Endothelial Notch signaling controls insulin transport in muscle.

The role of the endothelium is not just limited to acting as an inert barrier for facilitating blood transport. Endothelial cells (ECs), through expression of a repertoire of angiocrine molecules, regulate metabolic demands in an organ-specific manner. Insulin flux across the endothelium to muscle cells is a rate-limiting process influencing insulin-mediated lowering of blood glucose. Here, we demonstrate that Notch signaling in ECs regulates insulin transport to muscle. Notch signaling activity was higher in ECs isolated from obese mice compared to non-obese. Sustained Notch signaling in ECs lowered insulin sensitivity and increased blood glucose levels. On the contrary, EC-specific inhibition of Notch signaling increased insulin sensitivity and improved glucose tolerance and glucose uptake in muscle in a high-fat diet-induced insulin resistance model. This was associated with increased transcription of Cav1, Cav2, and Cavin1, higher number of caveolae in ECs, and insulin uptake rates, as well as increased microvessel density. These data imply that Notch signaling in the endothelium actively controls insulin sensitivity and glucose homeostasis and may therefore represent a therapeutic target for diabetes.

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.

Renal globotriaosylceramide facilitates tubular albumin absorption and its inhibition protects against acute kidney injury.

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S-/-) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S-/- mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin.

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.

Hyperosmolarity impedes the cross-priming competence of dendritic cells in a TRIF-dependent manner.

Tissue osmolarity varies among different organs and can be considerably increased under pathologic conditions. Hyperosmolarity has been associated with altered stimulatory properties of immune cells, especially macrophages and dendritic cells. We have recently reported that dendritic cells upon exposure to hypertonic stimuli shift their profile towards a macrophage-M2-like phenotype, resulting in attenuated local alloreactivity during acute kidney graft rejection. Here, we examined how hyperosmotic microenvironment affects the cross-priming capacity of dendritic cells. Using ovalbumin as model antigen, we showed that exposure of dendritic cells to hyperosmolarity strongly inhibits activation of antigen-specific T cells despite enhancement of antigen uptake, processing and presentation. We identified TRIF as key mediator of this phenomenon. Moreover, we detected a hyperosmolarity-triggered, TRIF-dependent clustering of MHCI loaded with the ovalbumin-derived epitope, but not of overall MHCI molecules, providing a possible explanation for a reduced T cell activation. Our findings identify dendritic cells as important players in hyperosmolarity-mediated immune imbalance and provide evidence for a novel pathway of inhibition of antigen specific CD8+ T cell response in a hypertonic micromilieu.

Tubular Dickkopf-3 promotes the development of renal atrophy and fibrosis.

Renal tubular atrophy and interstitial fibrosis are common hallmarks of etiologically different progressive chronic kidney diseases (CKD) that eventually result in organ failure. Even though these pathological manifestations constitute a major public health problem, diagnostic tests, as well as therapeutic options, are currently limited. Members of the dickkopf (DKK) family, DKK1 and -2, have been associated with inhibition of Wnt signaling and organ fibrosis. Here, we identify DKK3 as a stress-induced, tubular epithelia-derived, secreted glycoprotein that mediates kidney fibrosis. Genetic as well as antibody-mediated abrogation of DKK3 led to reduced tubular atrophy and decreased interstitial matrix accumulation in two mouse models of renal fibrosis. This was facilitated by an amplified, antifibrogenic, inflammatory T cell response and diminished canonical Wnt/ß-catenin signaling in stressed tubular epithelial cells. Moreover, in humans, urinary DKK3 levels specifically correlated with the extent of tubular atrophy and interstitial fibrosis in different glomerular and tubulointerstitial diseases. In summary, our data suggest that DKK3 constitutes an immunosuppressive and a profibrotic epithelial protein that might serve as a potential therapeutic target and diagnostic marker in renal fibrosis.

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.

Dickkopf-3, a tissue-derived modulator of local T-cell responses.

The adaptive immune system protects organisms from harmful environmental insults. In parallel, regulatory mechanisms control immune responses in order to assure preservation of organ integrity. Yet, molecules involved in the control of T-cell responses in peripheral tissues are poorly characterized. Here, we investigated the function of Dickkopf-3 in the modulation of local T-cell reactivity. Dkk3 is a secreted, mainly tissue-derived protein with highest expression in organs considered as immune-privileged such as the eye, embryo, placenta, and brain. While T-cell development and activation status in naïve Dkk3-deficient mice was comparable to littermate controls, we found that Dkk3 contributes to the immunosuppressive microenvironment that protects transplanted, class-I mismatched embryoid bodies from T-cell-mediated rejection. Moreover, genetic deletion or antibody-mediated neutralization of Dkk3 led to an exacerbated experimental autoimmune encephalomyelitis (EAE). This phenotype was accompanied by a change of T-cell polarization displayed by an increase of IFNγ-producing T cells within the central nervous system. In the wild-type situation, Dkk3 expression in the brain was up-regulated during the course of EAE in an IFNγ-dependent manner. In turn, Dkk3 decreased IFNγ activity and served as part of a negative feedback mechanism. Thus, our findings suggest that Dkk3 functions as a tissue-derived modulator of local CD4(+) and CD8(+) T-cell responses.

Sulfatides are required for renal adaptation to chronic metabolic acidosis.

Urinary ammonium excretion by the kidney is essential for renal excretion of sufficient amounts of protons and to maintain stable blood pH. Ammonium secretion by the collecting duct epithelia accounts for the majority of urinary ammonium; it is driven by an interstitium-to-lumen NH3 gradient due to the accumulation of ammonium in the medullary and papillary interstitium. Here, we demonstrate that sulfatides, highly charged anionic glycosphingolipids, are important for maintaining high papillary ammonium concentration and increased urinary acid elimination during metabolic acidosis. We disrupted sulfatide synthesis by a genetic approach along the entire renal tubule. Renal sulfatide-deficient mice had lower urinary pH accompanied by lower ammonium excretion. Upon acid diet, they showed impaired ammonuria, decreased ammonium accumulation in the papilla, and chronic hyperchloremic metabolic acidosis. Expression levels of ammoniagenic enzymes and Na(+)-K(+)/NH4(+)-2Cl(-) cotransporter 2 were higher, and transepithelial NH3 transport, examined by in vitro microperfusion of cortical and outer medullary collecting ducts, was unaffected in mutant mice. We therefore suggest that sulfatides act as counterions for interstitial ammonium facilitating its retention in the papilla. This study points to a seminal role of sulfatides in renal ammonium handling, urinary acidification, and acid-base homeostasis.

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.

Lipid droplet accumulation is associated with an increase in hyperglycemia-induced renal damage: prevention by liver X receptors.

Dyslipidemia is a frequent component of the metabolic disorder of diabetic patients contributing to organ damage. Herein, in low-density lipoprotein receptor-deficient hyperlipidemic and streptozotozin-induced diabetic mice, hyperglycemia and hyperlipidemia acted reciprocally, accentuating renal injury and altering renal function. In hyperglycemic-hyperlipidemic kidneys, the accumulation of Tip47-positive lipid droplets in glomeruli, tubular epithelia, and macrophages was accompanied by the concomitant presence of the oxidative stress markers xanthine oxidoreductase and nitrotyrosine, findings that could also be evidenced in renal biopsy samples of diabetic patients. As liver X receptors (LXRα,ß) regulate genes linked to lipid and carbohydrate homeostasis and inhibit inflammatory gene expression in macrophages, the effects of systemic and macrophage-specific LXR activation were analyzed on renal damage in hyperlipidemic-hyperglycemic mice. LXR stimulation by GW3965 up-regulated genes involved in cholesterol efflux and down-regulated proinflammatory/profibrotic cytokines, inhibiting the pathomorphology of diabetic nephropathy, renal lipid accumulation, and improving renal function. Xanthine oxidoreductase and nitrotyrosine levels were reduced. In macrophages, GW3965 or LXRα overexpression significantly suppressed glycated or acetylated low-density lipoprotein-induced cytokines and reactive oxygen species. Specifically, in mice, transgenic expression of LXRα in macrophages significantly ameliorated hyperlipidemic-hyperglycemic nephropathy. The results demonstrate the presence of lipid droplet-induced oxidative mechanisms and the pathophysiologic role of macrophages in diabetic kidneys and indicate the potent regulatory role of LXRs in preventing renal damage in diabetes.

Glycosphingolipids are essential for intestinal endocytic function.

Glycosphingolipids (GSLs) constitute major components of enterocytes and were hypothesized to be potentially important for intestinal epithelial polarization. The enzyme UDP-glucose ceramide glucosyltransferase (Ugcg) catalyzes the initial step of GSL biosynthesis. Newborn and adult mice with enterocyte-specific genetic deletion of the gene Ugcg were generated. In newborn mutants lacking GSLs at day P0, intestinal epithelia were indistinguishable from those in control littermates displaying an intact polarization with regular brush border. However, those mice were not consistently able to absorb nutritional lipids from milk. Between postnatal days 5 and 7, severe defects in intestinal epithelial differentiation occurred accompanied by impaired intestinal uptake of nutrients. Villi of mutant mice became stunted, and enterocytes lacked brush border. The defects observed in mutant mice caused diarrhea, malabsorption, and early death. In this study, we show that GSLs are essential for enterocyte resorptive function but are primarily not for polarization; GSLs are required for intracellular vesicular transport in resorption-active intestine.