Endogenous levels of 1-O-acylceramides increase upon acidic ceramidase deficiency and decrease due to loss of Dgat1 in a tissue-dependent manner.

Except for epidermis and liver, little is known about endogenous expression of 1-O-acylceramides (1-OACs) in mammalian tissue. Therefore, we screened several organs (brain, lung, liver, spleen, lymph nodes, heart, kidney, thymus, small intestine, and colon) from mice for the presence of 1-OACs by LC-MS2. In most organs, low levels of about 0.25-1.3 pmol 1-OACs/mg wet weight were recorded. Higher levels were detected in liver, small and large intestines, with about 4-13 pmol 1-OACs/mg wet weight. 1-OACs were esterified mainly with palmitic, stearic, or oleic acids. Esterification with saturated very long-chain fatty acids, as in epidermis, was not observed. Western-type diet induced 3-fold increased 1-OAC levels in mice livers while ceramides were unaltered. In a mouse model of Farber disease with a decrease of acid ceramidase activity, we observed a strong, up to 50-fold increase of 1-OACs in lung, thymus, and spleen. In contrast, 1-OAC levels were reduced 0.54-fold in liver. Only in lung 1-OAC levels correlated to changes in ceramide levels - indicating tissue-specific mechanisms of regulation. Glucosylceramide synthase deficiency in liver did not cause changes in 1-OAC or ceramide levels, whereas increased ceramide levels in glucosylceramide synthase-deficient small intestine caused an increase in 1-OAC levels. Deficiency of Dgat1 in mice resulted in a reduction of 1-OACs to 30% in colon, but not in small intestine and liver, going along with constant free ceramides levels. From these data, we conclude that Dgat1 as well as lysosomal lipid metabolism contribute in vivo to homeostatic 1-OAC levels in an organ-specific manner.

Gangliosides modulate insulin secretion by pancreatic beta cells under glucose stress.

In pancreatic beta cells, the entry of glucose and downstream signaling for insulin release is regulated by the glucose transporter 2 (Glut2) in rodents. Dysfunction of the insulin-signaling cascade may lead to diabetes mellitus. Gangliosides, sialic acid-containing glycosphingolipids (GSLs), have been reported to modulate the function of several membrane proteins.Murine islets express predominantly sialylated GSLs, particularly the simple gangliosides GM3 and GD3 having a potential modulatory role in Glut2 activity. Conditional, tamoxifen-inducible gene targeting in pancreatic islets has now shown that mice lacking the glucosylceramide synthase (Ugcg), which represents the rate-limiting enzyme in GSL biosynthesis, displayed impaired glucose uptake and showed reduced insulin secretion. Consequently, mice with pancreatic GSL deficiency had higher blood glucose levels than respective controls after intraperitoneal glucose application. High-fat diet feeding enhanced this effect. GSL-deficient islets did not show apoptosis or ER stress and displayed a normal ultrastructure. Their insulin content, size and number were similar as in control islets. Isolated beta cells from GM3 synthase null mice unable to synthesize GM3 and GD3 also showed lower glucose uptake than respective control cells, corroborating the results obtained from the cell-specific model. We conclude that in particular the negatively charged gangliosides GM3 and GD3 of beta cells positively influence Glut2 function to adequately respond to high glucose loads.

Bacterial immunogenic α-galactosylceramide identified in the murine large intestine: dependency on diet and inflammation.

The glycosphingolipid, α-galactosylceramide (αGalCer), when presented by CD1d on antigen-presenting cells, efficiently activates invariant natural killer T (iNKT) cells. Thereby, it modulates immune responses against tumors, microbial and viral infections, and autoimmune diseases. Recently, the production of αGalCer by Bacteroidetes from the human gut microbiome was elucidated. Using hydrophilic interaction chromatography coupled to MS2, we screened murine intestinal tracts to identify and quantify αGalCers, and we investigated the αGalCer response to different dietary and physiologic conditions. In both the cecum and the colon of mice, we found 1-15 pmol of αGalCer per milligram of protein; in contrast, mice lacking microbiota (germ-free mice) and fed identical diet did not harbor αGalCer. The identified αGalCer contained a ß(R)-hydroxylated hexadecanoyl chain N-linked to C18-sphinganine, which differed from what has been reported with Bacteroides fragilis Unlike ß-anomeric structures, but similar to αGalCers from B. fragilis, the synthetic form of the murine αGalCer induced iNKT cell activation in vitro. Last, we observed a decrease in αGalCer production in mice exposed to conditions that alter the composition of the gut microbiota, including Western type diet, colitis, and influenza A virus infection. Collectively, this study suggests that αGalCer is produced by commensals in the mouse intestine and reveals that stressful conditions causing dysbiosis alter its synthesis. The consequences of this altered production on iNKT cell-mediated local and systemic immune responses are worthy of future studies.

Deletion of Specific Sphingolipids in Distinct Neurons Improves Spatial Memory in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by progressive neurodegeneration and a concomitant loss of synapses and cognitive abilities. Recently, we have proposed that an alteration of neuronal membrane lipid microdomains increases neuronal resistance toward amyloid-ß stress in cultured neurons and protects from neurodegeneration in a mouse model of AD. Lipid microdomains are highly enriched in a specific subclass of glycosphingolipids, termed gangliosides. The enzyme glucosylceramide synthase (GCS) catalyzes the rate-limiting step in the biosynthesis of these gangliosides. The present work now demonstrates that genetic GCS deletion in subsets of adult forebrain neurons significantly improves the spatial memory and counteracts the loss of dendritic spines in the hippocampal dentate gyrus of 5x familial AD mice (5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP mice), when compared to 5xFAD//Ugcgf/f littermates (5xFAD mice). Aberrantly activated glial cells and their expression of pro-inflammatory cytokines have emerged as the major culprits for synaptic loss in AD. Typically, astrocytic activation is accompanied by a thickening of astrocytic processes, which impairs astrocytic support for neuronal synapses. In contrast to 5xFAD mice, 5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP display a less pronounced thickening of astrocytic processes and a lower expression of tumor necrosis factor-α and interleukin 1-α in the hippocampus. Thus, this work further emphasizes that GCS inhibition may constitute a potential therapeutic target against AD.

Lipid microdomain modification sustains neuronal viability in models of Alzheimer's disease.

Decreased neuronal insulin receptor (IR) signaling in Alzheimer's disease is suggested to contribute to synaptic loss and neurodegeneration. This work shows that alteration of membrane microdomains increases IR levels and signaling, as well as neuronal viability in AD models in vitro and in vivo. Neuronal membrane microdomains are highly enriched in gangliosides. We found that inhibition of glucosylceramide synthase (GCS), the key enzyme of ganglioside biosynthesis, increases viability of cortical neurons in 5xFAD mice, as well as in cultured neurons exposed to oligomeric amyloid-ß-derived diffusible ligands (ADDLs). We furthermore demonstrate a molecular mechanism explaining how gangliosides mediate ADDL-related toxic effects on IR of murine neurons. GCS inhibition increases the levels of functional dendritic IR on the neuronal surface by decreasing caveolin-1-mediated IR internalization. Consequently, IR signaling is increased in neurons exposed to ADDL stress. Thus, we propose that GCS inhibition constitutes a potential target for protecting neurons from ADDL-mediated neurotoxicity and insulin resistance in Alzheimer's disease.

Fasting-Induced Lipolysis and Hypothalamic Insulin Signaling Are Regulated by Neuronal Glucosylceramide Synthase.

Central nervous regulation of body weight and adipose tissue function is mainly conducted by hypothalamic neurons. Neuronal function depends on the integrity of the membrane lipid microenvironment. Lipid microdomains contain large quantities of cholesterol and glycosphingolipids, including glucosylceramide synthase (GCS) (gene Ugcg)-derived gangliosides. The current study demonstrates that Ugcgf/f//CamKCreERT2 mice with genetic GCS deletion in forebrain neurons, dominantly targeting mediobasal hypothalamus (MBH), display impaired fasting-induced lipolysis accompanied by a decreased norepinephrine content in white adipose tissue (WAT). MBH insulin receptor (IR) levels and signaling are increased in Ugcgf/f//CamKCreERT2 mice. These results are in concordance with reports stating that MBH insulin signaling restrains sympathetic nervous outflow to WAT in fasted mice. In line with the in vivo data, pharmacological GCS inhibition by Genz123346 also increases IR levels as well as IR phosphorylation in insulin-stimulated hypothalamic cells. In addition to studies suggesting that simple gangliosides like GM3 regulate peripheral IR signaling, this work suggests that complex neuronal gangliosides also modulate hypothalamic IR signaling and protein levels. For example, the complex ganglioside GD1a interacts dynamically with the IRs on adult hypothalamic neurons. In summary, our results suggest that neuronal GCS expression modulates MBH insulin signaling and WAT function in fasted mice.

MicroRNAs in the hypothalamus.

MicroRNAs (miRNAs) are short (∼22 nucleotides) non-coding ribonucleic acid (RNA) molecules that negatively regulate the expression of protein-coding genes. Posttranscriptional silencing of target genes by miRNA is initiated by binding to the 3'-untranslated regions of target mRNAs, resulting in specific cleavage and subsequent degradation of the mRNA or by translational repression resulting in specific inhibition of protein synthesis. An increasing amount of evidence shows that miRNAs control a large number of biological processes and there exists a direct link between miRNAs and disease. miRNA molecules are abundantly expressed in tissue-specific and regional patterns and have been suggested as potential biomarkers, disease modulators and drug targets. The central nervous system is a prominent site of miRNA expression. Within the brain, several miRNAs are expressed and/or enriched in the region of the hypothalamus and miRNAs have recently been shown to be important regulators of hypothalamic control functions. The aim of this review is to summarize some of the current knowledge regarding the expression and role of miRNAs in the hypothalamus.

Neuronal expression of glucosylceramide synthase in central nervous system regulates body weight and energy homeostasis.

Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.

G-protein-gated inwardly rectifying K+ channel 4 (GIRK4) immunoreactivity in chemically defined neurons of the hypothalamic arcuate nucleus that control body weight.

G-protein-gated inwardly rectifying K(+) channels (GIRKs; also called Kir3) are a family of K(+) channels, which are activated (opened) via a signal transduction cascade starting with ligand-stimulated G-protein-coupled receptors (GPCRs). Four GIRK genes have been identified (GIRK1-4). GIRK4 (Kir3.4) has a role in regulating energy homeostasis, since mice with a targeted mutation in the GIRK4 gene exhibit a predisposition to late-onset obesity. GIRK4 mRNA is expressed in hypothalamic regions that harbor neurons involved in the regulation of food intake and body weight. Using goat and rabbit antisera to the GIRK4 protein, the cellular localization and transmitter content of GIRK4-immunoreactive neurons was determined in the hypothalamic arcuate nucleus, a region that contains neurons which are accessible to circulating hormones and is intimately associated with the control of body weight. GIRK4-immunoreactive large cell bodies were demonstrated in the ventrolateral part of the arcuate nucleus, with smaller neuronal cell bodies in the ventromedial part of the nucleus. Double-labeling showed presence of GIRK4 immunoreactivity in large neurons of the ventrolateral arcuate nucleus containing the peptides α-melanocyte-stimulating hormone (α-MSH), a marker for pro-opiomelanocortin (POMC) neurons, and cocaine- and amphetamine-regulated transcript (CART). GIRK4 immunoreactivity was also seen in neurons of the ventromedial part of the arcuate nucleus containing agouti-regulated peptide (AgRP) and neuropeptide Y (NPY). The results suggest that the GIRK4 channel protein plays a role in regulating membrane excitability in chemically defined neurons of the arcuate nucleus that control body weight.