Aged mice require full transcription factor, Runx2/Cbfa1, gene dosage for cancellous bone regeneration after bone marrow ablation.

Runx2 is prerequisite for the osteoblastic differentiation in vivo. To elucidate Runx2 gene functions in adult bone metabolism, we conducted bone marrow ablation in Runx2 heterozygous knockout mice and found that aged (but not young) adult Runx2 heterozygous knockout mice have reduced new bone formation capacity after bone marrow ablation. We also found that bone marrow cells from aged Runx2 heterozygous knockout mice have reduced ALP(+) colony-forming potential in vitro. This indicates that full Runx2 dosage is needed for the maintenance of osteoblastic activity in adult mice. INTRODUCTION: Null mutation of the Runx2 gene results in total loss of osteoblast differentiation, and heterozygous Runx2 deficiency causes cleidocranial dysplasia in humans and mice. However, Runx2 gene functions in adult bone metabolism are not known. We therefore examined the effects of Runx2 gene function in adult mice with heterozygous loss of the Runx2 gene. MATERIALS AND METHODS: Bone marrow ablation was conducted in young adult (2.5 +/- 0.5 months old) or aged adult (7.5 +/- 0.5 months old) Runx2 heterozygous knockout mice and wildtype (WT) littermates. Cancellous bone regeneration was evaluated by 2D microCT. RESULTS: Although new bone formation was observed after bone marrow ablation in the operated bone marrow cavity of WT mice, such bone formation was significantly reduced in Runx2 heterozygous knockout mice. Interestingly, this effect was observed specifically in aged but not young adult mice. Runx2 heterozygous deficiency in aged mice significantly reduced the number of alkaline phosphatase (ALP)(+) cell colonies in the bone marrow cell cultures, indicating a reduction in the numbers of osteoprogenitor cells. Such effects of heterozygous Runx2 deficiency on osteoblasts in vitro was specific to the cells from aged adult mice, and it was not observed in the cultures of marrow cells from young adult mice. CONCLUSION: These results indicate that full gene dosage of Runx2 is required for cancellous bone formation after bone marrow ablation in adult mice.     

Intrauterine versus postnatal repair of created pulmonary artery stenosis in the lamb. Morphologic comparison

Experimental lamb models were used for intrauterine creation of pulmonary artery stenosis and later intrauterine repair or postnatal repair. Intrauterine creation of pulmonary artery stenosis was performed in 23 fetal lambs at 90 +/- 1 days of gestation. Eight lambs underwent intrauterine repair of pulmonary artery stenosis at 135 +/- 1 days of gestation and were studied 110 +/- 13 days after repair. Seven lambs underwent postnatal repair at 57 +/- 9 days after birth and were studied 162 +/- 32 days after repair. Eight fetal lambs with unrepaired pulmonary artery stenosis were studied 89 +/- 18 days after birth. All study lambs were compared with normal control lambs. The systolic right ventricular pressure was significantly higher after unrepaired stenosis (78.6 +/- 6.8 mm Hg) than in other lambs, but there was no statistically significant difference after intrauterine repair (23.3 +/- 2.9 mm Hg), postnatal repair (25.9 +/- 3.4 mm Hg), and normal lambs (21.6 +/- 1.1 mm Hg). The systolic pulmonary artery pressure was also not statistically different in these three groups. The weight measurements were age-adjusted for comparison of postnatal and intrauterine repair with normal lambs. The adjusted heart weights were similar in the three groups. The comparison of the adjusted heart weight/adjusted body weight ratio (10(-3) showed a significantly higher ratio in postnatal repair (7.4 +/- 0.1) than in intrauterine repair (6.1 +/- 0.1). The adjusted right ventricular weight/adjusted left ventricular weight ratio was significantly higher in the postnatal repair group (0.71 +/- 0.01) than in both the intrauterine repair group (0.59 +/- 0.01) and normal lambs (0.59 +/- 0.01). The transverse myocyte diameter was not statistically different in all groups of animals and there were no ultrastructural changes even when the pulmonary stenosis was unrepaired. We conclude that intrauterine repair was more satisfactory than postnatal repair in terms of age-adjusted heart weight results, but we did not find any advantages of intrauterine repair in terms of histologic and ultrastructural changes.     

Growth hormone and insulin reverse net whole body and skeletal muscle protein catabolism in cancer patients.

The authors examined the effect of recombinant-human growth hormone (r-hGH) and insulin (INS) administration on protein kinetics in cancer patients. Twenty-eight cancer patients either received r-hGH for 3 days (GH group, n = 12, weight loss = 6 +/- 2%) or were not treated (control [CTL] group, n = 16, weight loss = 11 +/- 2%) before metabolic study. Recombinant-human growth hormone dose was 0.1 mg/kg/day (n = 6) or 0.2 mg/kg/day (n = 6). Patients then underwent measurement of baseline protein kinetics (GH/B, CTL/B) followed by a 2-hour euglycemic insulin infusion (1 mU/kg/minute) and repeat kinetic measurements (GH/INS,CTL/INS). Whole-body protein net balance (mumol leucine/kg/minute) was higher (p less than 0.05) in GH/INS (0.20 +/- 0.06) than in CTL/INS (0.06 +/- 0.03) or GH/B (-0.19 +/- 0.03). Skeletal muscle protein net balance (nmol phenylalanine/100 g/minute) in GH/INS (25 +/- 6) and CTL/INS (19 +/- 5) was higher than CTL/B (-18 +/- 3). Recombinant-human growth hormone and insulin reduce whole-body and skeletal muscle protein loss in cancer patients. Simultaneous use of these agents during nutritional therapy may benefit the cancer patient.     

Preserved glucoregulation but attenuation of the vascular actions of insulin in mice heterozygous for knockout of the insulin receptor

Type 2 diabetes is preceded by years of insulin resistance and is characterized by reduced bioavailability of the antiatherosclerotic signaling molecule nitric oxide (NO) and premature atherosclerosis. The relationship between resistance to the glucoregulatory actions of insulin and its effects on the vasculature (in particular NO-dependent responses) is poorly characterized. We studied this relationship in mice heterozygous for knockout of the insulin receptor (IRKO), which have a mild perturbation of insulin signaling. Male heterozygous IRKO mice aged 8-12 weeks were compared with age- and sex-matched littermates. IRKO mice had fasting blood glucose, insulin, free fatty acid, and triglyceride levels similar to those of wild-type mice. Intraperitoneal glucose and insulin tolerance tests were also similar in the two groups. Insulin levels in response to a glucose load were approximately twofold higher in IRKO compared with wild-type mice (1.08 +/- 0.11 vs. 0.62 +/- 0.13 ng/ml; P = 0.004). Despite this mild metabolic phenotype, IRKO mice had increased systolic blood pressure (124 +/- 4 vs. 110 +/- 3 mmHg; P = 0.01). Basal NO bioactivity, assessed from the increase in tension of phenylephrine preconstricted aortic rings in response to the NO synthase inhibitor N(G)-monomethyl-l-arginine, was reduced in IRKO (61 +/- 14 vs. 152 +/- 30%; P = 0.005). Insulin-mediated NO release in aorta, assessed as the reduction in phenylephrine constrictor response after insulin preincubation, was lost in IRKO mice (5 +/- 8% change vs. 66 +/- 9% reduction in wild-type; P = 0.03). Insulin-stimulated aortic endothelial NO synthase phosphorylation was also significantly blunted in IRKO mice (P < 0.05). These data demonstrate that insulin-stimulated NO responses in the vasculature are exquisitely sensitive to changes in insulin-signaling pathways in contrast to the glucoregulatory actions of insulin. These findings underscore the importance of early intervention in insulin-resistant states, where glucose homeostasis may be normal but substantial abnormalities of the vascular effects of insulin may already be present.     

Maternal-fetal interactions and birth order influence insulin variable number of tandem repeats allele class associations with head size at birth and childhood weight gain

Polymorphism of the insulin gene (INS) variable number of tandem repeats (VNTR; class I or class III alleles) locus has been associated with adult diseases and with birth size. Therefore, this variant is a potential contributory factor to the reported fetal origins of adult disease. In the population-based Avon Longitudinal Study of Pregnancy and Childhood birth cohort, we have confirmed in the present study the association between the INS VNTR III/III genotype and larger head circumference at birth (odds ratio [OR] 1.92, 95% CI 1.23-3.07; P = 0.004) and identified an association with higher cord blood IGF-II levels (P = 0.05 to 0.0001). The genotype association with head circumference was influenced by maternal parity (birth order): the III/III OR for larger head circumference was stronger in second and subsequent pregnancies (OR 5.0, 95% CI 2.2-11.5; P = 0.00003) than in first pregnancies (1.2, 0.6-2.2; P = 0.8; interaction with birth order, P = 0.02). During childhood, the III/III genotype remained associated with larger head circumference (P = 0.004) and was also associated with greater BMI (P = 0.03), waist circumference (P = 0.03), and higher fasting insulin levels in girls (P = 0.02). In addition, there were interactions between INS VNTR genotype and early postnatal weight gain in determining childhood BMI (P = 0.001 for interaction), weight (P = 0.005), and waist circumference (P = 0.0005), such that in the approximately 25% of children (n = 286) with rapid early postnatal weight gain, class III genotype-negative children among this group gained weight more rapidly. Our results indicate that complex prenatal and postnatal gene-maternal/fetal interactions influence size at birth and childhood risk factors for adult disease.     

Mice with gene disruption of both endothelial and neuronal nitric oxide synthase exhibit insulin resistance

Studies from our laboratory using acute pharmacologic blockade of nitric oxide synthase (NOS) activity have suggested that nitric oxide (NO) has an important role in regulating carbohydrate metabolism. We now report on insulin sensitivity in mice with targeted disruptions in endothelial NOS (eNOS) and neuronal NOS (nNOS) genes compared with their wild-type (WT) counterparts. Mice underwent hyperinsulinemic-euglycemic clamp studies after a 24-h fast, during an insulin infusion of 20 mU x kg(-1) x min(-1). Glucose levels were measured at baseline and every 10 min during the clamp. Insulin levels were measured at baseline and at the end of the clamp study. Glucose infusion rates (GIRs) during the last 30 min of the clamp study were in a steady state. Tritiated glucose infusion was used to measure rates of endogenous glucose output (EGO) both at baseline and during steady-state euglycemia. Glucose disposal rates (GDRs) were computed from the GIR and EGO. Fasting and steady-state glucose and insulin levels were comparable in the 3 groups of mice. No differences in fasting EGO were noted between the groups. GIR was significantly reduced (37%, P = 0.001) in the eNOS knockout (KO) mice compared with the WT mice, with values for the nNOS mice being intermediate. EGO was completely suppressed in the nNOS and WT mice during insulin infusion, but not in the eNOS mice. Even so, the eNOS mice displayed significantly reduced whole-body GDRs compared with those of the WT mice (82.67+/-10.77 vs. 103.67+/-3.47 mg x kg(-1) x min(-1), P = 0.03). eNOS KO mice are insulin resistant at the level of the liver and peripheral tissues, whereas the nNOS KO mice are insulin resistant only in the latter. These data indicate that NO plays a role in modulating insulin sensitivity and carbohydrate metabolism and that the eNOS isoform may play a dominant role relative to nNOS.     

Racial disparities in the association between birth weight in the term infant and blood pressure at age 7 years: results from the collaborative perinatal project

BP has been inversely associated with birth weight in studies worldwide, but few studies have included black individuals. The US National Collaborative Perinatal Project followed 58,960 pregnant women and their resultant offspring for 7 yr. In this post hoc analysis, all term white or black children without kidney or heart disease were included (n = 29,710). The effect of birth weight and other risk factors on systolic (SBP) and diastolic BP (DBP) was evaluated at 7 yr. Mean birth weight and body mass index at 7 yr were slightly lower for black compared with white children (birth weight 3.14 +/- 0.48 versus 3.32 +/- 0.46 kg [P < 0.001]; body mass index 15.8 +/- 2.0 versus 16.3 +/- 2.0 [P < 0.001]). Compared with white mothers, black mothers were less likely to smoke (41 versus 52%), were more anemic (23 versus 7%), and were more likely to live in poverty (72 versus 39%). In linear regression, there was significant interaction between race and birth weight in predicting SBP (P = 0.002). In bivariate analysis, birth weight was positively associated with SBP (beta = 0.87) and DBP (beta = 1.14) in black children (P < 0.001) but not associated with either in white children. With maternal poverty, educational level, and anemia during pregnancy added to the model, birth weight remained a significant positive predictor of SBP (beta = 0.89, P < 0.001) in black but not in white children (beta = 0.02, P = 0.17). The association between birth weight and SBP differs between black and white children. The cause of intrauterine growth restriction-associated hypertension seems to be race sensitive; therefore, future studies of racial disparities in the "Barker hypothesis" may help in the understanding of the mechanism of fetal programming of hypertension.     

Nerve Injury Induces a Tonic Bilateral μ-Opioid Receptor-mediated Inhibitory Effect on Mechanical Allodynia in Mice

Background: Mice lacking the [mu]-opioid receptor gene have been used to characterize the role of [mu]-opioid receptors in nociception and the analgesic actions of opioid agonists. In this study, the authors determined the role of [mu]-opioid receptors in neuropathic pain behaviors and the effectiveness of [mu]- and [kappa]-opioid receptor agonists on this behavior in mice.

Methods: The authors studied the behavioral responses of [mu]-opioid receptor knockout and wild-type mice to thermal and mechanical stimuli before and after neuropathic pain induced by unilateral ligation and section of the L5 spinal nerve. Response to mechanical stimuli was evaluated by determining the frequency of hind paw withdrawal to repetitive stimulation using a series of von Frey monofilaments. Thermal hyperalgesia was assessed by determining the paw withdrawal latencies to radiant heat and frequency of hind paw withdrawal to cooling stimuli. The effects of systemic morphine, the [kappa]-opioid agonist U50488H, and naloxone on responses to mechanical and thermal stimuli were also studied in spinal nerve-injured mice.

Results: After spinal nerve injury, wild-type mice developed increased responsiveness to mechanical, heat, and cooling stimuli ipsilateral to nerve injury. [mu]-Opioid receptor knockout mice not only had more prominent mechanical allodynia in the nerve-injured paw, but also expressed contralateral allodynia to mechanical stimuli. Hyperalgesia to thermal stimuli was similar between [mu]-opioid knockout and wild-type animals. Morphine decreased mechanical allodynia dose dependently (3-30 mg/kg subcutaneous) in wild-type mice-an effect that was attenuated in the heterozygous mice and absent in the homozygous [mu]-opioid knockout mice. The [kappa]-opioid agonist U50488H (3-10 mg/kg subcutaneous) attenuated mechanical allodynia in wild-type, heterozygous, and homozygous [mu]-opioid mice. Naloxone in wild-type mice resulted in enhanced ipsilateral and contralateral allodynia to mechanical stimuli that resembled the pain behavior observed in [mu]-opioid receptor knockout mice.     

Relationship between weight at birth and the number and size of renal glomeruli in humans: a histomorphometric study

BACKGROUND: The number of nephrons in humans varies considerably under normal circumstances, and retarded intrauterine growth has been reported to be associated with a significant reduction in nephron number. Low nephron number may be an independent risk factor for the development of hypertension. We therefore decided to evaluate the relationship between body weight at birth and the number and size of nephron units. METHODS: We examined coronal sections of the kidneys of 35 neonates who died within two weeks of birth because of hyaline membrane, infectious complications, brain hemorrhage, or perinatal hypoxia and had no urinary congenital malformations. Nine of them (5 males and 4 females) were between 36 and 37 weeks of gestation, and the rest had 38 or more weeks of gestation. Eighteen neonates weighed less than 2500 g at birth [low birth weight (LBW); 9 females and 9 males], and 17 had weights above this value [normal birth weight (NBW); 8 females and 9 males]. In each section, glomeruli present in four sequential subcapsular microscopic fields, corresponding to 0.6 mm2, were counted; in addition, the area of each of 65 consecutive glomeruli was determined by a computerized measurement system. Glomerular volume was calculated from the glomerular area. Linear regression analysis was used to test the relationship between glomerular number and size and the weight at birth. RESULTS: The number of glomeruli per 0.6 mm2 of renal cortex was 92.9 +/- 4.85 in the LBW and 105.8 +/- 3.91 in NBW (P < 0.0001). Glomerular volume (micro(3) x 10(-3)) was 529.1 +/- 187.63 in the LBW group and 158.0 +/- 49.89 in the NBW group (P < 0.0001). The glomeruli occupied 8.59 +/- 1.38% of the kidney area under examination in the LBW group and 14.3 +/- 2.75% in the NBW group (P < 0.0001). There were significant direct correlations between the weight at birth and the number of glomeruli (r = 0.870, P < 0.0001) and area occupied by glomeruli (r = 0.935, P < 0.0001). There were inverse correlations between the number of glomeruli and the volume of the glomeruli (r = -0.816, P < 0.0001) and the weight at birth and glomerular volume (r = -0.848, P < 0.0001). These findings were independent of sex and race (black vs. white). Essential arterial hypertension existed in 38.9% of the mothers of children with LBW and in 5.9% of the mothers of children with NBW (P < 0.05). Smoking habits existed in 50% of the mothers of LBW children and in 11.8% of the mothers of NBW children (P < 0.05). CONCLUSION: There are strong correlations between glomerular number (direct) and size (inverse) with LBW in this cohort. Endowment with decreased nephron numbers may be a risk factor for hypertension and the rate of progression of renal disease.     

Neurokinin-1 Receptors Are Involved in Behavioral Responses to High-intensity Heat Stimuli and Capsaicin-induced Hyperalgesia in Mice

Background: The neurokinin-1 (NK-1) receptor and its ligand, substance P, are thought to play important roles in nociception and hyperalgesia. This study evaluated the role of the NK-1 receptor in processing noxious stimuli in normal and inflammatory states.

Methods: Behavioral responses to heat and mechanical and chemical stimuli were studied in NK-1 receptor knockout mice and wild-type control mice. Thermal nociception was evaluated by measuring paw lick or jump latencies to hot plate (52, 55, and 58[degree sign]C) and paw withdrawal latencies to radiant heat applied to the hind paws. Mechanical nociception was measured by von Frey monofilament applications to the hind paws. Intraplantar capsaicin-induced (10 [micro sign]g/20 [micro sign]l) paw licking and mechanical and heat hyperalgesia were compared in NK-1 knockout and wild-type mice.

Results: Withdrawal responses to radiant heat (4.3 +/- 0.18 s for knockout and 4.4 +/- 0.8 s for wild-type mice) and von Frey monofilaments were similar in knockout and wild-type mice. In the hot plate test, increasing the hot plate temperature from 52[degree sign]C to 58[degree sign]C resulted in a decrease in the response latency in the wild-type mice (30.4 +/- 17.5 s to 15.2 +/- 6.8 s, P < 0.05), whereas in the knockout mice the response latencies remained constant (28.2 +/- 19.8 s to 29 +/- 15.1 s, not significant). Capsaicin-induced paw licking (14.5 +/- 12.8 s for knockout and 41.3 +/- 37.3 s for wild-type mice, P < 0.05) and mechanical and heat hyperalgesia were attenuated in the knockout mice.     

The fetal environment: a critical phase that determines future renal outcomes in autosomal dominant polycystic kidney disease

Orskov and colleagues demonstrate the impact of birth weight on the mean age of end-stage renal disease (ESRD) in a large Danish ADPKD cohort. Each kilogram of birth weight extended the mean age of ESRD onset by 1.7 years. Placental insufficiency, activation of the renin-angiotensin-aldosterone system, increased fetal vasopressin levels, compensatory increases in insulin like growth factor-I, and a reduction in total nephron number may all contribute to this observation. Collectively, these changes result in an accelerated pace of cyst formation and expansion, and an inability to maintain glomerular hyperfiltration during kidney expansion which results in a more rapid progression to ESRD. Therefore the intrauterine environment may play a critical role in disease severity in ADPKD.     

Effects of overexpression of human GLUT4 gene on maternal diabetes and fetal growth in spontaneous gestational diabetic C57BLKS/J Lepr(db/+) mice.

During gestation, heterozygous C57BLKS/J-Lepr(db/+) mice develop spontaneous gestational diabetes mellitus (GDM), and the newborn fetuses are macrosomic compared with offspring from wild-type (+/+) mothers. To investigate the effects of the leptin receptor mutation on maternal metabolism and fetal growth during pregnancy, we studied +/+, db/+, and db/+ transgenic mice that overexpress the human GLUT4 gene two- to three-fold (db/+TG6). During pregnancy, fasting plasma glucose and hepatic glucose production were twofold greater in db/+ than +/+ mice, despite similar insulin levels. In skeletal muscle, insulin-stimulated tyrosine phosphorylation was decreased in pregnant +/+ mice, and even more so in db/+ mice: insulin receptor beta (IR-beta), +/+ 34%, db/+ 57% decrease, P<0.05; insulin receptor substrate 1 (IRS-1), +/+ 44%, db/+ 61% decrease, P<0.05; and phosphoinositol (PI) 3-kinase (p85alpha), +/+ 33%, db/+ 65% decrease, P<0.05. Overexpression of GLUT4 in db/+TG6 mice markedly improved glucose-stimulated insulin secretion, by 250%, and increased IRbeta, IRS-1, and p85alpha phosphorylation twofold, despite no change in concentration of these proteins. Plasma leptin concentration increased 40-fold during pregnancy, from 2.2+/-0.5 to 92+/-11 ng/ml and 3.6+/-0.1 to 178+/-34 ng/ml in +/+ and db/+ mice, respectively (P<0.01), but was increased to only 23+/-3 ng/ml in pregnant db/+TG6 mice (P<0.001). Maternal fat mass and energy intake were greater in db/+ mice, and fat mass was reduced by GLUT4 overexpression, independent of food intake. Fetal body weight was increased by 8.1 and 7.9% in db/+ and db/+TG6 mothers, respectively (P<0.05), regardless of fetal genotype, whereas fetuses from db/+TG8 mothers (four- to fivefold overexpression) weighed significantly less compared with pups from +/+ or db/+ mothers (P<0.05). These results suggest that the single mutant db allele effects susceptibility to GDM through abnormalities in insulin receptor signaling, defective insulin secretion, and greater nutrient availability. GLUT4 overexpression markedly improves insulin-signaling in GDM, resulting in increased insulin secretion and improved glycemic control. However, maternal hyperglycemia appears not to be the sole cause of fetal macrosomia. These data suggest that GDM is associated with defects in insulin receptor signaling in maternal skeletal muscle, and this may be an important factor provoking maternal and fetal perinatal complications.     

Insulin resistance syndrome in 8-year-old Indian children: small at birth, big at 8 years, or both?

We have studied 477 8-year-old Indian children to define the relationship between birth weight and cardiovascular risk factors, including insulin resistance syndrome (IRS) variables and plasma total and LDL cholesterol concentrations. All risk factors were strongly related to current weight. After adjustment for current weight, age, and sex, lower birth weight was associated with higher systolic blood pressure (P = 0.008), fasting plasma insulin and 32-33 split proinsulin concentrations (P = 0.08 and 0.02), glucose and insulin concentrations 30 min postglucose (P = 0.06 and 0.04), subscapular/triceps skinfold ratio (P = 0.003), and plasma total and LDL cholesterol concentrations (P = 0.002 and 0.001). Lower birth weight was associated with increased calculated insulin resistance (homeostasis model assessment [HOMA], P = 0.03), but was not related to the HOMA index of beta-cell function. The highest levels of IRS variables and total and LDL cholesterol were in children of low birth weight but high fat mass at 8 years. Taller height at 8 years predicted higher fasting plasma insulin concentrations, insulin resistance, and plasma total and LDL cholesterol concentrations. The most insulin-resistant children were those who had short parents but had themselves grown tall. Although the implications of our findings in relation to height are unclear, interventions to improve fetal growth and to control obesity in childhood are likely to be important factors in the prevention of cardiovascular disease and IRS in India.     

Variation at the insulin gene VNTR (variable number tandem repeat) polymorphism and early growth: studies in a large Finnish birth cohort

Variation at the insulin gene (INS-)VNTR (variable number of tandem repeats) minisatellite polymorphism has been reported to be associated with both early growth and adult metabolic phenotypes. However, the samples studied have been small and the relationship between INS-VNTR variation and parameters of early growth inconsistent, with four previous studies producing conflicting results. We have studied the relationship between INS-VNTR class (measured by genotyping the nearby -23HphI variant with which it is in tight linkage disequilibrium) and early growth in 5,646 members of the Northern Finnish Birth Cohort of 1966. Comparing class III homozygotes with other genotypes using multivariate linear regression analysis, we found no significant associations with any early growth measure (birth weight, birth length, ponderal index, and head circumference at 1 year), even after stratifying subjects by growth trajectory during infancy and/or birth order. For example, among infants with limited postnatal growth realignment (n = 2,470), class III/III infants were no heavier at birth (difference [+/-SE] in the means [fully adjusted], 58 +/- 51 g; P = 0.26) than class I/- infants. No significant associations were detected following reanalysis with an additive model (for example, for birth weight, beta = 20 g [95% CI -3 to 44], P = 0.09). Studies of this large population-based cohort have failed to generate convincing evidence that INS-VNTR variation influences early growth.     

Phosphorylated insulin-like growth factor binding protein 1 is increased in pregnant diabetic subjects.

During pregnancy, IGFs and their binding proteins (IGFBPs) are important for the growth of fetal and maternal tissues. IGFBP-1 normally circulates as a single, highly phosphorylated species (hpIGFBP-1). However, in pregnancy there are lesser phosphorylated isoforms (lpIGFBP-1) with decreased affinity for IGF-I, allowing for increased IGF bioavailability. Because regulation of IGFBP-1 is abnormal in type 1 diabetes, we examined the impact of this on IGFBP-1 and its phosphorylation status in diabetic pregnancy. We assessed IGFBP-1 in relation to birth weight, maternal weight gain, duration of diabetes, glycemic control, and the presence or absence of retinopathy in 44 diabetic and 11 nondiabetic subjects. We found that in type 1 diabetic patients there was a significant negative relationship between hpIGFBP-1 and birth weight (r = -0.42, P < 0.01) and between the ratio of hpIGFBP-1 to lpIGFBP-1 and birth weight (r = -0.38, P = 0.02) by week 18 of gestation. Multiple regression analysis confirmed that hpIGFBP-1 was the best single predictor of birth weight (R2 = 0.3, P = 0.001) in diabetic subjects using models including other parameters known to influence fetal size. In contrast to hpIGFBP-1 levels, lpIGFBP-1 levels were not associated with birth weight, but were significantly related to initial maternal BMI and maternal weight throughout gestation in diabetic subjects (r = -0.57, P < 0.001). hpIGFBP-1 levels were positively related to duration of diabetes (r = 0.38, P < 0.01). Diabetic subjects had significantly higher hpIGFBP-1 and lpIGFBP-1 levels than nondiabetic subjects (hpIGFBP-1: 215 +/- 21 vs. 108 +/- 13 microg/l, P = 0.01; lpIGFBP-1: 139 +/- 12 vs. 66 +/- 5 microg/l, P < 0.001), but the ratio of hpIGFBP-1 to lpIGFBP-1 was similar in both groups (2.1 +/- 0.3 [diabetic] vs. 1.7 +/- 0.2 [nondiabetic], NS). In summary, maternal IGFBP-1 levels were higher in diabetic than in normal pregnancies. Diabetic subjects with prolonged duration of diabetes and retinopathy had higher total IGFBP-1 levels than those with shorter disease duration. Thus hpIGFBP-1 in diabetic pregnancy is positively related to the duration of diabetes and inversely related to fetal growth, with lpIGFBP-1 being related to maternal weight and BMI. The ratio of hpIGFBP-1 to lpIGFBP-1 may be a more robust indicator of fetal outcome, since it was consistent between diabetic and nondiabetic subjects. Measurement of the different phosphorylated isoforms of IGFBP-1 may increase the usefulness of IGFBP-1 as a predictor of fetal growth in both normal and diabetic pregnancy.     

Mode of delivery in women after liver transplantation

AIM: A high rate of cesarean sections has been reported among high-risk pregnancies in liver transplant recipients. The aim of this study was to analyze the course of deliveries and the indications for cesarean sections in women after liver transplantation. MATERIALS AND METHODS: From 2001 to 2006, we noted 21 deliveries in 17 liver recipients. The mean age of women was 27.9 +/- 6.6 years and the mean time from transplantation to pregnancy was 4.3 +/- 3.6 years. Most patients were primigravidas on tacrolimus-based immunosuppressive regimens. We retrospectively analyzed obstetric data regarding the delivery and the early puerperium. RESULTS: We noted 6 vaginal deliveries (29%) and 15 cesarean sections (71%). Mean gestational age in the group of vaginal deliveries was 37.6 +/- 2.2 weeks. No labor complications were noted. All neonates were delivered in a good state (Apgar score from 8 to 10 points) with mean birth weight of 2725 g. All cesarean sections were performed for obstetric indications: fetal distress, breech presentation, intrauterine growth retardation, or complications related to premature labor. Mean gestational age was 37.0 +/- 1.9 weeks. The Apgar scores ranged from 4 to 10 points; mean birth weight was 2787 g. The mean period of hospitalization after surgical labor was 4 days longer compared with the vaginal delivery group. CONCLUSION: The high rate of cesarean sections (71%) in liver recipients is associated with a great incidence of obstetric complications of pregnancy. Safe and uneventful vaginal delivery is possible with growing experience in the management of pregnant transplanted women.     

Relative impact of androgen and estrogen receptor activation in the effects of androgens on trabecular and cortical bone in growing male mice: a study in the androgen receptor knockout mouse model.

The relative importance of AR and ER activation has been studied in pubertal male AR knockout and WT mice after orchidectomy and androgen replacement therapy, either with or without an aromatase inhibitor. AR activation dominates normal trabecular bone development and cortical bone modeling in male mice. Moreover, optimal periosteal bone expansion is only observed in the presence of both AR and ER activation. INTRODUCTION: Androgen receptor (AR)-mediated androgen action has traditionally been considered a key determinant of male skeletal growth. Increasing evidence, however, suggests that estrogens are also essential for normal male bone growth. Therefore, the relative importance of AR-mediated and estrogen receptor (ER)-mediated androgen action after aromatization remains to be clarified. MATERIALS AND METHODS: Trabecular and cortical bone was studied in intact or orchidectomized pubertal AR knockout (ARKO) and male wildtype (WT) mice, with or without replacement therapy (3-8 weeks of age). Nonaromatizable (dihydrotestosterone [DHT]) and aromatizable (testosterone [T]) androgens and T plus an aromatase inhibitor (anastrazole) were administered to orchidectomized ARKO and WT mice. Trabecular and cortical bone modeling were evaluated by static and dynamic histomorphometry, respectively. RESULTS: AR inactivation or orchidectomy induced a similar degree of trabecular bone loss (-68% and -71%, respectively). Both DHT and T prevented orchidectomy-induced bone loss in WT mice but not in ARKO mice. Administration of an aromatase inhibitor did not affect T action on trabecular bone. AR inactivation and orchidectomy had similar negative effects on cortical thickness (-13% and -8%, respectively) and periosteal bone formation (-50% and -26%, respectively). In orchidectomized WT mice, both DHT and T were found to stimulate periosteal bone formation and, as a result, to increase cortical thickness. In contrast, the periosteum of ARKO mice remained unresponsive to either DHT or T. Interestingly, administration of an aromatase inhibitor partly reduced T action on periosteal bone formation in orchidectomized WT mice (-34% versus orchidectomized WT mice on T), but not in ARKO mice. This effect was associated with a significant decrease in serum IGF-I (-21% versus orchidectomized WT mice on T). CONCLUSIONS: These findings suggest a major role for AR activation in normal development of trabecular bone and periosteal bone growth in male mice. Moreover, optimal stimulation of periosteal growth is only obtained in the presence of both AR and ER activation.     

Adipocyte metabolism in adipocyte fatty acid binding protein knockout mice (aP2-/-) after short-term high-fat feeding: functional compensation by the keratinocyte [correction of keritinocyte] fatty acid binding protein

Mice null for adipocyte fatty acid binding protein (AFABP) compensate by increasing expression of keratinocyte fatty acid binding protein (KFABP) (Hotamisligil et al. Science 274:1377-1379, 1996). In the present study, AFABP knockout (KO) and wild-type (WT) mice became equally obese on a high-fat diet, as judged by fat pad weights, adipocyte size, and body composition analysis. High-fat feeding led to moderate insulin resistance in both WT and AFABP knockout mice, as indicated by an approximately 2-fold increase in plasma insulin. However, in the high fat-fed mice, plasma glucose levels were approximately 15% lower in the AFABP-KO mice. Adipocytes isolated from AFABP-KO and WT mice fed high- or low-fat diets exhibited similar rates of basal and norepinephrine-stimulated lipolysis and insulin-stimulated rates of glucose conversion to fatty acids and glyceride-glycerol. However, basal glucose conversion to fatty acids was higher in adipocytes of AFABP-KO mice. Adipocyte tumor necrosis factor-alpha release was similarly increased by high-fat diet-induced obesity in both WT and AFABP-KO mice. As assessed by Western blot analysis, the level of KFABP protein in AFABP-KOs was approximately 40% of the level of AFABP in WT controls. The binding affinities of KFABP for long-chain fatty acids were 2- to 4-fold higher than those of AFABP, but the relative affinities for different fatty acids were similar. As for AFABP, the rate of fatty acid transfer from KFABP to model phospholipid vesicles was increased with acceptor membrane concentration and by inclusion of acidic phospholipids, indicating a similar mechanism of transfer. We conclude KFABP can functionally compensate for the absence of AFABP, resulting in no major alterations in adipocyte metabolism or fat accumulation in response to short-term feeding of high-fat diets that result in moderate hyperinsulinemia.     

Calorie restriction increases insulin-stimulated glucose transport in skeletal muscle from IRS-1 knockout mice.

Calorie restriction (CR), even for brief periods (4-20 days), results in increased whole-body insulin sensitivity, in large part due to enhanced insulin-stimulated glucose transport by skeletal muscle. Evidence suggests that the cellular alterations leading to this effect are postreceptor steps in insulin signaling. To determine whether insulin receptor substrate (IRS)-1 is essential for the insulin-sensitizing effect of CR, we measured in vitro 2-deoxyglucose (2DG) uptake in the presence and absence of insulin by skeletal muscle isolated from wild-type (WT) mice and transgenic mice lacking IRS-1 (knockout [KO]) after either ad libitum (AL) feeding or 20 days of CR (60% of ad libitum intake). Three muscles (soleus, extensor digitorum longus [EDL], and epitrochlearis) from male and female mice (4.5-6 months old) were studied. In each muscle, insulin-stimulated 2DG uptake was not different between genotypes. For EDL and epitrochlearis, insulin-stimulated 2DG uptake was greater in CR compared to AL groups, regardless of sex. Soleus insulin-stimulated 2DG uptake was greater in CR compared with AL in males but not females. The diet effect on 2DG uptake was not different for WT and KO animals. Genotype also did not alter the CR-induced decrease in plasma constituents (glucose, insulin, and leptin) or body composition (body weight, fat pad/body weight ratio). Consistent with previous studies in rats, IRS-1 protein expression in muscle was reduced in WT-CR compared with WT-AL mice, and muscle IRS-2 abundance was unchanged by diet. Skeletal muscle IRS-2 protein expression was significantly lower in WT compared with KO mice. These data demonstrate that IRS-1 is not essential for the CR-induced increase in insulin-stimulated glucose transport in skeletal muscle, and the absence of IRS-1 does not modify any of the characteristic adaptations of CR that were evaluated.     

Impact of genetic background on development of hyperinsulinemia and diabetes in insulin receptor/insulin receptor substrate-1 double heterozygous mice

Type 2 diabetes is a complex disease in which genetic and environmental factors interact to produce alterations in insulin action and insulin secretion, leading to hyperglycemia. To evaluate the influence of genetic background on development of diabetes in a genetically susceptible host, we generated mice that are double heterozygous (DH) for knockout of the insulin receptor and insulin receptor substrate-1 on three genetic backgrounds (C57BL/6 [B6], 129Sv, and DBA). Although DH mice on all backgrounds showed insulin resistance, their phenotypes were dramatically different. B6 DH mice exhibited marked hyperinsulinemia and massive islet hyperplasia and developed early hyperglycemia, with 85% overtly diabetic by 6 months. By contrast, 129Sv DH mice showed mild hyperinsulinemia and minimal islet hyperplasia, and < 2% developed diabetes. DBA mice had slower development of hyperglycemia, intermediate insulin levels, and evidence of islet degeneration, with 64% developing diabetes. Thus, mice carrying the same genetic defects on different backgrounds exhibited the full spectrum of abnormalities observed in humans with type 2 diabetes, which allowed for identification of potential loci that promote development of the diabetic phenotype.