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Genome-wide association studies (GWASs) have identified SNPs of the fat mass and obesity (FTO) gene as the most important risk alleles for obesity. However, how the presence of risk alleles affect phenotype is still a matter of intense investigation. In 2014, a study revealed that long-range enhancers from the intronic regions of the FTO gene regulate iroquois-class homeobox protein (IRX)3 expression. IRX3 is expressed in hypothalamic pro-opiomelanocortin (POMC) neurons and changes in its expression levels affect body adiposity by modifying food intake and energy expenditure. These findings have placed IRX3 as a potential target for the treatment of obesity. Here, we review studies that evaluated the roles of IRX3 in development, neurogenesis, and body energy homeostasis.

Leptin regulates both feeding and glycaemia primarily through its receptors expressed on agouti-related peptide (AgRP) and pro-opiomelanocortin-expressing (POMC) neurons; however, it is unknown whether activity of these neuronal populations mediates the regulation of these processes. To determine this, we injected Cre-dependent designer receptors exclusively activated by designer drugs (DREADD) viruses into the hypothalamus of normoglycaemic and diabetic AgRP-ires-cre and POMC-cre mice to chemogenetically activate or inhibit these neuronal populations. Despite robust changes in food intake, activation or inhibition of AgRP neurons did not affect glycaemia, while activation caused significant (P = 0.014) impairment in insulin sensitivity. Stimulation of AgRP neurons in diabetic mice reversed leptin’s ability to inhibit feeding but did not counter leptin’s ability to lower blood glucose levels. Notably, the inhibition of POMC neurons stimulated feeding while decreasing glucose levels in normoglycaemic mice. The findings suggest that leptin’s effects on feeding by AgRP neurons are mediated by changes in neuronal firing, while the control of glucose balance by these cells is independent of chemogenetic activation or inhibition. The firing-dependent glucose lowering mechanism within POMC neurons is a potential target for the development of novel anti-diabetic medicines.

Background The Iroquois homeobox 3 (Irx3) gene has been identified as a functional long-range target of obesity-associated variants within the fat mass and obesity-associated protein (FTO) gene. It is highly expressed in the hypothalamus, and both whole-body knockout and hypothalamic restricted abrogation of its expression results in a lean phenotype, which is mostly explained by the resulting increased energy expenditure in the brown adipose tissue. Because of its potential implication in the pathogenesis of obesity, we evaluated the hypothalamic cell distribution of Irx3 and the outcomes of inhibiting its expression in a rodent model of diet-induced obesity. Methods Bioinformatics tools were used to evaluate the correlations between hypothalamic Irx3 and neurotransmitters, markers of thermogenesis and obesity related phenotypes. Droplet-sequencing analysis in >20,000 hypothalamic cells was used to explore the types of hypothalamic cells expressing Irx3. Lentivirus was used to inhibit hypothalamic Irx3 and the resulting phenotype was studied. Findings IRX3 is expressed predominantly in POMC neurons. Its expression is inhibited during prolonged fasting, as well as when mice are fed a high-fat diet. The partial inhibition of hypothalamic Irx3 using a lentivirus resulted in increased diet-induced body mass gain and adiposity due to increased caloric intake and reduced energy expenditure. Interpretation Contrary to the results obtained when lean mice are submitted to complete inhibition of Irx3, partial inhibition of hypothalamic Irx3 in obese mice causes an exacerbation of the obese phenotype. These data suggest that at least some of the Irx3 functions in the hypothalamus are regulated according to a hormetic pattern, and modulation of its expression can be a novel approach to modifying the body's energy-handling regulation. Fund Sao Paulo Research Foundation grants 2013/07607-8 (LAV) and 2017/02983-2 (JDJ); NIH grants R01DK083567 (YBK).

Recent studies have indicated that systemic topiramate can induce an improvement on the aesthetic appearance of skin scars. Here, we evaluated topical topiramate as an agent to improve wound healing in C57/BL6 mice. Mice were inflicted with a 6.0 mm punch to create two wounds in the skin of the dorsal region. Thereafter, mice were randomly assigned to either vehicle or topical topiramate (20 µl of 2% cream) once a day for 14 days, beginning on the same day as wound generation. We analyzed the wound samples over real-time PCR, Western blotting, and microscopy. There was no effect of the topiramate treatment on the time for complete reepithelization of the wound. However, on microscopic analysis, topiramate treatment resulted in increased granulation tissue, thicker epidermal repair, and improved deposition of type I collagen fibers. During wound healing, there were increased expressions of anti-inflammatory markers, such as IL-10, TGF-β1, and reduced expression of the active form of JNK. In addition, topiramate treatment increased the expression of active forms of two intermediaries in the insulin-signaling pathway, IRS-1 and Akt. Finally, at the end of the wound-healing process, topiramate treatment resulted in increased expression of SOX-2, a transcription factor that is essential to maintain cell self-renewal of undifferentiated embryonic stem cells. We conclude that topical topiramate can improve the overall quality of wound healing in the healthy skin of mice. This improvement is accompanied by reduced expression of markers involved in inflammation and increased expression of proteins of the insulin-signaling pathway.

In outbred mice, susceptibility or resistance to diet-induced obesity is associated with rapid changes in hypothalamic proopiomelanocortin (POMC) levels. Here, we evaluated 3 hypotheses that potentially explain the development of the different obesity phenotypes in outbred Swiss mice. First, rapid and differential changes in the gut microbiota in obesity-prone (OP) and obesity-resistant (OR) mice fed on a high-fat diet (HFD) might cause differential efficiencies in fatty acid harvesting leading to changes in systemic fatty acid concentrations that in turn affect POMC expression and processing. Second, independently of the gut microbiota, OP mice might have increased blood fatty acid levels after the introduction of a HFD, which could affect POMC expression and processing. Third, fatty acids might act directly in the hypothalamus to differentially regulate POMC expression and/or processing in OP and OR mice. We evaluated OP and OR male Swiss mice using 16S rRNA sequencing for the determination of gut microbiota; gas chromatography for blood lipid determination; and immunoblot and real-time polymerase chain reaction for protein and transcript determination and indirect calorimetry. Some experiments were performed with human pluripotent stem cells differentiated into hypothalamic neurons. We did not find evidence supporting the first 2 hypotheses. However, we found that in OP but not in OR mice, palmitate induces a rapid increase in hypothalamic POMC, which is followed by increased expression of proprotein convertase subtilisin/kexin type 1 PC1/3. Lentiviral inhibition of hypothalamic PC1/3 increased caloric intake and body mass in both OP and OR mice. In human stem cell-derived hypothalamic cells, we found that palmitate potently suppressed the production of POMC-derived peptides. Palmitate directly regulates PC1/3 in OP mice and likely has a functional impact on POMC processing.

Leptin is an adipocyte-derived hormone that reduces food intake and stimulates energy expenditure. In order to achieve its physiological actions, it needs to enter the brain. The precise routes of brain entry of many metabolic signals have not been identified, but most studies suggest that transport across the BBB and CSF barrier are required. This study was designed to test the hypothesis that LRP1 mediates leptin’s transport across the blood-CSF barrier at the choroid plexus (ChP). To address this, we have first generated Foxj1-Cre; tdTomato mice to see the expression pattern of Foxj1 in the brain. The expression of tdTomato is restricted to the epithelial cells of the ChP and the ependymal cells of the ventricles. Mice lacking LRP1 in the epithelial cells and ependymal cells of the brain were thus generated and studied. Here we show that deletion of LRP1 in the epithelial cells of the ChP and the ependymal cells of the ventricles causes hyperleptinemia. This effect is independent of body weight and adiposity. In addition, mice lacking LRP1 in the epithelial cells of the ChP and the ependymal cells of the ventricles are insulin-resistant. Most importantly, leptin-induced food intake and hypothalamic Stat3 phosphorylation were significantly impaired in Foxj1-Cre; LRP1lox/lox mice compared with LRP1lox/lox mice. However, ICV leptin decreased food intake and increased hypothalamic Stat3 phosphorylation in Foxj1-Cre; LRP1lox/lox mice. There were no differences in both food intake and Stat3 phosphorylation between LRP1lox/lox mice and Foxj1-Cre; LRP1lox/lox mice. These data clearly demonstrate that LRP1 in the epithelial cells of the ChP mediates leptin transport across the blood-CSF barrier at the ChP and further implicate that defective leptin transport may not contribute to the regulation of body weight homeostasis. Disclosure J. Seo: None. M. Kang: None. N. Ferreira Mendes: None. T. Araujo: None. W. Yang: None. Y. Kim: None. Funding National Institutes of Health (R01DK106076); National Research Foundation of Korea (2018R1D1A1B07049123)

Recent studies show that the metabolic effects of fructose may vary depending on the phase of its consumption along with the light/dark cycle. Here, we investigated the metabolic outcomes of fructose consumption by rats during either the light (LPF) or the dark (DPF) phases of the light/dark cycle. This experimental approach was combined with other interventions, including restriction of chow availability to the dark phase, melatonin administration or intracerebroventricular inhibition of adenosine monophosphate-activated protein kinase (AMPK) with Compound C. LPF, but not DPF rats, exhibited increased hypothalamic AMPK phosphorylation, glucose intolerance, reduced urinary 6-sulfatoxymelatonin (6-S-Mel) (a metabolite of melatonin) and increased corticosterone levels. LPF, but not DPF rats, also exhibited increased chow ingestion during the light phase. The mentioned changes were blunted by Compound C. LPF rats subjected to dark phase-restricted feeding still exhibited increased hypothalamic AMPK phosphorylation but failed to develop the endocrine and metabolic changes. Moreover, melatonin administration to LPF rats reduced corticosterone and prevented glucose intolerance. Altogether, the present data suggests that consumption of fructose during the light phase results in out-of-phase feeding due to increased hypothalamic AMPK phosphorylation. This shift in spontaneous chow ingestion is responsible for the reduction of 6-S-Mel and glucose intolerance.

Seaweed, a popular and abundant food ingredient mainly consumed in Asian countries, is a good source of bioactive compounds with anti-obesity effects. However, the anti-obesity effects of Sargassum thunbergii have not yet been established. In this study, we isolated six indole derivatives (STCs)-indole-2-carboxaldehyde (STC-1), indole-3-carboxaldehyde (STC-2), indole-4-carboxaldehyde (STC-3), indole-5-carboxaldehyde (STC-4), indole-6-carboxaldehyde (STC-5), and indole-7-carboxaldehyde (STC-6)-from S. thunbergii and evaluated their inhibitory effects on adipocyte differentiation in 3T3-L1 cells. We found that STC-1 and STC-5 resulted in non-toxic inhibition of the differentiation of 3T3-L1 adipocytes and thus selected these compounds for further study. STC-1 and STC-5 significantly inhibited lipid accumulation and downregulated the expression of peroxisome proliferator-activated receptor-gamma (PPAR gamma), CCAAT/enhancer-binding protein alpha (C/EBP alpha), and sterol regulatory element-binding protein 1c (SREBP-1c) in a dose-dependent manner. The specific mechanism mediating the effects of STC-1 and STC-5 was shown to be AMP-activated protein kinase (AMPK) activation. Our results demonstrated the inhibitory effect of STC-1 and STC-5 on adipogenesis through the activation of the AMPK signal pathway. Together, these findings suggested that STC-1 and STC-5 may be effective candidates for the prevention of obesity or obesity-related diseases.

The Myrtaceae family is a common source of medicines used in the treatment of numerous diseases in South America. In Brazil, fruits of the Campomanesia species are widely used to make liqueurs, juices and sweets, whereas leaves are traditionally employed as a medicine for dysentery, stomach problems, diarrhea, cystitis and urethritis. Ethanol extracts of Campomanesia adamantium (Myrtaceae) leaves and fruits were evaluated against prostate cancer cells (PC-3). The compound (2E)-1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenylprop-2-en-1-one, cardamonin) was isolated from ethanol extracts of C. adamantium leaves in a bioactivity-guided study and quantified by UPLC-MS/MS. In vitro studies showed that the isolated chalcone cardamonin inhibited prostate cancer cell proliferation and decreased the expression of NFkB1. Moreover, analysis by flow cytometry showed that this compound induced DNA fragmentation, suggesting an effect on apoptosis induction in the PC-3 cell line.

Inflammatory cytokines have been associated with the pathophysiology of hypertension and target organ damage (TOD). Resistant hypertensive patients (RHTN) are characterized by poor blood pressure control and higher prevalence of TOD. This study evaluated the relationship between plasma levels of TNF-αand arterial stiffness (pulse wave velocity—PWV) in 32 RHTN and 19 normotensive subjects. Moreover, we investigated the effect of TNF-αinhibition on human endothelial cells (HUVECs) incubated with serum from RHTN and normotensive subjects. HUVECs containing serum obtained from normotensiven=8and hypertensiven=8individuals were treated with TNF-αinhibitor (infliximab). Cell suspensions were used for measurement of DNA fragmentation and reactive oxygen species (ROS) content. RHTN patients showed higher levels of TNF-αcompared to normotensive subjects, as well as higher PWV. Positive correlation was found between TNF-αlevels and PWV measures in the whole group. HUVECs incubated with serum from RHTN showed increased cell apoptosis and higher ROS content compared to normotensive subjects. Infliximab attenuated the apoptosis of HUVECs incubated with serum from RHTN, but no effect in ROS production was observed. Our findings suggest that TNF-αmight mediate, at least in part, vascular damage in resistant hypertension.

Deep vein thrombosis (DVT) is multi-causal disease associated to high morbidity and mortality due to complications, especially from pulmonary embolism. New factors of relevance on the DVT pathophysiology enhance our understanding of disease mechanisms and can be translated into improved management of patients, prevention of recurrence and development of new therapies. In this context, the precise role of platelets in the pathogenesis of DVT is not completely understood. Our objectives were to acquire and to analyze the whole platelet protein profile of samples from 3 DVT patients and to compare them to results obtained from 1 sibling and 1 neighbor from each patient (in order to minimize genetic and environmental interferences). These patients presented unprovoked and recurrent episodes of proximal DVT as well as a family history of DVT. Platelets were washed, lysed, and the proteins were hydrolyzed by trypsin. Peptides were first separated by HPLC and peptide fractions were further detected by LC-MS/MS. Five proteins was present on patients and absent in all the controls: Apolipoprotein A1 Binding-Protein, Coatomer (zeta1 sub-unit), 17β-hydroxysteroid dehydrogenase type XI, Leukotriene A-4 Hydrolase and Sorbitol Dehydrogenase. The analysis identified proteins that currently are not related to the pathophysiology of DVT, and the persistence of these inflammatory and lipid transportation-related proteins emphasize the relevance of these phenomena on DVT.

The aim of this work was to develop an efficient, biodegradable, biocompatible and safe controlled release system using insulin-loaded poly(epsilon-caprolactone) (PCL) nanoparticles. The insulin-loaded PCL nanoparticles were prepared by double emulsion method (water-in-oil-in-water) using Pluronic F68 as emulsifier. Using the double emulsion method a high insulin encapsulation efficiency (90.6 +/-1.6%) with a zeta potential of -29 +/-2.7 mV and average particle size of 796 +/-10.5 nm was obtained. Insulin-loaded PCL nanoparticles showed no toxicity to MIN6 cells. Insulin nanoparticles administered subcutaneously and intraperitoneally in rats reduced glycaemia of basal levels after 15 minutes, and presented a sustainable hypoglycemic effect on insulin-dependent type 1 diabetic rats, showing to be more efficient than unencapsulated insulin. Furthermore, these nanoparticles were not hepatotoxic, as evaluated by the effect over liver cell-death and oxidative stress scavenger system in rats. These results suggest that insulin-loaded PCL nanoparticles prepared by water-in-oil-in-water emulsion method are biocompatible, efficient and safe insulin-delivering system with controlled insulin release, which indicates that it may be a powerful tool for insulin-dependent patients care.

Melatonin can contribute to glucose homeostasis either by decreasing gluconeogenesis or by counteracting insulin resistance in distinct models of obesity. However, the precise mechanism through which melatonin controls glucose homeostasis is not completely understood. Male Wistar rats were administered an intracerebroventricular (icv) injection of melatonin and one of following: an icv injection of a phosphatidylinositol 3-kinase (PI3K) inhibitor, an icv injection of a melatonin receptor (MT) antagonist, or an intraperitoneal (ip) injection of a muscarinic receptor antagonist. Anesthetized rats were subjected to pyruvate tolerance test to estimate in vivo glucose clearance after pyruvate load and in situ liver perfusion to assess hepatic gluconeogenesis. The hypothalamus was removed to determine Akt phosphorylation. Melatonin injections in the central nervous system suppressed hepatic gluconeogenesis and increased hypothalamic Akt phosphorylation. These effects of melatonin were suppressed either by icv injections of PI3K inhibitors and MT antagonists and by ip injection of a muscarinic receptor antagonist. We conclude that melatonin activates hypothalamus-liver communication that may contribute to circadian adjustments of gluconeogenesis. These data further suggest a physiopathological relationship between the circadian disruptions in metabolism and reduced levels of melatonin found in type 2 diabetes patients.

Fructose consumption causes insulin resistance and favors hepatic gluconeogenesis through mechanisms that are not completely understood. Recent studies demonstrated that the activation of hypothalamic 5′-AMP-activated protein kinase (AMPK) controls dynamic fluctuations in hepatic glucose production. Thus, the present study was designed to investigate whether hypothalamic AMPK activation by fructose would mediate increased gluconeogenesis. Both ip and intracerebroventricular (icv) fructose treatment stimulated hypothalamic AMPK and acetyl-CoA carboxylase phosphorylation, in parallel with increased hepatic phosphoenolpyruvate carboxy kinase (PEPCK) and gluconeogenesis. An increase in AMPK phosphorylation by icv fructose was observed in the lateral hypothalamus as well as in the paraventricular nucleus and the arcuate nucleus. These effects were mimicked by icv 5-amino-imidazole-4-carboxamide-1-β-d-ribofuranoside treatment. Hypothalamic AMPK inhibition with icv injection of compound C or with injection of a small interfering RNA targeted to AMPKα2 in the mediobasal hypothalamus (MBH) suppressed the hepatic effects of ip fructose. We also found that fructose increased corticosterone levels through a mechanism that is dependent on hypothalamic AMPK activation. Concomitantly, fructose-stimulated gluconeogenesis, hepatic PEPCK expression, and glucocorticoid receptor binding to the PEPCK gene were suppressed by pharmacological glucocorticoid receptor blockage. Altogether the data presented herein support the hypothesis that fructose-induced hypothalamic AMPK activation stimulates hepatic gluconeogenesis by increasing corticosterone levels.