Mariana Portovedo

Saturated Fatty Acids Modulate Autophagy's Proteins in the Hypothalamus

Autophagy is an important process that regulates cellular homeostasis by degrading dysfunctional proteins, organelles and lipids. In this study, the hypothesis that obesity could lead to impairment in hypothalamic autophagy in mice was evaluated by examining the hypothalamic distribution and content of autophagic proteins in animal with obesity induced by 8 or 16 weeks high fat diet to induce obesity and in response to intracerebroventricular injections of palmitic acid. The results showed that chronic exposure to a high fat diet leads to an increased expression of inflammatory markers and downregulation of autophagic proteins. In obese mice, autophagic induction leads to the downregulation of proteins, such as JNK and Bax, which are involved in the stress pathways. In neuron cell- line, palmitate has a direct effect on autophagy even without inflammatory activity. Understanding the cellular and molecular bases of overnutrition is essential for identifying new diagnostic and therapeutic targets for obesity.

Defective Regulation of the Ubiquitin/Proteasome System in the Hypothalamus of Obese Male Mice

In both human and experimental obesity, inflammatory damage to the hypothalamus plays an important role in the loss of the coordinated control of food intake and energy expenditure. Upon prolonged maintenance of increased body mass, the brain changes the defended set point of adiposity, and returning to normal weight becomes extremely difficult. Here we show that in prolonged but not in short-term obesity, the ubiquitin/proteasome system in the hypothalamus fails to maintain an adequate rate of protein recycling, leading to the accumulation of ubiquitinated proteins. This is accompanied by an increased colocalization of ubiquitin and p62 in the arcuate nucleus and reduced expression of autophagy markers in the hypothalamus. Genetic protection from obesity is accompanied by the normal regulation of the ubiquitin/proteasome system in the hypothalamus, whereas the inhibition of proteasome or p62 results in the acceleration of body mass gain in mice exposed for a short period to a high-fat diet. Thus, the defective regulation of the ubiquitin/proteasome system in the hypothalamus may be an important mechanism involved in the progression and autoperpetuation of obesity.

Autophagy proteins are modulated in the liver and hypothalamus of the offspring of mice with diet-induced obesity ☆ ☆☆

Nutritional excess during pregnancy and lactation has a negative impact on offspring phenotype. In adulthood, obesity and lipid overload represent factors that compromise autophagy, a process of lysosomal degradation. Despite knowledge of the impact of obesity on autophagy, changes in offspring of obese dams have yet to be investigated. In this study, we tested the hypothesis that maternal obesity induced by a high fat diet (HFD) modulates autophagy proteins in the hypothalamus and liver of the offspring of mice. At birth (d0), offspring of obese dams (HFD-O) showed an increase in p62 protein and a decrease in LC3-II, but only in the liver. After weaning (d18), the offspring of HFD-O animals showed impairment of autophagy markers in both tissues compared to control offspring (SC-O). Between day 18 and day 42, both groups received a control diet and we observed that the protein content of p62 remained increased in the livers of the HFD-O offspring. However, after 82days, we did not find any modulation in offspring autophagy proteins. On the other hand, when the offspring of obese dams that received an HFD from day 42 until day 82 (OH-H) were compared with the offspring from the controls that only received an HFD in adulthood (OC-H), we saw impairment in autophagy proteins in both tissues. In conclusion, this study describes that HFD-O offspring showed early impairment of autophagy proteins. Although the molecular mechanisms have not been explored, it is possible that changes in autophagy markers could be associated with metabolic disturbances of offspring.

Defective apoptosis in intestinal and mesenteric adipose tissue of Crohn's disease patients.

Background Crohn’s disease (CD) is associated with complex pathogenic pathways involving defects in apoptosis mechanisms. Recently, mesenteric adipose tissue (MAT) has been associated with CD ethiopathology, since adipose thickening is detected close to the affected intestinal area. However, the potential role of altered apoptosis in MAT of CD has not been addressed. Aims To evaluate apoptosis in the intestinal mucosa and MAT of patients with CD. Methods Samples of intestinal mucosa and MAT from patients with ileocecal CD and from non-inflammatory bowel diseases patients (controls) were studied. Apoptosis was assessed by TUNEL assay and correlated with the adipocytes histological morphometric analysis. The transcriptional and protein analysis of selected genes and proteins related to apoptosis were determined. Results TUNEL assay showed fewer apoptotic cells in CD, when compared to the control groups, both in the intestinal mucosa and in MAT. In addition, the number of apoptotic cells (TUNEL) correlated significantly with the area and perimeter of the adipose cells in MAT. Transcriptomic and proteomic analysis reveal a significantly lower transcript and protein levels of Bax in the intestinal mucosa of CD, compared to the controls; low protein levels of Bax were found localized in the lamina propria and not in the epithelium of this tissue. Furthermore, higher level of Bcl-2 and low level of Caspase 3 were seen in the MAT of CD patients. Conclusion The defective apoptosis in MAT may explain the singular morphological characteristics of this tissue in CD, which may be implicated in the pathophysiology of the disease.

Autophagy is decreased in mesenteric fat tissue but not in intestinal mucosae of patients with Crohn’s disease

Crohn’s disease (CD) is a chronic intestinal disease with a multifactorial etiology. Recently, a role for mesenteric fat has been proposed in CD pathophysiology, since fat hypertrophy is detected close to the affected intestinal area; however, there are few studies regarding autophagy and the hypertrophied mesenteric tissue in CD. To evaluate autophagy-related proteins in intestinal mucosae and mesenteric fat of patients with CD and controls, patients with ileocecal CD (CD Group) and with non-inflammatory disease (FC Group) selected for surgery were studied. Expression of LC3-II was determined by immunoblotting of protein extracts. In addition, beclin-1, LC3 and Atg16-L1 RNA levels were measured using RT-PCR. The expression of LC3-II was significantly lower in the mesenteric tissue and higher in intestinal mucosae of CD when compared to controls. However, mRNA expression of autophagy-related proteins was similar when comparing the mesenteric fat groups. These findings suggest a defect in autophagy activation in the mesenteric fat tissue of CD individuals, which could be involved in the maintenance of the inflammatory process.

Transcriptional and Molecular Pathways Activated in Mesenteric Adipose Tissue and Intestinal Mucosa of Crohn’s Disease Patients

Crohns disease (CD) is a chronic inflammatory disorder, characterized by cytokine imbalance and transcription signaling pathways activation. In addition, the increase of mesenteric adipose tissue (MAT) near the affected intestinal area is a hallmark of CD. Therefore, we evaluated the transcription signaling pathways and cytokines expression in intestinal mucosa and MAT of active CD patients. Ten patients with ileocecal CD and eight with noninflammatory diseases were studied. The biopsies of intestinal mucosa and MAT were snap-frozen and protein expression was determined by immunoblotting. RNA levels were measured by qPCR. The pIkB/IkB ratio and TNFα level were significantly higher in intestinal mucosa of CD when compared to controls. However, STAT1 expression was similar between intestinal mucosa of CD and controls. Considering the MAT, the pIkB/IkB ratio was significantly lower and the anti-inflammatory cytokine IL10 was significantly higher in CD when compared to controls. Finally, the protein content of pSTAT1 was higher in MAT of CD compared to controls. These findings reinforce the predominance of the proinflammatory NF-kB pathway in CD intestinal mucosa. For the first time, we showed the activation of STAT1 pathway in MAT of CD patients, which may help to understand the physiopathology of this immune mediated disease.

Effects of maternal low-protein diet on parameters of locomotor activity in a rat model of cerebral palsy.

Children with cerebral palsy have feeding difficulties that can contribute to undernutrition. The aim of this study was to investigate the effect of early undernutrition on locomotor activity and the expression of the myofibrillar protein MuRF‐1 in an experimental model of cerebral palsy (CP). In order to achieve this aim, pregnant rats were divided into two groups according to the diet provided: Normal Protein (NP, n = 9) and Low Protein (LP, n = 12) groups. After birth, the pups were divided into four groups: Normal Protein Sham (NPS, n = 16), Normal Protein Cerebral Palsy (NPCP, n = 21), Low Protein Sham (LPS, n = 20) and Low Protein Cerebral Palsy (LPCP, n = 18) groups. The experimental cerebral palsy protocol consisted of two episodes of anoxia at birth and during the first days of life. Each day, nitrogen flow was used (9l/min during 12 min). After nitrogen exposure, sensorimotor restriction was performed 16 h per day, from the 2nd to the 28th postnatal day (PND). Locomotor activity was evaluated at 8th, 14th, 17th, 21th and 28th PND. At PND 29, soleus muscles were collected to analyse myofibrillar protein MuRF‐1. Our results show that CP animals decreased body weight (p < 0.001), which were associated with alterations of various parameters of locomotor activity (p < 0.05), compared to their control. Undernourished animals also showed a decrease (p < 0.05) in body weight and locomotor activity parameters. Moreover, CP decreased MuRF‐1 levels in nourished rats (p = 0.015) but not in undernourished rats. In summary, perinatal undernutrition exacerbated the negative effects of cerebral palsy on locomotor activity and muscle atrophy, but it appears not be mediated by changes in MuRF‐1 levels.

Ileal pouch of ulcerative colitis and familial adenomatous polyposis patients exhibit modulation of autophagy markers

Total retocolectomy with ileal pouch-anal anastomosis (IPAA) is the surgery of choice for patients with ulcerative colitis (UC) that are refractory to clinical treatment. Pouchitis is one of the most common complications after this procedure. Defects in autophagy have been reported in inflammatory bowel diseases. However, there are no studies on the IP. Therefore, we studied markers for autophagy in the IP mucosa of UC and FAP patients comparing them to controls with a normal distal ileum. Sixteen patients with IP in “J” shape, asymptomatic and with endoscopically normal IP were evaluated. The control group consisted of eight patients with normal colonoscopy. There was a significant decrease in the transcriptional levels of ATG5, MAP1LC3A and BAX in the FAP group. There was also a decrease in the protein level of Beclin-1 in the UC and FAP compared to the control group. Although the LC3II levels by immunoblot were higher in the UC group, LC3/p62 co-localization were lower in the immunofluorescence analysis in the UC and FAP compared to the control group. Corroborating these results, there was an increase of p62 by immunoblot in the UC group. These findings indicated a modulation of macroautophagy markers in the IP, which may explain the mucosa inflammation predisposition.

Beet Stalks and Leaves (Beta vulgaris L.) Protect Against High-Fat Diet-Induced Oxidative Damage in the Liver in Mice

Some flavonoids identified in beet stalks can help the antioxidant endogenous defenses during a chronic inflammation process. The current study investigates the effect of polyphenols present in beet stalks and leaves on liver oxidative damage in mice fed a high-fat diet (HF). The control (CT) or HF diet groups were supplemented with dehydrated beet stalks and leaves (SL) or beet stalk and leaf ethanolic extract (EX). In terms of Vitexin-rhaminoside equivalents (VRE), EX groups received ~5.91 mg of VRE·100 g−1 diet, while the SL groups received ~3.07 mg VRE·100 g−1 diet. After 8 weeks, we evaluated fasting blood glucose; cholesterol, hepatic Malondialdehyde (MDA) levels and hepatic Glutathione (GSH), Glutathione peroxidase (GPx), Glutathione reductase (GR) and Superoxide dismutase (SOD) activity. Dehydrated beet stalks and leaves (HFSL) attenuated the deleterious effects of a HF diet on lipid metabolism, reduced fasting blood glucose levels, ameliorated cholesterol levels and reduced GPx and GR activities (p < 0.05) compared to the HF group. However; the addition of ethanolic extract from beet stalks and leaves was unable (p > 0.05) to prevent the liver damage caused by HF diet in mice. The presence of flavonoids, such as Vitexin derivatives in beet stalks and leaves can help the liver damage induced by HF diet.

Interesterified soybean oil promotes weight gain, impaired glucose tolerance and increased liver cellular stress markers

Interesterified fats have largely replaced hydrogenated vegetable fat, which is rich in trans fatty acids, in the food industry as an economically viable alternative, generating interest to study their health effects. The aim of this study was to evaluate the effect that interesterification of oils and fat has on lipid-induced metabolic dysfunction, hepatic inflammation and ER stress. Five week-old male Wistar rats were randomly divided into three experimental groups, submitted to either normocaloric and normolipidic diet containing 10% of lipids from unmodified soybean oil (SO) or from interesterified soybean oil (ISO), and one more group submitted to a high fat diet (HFD) containing 60% of fat from lard as a positive control, for 8 or 16 weeks. Metabolic parameters and hepatic gene expression were evaluated. The HFD consumption led to increased body mass, adiposity and impaired glucose tolerance compared to SO and ISO at both time points of diet. However, the ISO group showed an increased body mass gain, retroperitoneal WAT mass, fasting glucose, and impaired glucose tolerance during ipGTT at 16 weeks compared to SO. Moreover, at 8 weeks, hepatic gene expression of Atf3 and Tnf were increased in the ISO group compared to the SO group. Thus, replacement of natural fat with interesterified fat on a normocaloric and normolipidic diet negatively modulated metabolic parameters and resulted in impaired glucose tolerance in rats.