Hypothalamic α7 nicotinic acetylcholine receptor (α7nAChR) is downregulated by TNFα-induced Let-7 overexpression driven by fatty acids.

The α7nAChR is crucial to the anti-inflammatory reflex, and to the expression of neuropeptides that control food intake, but its expression can be decreased by environmental factors. We aimed to investigate whether microRNA modulation could be an underlying mechanism in the α7nAchR downregulation in mouse hypothalamus following a short-term exposure to an obesogenic diet. Bioinformatic analysis revealed Let-7 microRNAs as candidates to regulate Chrna7, which was confirmed by the luciferase assay. Mice exposed to an obesogenic diet for 3 days had increased Let-7a and decreased α7nAChR levels, accompanied by hypothalamic fatty acids and TNFα content. Hypothalamic neuronal cells exposed to fatty acids presented higher Let-7a and TNFα levels and lower Chrna7 expression, but when the cells were pre-treated with TLR4 inhibitor, Let-7a, TNFα, and Chrna7 were rescued to normal levels. Thus, the fatty acids overload trigger TNFα-induced Let-7 overexpression in hypothalamic neuronal cells, which negatively regulates α7nAChR, an event that can be related to hyperphagia and obesity predisposition in mice.

Short-term flaxseed oil, rich in omega 3, protects mice against metabolic damage caused by high-fat diet, but not inflammation.

It is known that long-term high-fat diet (HF) feeding drastically affects the adipose tissue, contributing to metabolic disorders. Recently, short-term HF consumption was shown to affect different neuronal signaling pathways. Thus, we aimed to evaluate the inflammatory effects of a short-term HF and whether a diet containing omega-3 fatty acid fats from flaxseed oil (FS) has protective effects. Mice were divided into three groups for 3 d, according to their diets: Control group (CT), HF, or FS for 3 d. Lipid profiles were assessed through mass spectrometry and inflammatory markers by RT-qPCR and Western blotting. After short-term HF, mice increased food intake, body weight, adiposity, and fasting glucose. Increased mRNA content of Ccl2 and Tnf was demonstrated in the HF compared to CT in mesenteric adipose tissue. In the liver, TNFα protein was higher in the HF group than in CT, followed by a decreased polyunsaturated fatty acids tissue incorporation in HF. On the other hand, the consumption of FS reduced food intake and fasting glucose, as well as increased omega-3 fatty acid incorporation in MAT and the liver. However, short-term FS was insufficient to control the early inflammation triggered by HF in MAT and the liver. These data demonstrated that a 3-d HF diet is enough to damage glucose homeostasis and trigger inflammation. In contrast, short-term FS protects against increased food intake and fasting glucose but not inflammation in mice.

In vitro inhibition of glucose gastro-intestinal enzymes and antioxidant activity of hydrolyzed collagen peptides from different species.

The growing value of industrial collagen by-products has given rise to interest in extracting them from different species of animals. Intrinsic protein structure variation of collagen sources and its hydrolysis can bring about different bioactivities. This study aimed to characterize and evaluate the differences in vitro biological potential of commercial bovine (BH), fish (FH), and porcine hydrolysates (PH) regarding their antioxidant and hypoglycemic activities. All samples showed percentages above 90% of protein content, with high levels of amino acids (glycine, proline, and hydroxyproline), responsible for the specific structure of collagen. The BH sample showed a higher degree of hydrolysis (DH) (8.7%) and a higher percentage of smaller than 2 kDa peptides (74.1%). All collagens analyzed in vitro showed inhibition of pancreatic enzymes (α-amylase and α-glucosidase), with the potential to prevent diabetes mellitus. The PH sample showed higher antioxidant activities measured by ORAC (67.08 ± 4.23 µmol Trolox Eq./g) and ABTS radical scavenging (65.69 ± 3.53 µmol Trolox Eq./g) methods. For the first time, DNA protection was analyzed to hydrolyzed collagen peptides, and the FH sample showed a protective antioxidant action to supercoiled DNA both in the presence (39.51%) and in the absence (96.36%) of AAPH (reagent 2,2'-azobis(2-amidinopropane)). The results confirmed that the source of native collagen reflects on the bioactivity of hydrolyzed collagen peptides, probably due to its amino acid composition. PRACTICAL APPLICATIONS: Our data provide new application for collagen hydrolysates with hypoglycemiant and antioxidant activity. These data open discussion for future studies on the additional benefits arising from collagen peptide consumption for the prevention of aging complications or hyperglycemic conditions as observed in chronic diseases such as diabetes mellitus type II (DM 2). The confirmation of these results can open new market areas for the use of collagen with pharmacological applications or to produce new supplements. Furthermore, provides a solution for waste collagen from meat industries and adds value to the product.

Activation of the α7 Nicotinic Acetylcholine Receptor Prevents against Microglial-Induced Inflammation and Insulin Resistance in Hypothalamic Neuronal Cells.

Neuronal hypothalamic insulin resistance is implicated in energy balance dysregulation and contributes to the pathogenesis of several neurodegenerative diseases. Its development has been intimately associated with a neuroinflammatory process mainly orchestrated by activated microglial cells. In this regard, our study aimed to investigate a target that is highly expressed in the hypothalamus and involved in the regulation of the inflammatory process, but still poorly investigated within the context of neuronal insulin resistance: the α7 nicotinic acetylcholine receptor (α7nAchR). Herein, we show that mHypoA-2/29 neurons exposed to pro-inflammatory microglial conditioned medium (MCM) showed higher expression of the pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α, in addition to developing insulin resistance. Activation of α7nAchR with the selective agonist PNU-282987 prevented microglial-induced inflammation by inhibiting NF-κB nuclear translocation and increasing IL-10 and tristetraprolin (TTP) gene expression. The anti-inflammatory role of α7nAchR was also accompanied by an improvement in insulin sensitivity and lower activation of neurodegeneration-related markers, such as GSK3 and tau. In conclusion, we show that activation of α7nAchR anti-inflammatory signaling in hypothalamic neurons exerts neuroprotective effects and prevents the development of insulin resistance induced by pro-inflammatory mediators secreted by microglial cells.

Evidence for a neuromuscular circuit involving hypothalamic interleukin-6 in the control of skeletal muscle metabolism.

Hypothalamic interleukin-6 (IL6) exerts a broad metabolic control. Here, we demonstrated that IL6 activates the ERK1/2 pathway in the ventromedial hypothalamus (VMH), stimulating AMPK/ACC signaling and fatty acid oxidation in mouse skeletal muscle. Bioinformatics analysis revealed that the hypothalamic IL6/ERK1/2 axis is closely associated with fatty acid oxidation- and mitochondrial-related genes in the skeletal muscle of isogenic BXD mouse strains and humans. We showed that the hypothalamic IL6/ERK1/2 pathway requires the α2-adrenergic pathway to modify fatty acid skeletal muscle metabolism. To address the physiological relevance of these findings, we demonstrated that this neuromuscular circuit is required to underpin AMPK/ACC signaling activation and fatty acid oxidation after exercise. Last, the selective down-regulation of IL6 receptor in VMH abolished the effects of exercise to sustain AMPK and ACC phosphorylation and fatty acid oxidation in the muscle after exercise. Together, these data demonstrated that the IL6/ERK axis in VMH controls fatty acid metabolism in the skeletal muscle.

Taurine upregulates insulin signaling and mitochondrial metabolism in vitro but not in adipocytes of obese women.

OBJECTIVES: Based on taurine's beneficial roles in metabolic diseases in rodents and obese individuals, we investigated the effects of taurine supplementation on adipose tissue using transcriptome analysis, 3T3-L1 adipocytes, and subcutaneous white adipose tissue (scWAT) of obese women. METHODS: First, we applied bioinformatics analysis to evaluate the effect of the taurine synthesis pathway on the adipose tissue of several BXD mice strains. After that, using 3T3-L1 adipocytes, we investigated the effects of different taurine doses in proteins related to insulin signaling, lipid oxidation, and mitochondrial function. Finally, we evaluated the effects of taurine supplementation (3 grams, 8 wk) on the same proteins in the scWAT of obese women. RESULTS: The transcriptome analysis showed that the taurine biosynthesis pathway was positively associated with insulin signaling and mitochondrial metabolism in the scWAT of BXD mice. The experiments using 3T3-L1 cells highlighted that the taurine dosage has an essential function in taurine synthesis, insulin, and mitochondrial markers. In contrast, the 8-wk taurine administration did not change the basal insulin, proteins of the taurine synthesis or insulin pathways, lipid oxidation, or mitochondrial metabolism in the scWAT of obese women. CONCLUSIONS: For the first time, to our knowledge, we showed that supplementation with 3 g of taurine for 8 wk promoted no effect in the insulin signaling pathway in the scWAT of obese women. These findings bring new perspectives to investigate different taurine doses and the intervention period for human studies owing to the potential antiobesity activity of taurine.

TLR4-interactor with leucine-rich repeats (TRIL) is involved in diet-induced hypothalamic inflammation.

Obesity and high-fat diet (HFD) consumption result in hypothalamic inflammation and metabolic dysfunction. While the TLR4 activation by dietary fats is a well-characterized pathway involved in the neuronal and glial inflammation, the role of its accessory proteins in diet-induced hypothalamic inflammation remains unknown. Here, we demonstrate that the knockdown of TLR4-interactor with leucine-rich repeats (Tril), a functional component of TLR4, resulted in reduced hypothalamic inflammation, increased whole-body energy expenditure, improved the systemic glucose tolerance and protection from diet-induced obesity. The POMC-specific knockdown of Tril resulted in decreased body fat, decreased white adipose tissue inflammation and a trend toward increased leptin signaling in POMC neurons. Thus, Tril was identified as a new component of the complex mechanisms that promote hypothalamic dysfunction in experimental obesity and its inhibition in the hypothalamus may represent a novel target for obesity treatment.

Anticancer effects of root and beet leaf extracts (Beta vulgaris L.) in cervical cancer cells (HeLa).

Cervical cancer is the fourth leading cause of cancer mortality in women worldwide. Beetroot (Beta vulgaris L.) has bioactive compounds that can inhibit the progression of different types of cancer. To analyze the antiproliferative effects of beet leaf and root extracts, we performed MTT, clonogenic survival, cell cycle analysis, Annexin/PI labeling, and western blotting. Here, we report that 10 and 100 µg/ml of root and leaf extracts decreased cell viability and potentiated rapamycin and cisplatin effects while decreased the number of large colonies, especially at 10 µg/ml (293.6 of control vs. 200.0 of leaf extract, p = .0059; 138.6 of root extract, p = .0002). After 48 hr, 100 µg/ml of both extracts led to increased sub-G1 and G0/G1 populations. In accordance, 100 µg/ml of root extract induced early apoptosis (mean = 0.64 control vs. 1.56 root; p = .048) and decreased cell size (p < .0001). Both extracts decreased phosphorylation and expression of mechanistic Target of Rapamycin (mTOR) signaling, especially by inhibiting ribosomal protein S6 (S6) phosphorylation, increasing cleaved poly(ADP-ribose) polysomerase 1 (PARP1) and Bcl-2-like protein 11 (BIM), and decreasing cyclin D1 expression, which regulates cell cycle progression. Here, we demonstrate that beetroot and leaf extracts could be an efficient strategy against cervical cancer.

Beetroot and leaf extracts present protective effects against prostate cancer cells, inhibiting cell proliferation, migration, and growth signaling pathways.

Beet (Beta vulgaris L.) has high nutritional value, containing bioactive compounds such as betalains and flavonoids. Scientific evidence points to the use of these natural compounds in the treatment of several types of cancer, such as prostate cancer, one of the main causes of morbidity and mortality in men. Here, we compared beet roots and leaves extracts, and their main compounds, apigenin, and betanin, respectively, in DU-145 and PC-3 prostate cancer cell lines. Both cells presented the proliferation decreased for beetroot and beet leaves extracts. The apigenin treatment also reduced the proliferation of both cell lines. Regarding cell migration, beet leaves extract was able to decrease the scratch area in both cell lines, whereas apigenin affected only PC-3 cells' migration. In colony formation assay, both extracts were effective in reducing the number of colonies formed. Besides, the beet leaves extracts and apigenin presented strong inhibition of growth-related signaling pathways in both cell lines, and the beetroot extract and betanin presented effects only in DU-145 cells. Furthermore, the extracts and isolated compounds were able to reduce the levels of apoptotic and cell cycle proteins. This study reveals that beet extracts have important anti-cancer effects against prostate cancer cells.

Characterization of pomegranate peel extracts obtained using different solvents and their effects on cell cycle and apoptosis in leukemia cells.

Pomegranate (Punica granatum L.) has been used in traditional herbal medicine by several cultures as an anti-inflammatory, antioxidant, antihyperglycemic, and for treatment and prevention of cancer and other diseases. Different parts of the fruit, extraction methods, and solvents can define the chemical profile of the obtained extracts and their biological activities. This study aimed to characterize the chemical profile of peel extracts collected using different extraction solvents and their biological effects on the cell cycle and apoptosis of THP-1 leukemic cells. Aqueous extract presented the highest content of punicalagins (α pun = 562.26 ± 47.14 mg/L and ß pun = 1,251.13 ± 22.21 mg/L) and the lowest content of ellagic acid (66.38 ± 0.21 mg/L), and it promoted a significant impairment of the cell cycle S phase. In fact, punicalagin-enriched fraction, but not an ellagic acid-enriched fraction, caused an S phase cell cycle arrest. All extracts increased the number of apoptotic cells. Punicalagin-enriched fraction increased the percentage of cells with fragmented DNA, which was intensified by ellagic acid combination. The treatment combining punicalagin and ellagic acid fractions increased the apoptotic cleaved PARP1 protein and reduced the activation of the growth-related mTOR pathway. Thus, these results evidence that solvent choice is critical for the phenolic compounds profile of pomegranate peel extracts and their biological activities.

Aqueous extract from Luehea divaricata Mart. Leaves reduces nociception in rats with neuropathic pain.

ETHNOPHARMACOLOGICAL RELEVANCE: Luehea divaricata, popularly known in Brazil as "açoita-cavalo", has been widely explored by different ethnic groups native to Brazil to treat different pathologic conditions, including inflammatory pain. However, no report could be found on the effect that extract of L. divaricata has on neuropathic pain. This is an important topic because convergent and divergent mechanisms underlie inflammatory vs. neuropathic pain indicate that there may not always be a clear mechanistic delineation between these two conditions. AIM OF THE STUDY: The study aimed to determine antioxidant activity and macronutrient composition of aqueous extract from leaves of L. divaricata, and the effect of oral administration on nociception in rats with chronic constriction injury (CCI) of sciatic nerve-induced neuropathic pain, one of the most commonly employed animal models of neuropathic pain. MATERIALS AND METHODS: The antioxidant activity of the extract was evaluated by total phenolic content and DPPH, ABTS●+ and ORAC methods. Vitexin was determined by HPLC to show that the composition of the extract of the present study is similar to that used in previous studies with this genus. Total sugar and sucrose concentrations were assessed by the anthrone method, while glucose and triacilglycerides were determined using commercially available kits. Fructose concentration was calculated from values for total sugars, glucose and sucrose. Total protein was determined by Bradford assay. The effect on DNA strand breaking was investigated by inhibition of strand breaking of supercoiled DNA by hydroxyl radical. The antinociceptive effects of aqueous extract (100, 300, 500, and 1000 mg/kg, i.g.) were evaluated on thermal and mechanical thresholds for neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats. We also compared the antinociceptive effect of the extract (500 mg/kg, i.g.) with that induced by gabapentin (50 mg/kg, i.g.), a first-line clinical treatment for neuropathic pain. The effect of co-administration of extract (500 mg/kg, i.g.) and low-dose gabapentin (30 mg/kg, i.g.) was also assessed. In addition, the effect of the extract on body weight, and blood and hepatic parameters were investigated to reveal possible side effects of treatment. RESULTS: The extract showed high content of total phenol; good reducing capacity for DPPH, ABTS●+ and ORAC assays; presence of vitexin; and a high capacity to inhibit strand breaking of supercoiled DNA. The predominant sugar was sucrose, followed by glucose and fructose. Total protein was greater than triacylglycerides, with the latter being present in a trace amount in the extract. The extract increased the thermal and mechanical thresholds, which was reduced by CCI. The antinociceptive effect was comparable to gabapentin and was also found after co-administration of extract and low-dose gabapentin. No significant change was found in body weight and blood and hepatic indicators after extract treatment. CONCLUSIONS: Aqueous extract from L. divaricata leaves was as effective as gabapentin at attenuating CCI-induced neuropathic pain, indicating for first time the therapeutic potential of this species for this type of pain.

Protective effects of beet (Beta vulgaris) leaves extract against oxidative stress in endothelial cells in vitro.

Beetroot is an herb used worldwide as a food product, raw material for food industry, ethanol production and source of food coloring. Beet leaves are an unconventional food with antioxidant properties, which might neutralize reactive oxygen species (ROS) induced by oxidized Low-Density Lipoprotein (LDL) present in dyslipidemias. This study aimed to elucidate the effects of beet leaves on the suppression of LDL oxidative processes. Beet leaves extract was produced, characterized, and tested for its antioxidant capacity using endothelial cells in vitro. A model of human umbilical vein endothelial cells was used in various tests, including viability assay, molecular analysis of antioxidant genes, ROS labeling, and macrophage adhesion assay. The extract improved the antioxidative protection of endothelial cells against different agents including oxidized LDL-cholesterol and H2 O2 . It acted on ROS directly due to its high content of natural antioxidants, but also due to the activation and improvement of cellular defenses such as Superoxide dismutase 1, Superoxide dismutase 2, and catalase. The inhibition of LDL-mediated oxidative effects on endothelial cells may turn this unconventional food a functional food with great potential for phytotherapy of atherosclerosis as an adjuvant for medicinal treatments.

Pomegranate Juice and Peel Extracts are Able to Inhibit Proliferation, Migration and Colony Formation of Prostate Cancer Cell Lines and Modulate the Akt/mTOR/S6K Signaling Pathway.

Pomegranate (Punica granatum) is known to contain polyphenols with many potential health benefits, including anti-tumoral, anti-inflammatory, and anti-microbial properties. It has been used in popular medicine for cancer treatment, which still represents the major cause of cancer-related deaths in men worldwide. Importantly, pomegranate peels are valuable by-products of the food industry that are rich in polyphenols. Here we report a comparison between juice and peel aqueous extracts in prostate cancer DU-145 and PC-3 cell lines. Both extracts were able to inhibit the proliferation, migration and colony formation of those cells, although peel extracts presented more robust effects compared to juice. Besides, the growth-related mTOR/S6K signaling pathway presented strong inhibition after pomegranate extracts treatment. This study presents evidence that both juice and isolated peel extracts from promegate fruit have important anti-cancer effects against prostate cancer cells, modulating the mTOR/S6K signaling pathway.

Modulation of hypothalamic S6K1 and S6K2 alters feeding behavior and systemic glucose metabolism.

The mTOR/S6Ks signaling is one of the intracellular pathways important for metabolic control, acting both peripherally and centrally. In the hypothalamus, mTOR/S6Ks axis mediates the action of leptin and insulin and can modulate the expression of neuropeptides. We analyzed the role of different S6Ks isoforms in the hypothalamic regulation of metabolism. We observed decreased food intake and decreased expression of agouti-related peptide (AgRP) following intracerebroventricular (icv) injections of adenoviral-mediated overexpression of three different S6Ks isoforms. Moreover, mice overexpressing p70-S6K1 in undefined periventricular hypothalamic neurons presented changes in glucose metabolism, as an increase in gluconeogenesis. To further evaluate the hypothalamic role of a less-studied S6K isoform, p54-S6K2, we used a Cre-LoxP approach to specifically overexpress it in AgRP neurons. Our findings demonstrate the potential participation of S6K2 in AgRP neurons regulating feeding behavior.

Overexpression of Mitogen-activated protein kinase phosphatase-3 (MKP-3) reduces FoxO1 phosphorylation in mice hypothalamus.

The mitogen-activated kinase phosphatase-3 (MKP-3) has gained great importance in the scientific community by acting as a regulator of the cell cycle through dephosphorylation of FoxO1, an important transcription factor involved in the insulin intracellular signaling cascade. When dephosphorylated and translocated to the nuclei, FoxO1 can promote the transcription of orexigenic neuropeptides (NPY/AgRP) in the hypothalamus, whereas insulin signaling is responsible for the disruption of this process. However, it is not understood if the hypothalamic activation of MKP-3 affects FoxO1 phosphorylation, and we hypothesized that MKP-3 overexpression reduces the capacity of the insulin signal to phosphorylate FoxO1. In the present study, we overexpressed the DUSP6 gene through an injection of adenovirus directly into the hypothalamic third ventricle of Swiss mice. The colocalization of the adenovirus was confirmed by the immunofluorescence assay. Then, MKP-3 overexpression resulted in a significant reduction of hypothalamic FoxO1 phosphorylation after insulin stimulation. This effect was independent of changes in Akt phosphorylation. Thus, the role of MKP-3 in the hypothalamus is closely associated with FoxO1 dephosphorylation and may provide a potential therapeutic target against hypothalamic disorders related to obesity and unbalanced food intake control.

Combinatorial effect of non-steroidal anti-inflammatory drugs and NF-κB inhibitors in ovarian cancer therapy.

Several epidemiological studies have correlated the use of non-steroidal anti-inflammatory drugs (NSAID) with reduced risk of ovarian cancer, the most lethal gynecological cancer, diagnosed usually in late stages of the disease. We have previously established that the pro-apoptotic cytokine melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24) is a crucial mediator of NSAID-induced apoptosis in prostate, breast, renal and stomach cancer cells. In this report we evaluated various structurally different NSAIDs for their efficacies to induce apoptosis and mda-7/IL-24 expression in ovarian cancer cells. While several NSAIDs induced apoptosis, Sulindac Sulfide and Diclofenac most potently induced apoptosis and reduced tumor growth. A combination of these agents results in a synergistic effect. Furthermore, mda-7/IL-24 induction by NSAIDs is essential for programmed cell death, since inhibition of mda-7/IL-24 by small interfering RNA abrogates apoptosis. mda-7/IL-24 activation leads to upregulation of growth arrest and DNA damage inducible (GADD) 45 α and γ and JNK activation. The NF-κB family of transcription factors has been implicated in ovarian cancer development. We previously established NF-κB/IκB signaling as an essential step for cell survival in cancer cells and hypothesized that targeting NF-κB could potentiate NSAID-mediated apoptosis induction in ovarian cancer cells. Indeed, combining NSAID treatment with NF-κB inhibitors led to enhanced apoptosis induction. Our results indicate that inhibition of NF-κB in combination with activation of mda-7/IL-24 expression may lead to a new combinatorial therapy for ovarian cancer.