Ceramides Increase Fatty Acid Utilization in Intestinal Progenitors to Enhance Stemness and Increase Tumor Risk.

BACKGROUND & AIMS: Cancers of the alimentary tract, including esophageal adenocarcinomas, colorectal cancers, and cancers of the gastric cardia, are common comorbidities of obesity. Prolonged, excessive delivery of macronutrients to the cells lining the gut can increase one's risk for these cancers by inducing imbalances in the rate of intestinal stem cell proliferation vs differentiation, which can produce polyps and other aberrant growths. We investigated whether ceramides, which are sphingolipids that serve as a signal of nutritional excess, alter stem cell behaviors to influence cancer risk. METHODS: We profiled sphingolipids and sphingolipid-synthesizing enzymes in human adenomas and tumors. Thereafter, we manipulated expression of sphingolipid-producing enzymes, including serine palmitoyltransferase (SPT), in intestinal progenitors of mice, cultured organoids, and Drosophila to discern whether sphingolipids altered stem cell proliferation and metabolism. RESULTS: SPT, which diverts dietary fatty acids and amino acids into the biosynthetic pathway that produces ceramides and other sphingolipids, is a critical modulator of intestinal stem cell homeostasis. SPT and other enzymes in the sphingolipid biosynthesis pathway are up-regulated in human intestinal adenomas. They produce ceramides, which serve as prostemness signals that stimulate peroxisome-proliferator activated receptor-α and induce fatty acid binding protein-1. These actions lead to increased lipid utilization and enhanced proliferation of intestinal progenitors. CONCLUSIONS: Ceramides serve as critical links between dietary macronutrients, epithelial regeneration, and cancer risk.

Antioxidant Effects of N-Acetylcysteine Prevent Programmed Metabolic Disease in Mice.

An adverse maternal in utero and lactation environment can program offspring for increased risk for metabolic disease. The aim of this study was to determine whether N-acetylcysteine (NAC), an anti-inflammatory antioxidant, attenuates programmed susceptibility to obesity and insulin resistance in offspring of mothers on a high-fat diet (HFD) during pregnancy. CD1 female mice were acutely fed a standard breeding chow or HFD. NAC was added to the drinking water (1 g/kg) of the treatment cohorts from embryonic day 0.5 until the end of lactation. NAC treatment normalized HFD-induced maternal weight gain and oxidative stress, improved the maternal lipidome, and prevented maternal leptin resistance. These favorable changes in the in utero environment normalized postnatal growth, decreased white adipose tissue (WAT) and hepatic fat, improved glucose and insulin tolerance and antioxidant capacity, reduced leptin and insulin, and increased adiponectin in HFD offspring. The lifelong metabolic improvements in the offspring were accompanied by reductions in proinflammatory gene expression in liver and WAT and increased thermogenic gene expression in brown adipose tissue. These results, for the first time, provide a mechanistic rationale for how NAC can prevent the onset of metabolic disease in the offspring of mothers who consume a typical Western HFD.

Identification of a Paracrine Signaling Mechanism Linking CD34high Progenitors to the Regulation of Visceral Fat Expansion and Remodeling.

Accumulation of visceral (VIS) is a predictor of metabolic disorders and insulin resistance. This is due in part to the limited capacity of VIS fat to buffer lipids allowing them to deposit in insulin-sensitive tissues. Mechanisms underlying selective hypertrophic growth and tissue remodeling properties of VIS fat are not well understood. We identified subsets of adipose progenitors (APs) unique to VIS fat with differential Cd34 expression and adipogenic capacity. VIS low (Cd34 low) APs are adipogenic, whereas VIS high (Cd34 high) APs are not. Furthermore, VIS high APs inhibit adipogenic differentiation of SUB and VIS low APs in vitro through the secretion of soluble inhibitory factor(s). The number of VIS high APs increased with adipose tissue expansion, and their abundance in vivo caused hypertrophic growth, fibrosis, inflammation, and metabolic dysfunction. This study unveils the presence of APs unique to VIS fat involved in the paracrine regulation of adipogenesis and tissue remodeling.

FOXN3 controls liver glucose metabolism by regulating gluconeogenic substrate selection.

The FOXN3 gene locus is associated with fasting blood glucose levels in non-diabetic human population genetic studies. The blood glucose-modifying variation within this gene regulates the abundance of both FOXN3 protein and transcript in primary human hepatocytes, with the hyperglycemia risk allele causing increases in both FOXN3 protein and transcript. Using transgenic and knock-out zebrafish models, we showed previously that FOXN3 is a transcriptional repressor that regulates fasting blood glucose by altering liver gene expression of MYC, a  master transcriptional regulator of glucose utilization, and by modulating pancreatic α cell mass and function through an unknown mechanism. Since homozygous Foxn3 null mice die perinatally, and heterozygous carries of the null allele are smaller than wild-type siblings, we examine the metabolic effects of decreasing mouse liver Foxn3 expression in adult life, performing dynamic endocrine tests not feasible in adult zebrafish. Fasting glucose, glucagon, and insulin; and dynamic responses to glucose, insulin, pyruvate, glutamine, and glucagon were measured. Gluconeogenic and amino acid catabolic gene expression was examined in livers, as well. Knocking down liver Foxn3 expression via transduction with adeno-associated virus serotype 8 particles encoding a short hairpin RNA targeting Fonx3 decreases fasting glucose and increases Myc expression, without altering fasting glucagon or fasting insulin. Liver Foxn3 knock-down confers increases glucose tolerance, has no effect on insulin tolerance or response to glucagon challenge, blunts pyruvate and glutamine tolerance, and modulates expression of amino acid transporters and catabolic enzymes. We conclude that liver Foxn3 regulates substrate selection for gluconeogenesis.

Myeloid-Cell-Derived VEGF Maintains Brain Glucose Uptake and Limits Cognitive Impairment in Obesity.

High-fat diet (HFD) feeding induces rapid reprogramming of systemic metabolism. Here, we demonstrate that HFD feeding of mice downregulates glucose transporter (GLUT)-1 expression in blood-brain barrier (BBB) vascular endothelial cells (BECs) and reduces brain glucose uptake. Upon prolonged HFD feeding, GLUT1 expression is restored, which is paralleled by increased expression of vascular endothelial growth factor (VEGF) in macrophages at the BBB. In turn, inducible reduction of GLUT1 expression specifically in BECs reduces brain glucose uptake and increases VEGF serum concentrations in lean mice. Conversely, myeloid-cell-specific deletion of VEGF in VEGF(Δmyel) mice impairs BBB-GLUT1 expression, brain glucose uptake, and memory formation in obese, but not in lean mice. Moreover, obese VEGF(Δmyel) mice exhibit exaggerated progression of cognitive decline and neuroinflammation on an Alzheimer's disease background. These experiments reveal that transient, HFD-elicited reduction of brain glucose uptake initiates a compensatory increase of VEGF production and assign obesity-associated macrophage activation a homeostatic role to restore cerebral glucose metabolism, preserve cognitive function, and limit neurodegeneration in obesity.

Dihydroceramides: From Bit Players to Lead Actors.

Sphingolipid synthesis involves a highly conserved biosynthetic pathway that produces fundamental precursors of complex sphingolipids. The final reaction involves the insertion of a double bond into dihydroceramides to generate the more abundant ceramides, which are converted to sphingomyelins and glucosylceramides/gangliosides by the addition of polar head groups. Although ceramides have long been known to mediate cellular stress responses, the dihydroceramides that are transiently produced during de novo sphingolipid synthesis were deemed inert. Evidence published in the last few years suggests that these dihydroceramides accumulate to a far greater extent in tissues than previously thought. Moreover, they have biological functions that are distinct and non-overlapping with those of the more prevalent ceramides. Roles are being uncovered in autophagy, hypoxia, and cellular proliferation, and the lipids are now implicated in the etiology, treatment, and/or diagnosis of diabetes, cancer, ischemia/reperfusion injury, and neurodegenerative diseases. This minireview summarizes recent findings on this emerging class of bioactive lipids.

Signaling by IL-6 promotes alternative activation of macrophages to limit endotoxemia and obesity-associated resistance to insulin.

Obesity and resistance to insulin are closely associated with the development of low-grade inflammation. Interleukin 6 (IL-6) is linked to obesity-associated inflammation; however, its role in this context remains controversial. Here we found that mice with an inactivated gene encoding the IL-6Rα chain of the receptor for IL-6 in myeloid cells (Il6ra(Δmyel) mice) developed exaggerated deterioration of glucose homeostasis during diet-induced obesity, due to enhanced resistance to insulin. Tissues targeted by insulin showed increased inflammation and a shift in macrophage polarization. IL-6 induced expression of the receptor for IL-4 and augmented the response to IL-4 in macrophages in a cell-autonomous manner. Il6ra(Δmyel) mice were resistant to IL-4-mediated alternative polarization of macrophages and exhibited enhanced susceptibility to lipopolysaccharide (LPS)-induced endotoxemia. Our results identify signaling via IL-6 as an important determinant of the alternative activation of macrophages and assign an unexpected homeostatic role to IL-6 in limiting inflammation.

Cutting edge: Inhibition of IL-6 trans-signaling protects from malaria-induced lethality in mice.

Circulating IL-6 levels correlate with the severity of blood-stage malaria in humans and mouse models, but the impact of IL-6 classic signaling through membrane IL-6Rα, as well as IL-6 trans-signaling through soluble IL-6Rα, on the outcome of malaria has remained unknown. In this study, we created IL-6Rα-deficient mice that exhibit a 50% survival of otherwise lethal blood-stage malaria of the genus Plasmodium chabaudi. Inducing IL-6 trans-signaling by injection of mouse recombinant soluble IL-6Rα in IL-6Rα-deficient mice restores the lethal outcome to malaria infection. In contrast, inhibition of IL-6 trans-signaling via injection of recombinant sGP130Fc protein in control mice results in a 40% survival rate. Our data demonstrate that IL-6 trans-signaling, rather than classic IL-6 signaling, contributes to malaria-induced lethality in mice, preceded by an increased inflammatory response. Therefore, inhibition of IL-6 trans-signaling may serve as a novel promising therapeutic basis to combat malaria.

Phosphoinositide-dependent kinase 1 provides negative feedback inhibition to Toll-like receptor-mediated NF-kappaB activation in macrophages.

Phosphoinositide-dependent kinase 1 (PDK-1) represents an important signaling component in the phosphatidylinositol 3-kinase (PI3K) pathway, which plays an essential role in controlling a coordinated innate immune response. Here, we show that mice with conditional disruption of PDK-1 specifically in myeloid lineage cells (PDK-1(Deltamyel) mice) show enhanced susceptibility to lipopolysaccharide (LPS)-induced septic shock accompanied by exaggerated liver failure. Furthermore, primary macrophages derived from PDK-1(Deltamyel) mice lack LPS- and Pam3CSK4-stimulated AKT activity but exhibit increased mRNA expression and release of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). Moreover, LPS- and Pam3CSK4-stimulated primary macrophages exhibit enhanced phosphorylation and degradation of IkappaBalpha. While immediate upstream Toll-like receptor 4 (TLR-4)-induced signaling, including IL-1 receptor (IL-1R)-associated protein kinase (IRAK) phosphorylation, is unaltered in the absence of PDK-1, macrophages from PDK-1(Deltamyel) mice exhibit prolonged ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF-6) in response to LPS stimulation. These experiments reveal a novel PDK-1-dependent negative feedback inhibition of TLR-induced NF-kappaB activation in macrophages in vivo.

Myeloid cell-restricted insulin receptor deficiency protects against obesity-induced inflammation and systemic insulin resistance.

A major component of obesity-related insulin resistance is the establishment of a chronic inflammatory state with invasion of white adipose tissue by mononuclear cells. This results in the release of pro-inflammatory cytokines, which in turn leads to insulin resistance in target tissues such as skeletal muscle and liver. To determine the role of insulin action in macrophages and monocytes in obesity-associated insulin resistance, we conditionally inactivated the insulin receptor (IR) gene in myeloid lineage cells in mice (IR(Deltamyel)-mice). While these animals exhibit unaltered glucose metabolism on a normal diet, they are protected from the development of obesity-associated insulin resistance upon high fat feeding. Euglycemic, hyperinsulinemic clamp studies demonstrate that this results from decreased basal hepatic glucose production and from increased insulin-stimulated glucose disposal in skeletal muscle. Furthermore, IR(Deltamyel)-mice exhibit decreased concentrations of circulating tumor necrosis factor (TNF) alpha and thus reduced c-Jun N-terminal kinase (JNK) activity in skeletal muscle upon high fat feeding, reflecting a dramatic reduction of the chronic and systemic low-grade inflammatory state associated with obesity. This is paralleled by a reduced accumulation of macrophages in white adipose tissue due to a pronounced impairment of matrix metalloproteinase (MMP) 9 expression and activity in these cells. These data indicate that insulin action in myeloid cells plays an unexpected, critical role in the regulation of macrophage invasion into white adipose tissue and in the development of obesity-associated insulin resistance.