabolism, and postnatal growth.
Experimental studies have elucidated the molecular mechanisms by which the lactogens and somatogens exert their biological actions (1). But similarities in their biological activities have made it difficult to define precisely the respective roles of PL, PRL, and GH in whole-body metabolism. For example, both the lactogens and somatogens induce ?-cell proliferation and insulin production in pancreatic islets and insulinoma cells (2, 3, 4, 5). At supraphysiological concentrations the lactogens inhibit insulin-dependent glucose uptake in isolated adipocytes and reduce insulin sensitivity (6, 7, 8, 9, 10, 11), mimicking the effects of GH. Human PL, which is related structurally to human GH, is reported to stimulate lipogenesis and lipolysis in isolated fat cells (6, 7, 12, 13); the rodent PLs, related more closely to rodent PRLs, have little or no lipolytic activity in vitro but may promote lipolysis in vivo (14, 15, 16). Both PRL and GH induce adipogenesis in rodent preadipocyte cell lines (15, 17, 18, 19) and mitogenesis in fetal and neonatal hepatocytes (20) and astrocytes (21, 22). Finally, human PL, rat PL-1, and mammalian PRLs enhance the growth of isolated rat yolk sacs and embryos (23, 24, 25) in culture and may increase IGF-I production in fetal tissues (20, 24, 26, 27), whereas GH is the major determinant of linear growth and IGF-I production during the postnatal period (28).
Thus, the metabolic actions of the lactogens appear to overlap with those of GH in some systems and complement those of GH in others. We hypothesized that this redundancy of hormone action may enable one member of the somatolactogen family to compensate for the absence of another, thereby obscuring or modulating the effect of an isolated hormone deficiency. In a similar vein, we theorized that a combination of defects in lactogen and GH signaling might aggravate the metabolic dysfunction that accompanies an isolated signaling defect.
To test these hypotheses, we generated a novel mouse model that combines resistance to all lactogenic hormones with a severe deficiency of pituitary GH. The model was created by breeding PRL receptor (PRLR)-deficient male mice, which are resistant to the actions of mouse PL as well as mouse PRL (29), with GH-deficient little females, which have fetal and postnatal hyposomatotropism (GH levels 5% of normal) due to a mutation in the GHRH receptor (30, 31). The confluence of defects in lactogen and GH signaling abolishes compensatory effects arising from overlapping actions of the lactogenic and somatogenic hormones.
We compared the weight gain and fat deposition of lactogen-resistant/GH-deficient (double-mutant) mice with those of wild-type, PRLR-deficient, and GH-deficient mice in the perinatal (d 7) and postnatal (d 30–650) periods. We then assessed the relationships among weight gain; glucose tolerance; insulin production; insulin action; and the plasma concentrations of IGF-I, IGF-II, IGF binding protein (IGFBP)-3, and leptin.
Materials and Methods
Generation of PRLR-deficient/GH-deficient double-mutant mice
Homozygous PRLR-deficient males (129 background) were mated with homozygous GH-deficient little females (C57BL/6 background, from Jackson Labs, Bar Harbor, ME) to yield mice that were heterozygous at both loci on a hybrid 129/Bl6 background. Double-heterozygous males and females were bred to produce wild-type, PRLR-deficient, GH-deficient, and double-mutant mice. The various genotypes were identified through PCR analysis as described previously (30, 32). All subsequent experiments were performed in mice on the hybrid 129/Bl6 background.
The mice were housed under nonsterile conditions in rooms with 12-h light, 12-h dark cycles. Food and water were provided ad libitum. The mouse chow was a standard preparation (Laboratory Rodent diet 5001, Ralston Purina Co., St. Louis, MO) containing 12.1% of calories as fat, 28% as protein, and 59.8% as carbohydrate.
All animal protocols were approved by the Duke University Medical Center Institutional Animal Care and Use Committee and followed federal guidelines.
Auxologic measurements and body fat stores
Body weight measurements were obtained on d 7 of life, and the neonates were killed at 1000 h to obtain trunk blood. Additional measurements of body weight were obtained at various time points between 30 and 700 d of age in 10 wild-type females, 10 wild-type males, 13 PRLR-deficient females, eight PRLR-deficient males, eight GH-deficient females, 16 GH-deficient males, 18 double-mutant females, and 23 double-mutant males. All animals were virginal.
Body fat stores, femur lengths, and femoral bone mineral densities were estimated by dual-energy x-ray absorptiometry using the Lunar Piximus densitometer (General Electric Medical Systems); individual mice were anesthetized with pentobarbital and scanned three times. The Lunar software provides measurements of fat mass and lean tissue mass; values are expressed as a percent of bone-free body weight. Each measurement of body fat and lean tissue mass excluded the head. Measurements of fat mass in mice obtained with the Lunar Piximus are precise and reproducible and correlate very strongly (r2 = 0.97) with measurements of fat mass obtained by chemical extraction (33).
Food intake in double-mutant mice
Food intake was measured during a 7-d period under standard conditions (12-h light, 12-h dark cycles, free access to food and water, environmental temperature 23 C); experimental data were expressed as grams food per day per gram body weight. The feeding studies were performed three times to ensure the reproducibility of the results.
Glucose, insulin, and leptin concentrations and insulin sensitivity were measured using methods described previously (32, 34). Plasma glucose concentrations were measured using a One-Touch Ultra glucometer (Lifescan, Milpitas, CA). Plasma insulin concentrations were measured using the ultrasensitive rat insulin RIA from Linco Corp. (St. Louis, MO); the sensitivity of the assay is 0.2 ng/ml. Fasting glucose and insulin concentrations were measured at 0900 h after an overnight (16 h) fast. Plasma glucose and insulin concentrations were also measured 30 min after ip administration of 10% dextrose (10 ml/kg body weight). In separate experiments we assessed glucose tolerance by measuring plasma glucose concentrations every 10 min after the intraperitoneal injection of 10% dextrose in water (10 ml/kg body weight) or after the ip administration of glucagon (0.04 mg/kg body weight, Bedford Labs, Bedford, OH). The glucose tolerance and glucagon tolerance tests were performed after 2 h of fasting. Insulin sensitivity was assessed (34) in nonfasted mice by measuring the rate of fall in blood glucose concentrations after an ip injection of Humalog insulin (1 U/kg body weight, Eli Lilly Corp., Indianapolis, IN). Plasma leptin concentrations were measured in nonfasted male mice and fasted (16 h) female mice using the Linco assay; the sensitivity of this assay is 0.2 ng/ml. Intra- and interassay variations of all assays were less than 15%.
IGF-I, IGF-II, and IGFBP-3 concentrations
IGF-I and IGF-II were measured by RIA after extraction of IGF binding proteins on a BioGel P-10 (Bio-Rad, Hercules, CA) column equilibrated in 1 M acetic acid. The methods have been validated for complete separation of IGFBPs from IGFs in serum and other biological fluids (35). The IGF-I assay uses recombinant human IGF-I as standard and tracer and a rabbit polyclonal antiserum. The IGF-II assay uses recombinant human IGF-II as standard and tracer and a mouse monoclonal antibody. The assays can be performed using 12.5 μl plasma. The intra- and interassay coefficients of variation for each assay are less than 10%. The sensitivities of the IGF-I and -II assays are 20 and 50 pg/ml, respectively. Plasma IGFBP-3 concentrations were measured by densitometric analysis of Western ligand blots, as previously described (36)
Data analysis
The breeding of double heterozygotes is predicted to yield only one homozygous wild-type, one PRLR-deficient, one GH-deficient, and one double-mutant mouse for every 16 pups. Because litters typically contained 5–11 pups, no single litter contained both males and females of the four genotypes of interest. Consequently, it was impossible to perform direct statistical comparisons within a single litter. We therefore used animals from additional litters of comparable age as well as true littermates.
All auxologic data and measurements of glucose, insulin, and leptin concentrations were expressed as mean ± SE. Statistical differences in weight gain during development among the various groups of mice were assessed by two-way ANOVA followed by the Bonferroni test of comparisons. All biochemical studies were repeated at least three times, using duplicate or triplicate replicates in each experiment. Differences among sample means were assessed by ANOVA followed by the Newman-Keuls test of multiple comparisons. P < 0.05 was considered statistically significant.
Results
Intrauterine and postnatal viability
Breeding of double-heterozygous males and females yielded PRLR-deficient, GH-deficient, double-mutant, and wild-type animals at the expected Mendelian ratios. The postnatal mice thrived under nonsterile conditions.
Weight gain, plasma glucose, and insulin and IGF concentrations in the perinatal period
To assess the effects of lactogen resistance and GH deficiency on perinatal growth and carbohydrate tolerance, we compared the weights and plasma glucose, insulin, and IGF concentrations of 7-d-old double-mutant mice with those of PRLR-deficient, GH-deficient, and wild-type mice. Data from male and female mice on d 7 were combined because previous studies (Ref. 28 and our unpublished observations) demonstrated no significant gender-dependent differences in mouse weight gain at this age. In addition, data from homozygous wild-type mice on d 7 were combined with data from 7-d-old mice that were heterozygous for either the PRLR or GHRH receptors because we found no significant differences in body weight, blood glucose, or plasma insulin, IGF-I, or IGF-II concentrations between wild-type and heterozygous mice at this stage of development (Table 1 and Figs. 1 and 2).
TABLE 1. Perinatal (d 7) weight gain and blood glucose concentrations in wild-type and heterozygous (heteros) mice and mutant mice (males and females combined)
FIG. 1. Plasma insulin concentrations (A) and glucose to insulin ratios (B) in mice on postnatal d 7. Values represent the mean ± SE of wild-type/heterozygous controls (cont, n = 32), PRLR-deficient mice (n = 11), GH-deficient (GHD) mice (n = 18), and double-mutant mice (n = 13). Data from males and females were combined.
FIG. 2. Plasma IGF-I and IGF-II concentrations in mice on postnatal d 7. Values represent the mean ± SE of wild-type/heterozygous controls (cont, n = 11), PRLR-deficient (n = 6) mice, GH-deficient (GHD, n = 14) mice, and double-mutant mice (n = 14). Data from males and females were combined.
As shown in Table 1, the weights of PRLR-or GH-deficient mice were normal on d 7. However, body weights and plasma glucose concentrations were reduced 11 (P < 0.05) and 16% (P < 0.01), respectively, in perinatal mice with combined lactogen resistance and GH deficiency (double mutants).
Random plasma insulin concentrations were reduced in double-mutant mice as well as GH-deficient mice (Fig. 1A) on d 7 of life. Because blood glucose concentrations were normal in GH-deficient mice, the glucose to insulin ratio was increased (Fig. 1B); these findings suggest heightened insulin sensitivity. In double-mutant mice, both glucose and insulin levels were low; therefore, the glucose to insulin ratio was normal. Isolated resistance to lactogenic hormones (PRLR deficiency) had no effect on blood glucose, insulin, or the glucose to insulin ratio at this age.
The reductions in body weight in the double mutants on d 7 were accompanied by small reductions in plasma IGF-I and IGF-II concentrations (Fig. 2). IGF concentrations in GH-deficient mice were comparable with those in double-mutant mice. However, there was no correlation between IGF-I concentrations and body weight in the mice at this age (n = 82, r = 0.1), and IGF-II concentrations correlated inversely although weakly with body weight (r = –0.225, P < 0.05). In contrast, random insulin (n = 41, r = 0.51, P < 0.001) and glucose (n = 103, r = 0.263, P = 0.007) concentrations and pancreas weight (n = 103, r = 0.83, P < 0.001) correlated positively with body weight on d 7.
Weight gain, femur length, bone mineral density, and fat deposition during postnatal life and aging
We next compared the weight gain of double-mutant mice during postnatal life with that of PRLR-deficient, GH-deficient, and wild-type littermates (n = 8–23 in each gender and genotype). Our studies of postnatal weight gain and metabolic function used only homozygous wild-type mice and mice with homozygous deletions of the GH and/or PRL receptors. As shown in Fig. 3, females with isolated PRLR deficiency gained weight normally until approximately 4 months of age; thereafter, weight gain was blunted, and older PRLR-deficient females weighed on average 8–10% less than wild-type females. In contrast, weight gain in PRLR-deficient males was normal. The weight deficiency of GH-deficient females persisted through 500 d, whereas GH-deficient males had some catch-up weight gain beginning at 120–180 d of life. Weight gain of double-mutant females was comparable with that of GH-deficient females until approximately 1 yr of age; thereafter, body weight appeared to increase slightly. In contrast, catch-up weight gain in double-mutant males was more dramatic than in double-mutant females and began around 160 d of life. By 600 d of life, the weights of the double-mutant males were comparable with, and in some cases exceeded, the weights of wild-type males.
FIG. 3. Postnatal weight gain in wild-type and mutant mice. Measurements of body weight were obtained at various times between 30 and 700 d of age in 10 homozygous wild-type females, 10 wild-type males, 13 PRLR-deficient females, eight PRLR-deficient males, eight GHD females, 16 GHD males, 18 double-mutant females, and 23 double-mutant males; all animals were virginal. The regression lines were calculated with GraphPad software (GraphPad Inc., San Diego, CA) using a nonlinear one-site binding model. Analysis of goodness of fit showed correlation coefficients (r) ranging from 0.77 to 0.82 in the four groups of female mice and 0.84 to 0.91 in the male mice.
Two-way ANOVA demonstrated significant differences in rates of weight gain between PRLR-deficient and wild-type females (P < 0.02) and between GH-deficient and double-mutant males (P < 0.01) after 120 d of age. There were no significant differences between GH-deficient and double-mutant females or between PRLR-deficient and wild-type males.
The accelerated weight gain in double-mutant males did not reflect an increase in linear growth; femur lengths of adult double-mutant males and females (age 12 ± 2 months) were only 72% as great as those of wild-type males (P < 0.001), and plasma IGF-I and IGFBP-3 concentrations and femoral bone mineral density were markedly reduced (Table 2). The magnitude of reductions in femur length and IGF and IGFBP-3 levels in GH-deficient mice were comparable with (or more dramatic than) those in double-mutant mice, suggesting that lactogen resistance had little or no effect on postnatal linear growth or IGF/IGFBP-3 expression. Isolated PRLR deficiency, however, reduced bone mineralization slightly in females but not males (Table 2).
TABLE 2. Parameters of growth and bone mineralization in adult wild-type (WT) and mutant mice
Body fat, expressed as a percent of body weight, was normal in PRLR-deficient (23.0 ± 4.4%) and GH-deficient males (25.6 ± 3.3%) at 10 months of age but was increased in double-mutant males (38.9 ± 3.8%); the corresponding value in wild-type mice was 21.5 ± 3.4% (n = 3–5 in each group). At 500 d, body fat increased in all groups but remained highest in double-mutant males (wild type 32.0 ± 2.6%, GH deficient 40.2 ± 4.8%, double mutant 48.6 ± 2.7%; the number of PRLR-deficient males at this age was not adequate for statistical analysis). Percent body fat relative to femur length, an indirect measure of body mass index, was increased in double-mutant males (Fig. 4A). Conversely, lean body mass, expressed as a percent of body weight, was low (wild type 68.5 ± 2.5%; PRLR 72.1 ± 3.3%; GH deficient 59.8 ± 2.5%, P < 0.05 vs. wild type; double mutant 46.6 ± 4.2%, P < 0.02 vs. wild type, mean ± SE, n = 3–7 in each group, Fig. 4B). Relative fat deposition in males with isolated GH deficiency also exceeded that in wild-type and PRLR-deficient males, but adiposity was more severe in double-mutant males.
FIG. 4. A, Percent body fat relative to femur length in male mice. Percent body fat and femur length were estimated by dual-energy x-ray absorptiometry analysis at 10 ± 2 months of age. B, Lean body mass of male mice, expressed as a percent of body weight. Values represent the mean ± SE of eight wild-type males, three PRLR males, 11 GH-deficient (GHD) males, and nine double males.
There were no significant differences in body fat among the groups of female mice at 120 d (wild type 13.0 ± 1.5%, PRLR deficient 11.2 ± 2.3%, GH deficient 20.8 ± 2.1%, double mutant 20.9 ± 2.0%, n = 3–5 in each group). In aging females (500–600 d), however, percent body fat was increased in double-mutant mice (49.9 ± 9.8%, P < 0.05) relative to wild-type (33.5 ± 2.8%), PRLR-deficient (27.8 ± 2.2%), and GHD mice (31.2 ± 6.1%, n = 3–8 in each group).
Changes in plasma leptin concentrations mirrored changes in body fat content; leptin levels were increased in adult double-mutant males and aging double-mutant females (Fig. 5) but were reduced slightly in adult PRLR-deficient females. Food intake in double-mutant males was normal or slightly increased at 90 d of age but decreased at 15 months of age (Fig. 6).
FIG. 5. Plasma leptin concentrations in adult wild-type and mutant mice. Values represent the mean ± SE of fasted (16 h) female mice (n = 3–5 in each group) and nonfasted male mice (n = 5–12 in each group) at 15 ± 3 months of age.
FIG. 6. Food intake in wild-type and double-mutant male mice. Values represent the mean ± SE of groups of three to five mice in three experiments at 3 ± 1 months and three experiments at 15 ± 2 months.
Developmental changes in insulin sensitivity and glucose tolerance
The increase in adiposity in double-mutant males during aging was accompanied by striking changes in fasting glucose, insulin concentrations, and insulin sensitivity. At 3 months of age, fasting glucose levels in double-mutant males and females were lower than those in wild-type, PRLR-deficient, or GH-deficient mice (Fig. 7). By age 12 months, however, fasting glucose concentrations in double-mutant males were higher than those in wild-type, PRLR-deficient, or GH-deficient males. In contrast, fasting glucose levels in older double-mutant females were comparable with those in GH-deficient females and lower than in wild-type or PRLR-deficient females.
FIG. 7. Fasting glucose levels in wild-type and mutant mice. Values represent the mean ± SE of four to eight mice of each gender and genotype at 3 ± 1 months of age and 12 ± 2 months of age.
Fasting insulin levels in adult double-mutant females, like those in adult GH-deficient females, were lower than those in wild-type or PRLR-deficient mice (Fig. 8). In contrast, fasting insulin concentrations in adult double mutant males were comparable with those in wild-type or PRLR-deficient males and higher than those in GH-deficient males. Thirty minutes after the ip administration of 10% dextrose (1 g/kg), the ratio of glucose (mg%) to insulin (nanograms per milliliter) was increased in GH-deficient males (183 ± 12 mg/ng vs. wild types 112 ± 17 mg/ng, mean ± SE, P < 0.05) but was normal in PRLR-deficient (135 ± 15 mg/ng) and double-mutant (84 ± 26 mg/ng) males (n = 3–6 in each group).
FIG. 8. Fasting insulin levels in adult mice. Values represent the mean ± SE of four to eight mice of each gender and genotype at 10–12 months of age. GHD, GH deficient.
The combination of normoinsulinemia with fasting hyperglycemia in the double-mutant males suggests relative hypoinsulinemia and/or insulin resistance. That the acquired obesity of double-mutant male mice is accompanied by insulin resistance is suggested by the results of insulin tolerance tests (Fig. 9). At 3 months of age, the rate of decline in blood glucose after insulin administration was more precipitous in double-mutant (P < 0.001) or GH-deficient (P < 0.002) males than in wild-type males. The effect of PRLR deficiency (P = 0.08) did not reach statistical significance. The calculated half-time to reach maximal effect was less in double-mutant mice (12 min) than in GH- or PRLR-deficient mice (15 min) or wild-type mice (20.5 min). By 12 months, the hypoglycemic response to insulin in double-mutant mice was blunted (P < 0.01 vs. wild types), and the time to reach half-maximal effect was greater in double-mutant (16.4 min), PRLR-deficient (15.8 min), and wild-type mice (15.9 min) than in GH-deficient mice (12.5 min). By 18 months of age, the sensitivity to insulin had declined in all groups; the maximal effect of insulin was now blunted in GH-deficient mice but even more so in double-mutant mice. Studies in PRLR-deficient males at this age were not performed because the available number of mice was inadequate for statistical analysis.
FIG. 9. Developmental changes in insulin sensitivity in wild-type and mutant males. Tail blood glucose concentrations were measured before and after the administration of Humalog insulin (0.75 U/kg body weight). Values represent the mean ± SE of three to eight male mice at 3 ± 1, 12 ± 2, and 18 ± 2 months of age. Solid circles, Wild type; open squares, PRLR deficient; open triangles, GH deficient; stars, double mutant. Studies in PRLR-deficient males were not performed at 18 months because the number of mice available was inadequate for statistical analysis. The regression curves were calculated with GraphPad software using a one-phase exponential decay model.
The insulin resistance in double-mutant males was accompanied by glucose intolerance (Fig. 10). After the ip administration of 10% dextrose (Fig. 10A), blood glucose levels in wild-type males peaked (227.2 ± 21.3 mg%, n = 7) at 10 min and returned to baseline by 70–80 min. In PRLR-deficient males, peak blood glucose levels were higher (249.3 ± 19.0 mg%, n = 8, P < 0.05) and peaked later (20 min) than in wild-type males; glucose values returned to near baseline by 80 min. Similar findings were noted in GH-deficient mice (peak blood glucose levels 319.5 ± 16.3 mg% at 20 min, n = 7, P < 0.01). In contrast, glucose levels in double-mutant males (335.3 ± 30.3 mg% at 20 min, n = 8, P < 0.05–0.01 at 10–70 min) exceeded those in all other groups and remained elevated at 75–100 min.
FIG. 10. A, Glucose tolerance in adult wild-type and mutant males. Tail blood glucose concentrations were measured before and after the ip administration of 10% dextrose (1 g/kg). B, Glucagon tolerance in adult wild-type and mutant males. Tail blood glucose concentrations were measured before and after the ip administration of glucagon (0.04 mg/kg). Values represent mean ± SE of seven to eight mice in each group at 10 ± 2 months of age. GHD, GH deficient; GTT, glucose tolerance test.
Similar changes were noted after ip administration of glucagon (Fig. 10B). Blood glucose rose to higher levels in double-mutant males (306.5 ± 28.2, n = 13, P < 0.05) than in wild-type (214 ± 27.8 mg%, n = 3), PRLR-deficient (234.6 ± 34.4 mg%, n = 5), or GH-deficient (244.0 ± 4.5 mg%, n = 3) males and failed to decline significantly even after 30 min. Collectively, the measurements of plasma insulin concentrations, the glucose responses to insulin administration and the results of the glucose and glucagon tolerance tests suggest that double-mutant males have defective first-phase insulin secretion as well as decreased peripheral insulin sensitivity.
Discussion
The generation of lactogen-resistant/GH-deficient mice provided a means to compare the effects of combined prenatal and postnatal defects in lactogen and GH signaling with the effects of isolated defects in lactogen action or GH production. Eight findings from our studies of the mutant mice are of note. First, the proportions of viable PRLR-deficient, GH-deficient, and double-mutant mice at birth conformed to expected Mendelian ratios, suggesting that neither lactogens nor GH is required for intrauterine survival in the mouse. Second, the combination of lactogen resistance and GH deficiency reduced weight gain during the early postnatal period (d 7). In contrast, mice with isolated lactogen resistance or GH deficiency had normal weights on d 7 of life. Third, double-mutant mice had perinatal hypoglycemia despite low plasma insulin concentrations, suggesting increased insulin sensitivity. Fourth, PRLR-deficient mice had normal plasma IGF-I, IGF-II, and IGFBP-3 concentrations, and the reductions in IGF and IGFBP levels in double-mutant mice were no more severe than those in GH-deficient mice; these findings suggest that lactogens play little or no role in postnatal IGF/IGFBP-3 expression (at least at or after 1 wk of life). Fifth, double-mutant males developed insulin resistance and glucose intolerance by 8–12 months of age. In contrast, adult males with isolated GH deficiency remained insulin responsive through a year of age but had a blunted response to insulin by 18 months. Sixth, the development of insulin resistance and glucose intolerance in double-mutant males was accompanied by excess fat deposition and a reduction in lean body mass. Seventh, the rates of weight gain and fat accumulation in PRLR-deficient and double-mutant males exceeded the rates of weight gain and fat deposition in PRLR-deficient and double-mutant females during the first year of life. Finally, the maintenance of the obese phenotype in aging double-mutant mice could not be explained readily by excess food intake, suggesting that reduced energy expenditure plays an important role.
Despite numerous investigations, the roles of GH and the lactogens in perinatal growth remain highly controversial. PL and GH circulate in fetal mouse and rat serum, and PRL secretion begins soon after birth (37, 38, 39). GH and PRL receptors are expressed widely in rodent fetal tissues (40, 41, 42, 43), and the somatogens and lactogens exert anabolic effects in isolated fetal and neonatal hepatocytes, astrocytes, pancreatic islets, and mesenchymal tissues (20, 21, 22, 23, 24, 25, 26, 27). Nevertheless, weight gain is normal during the first 1–2 wk after birth in Little, Ames (Prop-1-deficient), Snell (Pit-1-deficient), and GH-resistant dwarf mice and transgenic mice that overexpress GH (30, 44, 45, 46, 47, 48).
It is not surprising, therefore, that our mice with isolated GH deficiency or lactogen resistance had normal body weights at 7 d of life. However, mice with combined lactogen resistance and GH deficiency were approximately 10% lighter than wild-type mice at this age. It is currently unclear whether the growth failure reflects in part the resistance to PL action in utero; to answer this question, it will be necessary to conduct detailed studies of fetal growth in the various groups of mice. In any case, the data suggest that the lactogens and GH may act in concert to facilitate mouse growth and/or weight gain in the perinatal period and that concurrent production of one or more members of the somatolactogen family may preserve mouse perinatal weight gain in the absence of another family member (39, 49).
These findings may have implications for human fetal and perinatal growth. In humans, PL, GH, and PRL circulate at high concentrations in the fetus (39, 50), and GH and PRL are overexpressed during the neonatal period (51). Moreover, human fetal tissues, like rodent fetal tissues, express GH and PRL receptors (52, 53, 54). Yet birth weight is often normal in patients with GH deficiency or GH resistance, although body mass index may be increased owing to excess fat deposition (55, 56). Systematic studies of birth weight and length in children with Pit-1 or Prop-1 mutations have not been completed, so the effects of combined GH and PRL deficiencies on human fetal and perinatal growth are unknown. Nevertheless, such patients continue to produce PL and decidual (amniotic fluid) PRL, which might obscure the effects of pituitary GH and PRL deficiencies (39). Moreover, PRL production does not decline in some cases until 5 yr or more after birth.
Linear growth during postnatal life is controlled by the GH/IGF axis. The findings of our studies in GH-deficient little mice are consistent with previous observations: the adult little mice had striking reductions in femur length, bone mineral density, and plasma IGF-I and IGFBP-3 concentrations (57). The defects in IGF/IGFBP expression in adult double-mutant mice were no more severe than those in GH-deficient mice; indeed, plasma IGF-I and IGFBP-3 concentrations were slightly higher in older (obese) double mutants than in GH-deficient mice. In addition, mice with isolated PRLR deficiency had normal femoral growth and plasma IGF-I and IGFBP-3 concentrations. Collectively, these findings suggest that lactogens have little or no effect on linear growth or IGF-I/IGFBP-3 production after the perinatal period. Moreover, concurrent PRL expression apparently cannot restore or maintain postnatal IGF/IGFBP production in the setting of GH deficiency. Bone mineral density was reduced to a small degree in PRLR-deficient females but not males; this finding may reflect the effects of longstanding hypoestrogenemia in female lactogen-resistant mice (58).
Human infants with GH deficiency are predisposed to fasting hypoglycemia, presumably because of increased insulin sensitivity (59). The findings of our studies are consistent with clinical observations: perinatal mice with GH deficiency were normoglycemic despite hypoinsulinemia. Interestingly, double-mutant mice were hypoglycemic on d 7 despite comparable hypoinsulinemia. The mechanism for this effect is unclear, but perinatal hypoglycemia could in theory result from defects in hepatic glycogen synthesis (60) and/or glucose production as well as heightened insulin action. In contrast to GH-deficient or double-mutant mice, PRLR-deficient mice were neither hypoglycemic nor hypoinsulinemic. Presumably, any effects of isolated lactogen resistance on hepatic glycogenesis, glucose production, or insulin sensitivity in the perinatal period might be obscured by concurrent GH action.
Striking changes in insulin secretion and insulin action were observed in the double-mutant mice during development. At 3 months of age, the double mutants had fasting hypoglycemia, hypoinsulinemia, and heightened insulin sensitivity. In contrast, older double-mutant males had fasting hyperglycemia, relative hypoinsulinemia, peripheral insulin resistance, and glucose intolerance. The relative hypoinsulinemia may reflect the loss of PRL and GH signaling in the pancreatic ?-cell (1, 2, 3, 4, 34) and/or the marked reduction in plasma IGF-I (61). In theory, the IGF-I deficiency may also contribute to defects in insulin action (62); however, GH-deficient males, whose levels of IGF-I were comparable with or lower than those of double-mutant males had fasting hypoinsulinemia and a normal hypoglycemic response to insulin at 12 months of age.
The reduction in insulin sensitivity in aging double-mutant mice may be caused, at least in part, by a progressive increase in fat deposition (63). The pathogenesis of fat deposition in the double mutants is currently unclear. GH stimulates white adipose tissue (WAT) lipolysis and inhibits lipogenesis, whereas GH deficiency limits fat mobilization and increases WAT stores. Lactogenic hormones have variable and inconsistent effects on WAT lipogenesis and lipolysis in vitro but may induce lipolysis in vivo (6, 7, 12, 13, 14, 15, 16); thus, the combination of lactogen resistance and GH deficiency may exacerbate fat deposition and weight gain.
The obesity of the double-mutant males cannot be explained readily by excess energy intake; food intake in older double mutants was lower than in wild-type mice, possibly a consequence of hyperleptinemia. The suppression of food intake suggests that maintenance of excess fat stores in older animals may involve reduced energy expenditure as well as impaired fat mobilization. A reduction in energy expenditure might be expected because percent lean body mass (the major determinant of resting energy expenditure) is decreased in obese double-mutant mice.
Interestingly, females with isolated PRLR deficiency, in contrast to males, had slightly reduced rates of weight gain after 4 months of age. Gender-dependent differences in metabolic responses to PRL have been described previously; for example, PRL stimulates food intake and reduces brown adipose tissue uncoupling protein 1 expression in female but not male rats (64, 65, 66, 67) and increases resistin expression in adipose tissue of male but not female mice (68). Differences in weight gain between PRLR-deficient males and females might reflect differences in gonadal function; PRLR-deficient males have normal testosterone levels, whereas females have severe hypoprogesteronemia and less severe hypoestrogenemia (58).
Like the effect of lactogen resistance, the effect of GH deficiency on weight gain appears to vary according to gender: relative to double-mutant and GH-deficient males, double-mutant and GH-deficient females have reduced rates of weight gain until later stages of development. In theory, effects of lactogen resistance or GH deficiency on adipogenesis (15, 17, 18, 19), lipolysis (69), and/or energy expenditure (67) might vary independently with age and gender. In addition, the magnitude of the defect in hormone signaling may be rate limiting; for example little mice, which produce GH at 5–10% of normal levels, accumulate excess WAT with age, whereas GH receptor knockout mice have reduced white adipose stores (48, 69). This finding suggests that low-level expression of GH may be adequate for adipogenesis in young (and possibly adult) mice but may not suffice for lipolysis or antilipogenesis in adulthood.
The mice used in our studies were developed on a hybrid background (C57BL6/129). We took a number of steps minimize the effects of genetic background on our results. These included: 1) the use of wild-type littermates of the same mixed genetic background as controls; 2) the use of mice from several litters of comparable age; and 3) the use of large numbers of mice in each experimental group. Variability in genetic background may explain in part the variability in weight gain and body fat deposition that we observed within individual groups of mice because the C57BL/6 background has been shown to facilitate weight gain in mice on high-fat diets (70). However, several lines of evidence suggest that metabolic defects in the lactogen-resistant and GH-deficient mice largely reflect differences in hormone production and action rather than genetic background per se. Like purebred GH-deficient little mice on a C57BL/6 background (44, 57), the hybrid GH-deficient mice had postnatal growth failure, catch-up weight gain, decreased bone mineral density, low plasma IGF-I and IGFBP-3 concentrations, fasting hypoglycemia and hypoinsulinemia, and increased insulin sensitivity. Like PRLR-deficient mice bred on a 129 background (29), the hybrid PRLR-deficient females are sterile and exhibit aberrant maternal behavior (our unpublished observations). PRLR-deficient females on the hybrid background, like PRLR-deficient females on a 129 background, have slightly decreased weight gain and fat deposition with age. Moreover, the mild glucose intolerance observed in our hybrid PRLR-deficient males is similar to or identical with that observed in purebred 129 PRLR-deficient mice (34). Thus, the mixed genetic background did not appear to alter markedly the phenotypes associated with lactogen resistance or GH deficiency.
The relevance of our studies for the pathogenesis of human metabolic disorders is currently unclear. Mounting evidence suggests that human infants and rodents that are growth retarded in utero or in infancy are predisposed to adult obesity, hypertension, dyslipidemia, and glucose intolerance (the metabolic syndrome) (71). The effects of perinatal growth failure appear to be most severe when there is rapid catch-up growth or weight gain during childhood (72, 73, 74). Because our double-mutant male mice have perinatal growth failure, catch-up weight gain, and late-onset obesity and glucose intolerance, the lactogen-resistant/GH-deficient mouse may serve as a model for the metabolic syndrome.
Acknowledgments
The authors thank Anne Petro and Rama Arumugam for technical assistance.
References
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Address all correspondence and requests for reprints to: Dr. Michael Freemark, Division of Pediatric Endocrinology and Diabetes, Box 3080, Duke University Medical Center, Durham, North Carolina 27710. E-mail: freem001@mc.duke.edu.
Abstract
To delineate the roles of the lactogens and GH in the control of perinatal and postnatal growth, fat deposition, insulin production, and insulin action, we generated a novel mouse model that combines resistance to all lactogenic hormones with a severe deficiency of pituitary GH. The model was created by breeding PRL receptor (PRLR)-deficient (knockout) males with GH-deficient (little) females. In contrast to mice with isolated GH or PRLR deficiencies, double-mutant (lactogen-resistant and GH-deficient) mice on d 7 of life had growth failure and hypoglycemia. These findings suggest that lactogens and GH act in concert to facilitate weight gain and glucose homeostasis during the perinatal period. Plasma insulin and IGF-I and IGF-II concentrations were decreased in both GH-deficient and double-mutant neonates but were normal in PRLR-deficient mice. Body weights of the double mutants were reduced markedly during the first 3–4 months of age, and adults had striking reductions in femur length, plasma IGF-I and IGF binding protein-3 concentrations, and femoral bone mineral density. By age 6–12 months, however, the double-mutant mice developed obesity, hyperleptinemia, fasting hyperglycemia, relative hypoinsulinemia, insulin resistance, and glucose intolerance; males were affected to a greater degree than females. The combination of perinatal growth failure and late-onset obesity and insulin resistance suggests that the lactogen-resistant/GH-deficient mouse may serve as a model for the development of the metabolic syndrome.
Introduction
PLACENTAL LACTOGEN (PL), prolactin (PRL), and GH constitute a somatolactogen superfamily of polypeptides that have similarities in structure and biological activity. Through binding to cognate PRL and GH receptors in target tissues, the lactogens (PL and PRL) and somatogens (GH) regulate reproductive behavior and function, mammary growth and milk protein production, carbohydrate, lipid, protein, and mineral metabolism, and postnatal growth.
Experimental studies have elucidated the molecular mechanisms by which the lactogens and somatogens exert their biological actions (1). But similarities in their biological activities have made it difficult to define precisely the respective roles of PL, PRL, and GH in whole-body metabolism. For example, both the lactogens and somatogens induce ?-cell proliferation and insulin production in pancreatic islets and insulinoma cells (2, 3, 4, 5). At supraphysiological concentrations the lactogens inhibit insulin-dependent glucose uptake in isolated adipocytes and reduce insulin sensitivity (6, 7, 8, 9, 10, 11), mimicking the effects of GH. Human PL, which is related structurally to human GH, is reported to stimulate lipogenesis and lipolysis in isolated fat cells (6, 7, 12, 13); the rodent PLs, related more closely to rodent PRLs, have little or no lipolytic activity in vitro but may promote lipolysis in vivo (14, 15, 16). Both PRL and GH induce adipogenesis in rodent preadipocyte cell lines (15, 17, 18, 19) and mitogenesis in fetal and neonatal hepatocytes (20) and astrocytes (21, 22). Finally, human PL, rat PL-1, and mammalian PRLs enhance the growth of isolated rat yolk sacs and embryos (23, 24, 25) in culture and may increase IGF-I production in fetal tissues (20, 24, 26, 27), whereas GH is the major determinant of linear growth and IGF-I production during the postnatal period (28).
Thus, the metabolic actions of the lactogens appear to overlap with those of GH in some systems and complement those of GH in others. We hypothesized that this redundancy of hormone action may enable one member of the somatolactogen family to compensate for the absence of another, thereby obscuring or modulating the effect of an isolated hormone deficiency. In a similar vein, we theorized that a combination of defects in lactogen and GH signaling might aggravate the metabolic dysfunction that accompanies an isolated signaling defect.
To test these hypotheses, we generated a novel mouse model that combines resistance to all lactogenic hormones with a severe deficiency of pituitary GH. The model was created by breeding PRL receptor (PRLR)-deficient male mice, which are resistant to the actions of mouse PL as well as mouse PRL (29), with GH-deficient little females, which have fetal and postnatal hyposomatotropism (GH levels 5% of normal) due to a mutation in the GHRH receptor (30, 31). The confluence of defects in lactogen and GH signaling abolishes compensatory effects arising from overlapping actions of the lactogenic and somatogenic hormones.
We compared the weight gain and fat deposition of lactogen-resistant/GH-deficient (double-mutant) mice with those of wild-type, PRLR-deficient, and GH-deficient mice in the perinatal (d 7) and postnatal (d 30–650) periods. We then assessed the relationships among weight gain; glucose tolerance; insulin production; insulin action; and the plasma concentrations of IGF-I, IGF-II, IGF binding protein (IGFBP)-3, and leptin.
Materials and Methods
Generation of PRLR-deficient/GH-deficient double-mutant mice
Homozygous PRLR-deficient males (129 background) were mated with homozygous GH-deficient little females (C57BL/6 background, from Jackson Labs, Bar Harbor, ME) to yield mice that were heterozygous at both loci on a hybrid 129/Bl6 background. Double-heterozygous males and females were bred to produce wild-type, PRLR-deficient, GH-deficient, and double-mutant mice. The various genotypes were identified through PCR analysis as described previously (30, 32). All subsequent experiments were performed in mice on the hybrid 129/Bl6 background.
The mice were housed under nonsterile conditions in rooms with 12-h light, 12-h dark cycles. Food and water were provided ad libitum. The mouse chow was a standard preparation (Laboratory Rodent diet 5001, Ralston Purina Co., St. Louis, MO) containing 12.1% of calories as fat, 28% as protein, and 59.8% as carbohydrate.
All animal protocols were approved by the Duke University Medical Center Institutional Animal Care and Use Committee and followed federal guidelines.
Auxologic measurements and body fat stores
Body weight measurements were obtained on d 7 of life, and the neonates were killed at 1000 h to obtain trunk blood. Additional measurements of body weight were obtained at various time points between 30 and 700 d of age in 10 wild-type females, 10 wild-type males, 13 PRLR-deficient females, eight PRLR-deficient males, eight GH-deficient females, 16 GH-deficient males, 18 double-mutant females, and 23 double-mutant males. All animals were virginal.
Body fat stores, femur lengths, and femoral bone mineral densities were estimated by dual-energy x-ray absorptiometry using the Lunar Piximus densitometer (General Electric Medical Systems); individual mice were anesthetized with pentobarbital and scanned three times. The Lunar software provides measurements of fat mass and lean tissue mass; values are expressed as a percent of bone-free body weight. Each measurement of body fat and lean tissue mass excluded the head. Measurements of fat mass in mice obtained with the Lunar Piximus are precise and reproducible and correlate very strongly (r2 = 0.97) with measurements of fat mass obtained by chemical extraction (33).
Food intake in double-mutant mice
Food intake was measured during a 7-d period under standard conditions (12-h light, 12-h dark cycles, free access to food and water, environmental temperature 23 C); experimental data were expressed as grams food per day per gram body weight. The feeding studies were performed three times to ensure the reproducibility of the results.
Glucose, insulin, and leptin concentrations and insulin sensitivity were measured using methods described previously (32, 34). Plasma glucose concentrations were measured using a One-Touch Ultra glucometer (Lifescan, Milpitas, CA). Plasma insulin concentrations were measured using the ultrasensitive rat insulin RIA from Linco Corp. (St. Louis, MO); the sensitivity of the assay is 0.2 ng/ml. Fasting glucose and insulin concentrations were measured at 0900 h after an overnight (16 h) fast. Plasma glucose and insulin concentrations were also measured 30 min after ip administration of 10% dextrose (10 ml/kg body weight). In separate experiments we assessed glucose tolerance by measuring plasma glucose concentrations every 10 min after the intraperitoneal injection of 10% dextrose in water (10 ml/kg body weight) or after the ip administration of glucagon (0.04 mg/kg body weight, Bedford Labs, Bedford, OH). The glucose tolerance and glucagon tolerance tests were performed after 2 h of fasting. Insulin sensitivity was assessed (34) in nonfasted mice by measuring the rate of fall in blood glucose concentrations after an ip injection of Humalog insulin (1 U/kg body weight, Eli Lilly Corp., Indianapolis, IN). Plasma leptin concentrations were measured in nonfasted male mice and fasted (16 h) female mice using the Linco assay; the sensitivity of this assay is 0.2 ng/ml. Intra- and interassay variations of all assays were less than 15%.
IGF-I, IGF-II, and IGFBP-3 concentrations
IGF-I and IGF-II were measured by RIA after extraction of IGF binding proteins on a BioGel P-10 (Bio-Rad, Hercules, CA) column equilibrated in 1 M acetic acid. The methods have been validated for complete separation of IGFBPs from IGFs in serum and other biological fluids (35). The IGF-I assay uses recombinant human IGF-I as standard and tracer and a rabbit polyclonal antiserum. The IGF-II assay uses recombinant human IGF-II as standard and tracer and a mouse monoclonal antibody. The assays can be performed using 12.5 μl plasma. The intra- and interassay coefficients of variation for each assay are less than 10%. The sensitivities of the IGF-I and -II assays are 20 and 50 pg/ml, respectively. Plasma IGFBP-3 concentrations were measured by densitometric analysis of Western ligand blots, as previously described (36)
Data analysis
The breeding of double heterozygotes is predicted to yield only one homozygous wild-type, one PRLR-deficient, one GH-deficient, and one double-mutant mouse for every 16 pups. Because litters typically contained 5–11 pups, no single litter contained both males and females of the four genotypes of interest. Consequently, it was impossible to perform direct statistical comparisons within a single litter. We therefore used animals from additional litters of comparable age as well as true littermates.
All auxologic data and measurements of glucose, insulin, and leptin concentrations were expressed as mean ± SE. Statistical differences in weight gain during development among the various groups of mice were assessed by two-way ANOVA followed by the Bonferroni test of comparisons. All biochemical studies were repeated at least three times, using duplicate or triplicate replicates in each experiment. Differences among sample means were assessed by ANOVA followed by the Newman-Keuls test of multiple comparisons. P < 0.05 was considered statistically significant.
Results
Intrauterine and postnatal viability
Breeding of double-heterozygous males and females yielded PRLR-deficient, GH-deficient, double-mutant, and wild-type animals at the expected Mendelian ratios. The postnatal mice thrived under nonsterile conditions.
Weight gain, plasma glucose, and insulin and IGF concentrations in the perinatal period
To assess the effects of lactogen resistance and GH deficiency on perinatal growth and carbohydrate tolerance, we compared the weights and plasma glucose, insulin, and IGF concentrations of 7-d-old double-mutant mice with those of PRLR-deficient, GH-deficient, and wild-type mice. Data from male and female mice on d 7 were combined because previous studies (Ref. 28 and our unpublished observations) demonstrated no significant gender-dependent differences in mouse weight gain at this age. In addition, data from homozygous wild-type mice on d 7 were combined with data from 7-d-old mice that were heterozygous for either the PRLR or GHRH receptors because we found no significant differences in body weight, blood glucose, or plasma insulin, IGF-I, or IGF-II concentrations between wild-type and heterozygous mice at this stage of development (Table 1 and Figs. 1 and 2).
TABLE 1. Perinatal (d 7) weight gain and blood glucose concentrations in wild-type and heterozygous (heteros) mice and mutant mice (males and females combined)
FIG. 1. Plasma insulin concentrations (A) and glucose to insulin ratios (B) in mice on postnatal d 7. Values represent the mean ± SE of wild-type/heterozygous controls (cont, n = 32), PRLR-deficient mice (n = 11), GH-deficient (GHD) mice (n = 18), and double-mutant mice (n = 13). Data from males and females were combined.
FIG. 2. Plasma IGF-I and IGF-II concentrations in mice on postnatal d 7. Values represent the mean ± SE of wild-type/heterozygous controls (cont, n = 11), PRLR-deficient (n = 6) mice, GH-deficient (GHD, n = 14) mice, and double-mutant mice (n = 14). Data from males and females were combined.
As shown in Table 1, the weights of PRLR-or GH-deficient mice were normal on d 7. However, body weights and plasma glucose concentrations were reduced 11 (P < 0.05) and 16% (P < 0.01), respectively, in perinatal mice with combined lactogen resistance and GH deficiency (double mutants).
Random plasma insulin concentrations were reduced in double-mutant mice as well as GH-deficient mice (Fig. 1A) on d 7 of life. Because blood glucose concentrations were normal in GH-deficient mice, the glucose to insulin ratio was increased (Fig. 1B); these findings suggest heightened insulin sensitivity. In double-mutant mice, both glucose and insulin levels were low; therefore, the glucose to insulin ratio was normal. Isolated resistance to lactogenic hormones (PRLR deficiency) had no effect on blood glucose, insulin, or the glucose to insulin ratio at this age.
The reductions in body weight in the double mutants on d 7 were accompanied by small reductions in plasma IGF-I and IGF-II concentrations (Fig. 2). IGF concentrations in GH-deficient mice were comparable with those in double-mutant mice. However, there was no correlation between IGF-I concentrations and body weight in the mice at this age (n = 82, r = 0.1), and IGF-II concentrations correlated inversely although weakly with body weight (r = –0.225, P < 0.05). In contrast, random insulin (n = 41, r = 0.51, P < 0.001) and glucose (n = 103, r = 0.263, P = 0.007) concentrations and pancreas weight (n = 103, r = 0.83, P < 0.001) correlated positively with body weight on d 7.
Weight gain, femur length, bone mineral density, and fat deposition during postnatal life and aging
We next compared the weight gain of double-mutant mice during postnatal life with that of PRLR-deficient, GH-deficient, and wild-type littermates (n = 8–23 in each gender and genotype). Our studies of postnatal weight gain and metabolic function used only homozygous wild-type mice and mice with homozygous deletions of the GH and/or PRL receptors. As shown in Fig. 3, females with isolated PRLR deficiency gained weight normally until approximately 4 months of age; thereafter, weight gain was blunted, and older PRLR-deficient females weighed on average 8–10% less than wild-type females. In contrast, weight gain in PRLR-deficient males was normal. The weight deficiency of GH-deficient females persisted through 500 d, whereas GH-deficient males had some catch-up weight gain beginning at 120–180 d of life. Weight gain of double-mutant females was comparable with that of GH-deficient females until approximately 1 yr of age; thereafter, body weight appeared to increase slightly. In contrast, catch-up weight gain in double-mutant males was more dramatic than in double-mutant females and began around 160 d of life. By 600 d of life, the weights of the double-mutant males were comparable with, and in some cases exceeded, the weights of wild-type males.
FIG. 3. Postnatal weight gain in wild-type and mutant mice. Measurements of body weight were obtained at various times between 30 and 700 d of age in 10 homozygous wild-type females, 10 wild-type males, 13 PRLR-deficient females, eight PRLR-deficient males, eight GHD females, 16 GHD males, 18 double-mutant females, and 23 double-mutant males; all animals were virginal. The regression lines were calculated with GraphPad software (GraphPad Inc., San Diego, CA) using a nonlinear one-site binding model. Analysis of goodness of fit showed correlation coefficients (r) ranging from 0.77 to 0.82 in the four groups of female mice and 0.84 to 0.91 in the male mice.
Two-way ANOVA demonstrated significant differences in rates of weight gain between PRLR-deficient and wild-type females (P < 0.02) and between GH-deficient and double-mutant males (P < 0.01) after 120 d of age. There were no significant differences between GH-deficient and double-mutant females or between PRLR-deficient and wild-type males.
The accelerated weight gain in double-mutant males did not reflect an increase in linear growth; femur lengths of adult double-mutant males and females (age 12 ± 2 months) were only 72% as great as those of wild-type males (P < 0.001), and plasma IGF-I and IGFBP-3 concentrations and femoral bone mineral density were markedly reduced (Table 2). The magnitude of reductions in femur length and IGF and IGFBP-3 levels in GH-deficient mice were comparable with (or more dramatic than) those in double-mutant mice, suggesting that lactogen resistance had little or no effect on postnatal linear growth or IGF/IGFBP-3 expression. Isolated PRLR deficiency, however, reduced bone mineralization slightly in females but not males (Table 2).
TABLE 2. Parameters of growth and bone mineralization in adult wild-type (WT) and mutant mice
Body fat, expressed as a percent of body weight, was normal in PRLR-deficient (23.0 ± 4.4%) and GH-deficient males (25.6 ± 3.3%) at 10 months of age but was increased in double-mutant males (38.9 ± 3.8%); the corresponding value in wild-type mice was 21.5 ± 3.4% (n = 3–5 in each group). At 500 d, body fat increased in all groups but remained highest in double-mutant males (wild type 32.0 ± 2.6%, GH deficient 40.2 ± 4.8%, double mutant 48.6 ± 2.7%; the number of PRLR-deficient males at this age was not adequate for statistical analysis). Percent body fat relative to femur length, an indirect measure of body mass index, was increased in double-mutant males (Fig. 4A). Conversely, lean body mass, expressed as a percent of body weight, was low (wild type 68.5 ± 2.5%; PRLR 72.1 ± 3.3%; GH deficient 59.8 ± 2.5%, P < 0.05 vs. wild type; double mutant 46.6 ± 4.2%, P < 0.02 vs. wild type, mean ± SE, n = 3–7 in each group, Fig. 4B). Relative fat deposition in males with isolated GH deficiency also exceeded that in wild-type and PRLR-deficient males, but adiposity was more severe in double-mutant males.
FIG. 4. A, Percent body fat relative to femur length in male mice. Percent body fat and femur length were estimated by dual-energy x-ray absorptiometry analysis at 10 ± 2 months of age. B, Lean body mass of male mice, expressed as a percent of body weight. Values represent the mean ± SE of eight wild-type males, three PRLR males, 11 GH-deficient (GHD) males, and nine double males.
There were no significant differences in body fat among the groups of female mice at 120 d (wild type 13.0 ± 1.5%, PRLR deficient 11.2 ± 2.3%, GH deficient 20.8 ± 2.1%, double mutant 20.9 ± 2.0%, n = 3–5 in each group). In aging females (500–600 d), however, percent body fat was increased in double-mutant mice (49.9 ± 9.8%, P < 0.05) relative to wild-type (33.5 ± 2.8%), PRLR-deficient (27.8 ± 2.2%), and GHD mice (31.2 ± 6.1%, n = 3–8 in each group).
Changes in plasma leptin concentrations mirrored changes in body fat content; leptin levels were increased in adult double-mutant males and aging double-mutant females (Fig. 5) but were reduced slightly in adult PRLR-deficient females. Food intake in double-mutant males was normal or slightly increased at 90 d of age but decreased at 15 months of age (Fig. 6).
FIG. 5. Plasma leptin concentrations in adult wild-type and mutant mice. Values represent the mean ± SE of fasted (16 h) female mice (n = 3–5 in each group) and nonfasted male mice (n = 5–12 in each group) at 15 ± 3 months of age.
FIG. 6. Food intake in wild-type and double-mutant male mice. Values represent the mean ± SE of groups of three to five mice in three experiments at 3 ± 1 months and three experiments at 15 ± 2 months.
Developmental changes in insulin sensitivity and glucose tolerance
The increase in adiposity in double-mutant males during aging was accompanied by striking changes in fasting glucose, insulin concentrations, and insulin sensitivity. At 3 months of age, fasting glucose levels in double-mutant males and females were lower than those in wild-type, PRLR-deficient, or GH-deficient mice (Fig. 7). By age 12 months, however, fasting glucose concentrations in double-mutant males were higher than those in wild-type, PRLR-deficient, or GH-deficient males. In contrast, fasting glucose levels in older double-mutant females were comparable with those in GH-deficient females and lower than in wild-type or PRLR-deficient females.
FIG. 7. Fasting glucose levels in wild-type and mutant mice. Values represent the mean ± SE of four to eight mice of each gender and genotype at 3 ± 1 months of age and 12 ± 2 months of age.
Fasting insulin levels in adult double-mutant females, like those in adult GH-deficient females, were lower than those in wild-type or PRLR-deficient mice (Fig. 8). In contrast, fasting insulin concentrations in adult double mutant males were comparable with those in wild-type or PRLR-deficient males and higher than those in GH-deficient males. Thirty minutes after the ip administration of 10% dextrose (1 g/kg), the ratio of glucose (mg%) to insulin (nanograms per milliliter) was increased in GH-deficient males (183 ± 12 mg/ng vs. wild types 112 ± 17 mg/ng, mean ± SE, P < 0.05) but was normal in PRLR-deficient (135 ± 15 mg/ng) and double-mutant (84 ± 26 mg/ng) males (n = 3–6 in each group).
FIG. 8. Fasting insulin levels in adult mice. Values represent the mean ± SE of four to eight mice of each gender and genotype at 10–12 months of age. GHD, GH deficient.
The combination of normoinsulinemia with fasting hyperglycemia in the double-mutant males suggests relative hypoinsulinemia and/or insulin resistance. That the acquired obesity of double-mutant male mice is accompanied by insulin resistance is suggested by the results of insulin tolerance tests (Fig. 9). At 3 months of age, the rate of decline in blood glucose after insulin administration was more precipitous in double-mutant (P < 0.001) or GH-deficient (P < 0.002) males than in wild-type males. The effect of PRLR deficiency (P = 0.08) did not reach statistical significance. The calculated half-time to reach maximal effect was less in double-mutant mice (12 min) than in GH- or PRLR-deficient mice (15 min) or wild-type mice (20.5 min). By 12 months, the hypoglycemic response to insulin in double-mutant mice was blunted (P < 0.01 vs. wild types), and the time to reach half-maximal effect was greater in double-mutant (16.4 min), PRLR-deficient (15.8 min), and wild-type mice (15.9 min) than in GH-deficient mice (12.5 min). By 18 months of age, the sensitivity to insulin had declined in all groups; the maximal effect of insulin was now blunted in GH-deficient mice but even more so in double-mutant mice. Studies in PRLR-deficient males at this age were not performed because the available number of mice was inadequate for statistical analysis.
FIG. 9. Developmental changes in insulin sensitivity in wild-type and mutant males. Tail blood glucose concentrations were measured before and after the administration of Humalog insulin (0.75 U/kg body weight). Values represent the mean ± SE of three to eight male mice at 3 ± 1, 12 ± 2, and 18 ± 2 months of age. Solid circles, Wild type; open squares, PRLR deficient; open triangles, GH deficient; stars, double mutant. Studies in PRLR-deficient males were not performed at 18 months because the number of mice available was inadequate for statistical analysis. The regression curves were calculated with GraphPad software using a one-phase exponential decay model.
The insulin resistance in double-mutant males was accompanied by glucose intolerance (Fig. 10). After the ip administration of 10% dextrose (Fig. 10A), blood glucose levels in wild-type males peaked (227.2 ± 21.3 mg%, n = 7) at 10 min and returned to baseline by 70–80 min. In PRLR-deficient males, peak blood glucose levels were higher (249.3 ± 19.0 mg%, n = 8, P < 0.05) and peaked later (20 min) than in wild-type males; glucose values returned to near baseline by 80 min. Similar findings were noted in GH-deficient mice (peak blood glucose levels 319.5 ± 16.3 mg% at 20 min, n = 7, P < 0.01). In contrast, glucose levels in double-mutant males (335.3 ± 30.3 mg% at 20 min, n = 8, P < 0.05–0.01 at 10–70 min) exceeded those in all other groups and remained elevated at 75–100 min.
FIG. 10. A, Glucose tolerance in adult wild-type and mutant males. Tail blood glucose concentrations were measured before and after the ip administration of 10% dextrose (1 g/kg). B, Glucagon tolerance in adult wild-type and mutant males. Tail blood glucose concentrations were measured before and after the ip administration of glucagon (0.04 mg/kg). Values represent mean ± SE of seven to eight mice in each group at 10 ± 2 months of age. GHD, GH deficient; GTT, glucose tolerance test.
Similar changes were noted after ip administration of glucagon (Fig. 10B). Blood glucose rose to higher levels in double-mutant males (306.5 ± 28.2, n = 13, P < 0.05) than in wild-type (214 ± 27.8 mg%, n = 3), PRLR-deficient (234.6 ± 34.4 mg%, n = 5), or GH-deficient (244.0 ± 4.5 mg%, n = 3) males and failed to decline significantly even after 30 min. Collectively, the measurements of plasma insulin concentrations, the glucose responses to insulin administration and the results of the glucose and glucagon tolerance tests suggest that double-mutant males have defective first-phase insulin secretion as well as decreased peripheral insulin sensitivity.
Discussion
The generation of lactogen-resistant/GH-deficient mice provided a means to compare the effects of combined prenatal and postnatal defects in lactogen and GH signaling with the effects of isolated defects in lactogen action or GH production. Eight findings from our studies of the mutant mice are of note. First, the proportions of viable PRLR-deficient, GH-deficient, and double-mutant mice at birth conformed to expected Mendelian ratios, suggesting that neither lactogens nor GH is required for intrauterine survival in the mouse. Second, the combination of lactogen resistance and GH deficiency reduced weight gain during the early postnatal period (d 7). In contrast, mice with isolated lactogen resistance or GH deficiency had normal weights on d 7 of life. Third, double-mutant mice had perinatal hypoglycemia despite low plasma insulin concentrations, suggesting increased insulin sensitivity. Fourth, PRLR-deficient mice had normal plasma IGF-I, IGF-II, and IGFBP-3 concentrations, and the reductions in IGF and IGFBP levels in double-mutant mice were no more severe than those in GH-deficient mice; these findings suggest that lactogens play little or no role in postnatal IGF/IGFBP-3 expression (at least at or after 1 wk of life). Fifth, double-mutant males developed insulin resistance and glucose intolerance by 8–12 months of age. In contrast, adult males with isolated GH deficiency remained insulin responsive through a year of age but had a blunted response to insulin by 18 months. Sixth, the development of insulin resistance and glucose intolerance in double-mutant males was accompanied by excess fat deposition and a reduction in lean body mass. Seventh, the rates of weight gain and fat accumulation in PRLR-deficient and double-mutant males exceeded the rates of weight gain and fat deposition in PRLR-deficient and double-mutant females during the first year of life. Finally, the maintenance of the obese phenotype in aging double-mutant mice could not be explained readily by excess food intake, suggesting that reduced energy expenditure plays an important role.
Despite numerous investigations, the roles of GH and the lactogens in perinatal growth remain highly controversial. PL and GH circulate in fetal mouse and rat serum, and PRL secretion begins soon after birth (37, 38, 39). GH and PRL receptors are expressed widely in rodent fetal tissues (40, 41, 42, 43), and the somatogens and lactogens exert anabolic effects in isolated fetal and neonatal hepatocytes, astrocytes, pancreatic islets, and mesenchymal tissues (20, 21, 22, 23, 24, 25, 26, 27). Nevertheless, weight gain is normal during the first 1–2 wk after birth in Little, Ames (Prop-1-deficient), Snell (Pit-1-deficient), and GH-resistant dwarf mice and transgenic mice that overexpress GH (30, 44, 45, 46, 47, 48).
It is not surprising, therefore, that our mice with isolated GH deficiency or lactogen resistance had normal body weights at 7 d of life. However, mice with combined lactogen resistance and GH deficiency were approximately 10% lighter than wild-type mice at this age. It is currently unclear whether the growth failure reflects in part the resistance to PL action in utero; to answer this question, it will be necessary to conduct detailed studies of fetal growth in the various groups of mice. In any case, the data suggest that the lactogens and GH may act in concert to facilitate mouse growth and/or weight gain in the perinatal period and that concurrent production of one or more members of the somatolactogen family may preserve mouse perinatal weight gain in the absence of another family member (39, 49).
These findings may have implications for human fetal and perinatal growth. In humans, PL, GH, and PRL circulate at high concentrations in the fetus (39, 50), and GH and PRL are overexpressed during the neonatal period (51). Moreover, human fetal tissues, like rodent fetal tissues, express GH and PRL receptors (52, 53, 54). Yet birth weight is often normal in patients with GH deficiency or GH resistance, although body mass index may be increased owing to excess fat deposition (55, 56). Systematic studies of birth weight and length in children with Pit-1 or Prop-1 mutations have not been completed, so the effects of combined GH and PRL deficiencies on human fetal and perinatal growth are unknown. Nevertheless, such patients continue to produce PL and decidual (amniotic fluid) PRL, which might obscure the effects of pituitary GH and PRL deficiencies (39). Moreover, PRL production does not decline in some cases until 5 yr or more after birth.
Linear growth during postnatal life is controlled by the GH/IGF axis. The findings of our studies in GH-deficient little mice are consistent with previous observations: the adult little mice had striking reductions in femur length, bone mineral density, and plasma IGF-I and IGFBP-3 concentrations (57). The defects in IGF/IGFBP expression in adult double-mutant mice were no more severe than those in GH-deficient mice; indeed, plasma IGF-I and IGFBP-3 concentrations were slightly higher in older (obese) double mutants than in GH-deficient mice. In addition, mice with isolated PRLR deficiency had normal femoral growth and plasma IGF-I and IGFBP-3 concentrations. Collectively, these findings suggest that lactogens have little or no effect on linear growth or IGF-I/IGFBP-3 production after the perinatal period. Moreover, concurrent PRL expression apparently cannot restore or maintain postnatal IGF/IGFBP production in the setting of GH deficiency. Bone mineral density was reduced to a small degree in PRLR-deficient females but not males; this finding may reflect the effects of longstanding hypoestrogenemia in female lactogen-resistant mice (58).
Human infants with GH deficiency are predisposed to fasting hypoglycemia, presumably because of increased insulin sensitivity (59). The findings of our studies are consistent with clinical observations: perinatal mice with GH deficiency were normoglycemic despite hypoinsulinemia. Interestingly, double-mutant mice were hypoglycemic on d 7 despite comparable hypoinsulinemia. The mechanism for this effect is unclear, but perinatal hypoglycemia could in theory result from defects in hepatic glycogen synthesis (60) and/or glucose production as well as heightened insulin action. In contrast to GH-deficient or double-mutant mice, PRLR-deficient mice were neither hypoglycemic nor hypoinsulinemic. Presumably, any effects of isolated lactogen resistance on hepatic glycogenesis, glucose production, or insulin sensitivity in the perinatal period might be obscured by concurrent GH action.
Striking changes in insulin secretion and insulin action were observed in the double-mutant mice during development. At 3 months of age, the double mutants had fasting hypoglycemia, hypoinsulinemia, and heightened insulin sensitivity. In contrast, older double-mutant males had fasting hyperglycemia, relative hypoinsulinemia, peripheral insulin resistance, and glucose intolerance. The relative hypoinsulinemia may reflect the loss of PRL and GH signaling in the pancreatic ?-cell (1, 2, 3, 4, 34) and/or the marked reduction in plasma IGF-I (61). In theory, the IGF-I deficiency may also contribute to defects in insulin action (62); however, GH-deficient males, whose levels of IGF-I were comparable with or lower than those of double-mutant males had fasting hypoinsulinemia and a normal hypoglycemic response to insulin at 12 months of age.
The reduction in insulin sensitivity in aging double-mutant mice may be caused, at least in part, by a progressive increase in fat deposition (63). The pathogenesis of fat deposition in the double mutants is currently unclear. GH stimulates white adipose tissue (WAT) lipolysis and inhibits lipogenesis, whereas GH deficiency limits fat mobilization and increases WAT stores. Lactogenic hormones have variable and inconsistent effects on WAT lipogenesis and lipolysis in vitro but may induce lipolysis in vivo (6, 7, 12, 13, 14, 15, 16); thus, the combination of lactogen resistance and GH deficiency may exacerbate fat deposition and weight gain.
The obesity of the double-mutant males cannot be explained readily by excess energy intake; food intake in older double mutants was lower than in wild-type mice, possibly a consequence of hyperleptinemia. The suppression of food intake suggests that maintenance of excess fat stores in older animals may involve reduced energy expenditure as well as impaired fat mobilization. A reduction in energy expenditure might be expected because percent lean body mass (the major determinant of resting energy expenditure) is decreased in obese double-mutant mice.
Interestingly, females with isolated PRLR deficiency, in contrast to males, had slightly reduced rates of weight gain after 4 months of age. Gender-dependent differences in metabolic responses to PRL have been described previously; for example, PRL stimulates food intake and reduces brown adipose tissue uncoupling protein 1 expression in female but not male rats (64, 65, 66, 67) and increases resistin expression in adipose tissue of male but not female mice (68). Differences in weight gain between PRLR-deficient males and females might reflect differences in gonadal function; PRLR-deficient males have normal testosterone levels, whereas females have severe hypoprogesteronemia and less severe hypoestrogenemia (58).
Like the effect of lactogen resistance, the effect of GH deficiency on weight gain appears to vary according to gender: relative to double-mutant and GH-deficient males, double-mutant and GH-deficient females have reduced rates of weight gain until later stages of development. In theory, effects of lactogen resistance or GH deficiency on adipogenesis (15, 17, 18, 19), lipolysis (69), and/or energy expenditure (67) might vary independently with age and gender. In addition, the magnitude of the defect in hormone signaling may be rate limiting; for example little mice, which produce GH at 5–10% of normal levels, accumulate excess WAT with age, whereas GH receptor knockout mice have reduced white adipose stores (48, 69). This finding suggests that low-level expression of GH may be adequate for adipogenesis in young (and possibly adult) mice but may not suffice for lipolysis or antilipogenesis in adulthood.
The mice used in our studies were developed on a hybrid background (C57BL6/129). We took a number of steps minimize the effects of genetic background on our results. These included: 1) the use of wild-type littermates of the same mixed genetic background as controls; 2) the use of mice from several litters of comparable age; and 3) the use of large numbers of mice in each experimental group. Variability in genetic background may explain in part the variability in weight gain and body fat deposition that we observed within individual groups of mice because the C57BL/6 background has been shown to facilitate weight gain in mice on high-fat diets (70). However, several lines of evidence suggest that metabolic defects in the lactogen-resistant and GH-deficient mice largely reflect differences in hormone production and action rather than genetic background per se. Like purebred GH-deficient little mice on a C57BL/6 background (44, 57), the hybrid GH-deficient mice had postnatal growth failure, catch-up weight gain, decreased bone mineral density, low plasma IGF-I and IGFBP-3 concentrations, fasting hypoglycemia and hypoinsulinemia, and increased insulin sensitivity. Like PRLR-deficient mice bred on a 129 background (29), the hybrid PRLR-deficient females are sterile and exhibit aberrant maternal behavior (our unpublished observations). PRLR-deficient females on the hybrid background, like PRLR-deficient females on a 129 background, have slightly decreased weight gain and fat deposition with age. Moreover, the mild glucose intolerance observed in our hybrid PRLR-deficient males is similar to or identical with that observed in purebred 129 PRLR-deficient mice (34). Thus, the mixed genetic background did not appear to alter markedly the phenotypes associated with lactogen resistance or GH deficiency.
The relevance of our studies for the pathogenesis of human metabolic disorders is currently unclear. Mounting evidence suggests that human infants and rodents that are growth retarded in utero or in infancy are predisposed to adult obesity, hypertension, dyslipidemia, and glucose intolerance (the metabolic syndrome) (71). The effects of perinatal growth failure appear to be most severe when there is rapid catch-up growth or weight gain during childhood (72, 73, 74). Because our double-mutant male mice have perinatal growth failure, catch-up weight gain, and late-onset obesity and glucose intolerance, the lactogen-resistant/GH-deficient mouse may serve as a model for the metabolic syndrome.
Acknowledgments
The authors thank Anne Petro and Rama Arumugam for technical assistance.
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