Daniel Castrogiovanni

Increased Male Offspring’s Risk of Metabolic-Neuroendocrine Dysfunction and Overweight after Fructose-Rich Diet Intake by the Lactating Mother

An adverse endogenous environment during early life predisposes the organism to develop metabolic disorders. We evaluated the impact of intake of an iso-caloric fructose rich diet (FRD) by lactating mothers (LM) on several metabolic functions of their male offspring. On postnatal d 1, ad libitum eating, lactating Sprague-Dawley rats received either 10% F (wt/vol; FRD-LM) or tap water (controls, CTR-LM) to drink throughout lactation. Weaned male offspring were fed ad libitum a normal diet, and body weight (BW) and food intake were registered until experimentation (60 d of age). Basal circulating levels of metabolic markers were evaluated. Both iv glucose tolerance and hypothalamic leptin sensitivity tests were performed. The hypothalamus was dissected for isolation of total RNA and Western blot analysis. Retroperitoneal (RP) adipose tissue was dissected and either kept frozen for gene analysis or digested to isolate adipocytes or for histological studies. FRD rats showed increased BW and decreased hypothalamic sensitivity to exogenous leptin, enhanced food intake (between 49-60 d), and decreased hypothalamic expression of several anorexigenic signals. FRD rats developed increased insulin and leptin peripheral levels and decreased adiponectinemia; although FRD rats normally tolerated glucose excess, it was associated with enhanced insulin secretion. FRD RP adipocytes were enlarged and spontaneously released high leptin, although they were less sensitive to insulin-induced leptin release. Accordingly, RP fat leptin gene expression was high in FRD rats. Excessive fructose consumption by lactating mothers resulted in deep neuroendocrine-metabolic disorders of their male offspring, probably enhancing the susceptibility to develop overweight/obesity during adult life.

Modulatory role of testosterone in plasma leptin turnover in rats.

We explored whether testosterone influences circulating leptin turnover in rats. Sham-operated male and female rats and 21-d gonadectomized rats treated or not treated with testosterone propionate were used. Anesthetized rats were implanted with an iv catheter, and then blood samples were drawn before and throughout a 60-min period following systemic leptin administration. Plasma testosterone, estradiol, and leptin concentrations were monitored. The results indicated that while gonadectomy blunted circulating concentrations of the homologous sex steroid, testosterone therapy, in gonadectomized rats, restored plasma testosterone concentrations to values found in normal male rats. Pharmacokinetic parameters evaluated during the test indicated the following: First, in the overall pharmacokinetic analyses, testosterone therapy in gonadectomized rats induced a more rapid disappearance of leptin from the circulation. Second, orchidectomy significantly enhanced the area under the curve (AUC) of circulating leptin, an effect fully reversed by testosterone treatment. Third, testosterone treatment in ovariectomized rats significantly decreased the AUC of leptin concentrations. Fourth, while gonadectomy alone did not modify circulating leptin half-life, conversely, testosterone therapy in gonadectomized rats decreased leptin half-life in the circulation. Finally, while orchidectomy reduced leptin body clearance, this parameter was increased by androgen therapy in gonadectomized rats. Our results strongly support that testosterone could play a main role in plasma leptin turnover by increasing leptin clearance rate and shortening plasma leptin half-life.

Parametrial Adipose Tissue and Metabolic Dysfunctions Induced by Fructose‐rich Diet in Normal and Neonatal‐androgenized Adult Female Rats

Hyperandrogenemia predisposes an organism toward developing impaired insulin sensitivity. The aim of our study was to evaluate endocrine and metabolic effects during early allostasis induced by a fructose‐rich diet (FRD) in normal (control; CT) and neonatal‐androgenized (testosterone propionate; TP) female adult rats. CT and TP rats were fed either a normal diet (ND) or an FRD for 3 weeks immediately before the day of study, which was at age 100 days. Energy intake, body weight (BW), parametrial (PM) fat characteristics, and endocrine/metabolic biomarkers were then evaluated. Daily energy intake was similar in CT and TP rats regardless of the differences in diet. When compared with CT‐ND rats, the TP‐ND rats were heavier, had larger PM fat, and were characterized by basal hypoadiponectinemia and enhanced plasma levels of non‐esterified fatty acid (NEFA), plasminogen activator inhibitor‐1 (PAI‐1), and leptin. FRD‐fed CT rats, when compared with CT‐ND rats, had high plasma levels of NEFA, triglyceride (TG), PAI‐1, leptin, and adiponectin. The TP‐FRD rats, when compared with TP‐ND rats, displayed enhanced leptinemia and triglyceridemia, and were hyperinsulinemic, with glucose intolerance. The PM fat taken from TP rats displayed increase in the size of adipocytes, decrease in adiponectin (protein/gene), and a greater abundance of the leptin gene. PM adipocyte response to insulin was impaired in CT‐FRD, TP‐ND, and TP‐FRD rats. A very short duration of isocaloric FRD intake in TP rats induced severe metabolic dysfunction at the reproductive age. Our study supports the hypothesis that the early‐androgenized female rat phenotype is highly susceptible to developing endocrine/metabolic dysfunction. In turn, these abnormalities enhance the risk of metabolic syndrome, obesity, type 2 diabetes, and cardiovascular disease.

Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

Constitutive and ligand-dependent GHSR1a activity attenuates CaV2 current and hypothalamic GABA release through distinct mechanisms and signaling pathways.

Oral metformin treatment prevents enhanced insulin demand and placental dysfunction in the pregnant rat fed a fructose-rich diet.

The intake of a fructose-rich diet (FRD) in the normal female rat induces features similar to those observed in the human metabolic syndrome phenotype. We studied the impact of FRD administration to mothers on pregnancy outcome. On gestational day (Gd) zero rats were assigned to either group: ad libitum drinking tap water alone (normal diet, ND) or containing fructose (10% w/vol; FRD) through pregnancy; all rats were fed a Purina chow diet ad libitum ND and FRD rats were daily cotreated or not with metformin (60 mg/Kg/day oral; ND + MF and FRD + MF) and submitted to a high glucose load test on Gd 14. Additionally, placentas from different groups were studied on Gd 20. Data indicated that: (1) although FRD rats well tolerated glucose overload, their circulating levels of insulin were significantly higher than in ND rats; (2) the mesometrial triangle blood vessel area was significantly lower in placentas from FRD than ND dams; (3) the detrimental effects of FRD administration to mothers were ameliorated by metformin cotreatment. Our study suggests that excessive intake of fructose during pregnancy enhanced the risk for developing gestational diabetes and subsequent preeclampsia, and that metformin prevented the poor pregnancy outcome induced by FRD.

Neuroendocrine, Metabolic, and Immune Functions during the Acute Phase Response of Inflammatory Stress in Monosodium L-Glutamate-Damaged, Hyperadipose Male Rat

In rats, neonatal treatment with monosodium L-glutamate (MSG) induces several metabolic and neuroendocrine abnormalities, which result in hyperadiposity. No data exist, however, regarding neuroendocrine, immune and metabolic responses to acute endotoxemia in the MSG-damaged rat. We studied the consequences of MSG treatment during the acute phase response of inflammatory stress. Neonatal male rats were treated with MSG or vehicle (controls, CTR) and studied at age 90 days. Pituitary, adrenal, adipo-insular axis, immune, metabolic and gonadal functions were explored before and up to 5 h after single sub-lethal i.p. injection of bacterial lipopolysaccharide (LPS; 150 µg/kg). Our results showed that, during the acute phase response of inflammatory stress in MSG rats: (1) the corticotrope-adrenal, leptin, insulin and triglyceride responses were higher than in CTR rats, (2) pro-inflammatory (TNFα) cytokine response was impaired and anti-inflammatory (IL-10) cytokine response was normal, and (3) changes in peripheral estradiol and testosterone levels after LPS varied as in CTR rats. These data indicate that metabolic and neroendocrine-immune functions are altered in MSG-damaged rats. Our study also suggests that the enhanced corticotrope-corticoadrenal activity in MSG animals could be responsible, at least in part, for the immune and metabolic derangements characterizing hypothalamic obesity.

Withdrawal of dietary phytoestrogens in adult male rats affects hypothalamic regulation of food intake, induces obesity and alters glucose metabolism

The absence of phytoestrogens in the diet during pregnancy has been reported to result in obesity later in adulthood. We investigated whether phytoestrogen withdrawal in adult life could alter the hypothalamic signals that regulate food intake and affect body weight and glucose homeostasis. Male Wistar rats fed from conception to adulthood with a high phytoestrogen diet were submitted to phytoestrogen withdrawal by feeding a low phytoestrogen diet, or a high phytoestrogen-high fat diet. Withdrawal of dietary phytoestrogens increased body weight, adiposity and energy intake through an orexigenic hypothalamic response characterized by upregulation of AGRP and downregulation of POMC. This was associated with elevated leptin and T4, reduced TSH, testosterone and estradiol, and diminished hypothalamic ERα expression, concomitant with alterations in glucose tolerance. Removing dietary phytoestrogens caused manifestations of obesity and diabetes that were more pronounced than those induced by the high phytoestrogen-high fat diet intake.

Melanocortin 4 receptor activation inhibits presynaptic N-type calcium channels in amygdaloid complex neurons

The melanocortin 4 receptor (MC4R) is a G protein‐coupled receptor involved in food intake and energy expenditure regulation. MC4R activation modifies neuronal activity but the molecular mechanisms by which this regulation occurs remain unclear. Here, we tested the hypothesis that MC4R activation regulates the activity of voltage‐gated calcium channels and, as a consequence, synaptic activity. We also tested whether the proposed effect occurs in the amygdala, a brain area known to mediate the anorexigenic actions of MC4R signaling. Using the patch‐clamp technique, we found that the activation of MC4R with its agonist melanotan II specifically inhibited 34.5 ± 1.5% of N‐type calcium currents in transiently transfected HEK293 cells. This inhibition was concentration‐dependent, voltage‐independent and occluded by the Gαs pathway inhibitor cholera toxin. Moreover, we found that melanotan II specifically inhibited 25.9 ± 2.0% of native N‐type calcium currents and 55.4 ± 14.4% of evoked inhibitory postsynaptic currents in mouse cultured amygdala neurons. In vivo, we found that the MC4R agonist RO27‐3225 increased the marker of cellular activity c‐Fos in several components of the amygdala, whereas the N‐type channel blocker ω conotoxin GVIA increased c‐Fos expression exclusively in the central subdivision of the amygdala. Thus, MC4R specifically inhibited the presynaptic N‐type channel subtype, and this inhibition may be important for the effects of melanocortin in the central subdivision of the amygdala.

Telomere instability is present in the progeny of mammalian cells exposed to bleomycin.

We analyzed the chromosomal aberrations involving telomeres in the progeny of mammalian cells exposed to the radiomimetic compound bleomycin (BLM) in order to determine if this antineoplastic drug induces long-term telomere instability. To this end, rat cells (ADIPO-P2 cell line, derived from adipose cells from Sprague-Dawley rat) were treated with a single concentration of BLM (2.5 μg/ml), and chromosomal aberrations were analyzed 18 h and 10 days after treatment by using PNA-FISH with a pan-telomeric probe [(TTAGGG)n repeats]. Cytogenetic analysis revealed a higher frequency of aberrations at 18 h and 10 days after treatment in BLM-exposed cultures vs. untreated cultures, although the yield of BLM-induced aberrations 10 days after treatment decreased about 25% compared with the one at 18 h after treatment. Moreover, the level of telomerase activity in BLM-treated cells compared with that of untreated control cells was significantly higher at 10 days after treatment, but did not differ at 18 h after treatment. These data indicate that in terms of unstable aberrations, the in vitro clastogenic effect of BLM on ADIPO-P2 cells persists for at least 10 days after exposure. In addition, our data demonstrate, for the first time, that BLM-induced telomere instability in mammalian cells (cytogenetically detectable as incomplete chromosome elements and telomere FISH signal loss and duplication) persists for several generations after exposure. Moreover, the appearance of telomere fusions in BLM-exposed cells 10 days after treatment suggests that this compound can induce delayed telomere instability. The increase in telomerase activity in BLM-exposed cells 10 days after treatment is accompanied by the presence of aberrations directly related to telomere dysfunction. This fact suggests that telomerase is not directly involved in BLM-induced telomere instability.

Enhanced proinflammatory cytokine response to bacterial lipopolysaccharide in the adult male rat after either neonatal or prepubertal ablation of biological testosterone activity.

A sex steroid-dependent modulation of the immune function in mammals is accepted, and evidence suggests that while estrogens enhance, androgens inhibit the immune response. The aim of this study was to explore in the adult male rat the effect of either neonatal flutamide (FTM) treatment or prepubertal orchidectomy (ODX) on endocrine markers in the basal condition and peripheral tumor necrosis factor alpha (TNFα) levels during inflammatory stress. For these purposes, (1) 5-day-old male rats were subcutaneously injected with either sterile vehicle alone or containing 1.75 mg FTM, and (2) 25-day-old male rats were sham operated or had ODX. Rats were sacrificed (at 100 days of age) in the basal condition for determination of peripheral metabolite levels. Additional rats were intravenously injected with bacterial lipopolysaccharide (LPS; 25 µg/kg body weight, i.v.) and bled for up to 4 h. Data indicate that (1) ODX increased peripheral glucocorticoid levels and reduced those of testosterone, whereas FTM-treated rats displayed low circulating leptin concentrations, and (2) LPS-induced TNFα secretion in plasma was significantly enhanced in the FTM and ODX groups. Our study supports that neonatal FTM treatment affected adiposity function, and adds data maintaining that androgens have a suppressive role in proinflammatory cytokine release in plasma during inflammation.