Andrés Giovambattista

Fructose-rich diet-induced abdominal adipose tissue endocrine dysfunction in normal male rats.

We have currently studied the changes induced by administration of a fructose-rich diet (FRD) to normal rats in the mass and the endocrine function of abdominal (omental) adipose tissue (AAT). Rats were fed ad libitum a standard commercial chow and tap water, either alone (control diet, CD) or containing fructose (10%, w/vol) (FRD). Three weeks after treatment, circulating metabolic markers and leptin release from adipocytes of AAT were measured. Plasma free fatty acids (FFAs), leptin, adiponectin, and plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in FRD than in CD rats. AAT mass was greater in FRD than in CD rats and their adipocytes were larger, they secreted more leptin and showed impaired insulin sensitivity. While leptin mRNA expression increased in AAT from FRD rats, gene expression of insulin receptor substrate, IRS1 and IRS2 was significantly reduced. Our study demonstrates that administration of a FRD significantly affects insulin sensitivity and several AAT endocrine/metabolic functions. These alterations could be part of a network of interacting abnormalities triggered by FRD-induced oxidative stress at the AAT level. In view of the impaired glucose tolerance observed in FRD rats, these alterations could play a key role in both the development of metabolic syndrome (MS) and β-cell failure.

Food Intake-Induced Leptin Secretion Modulates Hypothalamo-Pituitary-Adrenal Axis Response and Hypothalamic Ob-Rb Expression to Insulin Administration

The mutation of the ob gene is known to induce a phenotype of obesity accompanied by symptoms including enhanced production of glucocorticoid. Chronic administration to ob/ob mice of leptin, the ob gene product, reverses hypercorticosteronemia. This establishes a clear relation between adipocyte and hypothalamo-pituitary-adrenal (HPA) axis functions. In the present study we investigated the acute modulatory effects of food intake-stimulated leptin secretion on HPA axis activity and hypothalamic leptin receptor (Ob-Rb) expression in 24-hour fasting, adult female, BALB/c mice after insulin-induced hypoglycemia. Our results indicate that: (1) food supply for 45 min to 24-hour fasting mice increased plasma glucose levels and reversed both hypercorticosteronemia and hypoleptinemia; (2) the insulin-induced hypoglycemia produced a marked HPA axis activation in animals with no access to food but this response was fully prevented by food intake and the consecutive increase in plasma leptin levels; (3) the inhibitory effect of leptin on the HPA axis response to insulin-induced hypoglycemia was corroborated by i.p. administration of murine leptin, and (4) fasting-induced hypothalamic Ob-Rb overexpression is not modulated by insulin itself but by leptin, since increase in leptin levels by food intake or by administration of exogenous leptin completely reversed this Ob-Rb overexpression. These results confirm the inhibitory effect of leptin on the HPA axis response to various stress stimuli. They clearly demonstrate that acute food intake in 24-hour fasting mice: (a) rapidly reduced fasting-induced hypercorticosteronemia by enhancing both spontaneous and insulin-elicited endogenous leptin secretion; (b) fully prevented HPA axis response to insulin administration, by rapidly increasing endogenous leptin secretion and probably also by diminishing the extent and the duration of insulin-induced hypoglycemia, and (c) abolished hypothalamic Ob-Rb overexpression induced by fasting itself combined with insulin treatment. The present data strongly suggests an inhibitory effect of endogenous leptin on insulin-induced HPA axis response, an interaction relevant to the physiological adaptation to starvation and caloric excess, and further supports the pivotal role played by the hypothalamus in restoring homeostasis in different allostatic states.

Ghrelin gene-related peptides modulate rat white adiposity.

It is known that ghrelin and des-N-octanoyl (desacyl) ghrelin modulate food intake and adipogenesis in vivo. However, desacyl ghrelin represents the majority of ghrelin forms found in the circulation. The present study explored whether ghrelin gene-derived compounds could modulate, in vitro, adipocyte endocrine function and preadipocyte differentiation. Retroperitoneal (RP) adipocytes were cultured in the absence or presence of either ghrelin or desacyl ghrelin and in combination with either inhibitors of protein synthesis, insulin, dexamethasone (DXM), or GHSR1a antagonist. The results indicate that both ghrelin forms possess a direct leptin-releasing activity (LRA) on RP adipocytes and significantly enhanced adipocyte ob mRNA expression. These activities were related and unrelated to the activation of GHSR1a after coincubation with ghrelin and desacyl ghrelin, respectively. Moreover, desacyl ghrelin facilitated RP preadipocyte differentiation. Desacyl ghrelin enhanced cell lipid content, and PPARgamma2, and LPL mRNAs expression. The LRAs developed by different substances tested followed a rank order: ghrelin > desacyl ghrelin = insulin > or = DXM. Additionally, desacyl ghrelin was able to enhance medium glucose consumption by mature adipocytes in culture. These data strongly support that adipogenesis and adipocyte function are processes directly and positively modulated by ghrelin gene-derived peptides, thus further indicating that, besides their effects on food intake, ghrelin gene-derived peptides could play an important role on adiposity for maintaining homeostasis.

Antioxidant treatment prevents the development of fructose-induced abdominal adipose tissue dysfunction

In the present study, we tested the effect of OS (oxidative stress) inhibition in rats fed on an FRD [fructose-rich diet; 10% (w/v) in drinking water] for 3 weeks. Normal adult male rats received a standard CD (commercial diet) or an FRD without or with an inhibitor of NADPH oxidase, APO (apocynin; 5 mM in drinking water; CD-APO and FRD-APO). We thereafter measured plasma OS and metabolic-endocrine markers, AAT (abdominal adipose tissue) mass and cell size, FA (fatty acid) composition (content and release), OS status, LEP (leptin) and IRS (insulin receptor substrate)-1/IRS-2 mRNAs, ROS (reactive oxygen species) production, NADPH oxidase activity and LEP release by isolated AAT adipocytes. FRD-fed rats had larger AAT mass without changes in body weight, and higher plasma levels of TAG (triacylglycerol), FAs, TBARS (thiobarbituric acid-reactive substance) and LEP. Although no significant changes in glucose and insulin plasma levels were observed in these animals, their HOMA-IR (homoeostasis model assessment of insulin resistance) values were significantly higher than those of CD. The AAT from FRD-fed rats had larger adipocytes, higher saturated FA content, higher NADPH oxidase activity, greater ROS production, a distorted FA content/release pattern, lower insulin sensitivity together with higher and lower mRNA content of LEP and IRS-1-/2 respectively, and released a larger amount of LEP. The development of all the clinical, OS, metabolic, endocrine and molecular changes induced by the FRD were significantly prevented by APO co-administration. The fact that APO treatment prevented both changes in NADPH oxidase activity and the development of all the FRD-induced AAT dysfunctions in normal rats strongly suggests that OS plays an important role in the FRD-induced MS (metabolic syndrome) phenotype.

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.

Impact of Maternal Undernutrition on Hypothalamo-Pituitary-Adrenal Axis and Adipocyte Functions in Male Rat Offspring

Malnutrition induces profound deleterious effects on several metabolic and neuroendocrine functions. In the present study, we examined the impact of maternal food restriction, during gestation and lactation, on the metabolic-neuroendocrine function of their male offspring at 21 and 60 d of age. Well-nourished (WN) and undernourished (UN) pregnant rats were used, during gestation and lactation, until pups were weaned. Twenty-one-day-old WN and UN male pups were studied in basal and postinsulin conditions. Additional groups of weaned (WN and UN) male rats were fed either ad libitum (WN-WN and UN-WN) or in a restricted fashion (WN-UN and UN-UN) until experimentation at age 60 d. Body weights of mothers and their male offspring were monitored. Basal and post-insulin plasma concentrations of several metabolic fuels were evaluated. Our results indicate that 21-d-old UN male rats exhibited (vs their WN counterparts), decreased body weights, similar basal and postinsulin glycemia, similar basal plasma adrenocorticotropic hormone (ACTH) and corticosterone levels but diminished ACTH response to insulin treatment, and basal hypoleptinemia and significant insulin-induced leptin release. Finally, at 60 d of age, long-term UN (WN-UN and UN-UN) rats showed lower plasma (basal and postinsulin) glucose, and basal triglyceride levels than their counterparts (WN-WN and UN-WN). Sixty-day-old rats submitted to either food restriction protocol also showed a reduced hypothalamo-pituitary-adrenalaxis response to insulin-induced hypoglycemia and basal hypoleptinemia, in spite of restoration of normal body weights. These results further indicate a clear metabolic-neuroendocrine dysfunction in male pups of UN mothers, with the abnormality partially present at weaning and deteriorated by adulthood, even after the recovery of normal body weight. Our study strongly supports the importance of the irreversibility of a deleterious allostatic state resulting from fetal and early postnatal undernutrition.

Modulatory Role of the Epinergic System in the Neuroendocrine-Immune System Function

It is well recognized that the reciprocal interaction established between the immune and neuroendocrine systems is crucial for the homeostatic adaptation of individuals during septicemia. In the present study, using an in vivo rat model, we investigated the degree of participation of central and peripheral epinergic systems in the modulation of the hypothalamic-pituitary-adrenal and immune axes’ functions during endotoxemia. For this purpose, acute endotoxemia was induced in adult male rats pretreated intraperitoneally with either different inhibitors of phenylethanolamine-N-methyltransferase (PNMT) [which are active either peripherally (SKF 29661) or both peripherally and centrally (SKF 64139), thus lowering epinephrine (EPI) synthesis] or vehicle only (CTRL). Twelve hours after pretreatment, animals were intraperitoneally injected with vehicle alone (basal) or vehicle containing bacterial lipopolysaccharide (LPS) and sacrificed 2 h later. A significant (p < 0.05 vs. the respective basal value) hypoglycemia was found in all groups studied. No pretreatment modified basal plasma adrenocorticotropic hormone (ACTH), glucocorticoid and cytokine concentrations. Endotoxin-stimulated ACTH secretion was severalfold (p < 0.05) higher than the respective basal value in CTRL and in SKFs-pretreated rats; however, the plasma ACTH levels after LPS were significantly (p < 0.05 vs. CTRL and SKF-29661 values) reduced in SKF-64139-pretreated rats. All groups studied showed an appropriate adrenal response to endotoxin challenge. Although no differences were found in basal anterior pituitary (AP) ACTH content among groups, LPS treatment significantly (p < 0.05 versus the respective basal value) decreased AP ACTH in CTRL and SKF 29661 groups. No pretreatment modified the basal medial basal hypothalamus (MBH) corticotropin-releasing hormone (CRH) content. Conversely, SKF 64139 pretreatment significantly (p < 0.05 vs. CTRL and SKF 29661 values) reduced basal median eminence (ME) CRH content, and LPS administration significantly (p < 0.05) decreased ME CRH in CTRL and SKF-29661-pretreated rats. SKF 64139 pretreatment significantly (p < 0.05) enhanced basal MBH and ME arginine vasopressin (AVP) contents. LPS administration significantly (p < 0.05) decreased MBH AVP in CTRL and SKF-29661-pretreated rats and diminished (p < 0.05 vs. basal values) ME AVP in all groups. The plasma tumor necrosis factor alpha (TNFα) concentrations were enhanced severalfold (p < 0.05 vs. basal values) after LPS treatment in CTRL rats, but not in SKFs-treated animals. In order to explore the reduced cytokine release after LPS in PNMT-inhibited rats, additional ex vivo experiments were performed by using peripheral mononuclear cells (PMNC) from both CTRL and SKF-29661-pretreated rats. The results of these experiments confirmed an immune dysfunction after inhibition of peripheral EPI synthesis; in fact, basal and concanavalin-A-stimulated TNFα secretions were significantly (p < 0.05) lower in SKF-29661-treated than in CTRL PMNC, while, interestingly, addition of EPI (10–7 M) to the medium fully restored these effects. These data demonstrate that: (1) the mechanism whereby LPS-induced hypoglycemia was independent of epinergic activity; (2) selective central inhibition of epinergic function reduced endotoxin-stimulated ACTH secretion, an effect that could mainly be due to a decrease in CRH-ergic activity; (3) the central epinergic system positively and negatively modulates CRH- and AVPergic functions, respectively, and (4) inhibition of peripheral PNMT activity reduced immune system function in vivo and ex vivo. It is further suggested that low peripheral levels of EPI could be beneficial for the body’s defense mechanisms during endotoxic shock.

Excess fructose intake‐induced hypertrophic visceral adipose tissue results from unbalanced precursor cell adipogenic signals

We studied the effect of feeding normal adult male rats with a commercial diet supplemented with fructose added to the drinking water (10% w/v; fructose‐rich diet, FRD) on the adipogenic capacity of stromal‐vascular fraction (SVF) cells isolated from visceral adipose tissue (VAT) pads. Animals received either the commercial diet or FRD ad libitum for 3 weeks; thereafter, we evaluated the in vitro proliferative and adipogenic capacities of their VAT SVF cells. FRD significantly increased plasma insulin, triglyceride and leptin levels, VAT mass/cell size, and the in vitro adipogenic capacity of SVF cells. Flow cytometry studies indicated that the VAT precursor cell population number did not differ between groups; however, the accelerated adipogenic process could result from an imbalance between endogenous pro‐ and anti‐adipogenic SVF cell signals, which are clearly shifted towards the former. The increased insulin milieu and its intracellular mediator (insulin receptor substrate‐1) in VAT pads, as well as the enhanced SVF cell expression of Zpf423 and peroxisome proliferator receptor‐γ2 (all pro‐adipogenic modulators), together with a decreased SVF cell concentration of anti‐adipogenic factors (pre‐adipocyte factor‐1 and wingless‐type MMTV‐10b), strongly supports this assumption. We hypothesize that the VAT mass expansion recorded in FRD rats results from the combination of initial accelerated adipogenesis and final cell hypertrophy. It remains to be determined whether FRD administration over longer periods could perpetuate both processes, or whether cell hypertrophy itself remains responsible for a further VAT mass expansion, as observed in advanced/morbid obesity.

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.

Maternal Undernutrition Induces Neuroendocrine Immune Dysfunction in Male Pups at Weaning

The present study was designed to assess the effect of maternal undernutrition, during gestation and lactation, on the neuroendocrine [hypothalamo-pituitary-adrenal (HPA)]-immune axis response to endotoxin (LPS) administration. For this purpose, 21-day-old male rats from both well-nourished (WN) and undernourished (UN) mothers were examined 2 h after injection (i.p.) of vehicle alone (VEH) or containing LPS (130 µg/kg BW). Circulating levels of glucose (GLU), ACTH, corticosterone (B), tumor necrosis factor-alpha (TNFα) and leptin were explored. The results indicate that: (a) mother body weight was significantly (p < 0.05) reduced, as a consequence of UN, at the second and third weeks of pregnancy; (b) no differences in basal glycemia were found in the two groups of pups, and LPS treatment did not induce hypoglycemia, in either group; (c) basal plasma ACTH, B and TNFα levels were similar in the two groups, and LPS-induced ACTH, B and TNFα secretions, although severalfold higher than respective VEH values (p < 0.05) in pups from WN mothers, were fully (ACTH and B) and partially (TNFα) abolished in products from UN mothers; (d) both mean body weights and basal plasma leptin levels were significantly (p < 0.05) lower in pups from UN than from WN mothers, and LPS administration did not modify plasma leptin values in products from both groups. In addition, results of dispersed total adrenal cells incubated in vitro indicate that: (a) both basal and ACTH (22 pM)-induced B secretion were significantly (p < 0.05) lower in cells from UN than WN pups, and (b) leptin (100 nM) was able to inhibit partially ACTH-stimulated B output by adrenal gland (AG) cells from WN pups; however, it failed to inhibit ACTH-stimulated glucocorticoid release by AG cells from UN pups. The present results indicate that undernutrition in mothers, during the very critical periods of gestation and lactation, induces in their male pups at weaning: (a) reduced circulating leptin levels and body weight values; (b) metabolic adaptation to normal carbohydrate metabolism; (c) hyporesponsiveness of the HPA and immune (TNFα) axes during endotoxemia, and (d) leptin resistance at the adrenocortical level. This study strongly supports that undernutrition of mothers results in neuroendocrine immune dysfunction of their pups; however, adrenal resistance to the inhibitory effect of leptin on glucocorticoid output is developed, probably as an adaptive mechanism to counteract unfavorable metabolic conditions.