Hemodynamic effects of parathyroid hormone-related peptide-(1-34) in humans.

It has been suggested that PTH-related peptide-(1-34) (PTHrP) is a regulator or modulator of regional or systemic cardiovascular function with varying vasodilating actions in different species. We have studied the cardiovascular pharmacodynamic profile of PTHrP in healthy humans. In a double blind, placebo-controlled, cross-over study design, eight healthy subjects were assigned to stepwise increased i.v. doses of PTHrP. In addition, a dose-response curve to PTHrP was constructed in a dorsal hand vein in eight subjects. PTHrP dose-dependently increased pulse rate and renal plasma flow by more than 50% (P < 0.0001 for both parameters, by ANOVA), but only a small venodilating response was seen in hand vein experiments, and no effect was noted on mean arterial blood pressure or cardiac inotropic performance. Although it is unlikely that PTHrP regulates systemic hemodynamics, its chronotropic effect and its potent action on renal plasma flow may represent the primary cardiovascular physiological targets of action.

Diclofenac concentrations in defined tissue layers after topical administration.

INTRODUCTION: To date it is unclear whether therapeutic concentrations are attained in target tissues after topical administration of nonsteroidal anti-inflammatory drugs. Therefore this study in healthy volunteers was undertaken to measure diclofenac concentrations attained in defined tissue layers directly underlying the site of topical diclofenac application by in vivo microdialysis. METHODS: In each experiment two microdialysis probes were inserted, one into a superficial (3.9 +/- 0.3 mm) and one into a deep (9.3 +/- 0.5 mm) tissue layer, in 20 healthy volunteers and calibrated in vivo. The distance between the surface of the skin and the tips of the microdialysis probes was measured by 7.5 MHz ultrasound. Diclofenac was administered topically as a single dose of approximately 300 mg/100 cm2. Concentration versus time profiles in tissue layers were monitored for 5 hours. RESULTS: Concentration versus time profiles were obtained in 11 of 20 experiments. However, there was no correlation between area under the concentration-time curve (AUC) in a defined layer and the depth of probe insertion. In those experiments where concentration versus time profiles were obtained for both probes mean AUC was 532 +/- 197 microg x min x ml(-1) for superficial layers, and 438 +/- 249 microg x min x ml(-1) for deep layers. CONCLUSION: We conclude that transdermal penetration of diclofenac, at least after single doses, is not predictable and may strongly be influenced by individual skin properties.

Hemodynamic effects of continuous urodilatin infusion: a dose-finding study.

OBJECTIVE: To evaluate the effects of urodilatin (INN, ularitide) on systemic and renal hemodynamic parameters. METHODS: Twenty healthy male subjects were included in this double-blind, randomized placebo-controlled trial and assigned to receive either continuous intravenous infusion of different doses of 7.5, 15, or 22.5 ng/kg body weight/min urodilatin or placebo over 300 minutes. Cardiac performance, systolic time intervals, and airway function were measured noninvasively. The effects on renal hemodynamic values were assessed with para-aminohippurate and inulin clearance techniques. RESULTS: Urodilatin was well tolerated by all subjects at doses of 7.5 and 15 ng/kg/min. Infusion was stopped prematurely for the group that received 22.5 ng/kg/min urodilatin group because of systemic hypotensive responses with nausea and dizziness. Infusion of 15 ng/kg/min urodilatin significantly increased urine flow by a maximum of 165%, filtration fraction by 46%, renal resistance by 49%, and systemic vascular resistance by 45%. It decreased renal plasma flow by a maximum of 31% from baseline value. No change in cardiac inotropic function was detectable, but cardiac output decreased in all dose groups. Effects on glomerular filtration rate, forced expiratory volume, blood pressure, and pulse were not different from those with placebo. CONCLUSION: Continuous infusion of 7.5 ng/kg/min and 15 ng/kg/min urodilatin exerts a significant increase in systemic and renal vascular resistance. Results of our experiments suggested that the therapeutic window for continuous urodilatin infusion is small and that doses higher than approximately 20 ng/kg/min urodilatin carry high risk for adverse drug reactions.

In vivo drug-response measurements in target tissues by microdialysis.

To study the suitability of the microdialysis technique for the measurement of target tissue pharmacodynamics in humans, the model compounds theophylline, milrinone, and compound 48/80 were administered locally by means of reversed microdialysis to the interstitial space of skeletal muscle or skin in 24 healthy volunteers. Simultaneously, interstitial concentrations of cyclic adenosine monophosphate (cAMP; as an indicator of phosphodiesterase activity) were measured in skeletal muscle, and interstitial concentrations of histamine (as an indicator of mast cell release) were measured in skin. In muscle, reversed microdialysis with milrinone led to a dose-dependent increase in interstitial cAMP concentrations (n = 8), whereas no significant effect on cAMP was observed for theophylline versus placebo (1.63 +/- 0.53 nmol/L; n = 6), even at local concentrations exceeding those attained after therapeutic doses. In skin, reversed microdialysis with compound 48/80 increased interstitial histamine concentration dose dependently versus placebo (5.99 +/- 2.74 nmol/L; n = 10). From our experiments in human skeletal muscle and skin, we concluded that microdialysis was a suitable technique for the characterization of in vivo drug response at the relevant target site. Extension of these measurements to several other human tissues is readily feasible.

Indium-111-DOTA-lanreotide: biodistribution, safety and radiation absorbed dose in tumor patients.

UNLABELLED: Imaging with radiolabeled somatostatin/vasoactive intestinal peptide analogs has recently been established for the localization diagnosis of a variety of human tumors including neuroendocrine tumors, intestinal adenocarcinomas and lymphomas. This study reports on the biodistribution, safety and radiation absorbed dose of 111In-1,4,7,10-tetraazacyclododecane-N,N',N",N'''-tetraacetic acid (DOTA)-lanreotide, a novel peptide tracer, which identifies hSST receptor (R) subtypes 2 through 5 with high affinity, and hSSTR1 with low affinity. METHODS: The tumor localizing capacity of 111In-DOTA-lanreotide was initially investigated in 10 patients (3 lymphomas, 5 carcinoids and 2 intestinal adenocarcinomas). Indium-111 -DOTA-lanreotide was then administered to 14 cancer patients evaluated for possible radiotherapy with 90Y-DOTA-lanreotide (8 neuroendocrine tumors, 4 intestinal adenocarcinomas, 1 Hodgkin lymphoma and 1 prostate cancer). After intravenous administration of 111In-DOTA-lanreotide (approximately = 150 MBq; 10 nmol/patient), sequential images over one-known tumor site were recorded during the initial 30 min after peptide application. Thereafter, whole-body images were acquired in anterior and posterior views up to 72 hr postinjection. Dosimetry calculations were performed on the basis of scintigraphic data, urine, feces and blood activities. A comparison with 111In-DTPA-D-Phe1-octreotide (111In-OCT) scintigraphy was performed in 8 of the patients. RESULTS: After an initial rapid blood clearance [results of biexponential fits: T(eff1) 0.4 min (fraction a1 80%) and T(eff2) 13 min (fraction a2 14%)], the radioactivity was excreted into the urine and amounted to 42% +/- 3% of the injected dose (%ID) within 24 hr and 62% +/- 6%ID within 72 hr after injection of 111In-DOTA-lanreotide. In all patients, tumor sites were visualized during the initial minutes after injection of 111In-DOTA-lanreotide. The mean radiation absorbed dose amounted to 1.2 (range 0.21-5.8) mGy/MBq for primary tumors and/or metastases. The effective half-lives of 111In-DOTA-lanreotide in the tumors were T(eff1) 4.9 +/- 2.2 and T(eff2) 37.6 +/- 6.6 hr, and the mean residence time tau was 1.8 hr. The highest radiation absorbed doses were calculated for the spleen (0.39 +/- 0.13 mGy/MBq), kidneys (0.34 +/- 0.08 mGy/MBq), urinary bladder (0.21 +/- 0.03 mGy/MBq) and liver (0.16 +/- 0.04 mGy/MBq). The effective dose was 0.11 +/- 0.01 (range 0.09-0.12) mSv/MBq. During the observation period of 72 hr, no side effects were noted after 111In-DOTA-lanreotide application. The 111In-DOTA-lanreotide radiation absorbed tumor dose was significantly higher (ratio 2.25 +/- 0.60, p < 0.01) when directly compared with 111In-OCT. CONCLUSION: Indium-111 -DOTA-lanreotide shows a high tumor uptake for a variety of different human tumor types, has a favorable dosimetry over 111In-OCT and is clinically safe.

Effects of systemic NO synthase inhibition on choroidal and optic nerve head blood flow in healthy subjects.

PURPOSE: There is evidence from animal studies that nitric oxide (NO) is a major determinant of ocular blood flow. In humans NO synthase inhibition reduces pulsatile choroidal blood flow, but no data on optic nerve head (ONH) vasculature are available yet. The goal of this study was to investigate the effects of NO synthase inhibition on human choroidal and ONH blood flow using laser Doppler flowmetry. METHODS: The study design was a randomized, placebo-controlled, double-masked, balanced three-way crossover. On separate study days 12 healthy male subjects received infusions of N:(G)-nitro-L-arginine (L-NMMA; either 3 mg/kg over 5 minutes followed by 30 microg/kg per minute over 55 minutes or 6 mg/kg over 5 minutes followed by 60 microg/kg per minute over 55 minutes) or placebo. The effects of L-NMMA or placebo on choroidal and ONH blood flow were measured with laser Doppler flowmetry. In addition, laser interferometric measurement of fundus pulsation was performed in the macula to assess pulsatile choroidal blood flow. RESULTS: L-NMMA reduced all outcome parameters in the choroid and the ONH. The higher dose of L-NMMA caused a significant decrease in blood flow in the choroid (-26% +/- 9%; P: < 0.001) and the ONH (-20% +/- 16%; P: < 0.001) as evidenced from laser Doppler flowmetry and a significant decrease in fundus pulsation amplitude (-26% +/- 5%; P: < 0.001). CONCLUSIONS: These results indicate that NO is continuously released in human choroidal and ONH vessels.

Endothelin-1 contributes to hyperoxia-induced vasoconstriction in the human retina.

PURPOSE: There is evidence that ocular blood flow strongly depends on arterial oxygen tension. Results from recent animal studies indicate that the vasoconstrictor response to hyperoxia may be mediated in part by an increased production of endothelin (ET)-1. In an effort to answer the question whether the retinal vasoconstrictive response to hyperoxia in humans is mediated through ET-1, changes in ocular hemodynamics induced by 100% O2 breathing were studied in the absence and presence of an ET(A) receptor antagonist (BQ-123). METHODS: The study was a randomized, placebo-controlled, double-masked, balanced, three-way crossover design. On separate study days 15 healthy male subjects received infusions of BQ-123 (either 60 microg/min or 120 microg/min) or placebo. The effects of BQ-123 or placebo on hyperoxia-induced (100% O2 breathing) changes in retinal and pulsatile choroidal blood flow were assessed with the blue-field entoptic technique and with laser interferometric measurement of fundus pulsation, respectively. RESULTS: During baseline conditions, hyperoxia caused a decrease in retinal blood flow between -29% and -34% (P<0.001) and a decrease in fundus pulsation amplitude between -7% and -8% (P<0.001). BQ-123 dose dependently blunted the response to hyperoxia in the retina (60 microg/min: -25%, 120 microg/min: -20%; P = 0.003), but not in the choroid. CONCLUSIONS: These results indicate that ET-1 contributes to hyperoxia-induced retinal vasoconstriction in the human retina.

Similar effects of atorvastatin, simvastatin and pravastatin on thrombogenic and inflammatory parameters in patients with hypercholesterolemia.

BACKGROUND: Previous studies have suggested that statins exert beneficial effects beyond their favorable lipid lowering effect. Particularly, the modification of thrombus formation and degradation, alteration in inflammatory response, plaque stabilization and improved endothelial function are thought to be responsible for additional reduction of morbidity and mortality due to cardiovascular events. To date, however, it is still unclear whether these effects are elicited by all statins. METHODS AND RESULTS: We set out to compare in a controlled, randomized, double-blind study design the effects of almost equieffective cholesterol lowering doses of three chemically and pharmacokinetically different statins (atorvastatin, simvastatin, pravastatin) on hemostatic and inflammatory markers in 99 hypercholesterolemic patients. At entry and 3 months after onset of statin therapy plasma cholesterol and von Willebrand factor antigen (vWf-Ag), fibrinogen, d-dimer, prothrombin fragment 1+2 (F1.2) and C-reactive protein (CRP) were measured. The effect on plasma values of F1.2, vWf-Ag, d-dimer and CRP was not significantly different between the three treatment groups. The effect of simvastatin on fibrinogen (p = 0.005) was more pronounced than the effects of atorvastatin (p = 0.48 n.s.) and pravastatin (p = 0.15 n.s.). Plasma levels of F1.2 and vWf-Ag (when data of all statins were pooled) were significantly reduced by 7% and 10% versus baseline, respectively. No significant reduction was observed for d-dimer (p = 0.26) and CRP (p = 0.5). Total plasma cholesterol levels decreased significantly (p < 0.0001 in all groups) between 22% and 29% compared to baseline. CONCLUSION: The present study shows similar short-term (3 months) effects of atorvastatin, simvastatin and pravastatin on selected hemostatic and inflammatory parameters in plasma in patients with hypercholesterolemia. Thus, chemical and pharmacological differences between statins appear to exert no major influence on these parameters.

Varying sensitivity to the negative oestrogen feedback during the ovarian cycle of female rats: evidence for the involvement of oestrogen and the medial preoptic area.

The gonadotrophic response to a single injection of oestradiol benzoate (OB) was studied in acutely ovariectomized adult rats during the different stages of a 4-day ovarian cycle. The results showed a sudden decline of the sensitivity to the gonadotrophin-inhibiting effect of OB between metoestrus and dioestrus. This desensitization to the negative oestrogen feedback was probably caused by an oestrogen action on the medial preoptic area (MPOA). In rats ovariectomized and implanted with OB in the MPOA in metoestrus, an s.c. injection of OB on the presumptive day of pro-oestrus did not lower the circulating LH and FSH levels, whereas a clear suppression of gonadotrophin secretion was seen in females implanted with cholesterol in the MPOA or implanted with OB in the hypothalamic ventromedial-arcuate region. Similar findings were obtained in rats which had been ovariectomized 3-4 weeks before implantation. A final experiment demonstrated that bilateral lesioning of the MPOA also reduced the sensitivity to the negative feedback action of oestrogen in long-term ovariectomized rats. In all experiments performed, diminution of the oestrogen-induced inhibition of LH secretion was more marked than that of suppression of FSH secretion. It is proposed that desensitization to the negative oestrogen feedback, probably resulting from an inhibitory oestrogen action on medial preoptic neurones, is a prerequisite for adequate gonadotrophic support of preovulatory follicle maturation in the presence of a continuously rising oestrogen concentration in the blood.

Evidence that desensitization of the negative feedback of oestrogen and priming for the positive feedback of oestrogen depend upon a common mechanism of oestrogen action in rats.

Oestrogen priming of the central nervous system is required for the positive feedback of oestrogen, and the sensitivity of the negative feedback of oestrogen can be reduced by oestrogen itself. Using adult female and male rats we examined the possibility that these effects depend upon a common mechanism of oestrogen action that is mediated by the medial preoptic area (MPOA). Guide cannulae were implanted in the MPOA of 4-day cyclic rats which were ovariectomized during the evening of day 1 of dioestrus. Glass capillary tubes containing different substances were placed in the cannulae between 09.00 and 12.00 h on the presumptive day 2 of dioestrus. The effectiveness of oestrogen priming was evaluated by examining whether an s.c. implant of oestradiol-17 beta (OE2) induced an LH surge, and the inhibitory effect of oestrogen on tonic LH secretion was investigated by injecting the rats with 3 micrograms oestradiol benzoate (OB)/100 g body weight. The priming effect of an s.c. implant of OE2 could be imitated by the bilateral implantation in the MPOA of a mixture of OB and cholesterol at a ratio of 1:360 for 3 h only. Similar medial preoptic oestrogen implantation also significantly reduced the LH-inhibiting effect of OB. In accord with findings obtained in former studies on desensitization of the negative oestrogen feedback, oestrogen priming resulting from the s.c. administration of OE2 could be suppressed by short-term medial preoptic implantation of clomiphene citrate or apomorphine; bilateral electrical stimulation of the medial amygdaloid nucleus induced an increase in the serum concentration of LH in ovariectomized females implanted with OB in the MPOA, but not in castrated males pretreated and implanted with OB.(ABSTRACT TRUNCATED AT 250 WORDS)

Retardation of preovulatory desensitization to negative oestrogen feedback: mechanism of the effect of progesterone in 5-day cyclic rats.

Recent studies have shown that oestrogen can induce desensitization to its own gonadotrophin-inhibiting effect in female rats by an action on the medial preoptic area (MPOA). Probably as a consequence of this action, sensitivity to the negative oestrogen feedback declines markedly between metoestrus and dioestrus of the 4-day ovarian cycle. To study this desensitization process in 5-day cyclic rats, females exhibiting regular 5-day vaginal cyclicity were ovariectomized on consecutive days of the cycle, injected with oestradiol benzoate (OB) or oil on the day of ovariectomy and autopsied 24 h after the injection. Estimation of the serum concentration of LH revealed that desensitization to negative oestrogen feedback occurred only between day 2 of dioestrus and pro-oestrus, i.e. 2 days later than in females with a 4-day cycle. In the latter animals, an injection of progesterone in metoestrus or early dioestrus, which induced lengthening of the ovarian cycle for 1 day, delayed the onset of desensitization to a degree similar to that found in spontaneously 5-day cyclic rats. In acutely ovariectomized females, progesterone implants placed in the MPOA, but not those located in the mediobasal hypothalamus, increased the LH-inhibiting effect of low doses of OB. The results suggest that the prolonged secretion of progesterone recorded in 5-day cyclic rats retards follicle maturation and delays the forthcoming ovulation by acting, at least partly, on the MPOA and antagonizing the desensitizing effect of oestrogen. In this way, inhibition of gonadotrophin secretion by oestrogen is enhanced and the increase in tonic LH secretion necessary for the completion of follicle maturation is retarded.

Hormone-dependent brain development.

Hormones may be defined as chemical messengers that are produced in specialized cells and exert biological effects on other cells of the same organism by acting either locally (as local hormones) or on distant target cells (as systemic hormones). Hence, neurotransmitters may be regarded as local hormones of the brain, and two different hormonal actions can be distinguished for neurotransmitters as well as for systemic hormones: (1) transient, i.e. reversible activational or inactivational effects and (2) persistent, i.e. more or less irreversible differentiational or organizational effects, if the hormones act during critical developmental periods, especially of the brain. Abnormal levels of systemic hormones and neurotransmitters produced by genetic defects or deficient environments and occurring during brain differentiation can act as 'teratogens'. They lead to malorganizations of the brain and permanent dysfunctions of fundamental processes of life, such as reproduction, metabolism, and/or information processing. Such malorganizations of the brain appear to be preventable by improving the external environment and/or by diagnosing and correcting abnormalities of systemic hormones and neurotransmitters during brain development. These principles of 'teratophysiology and teratopsychology' open new possibilities for preventive therapy.

Observations on the orexigenic hypothalamic neuropeptide Y-system in neonatally overfed weanling rats.

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.

Syndrome X-like alterations in adult female rats due to neonatal insulin treatment.

Hypothalamic structures are decisively involved in the regulation of body weight and metabolism. In syndrome X, complex metabolic alterations are present, which in women are found to be associated with disturbances of reproductive function and altered androgen levels. In previous experiments in rats, it was shown that a temporary intrahypothalamic hyperinsulinism during early life predisposes to overweight and diabetogenic disturbances later in life, associated with disorganization of hypothalamic regulatory centers. To investigate the possible long-term consequences of elevated peripheral insulin levels during ontogenesis, the following experiment was performed. Newborn female Wistar rats were treated during neonatal life with daily subcutaneous injections of long-acting insulin ([IRI group] 0.3 IU on days 8 and 9 of life and 0.1 IU on days 10 and 11 of life), whereas control animals (CO) received daily NaCl injections. This temporary exposure to increased insulin levels during a critical developmental period resulted in an increased body weight gain including juvenile life and adulthood (P < .01), accompanied by hyperinsulinemia (P < .01), impaired glucose tolerance (P < .05), and increased systolic blood pressure in adulthood (P < .025). No significant alterations were detected either in cyclicity and fertility or in the levels of testosterone, androstenedione, or dehydroepiandrosterone (DHEA) in IRI rats. Morphometric evaluation of hypothalamic nuclei showed a reduced numerical density of neurons (P < .025) and a decreased neuronal volume density (P < .025) within the ventromedial hypothalamic nucleus (VMN) of the IRI rats, whereas the antagonistic lateral hypothalamic area (LHA) was morphometrically unchanged. Newborn offspring of IRI rats (F1 generation) were overweight (P < .05) and had an increased pancreatic insulin concentration (P < .02). In conclusion, perinatal hyperinsulinism seems to predispose to the later development of syndrome X-like changes in female rats, possibly due to impaired organization of hypothalamic regulators of body weight and metabolism.

Hypothalamic insulin and neuropeptide Y in the offspring of gestational diabetic mother rats.

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.

Elevation of hypothalamic neuropeptide Y-neurons in adult offspring of diabetic mother rats.

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.

Increased number of galanin-neurons in the paraventricular hypothalamic nucleus of neonatally overfed weanling 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.

Perinatal elevation of hypothalamic insulin, acquired malformation of hypothalamic galaninergic neurons, and syndrome x-like alterations in adulthood of neonatally overfed rats.

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.

Morphological alterations of hypothalamic nuclei due to intrahypothalamic hyperinsulinism in newborn rats.

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.

Alterations of hypothalamic catecholamines in the newborn offspring of gestational diabetic mother rats.

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.