Maternal low protein malnutrition during gestation and lactation (LP) is an animal model frequently used for the investigation of long-term deleterious consequences of perinatal growth retardation. Both perinatal malnutrition and growth retardation at birth are risk factors for diabetic and cardiovascular disturbances in later life. The pathophysiologic mechanisms responsible are unknown. Hypothalamic nuclei are decisively involved in the central nervous regulation of food intake, body weight and metabolism. We investigated effects of a low protein diet (8% protein; control diet, 17% protein) during gestation and lactation in rat dams on the organization of hypothalamic regulators of body weight and metabolism in the offspring at weaning (d 20 of life). LP offspring had significantly lower body weight than control offspring (CO; P: < 0.001), associated with hypoglycemia and hypoinsulinemia (P: < 0. 005) on d 20 of life. This was accompanied by a greater relative volume of the ventromedial hypothalamic nucleus (P: < 0.01) and a greater numerical density of Nissl-stained neurons in this nucleus (P: < 0.01) as well as in the paraventricular hypothalamic nucleus (PVN; P: < 0.001). In contrast, no significant differences in neuronal densities were observed generally in the lateral hypothalamic area, arcuate hypothalamic nucleus (ARC), and dorsomedial hypothalamic nucleus between LP offspring and CO offspring. On the other hand, LP offspring displayed fewer neurons immunopositive for neuropeptide Y in the ARC (P: < 0.05), whereas in the PVN, lower neuronal densities of neurons immunopositive for galanin were found in LP offspring compared with CO offspring (P: < 0.001). On the contrary, in the PVN, no significant group difference in the numerical density of cholecystokinin-8S-positive neurons was present. A long-term effect of these specific hypothalamic alterations on body weight and metabolism in LP offspring during later life is suggested.

We recently reported on an elevation of neurons expressing the main orexigenic peptide neuropeptide Y (NPY) in the arcuate hypothalamic nucleus (ARC) of neonatally hyperinsulinaemic offspring of gestational diabetic mother rats (GD) at weaning. To investigate possible consequences, the long-term outcome of those animals was examined. At adult age, GD offspring showed hyperphagia (p < 0.001), basal hyperinsulinaemia (p < 0.05) and impaired glucose tolerance (p < 0.05), and were overweight (p < 0.01). This was accompanied by an elevated number of NPY neurons (p < 0.001) and galanin neurons (p < 0.001) in the ARC in adult GD offspring under basal conditions. These findings support our hypothesis on perinatally acquired, persisting malformation and/or malprogramming of peptidergic hypothalamic neurons in the offspring of GD mothers, possibly promoting the development of overweight and diabetogenic disturbances during life.

In former studies, a temporary, intrahypothalamically localized hyperinsulinism during brain development was shown to result in overweight and metabolic disturbances during later life in rats. Therefore, we tested the hypothesis whether intrahypothalamic insulin treatment during early postnatal life may lead to hypothalamic morphological alterations, i.e., of numerical density of neurons and area of neuronal nuclei or area of neuronal cytoplasm, in this animal model. For this purpose, on the 8th day of age in Wistar rats a long-acting insulin was bilaterally applicated stereotactically into the hypothalamus (12 mIU on each side), while in controls the insulin-free agar-vehicle was given only. By computer-assisted morphometric analysis on the 15th day of life a decrease of the mean area of neuronal nuclei and the mean nucleus-cytoplasm-ratio within the VMN of the insulin-treated animals was observed, as compared to control rats (P < 0.05), while no significant alterations were found in the lateral hypothalamic area (LHA). Analysis of topographically distinct parts of the VMN revealed significant reductions of the mean area of neuronal nuclei (P < 0.001) and nucleus-cytoplasm-ratio (P < 0.05) in the anterior part of the VMN (VMNpa). Furthermore, in the ventrolateral part (VMNpv) a decreased mean neuronal density was observed in the insulin group (P < 0.01). In contrast, the dorsomedial part of the VMN (VMNpd) displayed an increased mean neuronal density in the insulin-treated animals (P < 0.05). In the dorsomedial hypothalamic nucleus (DMN) a significant increase of the mean area of neuronal nuclei (P < 0.01) and the area of neuronal cytoplasm were observed (P < 0.001). These alterations were accompanied by a significantly elevated mean numerical density of astrocytes (positive for glial fibriallary acidic protein; GFAP+) within the periventricular hypothalamic area (PER) of the insulin-treated rats (P < 0.05). These observations speak for a varying vulnerability of LHA, DMN and distinct parts of the VMN to hyperinsulinism during early development, possibly leading to a disturbed organization and, consecutively, permanent dysfunction of these morphologically connected and functionally interacting hypothalamic nuclei.

Early postnatal overnutrition is a risk factor for obesity in juvenile and adult life. Underlying pathophysiological mechanisms are still unclear. Hypothalamic neuropeptides are decisively involved in the regulation of body weight and food intake. In this study, we investigated consequences of early postnatal overnutrition, as compared to normo‐and undernutrition, on NPY within the arcuate nucleus and paraventricular nucleus (PVN). The normal litter size of Wistar rats was adjusted on the third day of life from 10 pups (normal litters, NL; normonutrition) to only three newborns (small litters, SL; overnutrition) or 18 pups per mother (large litters, LL; undernutrition). SL rats developed clear overweight until the day 21 of life (P<0.0001), as well as hyperleptinaemia (P<0.001), and hyperinsulinaemia (P<0.01). LL rats were underweight and had decreased leptin and insulin concentrations. Using radioimmunoassay, NPY contents were determined in hypothalamic micropunches, and immunocytochemistry for NPY was performed in serial hypothalamic sections on day 21 of life. While in the underweight, hypoleptinaemic, and hypoinsulinaemic LL rats increased concentrations of NPY in the arcuate nucleus and PVN were observed, no decrease in NPY content was found in the overweight, hyperleptinaemic, and hyperinsulinaemic SL rats. Moreover, the percentage of NPY‐immunopositive neurones per total number of neurones was increased not only in the LL rats, but also in the SL rats. Since the NPY system is functionally mature already at this age, these findings might indicate an acquired resistance of the hypothalamic NPY system to increased levels of insulin and/or leptin in early postnatally overfed SL rats.

OBJECTIVE:Since the offspring of gestational diabetic mothers (GD) is at increased risk to develop obesity and diabetogenic disturbances later in life, while pathophysiological mechanisms responsible are unclear, to investigate long-term consequences of neonatal hyperinsulinism occurring characteristically in GD offspring.METHODS:Newborn Wistar rats received daily subcutaneous injections of a long-acting insulin from the 8th to 11th day of life (IRI), while in controls (CO) NaCl was applied. Body weight was recorded throughout life. Glucose tolerance test was performed on the 140th day of life (1.5 g/kg glucose injected i.p. after an overnight fast and blood samples were taken up to 90 min from retroorbital plexus). On the 240th day of life, the vulnerability to a single "subdiabetogenic" dose of streptozotocin (STZ; 25 mg/kg body weight) was tested. Blood samples for estimating glucose levels were taken before STZ, and subsequently on days 2, 7, 14, 21, and 28 after STZ.RESULTS:IRI rats developed overweight during juvenile life until adulthood (P<0.001), characterized by a clear elevation of the Lee obesity index (P<0.005), and associated with basal hyperglycaemia (P<0.05), hyperinsulinaemia (P<0.05), as well as an increased insulin/glucose-ratio as a measure of insulin resistance (P<0.005). Impaired glucose tolerance occurred in early adulthood, and increased vulnerability to a "subdiabetogenic" dose of streptozotocin (see above), leading to significant hyperglycaemia (P<0.05), was evaluated in the 9th month of age. Accompanied by a transient reduction of hyperinsulinaemia during a period of 21 days, Lee obesity index and insulin/glucose-ratio decreased significantly after STZ treatment in IRI rats (P<0.01).CONCLUSIONS:Overweight and increased diabetes susceptibility in adulthood due to temporary hyperinsulinism during a critical period of postnatal life are suggested to be a consequence of acquired dysregulation and overstimulation, respectively, of the pancreatic insulin secretion in rats.

Perinatal overfeeding is a risk factor for overweight and diabetes during life. Underlying pathophysiological mechanisms are unclear. The peptide galanin is suggested to stimulate food intake by acting within the paraventricular hypothalamic nucleus (PVN). In early postnatally overfed rats overweight and hyperinsulinemia were observed, accompanied by an increased number of galanin-positive neurons in the PVN at weaning. Our results might indicate malformation of hypothalamic galaninergic neurons due to neonatal overfeeding and hyperinsulinism, respectively, in rats.

Insulin is a potent modulator of central nervous development and is suggested to influence the differentiation and maturation of hypothalamic structures involved in the regulation of body weight and metabolism. Hyperinsulinemic offspring of mothers with impaired glucose tolerance during pregnancy (gestational diabetes, GD) have an increased risk to develop overweight and diabetes mellitus during life, while the underlying pathophysiological mechanisms are still unknown. To investigate the effects of perinatal hyperinsulinism on the organization of hypothalamic regulators of body weight and metabolism, GD was induced in rats by application of streptozotocin on the day of conception (25 mg/kg, i.p.). On the 21st day of life, offspring of GD rats were overweight (p < 0.05) and hyperinsulinemic (p < 0.01). Using computer-assisted morphometric measurements, significantly decreased mean areas of neuronal nuclei and neuronal cytoplasm within the paraventricular hypothalamic nucleus (PVN; p < 0.01) and the ventromedial hypothalamic nucleus (VMN; p < 0.05) were observed in GD offspring. Analysis of topographically distinct parts revealed that these alterations particularly occurred in the parvocellular part of the PVN, as well as in the anterior, central, and dorsomedial part of the VMN. No morphometric alterations were found with in the lateral hypothalamic area and the dorsomedial hypothalamic nucleus. In the arcuate hypothalamic nucleus, the mean area of neuronal cytoplasm was decreased (p < 0.05), while the number of neurons expressing tyrosine hydroxylase was clearly elevated (p < 0.002). For astrocytes, a tendency towards an increased glia/neuron ratio was observed in the periventricular hypothalamic area. These observations suggest disturbed differentiation and organization of distinct hypothalamic nuclei and subnuclei, respectively, in hyperinsulinemic offspring of GD rats, possibly leading to dysfunctions of hypothalamic regulators of body weight and metabolism which might contribute to the lifelong increased risk to develop overweight and diabetogenic disturbances.

Overnutrition during critical developmental periods is suggested to be a risk factor for obesity and associated metabolic disorders in later life. Underlying mechanisms are unknown. Neuropeptides are essentially involved in the central nervous regulation of body weight. For instance, hypothalamic galanin (GAL) is a stimulator of food intake and body weight gain. To investigate long-term consequences of early postnatal overfeeding, the normal litter size of Wistar rats (n=10; controls) was reduced from day 3 to day 21 of life to only 3 pups per mother (small litters, SL; overnutrition). Throughout life, SL rats displayed hyperphagia (p<0.01), overweight (p<0.0001), hyperinsulinemia (p<0.01), impaired glucose tolerance (p<0.001), elevated triglycerides (p<0.001), and an increased systolic blood pressure (p<0.05). In adulthood, an increase of GAL-neurons in the arcuate hypothalamic nucleus (ARC) was found (p<0.001), positively correlated to body weight (p<0.001). A second experiment revealed hyperinsulinemia (p<0.001) and increased hypothalamic insulin levels (p<0.05) in SL rats during early postnatal life. Already on day 21 of life, i.e., at the end of the critical hypothalamic differentiation period, in SL rats the number of GAL-neurons was increased in the ARC (p<0.001), showing a positive correlation to body weight and insulin (p<0.05). In conclusion, neonatally acquired persisting malformation of hypothalamic galaninergic neurons, induced by early overfeeding and hyperinsulinism, might promote the development of overweight and syndrome X-like alterations during life.

Catecholamines are essential organizers of the developing brain. Throughout life, they are involved, e.g., in the regulation of body weight and metabolism by specific hypothalamic nuclei, which are suggested to be highly vulnerable to maternal gestational hyperglycemia. By application of streptozotocin (30 mg/kg, i.p.) gestational diabetes (GD) was induced in female rats. On the 1st day of life, male GD offspring were underweight (P<0.05) and hyperglycemic (P<0.05), while on the 21st day of life decreased body weight (P<0.001) and elevated pancreatic insulin (P<0.01) were observed. Using HPLC with electrochemical detection, hypothalamic catecholamines were determined in the newborns, and quantitative immunocytochemistry for tyrosine hydroxylase (TH) was performed. At birth, a tendency towards increased levels of norepinephrine (NE) and dopamine (DA) in the whole hypothalami of GD offspring was observed. In the 21-day-old offspring of GD mothers, NE was significantly increased in the ventromedial hypothalamic nucleus (VMN; P<0.05) and the lateral hypothalamic area (LHA; P<0.05), while DA was significantly elevated in the paraventricular hypothalamic nucleus (PVN; P<0.05) and the LHA (P<0.05). The NE/DA-ratio was found to be decreased in the PVN of GD offspring (P<0.01). Moreover, numerical density of TH-positive neurons was clearly increased within the parvocellular division of the PVN (P<0.0001) as well as in the periventricular hypothalamic area (PER; P<0.05). These data suggest specific alterations of catecholaminergic systems within hypothalamic regulators of body weight and metabolism during early development in the offspring of gestational diabetic mother rats.

Catecholamines are important organizers of the developing brain. During life, they are involved, e.g., in the regulation of body weight and metabolism by hypothalamic nuclei which are suggested to be highly vulnerable to malorganization by alterations of perinatal metabolism, as in early postnatal overnutrition. By reducing the litter size on the 3rd day of life (small litters, SL) early postnatal overnutrition was induced in Wistar rats. SL rats developed overweight (p < 0.001), associated with increased plasma insulin on the 21st day of life (p < 0.01). Using HPLC with ECD, catecholamines in various hypothalamic nuclei were determined, and quantitative immunocytochemistry for tyrosine hydroxylase (TH) was performed. In the 21-day-old SL rats, norepinephrine ((NE) was significantly increased in the paraventricular hypothalamic nucleus (PVN), while a significant decrease of dopamine (DA) appeared in the ventromedial hypothalamic nucleus (VMN). In the PVN of SL rats, this was accompanied by a reduction of the mean numerical density of neurons in general (p < 0.05) and an increased numerical density of neurons expressing TH (p < 0.05). Both parameters were unchanged within the periventricular hypothalamic area (PER). These data suggest specific disturbances of catecholaminergic systems within hypothalamic regulators of body weight and metabolism during development due to early postnatal overnutrition in rats.

Cholecystokinin (CCK) is suggested to be involved, e.g. in the central nervous modulation of food intake, possibly by acting within specific hypothalamic nuclei. Perinatal overnutrition predisposes to permanent obesity and hyperphagia, while underlying mechanisms are unclear. By reducing the litter size from the 3rd to 21st day of life, early overnutrition was induced in newborn rats. At weaning, clear overweight (P<0.001), hyperglycaemia (P<0.05), hyperinsulinaemia (P<0.001), and insulin resistance (P<0.001) occured. These early signs of obesity were associated with a significantly decreased number of CCK-positive neurons in the paraventricular hypothalamic nucleus (P<0.002). In conclusion, due to neonatal overfeeding malformation of CCKergic neurons at the end of the critical hypothalamic differentiation period occurs. Long-term consequences on CCK-related neuroendocrine regulations could be suggested, including those affecting food intake and body weight gain.

THE offspring of diabetic mothers is at increased risk to develop obesity and diabetogenic disturbances during life. Pathophysiological mechanisms responsible are unclear. Neuropeptide Y (NPY) is an important hypothalamic stimulator of food intake and body weight gain, and its levels are decreased by elevated insulin. In neonatally hyperinsulinaemic offspring of diabetic mother rats, hypothalamic insulin level was significantly increased at birth (p < 0.01). At weaning, i.e. at the end of the critical hypothalamic differentiation period, a significantly increased number of NPY-positive neurons (p < 0.01) appeared in the arcuate hypothalamic nucleus. In conclusion, an increase in the number of NPYergic neurons in the hypothalamus, possibly due to hypothalamic malformation and/or perinatally acquired hypothalamic insulin resistance, might contribute to the development of obesity and metabolic disturbances in the offspring of diabetic mothers.

Objective. Since perinatal stress events are well known to exert long-term influences on the function of hypothalamic-pituitary-adrenal (HPA) axis in rats, to investigate the consequences of exposure to IL-1ß, a potent stimulator of this axis, during early postnatal life. Methods. Wistar rats were treated twice a day with 0.02 µg human recombinant IL-1ß from day 1-4 of age, while controls received the vehicle only. Results. IL-1ß-treatment had no significant influence on the mortality and body weight. How- ever, at the end of treatment period on the 4th day of life, the thymus weight was decreased in the IL-1ß-treated group (P<0.01), while the adrenals were clearly enlarged (P<0.0002). These responses were associated with a nearly 4-fold elevation of the plasma corticosterone (CS) level as compared to vehicle-treated controls (P<0.001). At the age of seven months the stimulated CS levels induced by an acute stress (novel environment) were lower in rats treated neonatally with IL-1ß than in controls (P<0.01). This functional disturbance was associated with morphological alterations in the parvicellular part of the paraventricular nucleus (PVN) which is the main hypothalamic regula- tion centre of the HPA axis. A strong reduction of the numerical density of neurons was found in the neonatally IL-1ß-treated rats (P<0.005) while the neuronal nuclei were clearly enlarged (P<0.0005). Conclusion. As a part of an infection-induced stress response during critical periods of devel- opment, IL-1ß might be capable of inducing a permanent structural malorganization of the PVN and, consequently, functional malprogramming of the HPA axis in rats.