Beatriz de Carvalho Borges

Leptin resistance and desensitization of hypophagia during prolonged inflammatory challenge

Acute exposure to bacterial lipopolysaccharide (LPS) is a potent inducer of immune response as well as hypophagia. Nevertheless, desensitization of responses to LPS occurs during long-term exposure to endotoxin. We induced endotoxin tolerance, injecting repeated (6LPS) LPS doses compared with single (1LPS) treatment. 1LPS, but not 6LPS group, showed decreased food intake and body weight, which was associated with an increased plasma leptin and higher mRNA expression of OB-Rb, MC4R, and SOCS3 in the hypothalamus. Hypophagia induced by 1LPS was associated with lower levels of 2-arachidonoylglycerol (2-AG), increased number of p-STAT3 neurons, and decreased AMP-activated protein kinase (AMPK) activity. Desensitization of hypophagia in the 6LPS group was related to high 2-AG, with no changes in p-STAT3 or increased p-AMPK. Leptin decreased food intake, body weight, 2-AG levels, and AMPK activity and enhanced p-STAT3 in control rats. However, leptin had no effects on 2-AG, p-STAT3, or p-AMPK in the 1LPS and 6LPS groups. Rats treated with HFD to induce leptin resistance showed neither hypophagia nor changes in p-STAT3 after 1LPS, suggesting that leptin and LPS recruit a common signaling pathway in the hypothalamus to modulate food intake reduction. Desensitization of hypophagia in response to repeated exposure to endotoxin is related to an inability of leptin to inhibit AMPK phosphorylation and 2-AG production and activate STAT3. SOCS3 is unlikely to underlie this resistance to leptin signaling in the endotoxin tolerance. The present model of prolonged inflammatory challenge may contribute to further investigations on mechanisms of leptin resistance.

Expression of hypothalamic neuropeptides and the desensitization of pituitary–adrenal axis and hypophagia in the endotoxin tolerance

Repeated exposure to lipopolysaccharide (LPS) induces desensitization of hypothalamus-pituitary-adrenal axis (HPA) responses and hypophagia. We investigated the interplay between the neural circuitries involved in the control of food intake and HPA axis activity following single or repeated LPS injections. Male Wistar rats received a single or repeated i.p. injection of LPS (100 microg/kg) for 6 days and were subdivided into four groups: 6 saline, 5 saline+1 LPS, 5 LPS+1 saline and 6 LPS. Animals with a single exposure to LPS showed increased plasma levels of ACTH, CORT, PRL, TNF-alpha and also CRF mRNA in the paraventricular nucleus of the hypothalamus. These animals exhibited a reduced food intake and body weight associated with an increase of CART expression in the arcuate nucleus (ARC). Leptin plasma levels were not altered. On the other hand, repeated LPS administration did not alter ACTH, CORT, PRL and TNF-alpha, but it reduced leptin level, compared to single LPS or saline treatment. Furthermore, repeated LPS administration did not increase CRF or CART mRNA expression. Food intake and weight gain after repeated LPS injections were not different from saline-treated animals. There was no change in NPY and POMC mRNA expression in the ARC after single or repeated injections of LPS. In conclusion, desensitization induced by repeated exposure to LPS involves the blockade of HPA axis activation and anorexigenic response, which are both associated with an unresponsiveness of TNF-alpha production and CRF and CART expression in the hypothalamus.

Adrenalectomy enhances endotoxemia-induced hypophagia: higher activation of corticotrophin-releasing-factor and proopiomelanocortin hypothalamic neurons

Inflammatory and infectious processes evoke neuroendocrine and behavioral changes known as acute-phase response that includes activation of the hypothalamo-pituitary-adrenal (HPA) axis and reduction of food intake. Besides its action as the most important ACTH secretagogue, corticotrophin-releasing factor (CRF), synthesized in the paraventricular nucleus (PVN), is also involved in the control of food intake. Alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus also plays a role in the energy homeostasis, possessing anorexigenic effects. To investigate the participation of neuropeptides involved in the regulation of food intake during endotoxemia, we administrated lipopolysaccharide (LPS) in sham-operated and adrenalectomized (ADX) male Wistar rats to evaluate food intake, hormone responses and Fos-CRF and Fos-alpha-MSH immunoreactivity in the PVN and arcuate nucleus, as well as CRF and POMC mRNA expression in these hypothalamic nuclei. In sham-operated rats, treatment with LPS (100 microg/kg) showed lower food intake, higher plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF double labeled neurons and CRF mRNA expression in the PVN, with no changes in Fos-alpha-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to saline treated rats. After LPS treatment, ADX rats showed further increase in plasma ACTH levels, marked decrease of food intake, higher Fos-CRF immunoreactive neurons in the PVN and CRF mRNA expression, as well as an increase in Fos-alpha-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to sham-operated rats treated with LPS. In conclusion, the present data indicate that the marked hypophagia during endotoxemia following ADX is associated with an increased activation of CRF and POMC neurons in the hypothalamus and an increased mRNA expression of these neuropeptides.

Glial cell activity is maintained during prolonged inflammatory challenge in rats

We evaluated the expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), ionized calcium binding adaptor protein-1 (Iba-1), and ferritin in rats after single or repeated lipopolysaccharide (LPS) treatment, which is known to induce endotoxin tolerance and glial activation. Male Wistar rats (200-250 g) received ip injections of LPS (100 µg/kg) or saline for 6 days: 6 saline (N = 5), 5 saline + 1 LPS (N = 6) and 6 LPS (N = 6). After the sixth injection, the rats were perfused and the brains were collected for immunohistochemistry. After a single LPS dose, the number of GFAP-positive cells increased in the hypothalamic arcuate nucleus (ARC; 1 LPS: 35.6 ± 1.4 vs control: 23.1 ± 2.5) and hippocampus (1 LPS: 165.0 ± 3.0 vs control: 137.5 ± 2.5), and interestingly, 6 LPS injections further increased GFAP expression in these regions (ARC = 52.5 ± 4.3; hippocampus = 182.2 ± 4.1). We found a higher GS expression only in the hippocampus of the 6 LPS injections group (56.6 ± 0.8 vs 46.7 ± 1.9). Ferritin-positive cells increased similarly in the hippocampus of rats treated with a single (49.2 ± 1.7 vs 28.1 ± 1.9) or repeated (47.6 ± 1.1 vs 28.1 ± 1.9) LPS dose. Single LPS enhanced Iba-1 in the paraventricular nucleus (PVN: 92.8 ± 4.1 vs 65.2 ± 2.2) and hippocampus (99.4 ± 4.4 vs 73.8 ± 2.1), but had no effect in the retrochiasmatic nucleus (RCA) and ARC. Interestingly, 6 LPS increased the Iba-1 expression in these hypothalamic and hippocampal regions (RCA: 57.8 ± 4.6 vs 36.6 ± 2.2; ARC: 62.4 ± 6.0 vs 37.0 ± 2.2; PVN: 100.7 ± 4.4 vs 65.2 ± 2.2; hippocampus: 123.0 ± 3.8 vs 73.8 ± 2.1). The results suggest that repeated LPS treatment stimulates the expression of glial activation markers, protecting neuronal activity during prolonged inflammatory challenges.

Fish oil promotes survival and protects against cognitive decline in severely undernourished mice by normalizing satiety signals.

Severe malnutrition resulting from anorexia nervosa or involuntary starvation leads to low weight, cognitive deficits and increased mortality rates. In the present study, we examined whether fish oil supplementation, compared with that of canola oil, would ameliorate the morbidity and mortality associated with these conditions by normalizing endocannabinoid and monoaminergic systems as well as other systems involved in satiety and cognitive function within the hypothalamus and hippocampus. Female Sabra mice restricted to 40% of their daily food intake exhibited decreased body weight, were sickly in appearance, displayed cognitive deficits and had increased mortality rates. Strikingly, fish oil supplementation that contains high omega-3 fatty acids levels decreased mortality and morbidity, and normalized the expression of genes and neurotransmitters in the hippocampus and hypothalamus. Fish oil supplementation, but not canola oil, increased survival rates, improved general appearance and prevented cognitive decline, despite the facts that both diets contained an equivalent number of calories and that there were no differences in weight between mice maintained on the two diets in 100% but decrease in the 40%. In the hypothalamus, the beneficial effects of fish oil supplementation were related to normalization of the endocannabinoid 2-arachidonylglycerol, serotonin (5-HT) (P<.056), dopamine, neuropeptide Y (NPY) and Ca(2+)/calmodulin (CaM)-dependent protein kinase (Camkk2). In the hippocampus, fish oil supplementation normalized 5-HT, Camkk2, silent mating type information regulation 1 and brain-derived neurotrophic factor. In conclusion, dietary supplements of fish oil, as source of omega-3 fatty acids, may alleviate cognitive impairments associated with severe diet restriction and prolong survival independently of weight gain by normalizing neurochemical systems.

Lesion of the anteroventral third ventricle (AV3V) reduces hypothalamic activation and hypophyseal hormone secretion induced by lipopolysaccharide in rats

This study examined whether electrolytic ablation of the periventricular anteroventral third ventricle (AV3V) region would affect the hypothalamic activation and the increase of hypophysial hormone secretion induced by systemic injection of lipopolysaccharide (LPS) in rats. LPS significantly increased the number of cells showing Fos immunoreactivity in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus (P<0.05) and also increased plasma levels of vasopressin, oxytocin, adrenocorticotropin and corticosterone (P<0.05). AV3V lesion significantly reduced LPS-induced Fos immunoreactivity (P<0.05) and vasopressin and oxytocin secretion (P<0.05). Elevations in adrenocorticotropin but not in plasma corticosterone after LPS were affected by prior AV3V lesions. These findings demonstrate that LPS-induced Fos expression in the PVN and SON, and hypophysial hormone secretion is dependent on the integrity of the AV3V region.

High-fat diet induces site-specific unresponsiveness to LPS-stimulated STAT3 activation in the hypothalamus

Hypophagia induced by inflammation is associated with Janus kinase (JAK)-2/signal transducer and activator of transcription (STAT) 3 signaling pathway, and leptin-mediated hypophagia is also mediated by JAK2-STAT3 pathway. We have previously reported that lipopolysaccharide (LPS) did not reduce food intake in leptin-resistant high-fat diet (HFD) rats but maintained body weight loss. We investigated whether changes in p-STAT3 expression in the hypothalamus and brain stem could account for the desensitization of hypophagia in HFD animals after a low LPS dose (100 μg/kg). Wistar rats fed standard diet (3.95 kcal/g) or HFD (6.3 kcal/g) for 8 wk were assigned into control diet-saline, control diet-LPS, HFD-saline, and HFD-LPS groups. LPS reduced feeding in the control diet but not HFD. This group showed no p-STAT3 expression in the paraventricular nucleus (PVN) and ventromedial hypothalamic nucleus (VMH), but sustained, though lower than control, p-STAT3 in the nucleus of the solitary tract (NTS) and raphe pallidus (RPa). LPS decreased body weight in HFD rats and increased Fos expression in the NTS. LPS increased body temperature, oxygen consumption, and energy expenditure in both control diet and HFD rats, and this response was more pronounced in HFD-LPS group. Brown adipose tissue (BAT) thermogenesis and increased energy expenditure seem to contribute to body weight loss in HFD-LPS. This response might be related with increased brain stem activation. In conclusion, LPS activates STAT3-mediated pathway in the hypothalamus and brain stem, leading to hypophagia, however, LPS effects on food intake, but not body weight loss, are abolished by leptin resistance induced by HFD. The preserved STAT3 phosphorylation in the brain stem suggests that unresponsiveness to LPS on STAT3 activation under HFD might be selective to the hypothalamus.

Oxytocin projections to the nucleus of the solitary tract contribute to the increased meal‐related satiety responses in primary adrenal insufficiency

•  What is the central question of this study? Adrenalectomy‐induced hypophagia is related to enhanced activation of satiety responses in the nucleus of the solitary tract (NTS) and is reversed by oxytocin receptor antagonist. The potential role of hypothalamic oxytocin projections to the NTS in the satiety‐related responses following adrenalectomy has not been reported. •  What is the main finding and its importance? Our study shows that adrenalectomy increases oxytocin projections to the NTS, and oxytocin receptor antagonist reverses the increased activation of NTS neurons induced by feeding after adrenalectomy. These data indicate that oxytocin pathways to the NTS contribute to higher satiety‐related responses, indicating that oxytocin is a mediator of hypophagia following adrenalectomy through its stimulatory effects on the NTS.

Protein tyrosine phosphatase-1B contributes to LPS-induced leptin resistance in male rats

Leptin resistance is induced by the feedback inhibitors tyrosine phosphatase-1B (PTP1B) and decreased Src homology 2 domain-containing tyrosine phosphatase-2 (SHP-2) signaling. To investigate the participation of PTP1B and SHP-2 in LPS-induced leptin resistance, we injected repeated (6-LPS) intraperitoneal LPS doses (100 μg/kg ip) for comparison with a single (1-LPS) treatment and evaluated the expression of SHP-2, PTP1B, p-ERK1/2, and p-STAT3 in the hypothalamus of male Wistar rats. The single LPS treatment increased the expression of p-STAT3 and PTP1B but not SHP-2. The repeated LPS treatment reduced SHP-2, increased PTP1B, and did not change p-STAT3. We observed that the PTP1B expression induced by the endotoxin was highly colocalized with leptin receptor cells in the hypothalamus of LepRb-IRES-Cre-tdTomato reporter mice. The single, but not the repeated, LPS treatment decreased the food intake and body weight. Leptin had no stimulatory effect on the hypophagia, body weight loss, or pSTAT3 expression in 6-LPS rats, indicating leptin unresponsiveness. Notably, the PTP1B inhibitor (3.0 nmol/rat in 5 μl icv) restored the LPS-induced hypophagia in 6-LPS rats and restored the ability of leptin to reduce food intake and body weight as well as to phosphorylate STAT3 in the arcuate, paraventricular, and ventromedial nuclei of the hypothalamus. The present data suggest that an increased PTP1B expression in the hypothalamus underlies the development of leptin resistance during repeated exposure to LPS. Our findings contribute to understanding the mechanisms involved in leptin resistance during low-grade inflammation as seen in obesity.

PI3Kα inactivation in leptin receptor cells increases leptin sensitivity but disrupts growth and reproduction

The role of PI3K in leptin physiology has been difficult to determine due to its actions downstream of several metabolic cues, including insulin. Here, we used a series of mouse models to dissociate the roles of specific PI3K catalytic subunits and of insulin receptor (InsR) downstream of leptin signaling. We show that disruption of p110α and p110β subunits in leptin receptor cells (LRΔα+β) produces a lean phenotype associated with increased energy expenditure, locomotor activity, and thermogenesis. LRΔα+β mice have deficient growth and delayed puberty. Single subunit deletion (i.e., p110α in LRΔα) resulted in similarly increased energy expenditure, deficient growth, and pubertal development, but LRΔα mice have normal locomotor activity and thermogenesis. Blunted PI3K in leptin receptor (LR) cells enhanced leptin sensitivity in metabolic regulation due to increased basal hypothalamic pAKT, leptin-induced pSTAT3, and decreased PTEN levels. However, these mice are unresponsive to leptins effects on growth and puberty. We further assessed if these phenotypes were associated with disruption of insulin signaling. LRΔInsR mice have no metabolic or growth deficit and show only mild delay in pubertal completion. Our findings demonstrate that PI3K in LR cells plays an essential role in energy expenditure, growth, and reproduction. These actions are independent from insulin signaling.