Recent evidence indicates that the gut microbiota plays a key role in the pathophysiology of obesity. Indeed, diet-induced obesity (DIO) has been associated to substantial changes in gut microbiota composition in rodent models. In the context of obesity, enhanced adiposity is accompanied by low-grade inflammation of this tissue but the exact link with gut microbial community remains unknown. In this report, we studied the consequences of high-fat diet (HFD) administration on metabolic parameters and gut microbiota composition over different periods of time. We found that Akkermansia muciniphila abundance was strongly and negatively affected by age and HFD feeding and to a lower extend Bilophila wadsworthia was the only taxa following an opposite trend. Different approaches, including multifactorial analysis, showed that these changes in Akkermansia muciniphila were robustly correlated with the expression of lipid metabolism and inflammation markers in adipose tissue, as well as several circulating parameters (i.e., glucose, insulin, triglycerides, leptin) from DIO mice. Thus, our data shows the existence of a link between gut Akkermansia muciniphila abundance and adipose tissue homeostasis on the onset of obesity, thus reinforcing the beneficial role of this bacterium on metabolism.

Recent data suggest that ghrelin exerts its orexigenic action through regulation of hypothalamic AMP-activated protein kinase pathway, leading to a decline in malonyl-CoA levels and desinhibition of carnitine palmitoyltransferase 1A (CPT1A), which increases mitochondrial fatty acid oxidation and ultimately enhances the expression of the orexigenic neuropeptides agouti-related protein (AgRP) and neuropeptide Y (NPY). However, it is unclear whether the brain-specific isoform CPT1C, which is located in the endoplasmic reticulum of neurons, may play a role in this action. Here, we demonstrate that the orexigenic action of ghrelin is totally blunted in CPT1C knockout (KO) mice, despite having the canonical ghrelin signaling pathway activated. We also demonstrate that ghrelin elicits a marked upregulation of hypothalamic C18:0 ceramide levels mediated by CPT1C. Notably, central inhibition of ceramide synthesis with myriocin negated the orexigenic action of ghrelin and normalized the levels of AgRP and NPY, as well as their key transcription factors phosphorylated cAMP-response element–binding protein and forkhead box O1. Finally, central treatment with ceramide induced food intake and orexigenic neuropeptides expression in CPT1C KO mice. Overall, these data indicate that, in addition to formerly reported mechanisms, ghrelin also induces food intake through regulation of hypothalamic CPT1C and ceramide metabolism, a finding of potential importance for the understanding and treatment of obesity.

The brain-specific isoform carnitine palmitoyltransferase 1C (CPT1C) has been implicated in the hypothalamic regulation of food intake and energy homeostasis. Nevertheless, its molecular function is not completely understood, and its role in other brain areas is unknown. We demonstrate that CPT1C is expressed in pyramidal neurons of the hippocampus and is located in the endoplasmic reticulum throughout the neuron, even inside dendritic spines. We used molecular, cellular, and behavioral approaches to determine CPT1C function. First, we analyzed the implication of CPT1C in ceramide metabolism. CPT1C overexpression in primary hippocampal cultured neurons increased ceramide levels, whereas in CPT1C-deficient neurons, ceramide levels were diminished. Correspondingly, CPT1C knock-out (KO) mice showed reduced ceramide levels in the hippocampus. At the cellular level, CPT1C deficiency altered dendritic spine morphology by increasing immature filopodia and reducing mature mushroom and stubby spines. Total protrusion density and spine head area in mature spines were unaffected. Treatment of cultured neurons with exogenous ceramide reverted the KO phenotype, as did ectopic overexpression of CPT1C, indicating that CPT1C regulation of spine maturation is mediated by ceramide. To study the repercussions of the KO phenotype on cognition, we performed the hippocampus-dependent Morris water maze test on mice. Results show that CPT1C deficiency strongly impairs spatial learning. All of these results demonstrate that CPT1C regulates the levels of ceramide in the endoplasmic reticulum of hippocampal neurons, and this is a relevant mechanism for the correct maturation of dendritic spines and for proper spatial learning.

Background: The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). Methods: Seven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software. Results: Dikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. Conclusions: Biological association networks identified DKK1, UGT1A s and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX.