High-fat but not normal-fat intake of extra virgin olive oil modulates the liver proteome of mice.

PURPOSE: The metabolic benefits of the Mediterranean diet have been largely attributed to its olive oil content. Whether the ingested fat amount is relevant to these effects is not clear. We thus compared the effects of high-fat and normal-fat intake of extra-virgin olive oil (EVOO) on the liver proteome. METHODS: Three groups of mice were fed for 12 weeks with either normal-fat diets containing either soybean oil (control, C) or EVOO (NO) or a high-fat EVOO diet (HO). Body weight and food intake were measured weekly and serum parameters were analyzed. The liver was processed for data-independent acquisition mass spectrometry-based proteomics. The differentially expressed proteins among the groups were submitted to pathway enrichment analysis. RESULTS: The consumption of HO diet reduced food intake and serum triglycerides, while it preserved body weight gain, adiposity, and glycemia. However, it increased serum cholesterol and liver mass. The proteomic analysis showed 98 altered proteins, which were allocated in 27 significantly enriched pathways. The pathway analysis suggested stimulation of mitochondrial and peroxissomal ß-oxidation, and inhibition of lipid synthesis and gluconeogenesis in the HO group. Although the NO group failed to show significant liver proteome alterations, it presented reduced body fat, body weight gain, and serum triglycerides and glucose levels. CONCLUSION: The data indicate that the intake of the HO diet induced hepatic adjustments, which were partially successful in counteracting the detrimental outcomes of a high-fat feeding. Contrastingly, the NO diet had beneficial effects which were not accompanied by significant modifications on hepatic proteome.

Ovariectomy modifies lipid metabolism of retroperitoneal white fat in rats: a proteomic approach.

Menopause is often accompanied by visceral obesity. With the aim of exploring the consequences of ovarian failure on visceral fat, we evaluated the effects of ovariectomy and estrogen replacement on the proteome/phosphoproteome and on the fatty acid profile of the retroperitoneal adipose depot (RAT) of rats. Eighteen 3-mo-old female Wistar rats were either ovariectomized or sham operated and fed with standard chow for 3 mo. A subgroup of ovariectomized rats received estradiol replacement. RAT samples were analyzed with data-independent acquisitions LC-MS/MS, and pathway analysis was performed with the differentially expressed/phosphorylated proteins. RAT lipid profile was analyzed by gas chromatography. Ovariectomy induced high adiposity and insulin resistance and promoted alterations in protein expression and phosphorylation. Pathway analysis showed that five pathways were significantly affected by ovariectomy, namely, metabolism of lipids (including fatty acid metabolism and mitochondrial fatty acid ß-oxidation), fatty acyl-CoA biosynthesis, innate immune system (including neutrophil degranulation), metabolism of vitamins and cofactors, and integration of energy metabolism (including ChREBP activates metabolic gene expression). Lipid profile analysis showed increased palmitic and palmitoleic acid content. The analysis of the data indicated that ovariectomy favored lipogenesis whereas it impaired fatty acid oxidation and induced a proinflammatory state in the visceral adipose tissue. These effects are consistent with the findings of high adiposity, hyperleptinemia, and impaired insulin sensitivity. The observed alterations were partially attenuated by estradiol replacement. The data point to a role of disrupted lipid metabolism in adipose tissue in the genesis of obesity after menopause.

Effect of fish oil intake on glucose levels in rat prefrontal cortex, as measured by microdialysis.

BACKGROUND: Brain glucose sensing may contribute to energy homeostasis control. The prefrontal cortex (PFC) participates in the hedonic component of feeding control. As high-fat diets may disrupt energy homeostasis, we evaluated in male Wistar rats whether intake of high-fat fish-oil diet modified cortical glucose extracellular levels and the feeding induced by intracerebroventricular glucose or PFC glucoprivation. METHODS: Glucose levels in PFC microdialysates were measured before and after a 30-min meal. Food intake was measured in animals receiving intracerebroventricular glucose followed, 30-min. later, by 2-deoxy-D-glucose injected into the PFC. RESULTS: The fish-oil group showed normal body weight and serum insulin while fat pads weight and glucose levels were increased. Baseline PFC glucose and 30-min. carbohydrates intake were similar between the groups. Feeding-induced PFC glucose levels increased earlier and more pronouncedly in fish-oil than in control rats. Intracerebroventricular glucose inhibited feeding consistently in the control but not in the fish-oil group. Local PFC glucoprivation with 2-DG attenuated glucose-induced hypophagia. CONCLUSIONS: The present experiments have shown that, following food intake, more glucose reached the prefrontal cortex of the rats fed the high-fat fish-oil diet than of the rats fed the control diet. However, when administered directly into the lateral cerebral ventricle, glucose was able to consistently inhibit feeding only in the control rats. The findings indicate that, an impairment of glucose transport into the brain does not contribute to the disturbances induced by the high-fat fish-oil feeding.

Plasma signatures of Congenital Generalized Lipodystrophy patients identified by untargeted lipidomic profiling are not changed after a fat-containing breakfast meal.

BACKGROUND: The incapacity to store lipids in adipose tissue in Congenital Generalized Lipodystrophy (CGL) causes hypoleptinemia, increased appetite, ectopic fat deposition and lipotoxicity. CGL patients experience shortened life expectancy. The plasma lipidomic profile has not been characterized fully in CGL, nor has the extent of dietary intake in its modulation. The present work investigated the plasma lipidomic profile of CGL patients in comparison to eutrophic individuals at the fasted state and after a breakfast meal. METHOD: Blood samples from 11 CGL patients and 10 eutrophic controls were collected after 12 h fasting (T0) and 90 min after an ad libitum fat-containing breakfast (T90). The lipidomic profile of extracted plasma lipids was characterized by non-target liquid chromatography mass spectrometry. RESULTS: Important differences between groups were observed at T0 and at T90. Several molecular species of fatty acyls, glycerolipids, sphingolipids and glycerophospholipids were altered in CGL. All the detected fatty acyl molecular species, several diacylglycerols and one triacylglycerol species were upregulated in CGL. Among sphingolipids, one sphingomyelin and one glycosphingolipid species showed downregulation in CGL. Alterations in the glycerophospholipids glycerophosphoethanolamines, glycerophosphoserines and cardiolipins were more complex. Interestingly, when comparing T90 versus T0, the lipidomic profile in CGL did not change as intensely as it did for control participants. CONCLUSIONS: The present study found profound alterations in the plasma lipidomic profile of complex lipids in CGL patients as compared to control subjects. A fat-containing breakfast meal did not appear to significantly influence the CGL profile observed in the fasted state. Our study may have implications for clinical practice, also aiding to a deeper comprehension of the role of complex lipids in CGL in view of novel therapeutic strategies.

High-fat diet intake induces depressive-like behavior in ovariectomized rats.

This study tested the effects of ovariectomy, allied or not to high-fat feeding and estradiol replacement, on hormonal, metabolic and behavioral parameters, to explore the connection of obesity and depression after menopause. Wistar rats were either ovariectomized or sham-operated and fed with either standard chow or lard-enriched diet for twelve weeks. Sub-groups of ovariectomized rats received estradiol replacement. Depressive-like behaviors were assessed by the forced swim test and locomotor activity was assessed by the elevated plus maze test. Ovariectomy alone increased body weight gain and feed efficiency and induced hyperleptinemia and glucose intolerance while it increased caloric intake and body adiposity only marginally. High-fat intake alone induced obesity and, in combination with ovariectomy, accentuated the ovariectomy-induced alterations. Estradiol replacement attenuated the hormonal alterations only in chow-fed rats. Ovariectomy combined with high-fat intake induced depressive-like behaviors, which were marginally attenuated by estradiol. Depressive-like behaviors were associated with metabolic and body composition parameters and with estrogen status. The data indicate that the vulnerability to develop depression after menopause is influenced by high-fat intake. It is suggested that weight management is a crucial issue in postmenopausal women, probably having a beneficial role in preventing the appearance of mental health problems.

A diet including xanthan gum triggers a pro-inflammatory response in Wistar rats inoculated with Walker 256 cells.

OBJECTIVE: The aim of this study was to evaluate the effect of adding xanthan gum to the diet of rats on the production of cytokines and pro-inflammatory factors and on tumor development in rats inoculated with Walker 256 tumor cells. METHODS: Fifty-six rats were divided into 4 groups: control diet (C), control diet with tumor (TC), xanthan gum diet (XG), xanthan gum diet with tumor (TXG). RESULTS: The ingestion of xanthan gum promotes changes in cytokine content: increasing IL-6 TNF-α and IL-10 in retroperitoneal adipose tissue compared to the control group; and increasing TNF-α in the mesenteric adipose tissue compared to the C and TXG groups. On the contrary, the addition of xanthan gum to the diet did not affect the development of Walker 256 tumors in rats. CONCLUSION: The continuous use of xanthan gum triggered a pro-inflammatory response, promoting an increase in pro-inflammatory cytokines in the adipose tissue, but it did not have an effect on the tumor development in the animals inoculated with Walker 256 tumor cells.

Maternal consumption of green tea extract during pregnancy and lactation alters offspring's metabolism in rats.

INTRODUCTION: Green tea extract has anti-inflammatory and antioxidant effects which improve dyslipidemia and decrease adipose tissue depots associated with hyperlipidic diet consumption. OBJECTIVE: To evaluate the effect of green tea extract consumption by rats during pregnancy and lactation on the metabolism of their offspring that received control or high-fat diet with water during 10 weeks after weaning. METHODS: Wistar rats received water (W) or green tea extract diluted in water (G) (400 mg/kg body weight/day), and control diet (10 animals in W and G groups) during pregnancy and lactation. After weaning, offspring received water and a control (CW) or a high-fat diet (HW), for 10 weeks. One week before the end of treatment, oral glucose tolerance test was performed. The animals were euthanized and the samples were collected for biochemical, hormonal and antioxidant enzymes activity analyses. In addition, IL-10, TNF-α, IL-6, and IL-1ß were quantified by ELISA while p-NF-κBp50 was analyzed by Western Blotting. Repeated Measures ANOVA, followed by Tukey's test were used to find differences between data (p < 0.05). RESULTS: The consumption of high-fat diet by rats for 10 weeks after weaning promoted hyperglycemia and hyperinsulinemia, and increased fat depots. The ingestion of a high-fat diet by the offspring of mothers who consumed green tea extract during pregnancy and lactation decreased the inflammatory cytokines in adipose tissue, while the ingestion of a control diet increased the same cytokines. CONCLUSION: Our results demonstrate that prenatal consumption of green tea associated with consumption of high-fat diet by offspring after weaning prevented inflammation. However, maternal consumption of the green tea extract induced a proinflammatory status in the adipose tissue of the adult offspring that received the control diet after weaning.

High-Fat Feeding Improves Anxiety-Type Behavior Induced by Ovariectomy in Rats.

Menopause-induced changes may include increased incidence of both depression/anxiety and obesity. We hypothesized that behavioral changes that may develop after ovarian failure could be related to neurochemical and metabolic aspects affected by this condition and that high-fat intake may influence these associations. The present study investigated in rats the effects of ovariectomy, either alone or combined with high-fat diets enriched with either lard or fish-oil, on metabolic, behavioral and monoaminergic statuses, and on gene expression of neuropeptides and receptors involved in energy balance and mood regulation. Female rats had their ovaries removed and received either standard chow (OvxC) or high-fat diets enriched with either lard (OvxL) or fish-oil (OvxF) for 8 weeks. The Sham group received only chow diet. Ovariectomy increased feed efficiency and body weight gain and impaired glucose homeostasis and serotonin-induced hypophagia, effects either maintained or even accentuated by the lard diet but counteracted by the fish diet. The OvxL group developed obesity and hyperleptinemia. Regarding components of hypothalamic serotonergic system, both ovariectomy alone or combined with the fish diet increased 5-HT2C expression while the lard diet reduced 5-HT1B mRNA. Ovariectomy increased the anxiety index, as derived from the elevated plus maze test, while both high-fat groups showed normalization of this index. In the forced swimming test, ovariectomy allied to high-lard diet, but not to fish-oil diet, reduced the latency to immobility, indicating vulnerability to a depressive-like state. Linear regression analysis showed hippocampal AgRP to be negatively associated with the anxiety index and hypothalamic AgRP to be positively associated with the latency to immobility. These AgRp gene expression associations are indicative of a beneficial involvement of this neuropeptide on both depression and anxiety measures. The present findings demonstrate metabolic, neurochemical and behavioral alterations after ovaries removal and highlight a positive effect of high-fat feeding on the anxiety-like behavior shown by ovariectomized animals. Since the polyunsaturated ômega-3 intake (fish diet), unlike the saturated fat intake (lard diet), failed to induce deleterious metabolic or neurochemical consequences, further studies are needed focusing on the potential of this dietary component as an adjuvant anxiolytic agent after menopause.

Corrigendum: High-Fat Feeding Improves Anxiety-Type Behavior Induced by Ovariectomy in Rats.

[This corrects the article DOI: 10.3389/fnins.2018.00557.].

Molecular analysis of PROP1, PIT1, HESX1, LHX3, and LHX4 shows high frequency of PROP1 mutations in patients with familial forms of combined pituitary hormone deficiency.

UNLABELLED: Combined Pituitary Hormone Deficiency (CPHD) is a prevalent disease in Neuroendocrinology services. The genetic form of CPHD may originate from mutations in pituitary transcription factor (PTF) genes and the pituitary image in these cases may give a clue of what PTF is most probably mutated: defects in LHX4 are usually associated with ectopic posterior pituitary (EPP); defects in LHX3, PIT1, and PROP1, with normally placed posterior pituitary (NPPP); HESX1 mutations are associated with both. OBJECTIVE: To identify mutations in PTF genes in patients with idiopathic hypopituitarism followed in our service, based on the presence or absence of EPP on sellar MRI. METHODS: Forty patients with idiopathic hypopituitarism (36 families, 9 consanguineous), followed in the Neuroendocrinology Outpatient Clinic of UNIFESP, Brazil, were submitted to sequencing analyses of PTF genes as follows: LHX3, HESX1, PIT1, and PROP1 were sequenced in patients with NPPP (26/40) and HESX1 and LHX4 in patients with EPP (14/40). RESULTS: We identified only PROP1 mutations in 9 out of 26 patients with CPHD and NPPP (35%). Since eight of them came from 4 consanguineous families, the prevalence of PROP1 mutations was higher when only consanguineous families were considered (44%, 4/9). At the end of the study, we decided to sequence PROP1 in patients with EPP, just to confirm that they were not candidates for PROP1 mutations. No patients with EPP had PROP1 or other PTF mutations. CONCLUSIONS: Patients with idiopathic CPHD and NPPP, born from consanguineous parents, are the strong candidates for PROP1 mutations. Other developmental gene(s) may be involved in the genesis of idiopathic hypopituitarism associated with EPP.

High-fat diets rich in soy or fish oil distinctly alter hypothalamic insulin signaling in rats.

Hypothalamic insulin inhibits food intake, preventing obesity. High-fat feeding with polyunsaturated fats may be obesogenic, but their effect on insulin action has not been elucidated. The present study evaluated insulin hypophagia and hypothalamic signaling after central injection in rats fed either control diet (15% energy from fat) or high-fat diets (50% energy from fat) enriched with either soy or fish oil. Soy rats had increased fat pad weight and serum leptin with normal body weight, serum lipid profile and peripheral insulin sensitivity. Fish rats had decreased body and fat pad weight, low leptin and corticosterone levels, and improved serum lipid profile. A 20-mU dose of intracerebroventricular (ICV) insulin inhibited food intake in control and fish groups, but failed to do so in the soy group. Hypothalamic protein levels of IR, IRS-1, IRS-2, Akt, mTOR, p70S6K and AMPK were similar among groups. ICV insulin stimulated IR tyrosine phosphorylation in control (68%), soy (36%) and fish (34%) groups. Tyrosine phosphorylation of the pp185 band was significantly stimulated in control (78%) and soy (53%) rats, but not in fish rats. IRS-1 phosphorylation was stimulated only in control rats (94%). Akt serine phosphorylation was significantly stimulated only in control (90%) and fish (78%) rats. The results showed that, rather than the energy density, the fat type was a relevant aspect of high-fat feeding, since blockade of hypothalamic insulin signal transmission and insulin hypophagia was promoted only by the high-fat soy diet, while they were preserved in the rats fed with the high-fat fish diet.

Development of a sensitive and specific quantitative reverse transcription-polymerase chain reaction assay for blood thyroglobulin messenger ribonucleic acid in the follow-up of patients with differentiated thyroid carcinoma.

CONTEXT: Serum thyroglobulin is a sensitive tumor marker in the follow-up of patients with differentiated thyroid carcinoma (DTC), but the presence of endogenous anti-thyroglobulin antibodies (TgAb) can interfere on its measurement. To prevent interference by TgAb, several investigators have tried to quantify blood mRNA Tg by real-time RT-PCR, but the results have been variable, not reporting a correlation between mRNA Tg and the presence of metastases. OBJECTIVE: The aim of the study was to evaluate the development of a sensitive and specific quantitative RT-PCR assay for blood mRNA Tg in the follow-up of patients with DTC. DESIGN AND PATIENTS: An assay employing primers located in a region not affected by alternative splicing or single nucleotide polymorphisms was developed to study 104 DTC patients (13 of 104 with positive TgAb). RESULTS: The assay is specific for thyroid tissue because we found mRNA Tg expression in normal thyroid tissue, but we did not find any mRNA Tg expression in any extrathyroidal tissues. Quantitative mRNA Tg levels were significantly different between patients "free of disease" (82 of 104) and those with metastases (22 of 104) (2.61 +/- 0.26 vs. 27.58 +/- 1.62 pg mRNA Tg/microg RNA) (P < 0.0001). A cutoff point of 5.51 was able to discriminate between the two groups. In addition, the measurement of mRNA Tg was not affected by the presence of TgAb. CONCLUSION: This new mRNA Tg quantification is a reliable method that allowed us to differentiate patients free of disease from those with metastases, and it could represent an appropriate molecular marker for the follow-up of patients with DTC, especially those with positive TgAb.

Clinical and molecular analysis of thyroid hypoplasia: a population-based approach in southern Brazil.

BACKGROUND: Congenital hypothyroidism (CH) is mainly due to developmental abnormalities leading to thyroid dysgenesis (TD). TD encompasses very distinct morphologic subtypes of disease. This study examined and compared the phenotype in TD variants and searched for genetic alterations in sporadic thyroid hypoplasia (TH), the most misdiagnosed form of CH. This was a longitudinal study over a 14-year period (1990-2004). METHODS: A continuous series of 353 children with TD was identified using thyroid function tests [thyroxine (T4) and TSH], scintigraphy, and ultrasound as diagnostic tools. Individual phenotypes were analyzed in 253 children with TD. Mutations in the most likely candidate genes were studied in 35 cases of TH. RESULTS: The overall birth prevalence of permanent CH was 1:4795. Ectopy represented 37% of all cases of permanent primary CH, dyshormonogenesis 28%, agenesis 24%, hypoplasia 10%, and hemiagenesis 1%. The lowest screening T4 level and the highest TSH level were in the agenetic group, followed by TH. The TH group had an improvement in the thyroid function showing less-severe phenotype with aging. In the molecular analysis, one patient was identified with a mutation in the PAX8 gene (155G>C; R52P); four patients had a heterozygous G>C substitution in position -569; two patients showed a (234C>A; P52T) or (2181C>G; D727E) polymorphic variants of the TSH-R gene; and one patient presented a novel heterozygous nonsynonymous substitution, 293G>A; S98N, in the NKX2.5 gene. CONCLUSIONS: The prevalence of CH was within the previously reported range of 1:3000-4000. Ectopy was the most common etiology. Clinical analysis revealed distinct hormonal patterns in TH subgroup when compared with other variants of TD, with genetic abnormalities identified only in few cases in the TSH-R, PAX8, and NKX2.5 genes.

Molecular analysis of PROP1, PIT1, HESX1, LHX3, and LHX4 shows high frequency of PROP1 mutations in patients with familial forms of combined pituitary hormone deficiency

Combined Pituitary Hormone Deficiency (CPHD) is a prevalent disease in Neuroendocrinology services. The genetic form of CPHD may originate from mutations in pituitary transcription factor (PTF) genes and the pituitary image in these cases may give a clue of what PTF is most probably mutated: defects in LHX4 are usually associated with ectopic posterior pituitary (EPP); defects in LHX3, PIT1, and PROP1, with normally placed posterior pituitary (NPPP); HESX1 mutations are associated with both. OBJECTIVE: To identify mutations in PTF genes in patients with idiopathic hypopituitarism followed in our service, based on the presence or absence of EPP on sellar MRI. METHODS: Forty patients with idiopathic hypopituitarism (36 families, 9 consanguineous), followed in the Neuroendocrinology Outpatient Clinic of UNIFESP, Brazil, were submitted to sequencing analyses of PTF genes as follows: LHX3, HESX1, PIT1, and PROP1 were sequenced in patients with NPPP (26/40) and HESX1 and LHX4 in patients with EPP (14/40). RESULTS: We identified only PROP1 mutations in 9 out of 26 patients with CPHD and NPPP (35 percent). Since eight of them came from 4 consanguineous families, the prevalence of PROP1 mutations was higher when only consanguineous families were considered (44 percent, 4/9). At the end of the study, we decided to sequence PROP1 in patients with EPP, just to confirm that they were not candidates for PROP1 mutations. No patients with EPP had PROP1 or other PTF mutations. CONCLUSIONS: Patients with idiopathic CPHD and NPPP, born from consanguineous parents, are the strong candidates for PROP1 mutations. Other developmental gene(s) may be involved in the genesis of idiopathic hypopituitarism associated with EPP.

Deficiência Combinada de Hormônios Hipofisários (DCHH) é uma doença prevalente em todos os serviços de Neuroendocrinologia. A DCHH de origem genética pode resultar de mutações nos genes de fatores de transcrição hipofisários (FTH), e a ressonância magnética (RM) de sela desses pacientes pode indicar qual FTH tem maior probabilidade de estar mutado: mutações no LHX4 estão geralmente associadas a neuro-hipófise ectópica (NHE); mutações no LHX3, PIT1 e PROP1, a neuro-hipófise tópica (NHT); mutações no HESX1 podem estar associadas a NHE e NHT. OBJETIVO: Identificar mutações nos FTH em pacientes acompanhados em nosso serviço, portadores de hipopituitarismo idiopático, selecionando os genes a serem estudados de acordo com a presença ou ausência de NHE à RM sela. MÉTODOS: Os genes dos FTH foram seqüenciados em 40 pacientes com hipopituitarismo idiopático (36 famílias, 9 consangüíneas), acompanhados na unidade de Neuroendocrinologia da UNIFESP, SP, Brasil: LHX3, HESX1, PIT1 e PROP1 foram seqüenciados nos pacientes com NHT (26/40) e HESX1 e LHX4, nos pacientes com NHE (14/40). RESULTADOS: Somente mutações PROP1 foram identificadas em 9 de 26 pacientes (35 por cento) com NHT, 8 deles provenientes de 4 famílias consangüíneas (4/9, 44 por cento). Uma vez que mutações no PROP1 foram tão freqüentes, decidimos, ao final do estudo, seqüenciá-lo também nos pacientes com NHE. Nenhum paciente com NHE apresentou mutações no PROP1 ou em outro FTH. CONCLUSÃO: Mutações no gene PROP1 foram encontradas em 22,5 por cento (9/40) de todos os pacientes, em 35 por cento (9/26) dos pacientes com NHT e em 44 por cento (4/9) se considerarmos somente as famílias consangüíneas. Portanto, pacientes com DCHH idiopática e NHT, provenientes de famílias de pais consangüíneos, são os melhores candidatos a mutações PROP1.