Sinju Sundaresan

CD36- and GPR120-Mediated Ca2+ Signaling in Human Taste Bud Cells Mediates Differential Responses to Fatty Acids and Is Altered in Obese Mice

BACKGROUND & AIMS It is important to increase our understanding of gustatory detection of dietary fat and its contribution to fat preference. We studied the roles of the fat taste receptors CD36 and GPR120 and their interactions via Ca(2+) signaling in fungiform taste bud cells (TBC). METHODS We measured Ca(2+) signaling in human TBC, transfected with small interfering RNAs against messenger RNAs encoding CD36 and GPR120 (or control small interfering RNAs). We also studied Ca(2+) signaling in TBC from CD36(-/-) mice and from wild-type lean and obese mice. Additional studies were conducted with mouse enteroendocrine cell line STC-1 that express GPR120 and stably transfected with human CD36. We measured release of serotonin and glucagon-like peptide-1 from human and mice TBC in response to CD36 and GPR120 activation. RESULTS High concentrations of linoleic acid induced Ca(2+) signaling via CD36 and GPR120 in human and mice TBC, as well as in STC-1 cells, and low concentrations induced Ca(2+) signaling via only CD36. Incubation of human and mice fungiform TBC with lineoleic acid down-regulated CD36 and up-regulated GPR120 in membrane lipid rafts. Obese mice had decreased spontaneous preference for fat. Fungiform TBC from obese mice had reduced Ca(2+) and serotonin responses, but increased release of glucagon-like peptide-1, along with reduced levels of CD36 and increased levels of GPR120 in lipid rafts. CONCLUSIONS CD36 and GPR120 have nonoverlapping roles in TBC signaling during orogustatory perception of dietary lipids; these are differentially regulated by obesity.

CD36-dependent signaling mediates fatty acid-induced gut release of secretin and cholecystokinin

Genetic variants in the fatty acid (FA) translocase FAT/CD36 associate with abnormal postprandial lipids and influence risk for the metabolic syndrome. CD36 is abundant on apical enterocyte membranes in the proximal small intestine, where it facilitates FA uptake and FA‐initiated signaling. We explored whether CD36 signaling influences FA‐mediated secretion of cholecystokinin (CCK) and secretin, peptides released by enteroendocrine cells (EECs) in the duodenum/jejunum, which regulate events important for fat digestion and homeostasis. CD36 was immunodetected on apical membranes of secretin‐ and CCK‐positive EECs and colocalized with cytosolic granules. Intragastric lipid administration to CD36–/– mice released less secretin (–60%) and CCK (–50%) compared with wild‐type mice. Likewise, diminished secretin and CCK responses to FA were observed with CD36–/– intestinal segments in vitro, arguing against influence of alterations in fat absorption. Signaling mechanisms underlying peptide release were examined in STC‐1 cells stably expressing human CD36 or a signaling‐impaired mutant (CD36K/A). FA stimulation of cells expressing CD36 (vs. vector or CD36K/A) released more secretin (3.5‐ to 4‐fold) and CCK (2‐ to 3‐fold), generated more cAMP (2‐ to 2.5‐fold), and enhanced protein kinase A activation. Protein kinase A inhibition (H‐89) blunted secretin (80%) but not CCK release, which was reduced (50%) by blocking of calmodulin kinase II (KN‐62). Coculture of STC‐1 cells with Caco‐2 cells stably expressing CD36 did not alter secretin or CCK release, consistent with a minimal effect of adjacent enterocytes. In summary, CD36 is a major mediator of FA‐induced release of CCK and secretin. These peptides contribute to the role of CD36 in fat absorption and to its pleiotropic metabolic effects.—Sundaresan, S., Shahid, R., Riehl, T. E., Chandra, R., Nassir, F., Stenson, W. F., Liddle, R. A., Abumrad, N. A. CD36‐dependent signaling mediates fatty acid‐induced gut release of secretin and cholecystokinin. FASEB J. 27, 1191–1202 (2013). www.fasebj.org

Adipocyte-induced CD36 expression drives ovarian cancer progression and metastasis

Ovarian cancer (OvCa) is characterized by widespread and rapid metastasis in the peritoneal cavity. Visceral adipocytes promote this process by providing fatty acids (FAs) for tumour growth. However, the exact mechanism of FA transfer from adipocytes to cancer cells remains unknown. This study shows that OvCa cells co-cultured with primary human omental adipocytes express high levels of the FA receptor, CD36, in the plasma membrane, thereby facilitating exogenous FA uptake. Depriving OvCa cells of adipocyte-derived FAs using CD36 inhibitors and short hairpin RNA knockdown prevented development of the adipocyte-induced malignant phenotype. Specifically, inhibition of CD36 attenuated adipocyte-induced cholesterol and lipid droplet accumulation and reduced intracellular reactive oxygen species (ROS) content. Metabolic analysis suggested that CD36 plays an essential role in the bioenergetic adaptation of OvCa cells in the adipocyte-rich microenvironment and governs their metabolic plasticity. Furthermore, the absence of CD36 affected cellular processes that play a causal role in peritoneal dissemination, including adhesion, invasion, migration and anchorage independent growth. Intraperitoneal injection of CD36-deficient cells or treatment with an anti-CD36 monoclonal antibody reduced tumour burden in mouse xenografts. Moreover, a matched cohort of primary and metastatic human ovarian tumours showed upregulation of CD36 in the metastatic tissues, a finding confirmed in three public gene expression data sets. These results suggest that omental adipocytes reprogram tumour metabolism through the upregulation of CD36 in OvCa cells. Targeting the stromal-tumour metabolic interface via CD36 inhibition may prove to be an effective treatment strategy against OvCa metastasis.

Ubiquitination and degradation of the hominoid-specific oncoprotein TBC1D3 is regulated by protein palmitoylation

Expression of the hominoid-specific oncoprotein TBC1D3 promotes enhanced cell growth and proliferation by increased activation of signal transduction through several growth factors. Recently we documented the role of CUL7 E3 ligase in growth factors-induced ubiquitination and degradation of TBC1D3. Here we expanded our study to discover additional molecular mechanisms that control TBC1D3 protein turnover. We report that TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. The expression of double palmitoylation mutant TBC1D3:C318/325S resulted in protein mislocalization and enhanced growth factors-induced TBC1D3 degradation. Moreover, ubiquitination of TBC1D3 via CUL7 E3 ligase complex was increased by mutating the palmitoylation sites, suggesting that depalmitoylation of TBC1D3 makes the protein more available for ubiquitination and degradation. The results reported here provide novel insights into the molecular mechanisms that govern TBC1D3 protein degradation. Dysregulation of these mechanisms in vivo could potentially result in aberrant TBC1D3 expression and promote oncogenesis.

CD36 Deficiency Impairs the Small Intestinal Barrier and Induces Subclinical Inflammation in Mice

Background & Aims CD36 has immunometabolic actions and is abundant in the small intestine on epithelial, endothelial, and immune cells. We examined the role of CD36 in gut homeostasis by using mice null for CD36 (CD36KO) and with CD36 deletion specific to enterocytes (Ent-CD36KO) or endothelial cells (EC-CD36KO). Methods Intestinal morphology was evaluated by using immunohistochemistry and electron microscopy. Intestinal inflammation was determined from neutrophil infiltration and expression of cytokines, toll-like receptors, and cyclooxygenase-2. Barrier integrity was assessed from circulating lipopolysaccharide and dextran administered intragastrically. Epithelial permeability to luminal dextran was visualized by using two-photon microscopy. Results The small intestines of CD36KO mice fed a chow diet showed several abnormalities including extracellular matrix accumulation with increased expression of extracellular matrix proteins, evidence of neutrophil infiltration, inflammation, and compromised barrier function. Electron microscopy showed shortened desmosomes with decreased desmocollin 2 expression. Systemically, leukocytosis and neutrophilia were present together with 80% reduction of anti-inflammatory Ly6Clow monocytes. Bone marrow transplants supported the primary contribution of non-hematopoietic cells to the inflammatory phenotype. Specific deletion of endothelial but not of enterocyte CD36 reproduced many of the gut phenotypes of germline CD36KO mice including fibronectin deposition, increased interleukin 6, neutrophil infiltration, desmosome shortening, and impaired epithelial barrier function. Conclusions CD36 loss results in chronic neutrophil infiltration of the gut, impairs barrier integrity, and systemically causes subclinical inflammation. Endothelial cell CD36 deletion reproduces the major intestinal phenotypes. The findings suggest an important role of the endothelium in etiology of gut inflammation and loss of epithelial barrier integrity.

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of >95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

671 Acid Suppression Results in Increased Expression of Gastrin-Positive Cells in Duodenal Lamina Propria of Mice With Targeted Deletion of Men1 and Somatostatin Gene Loci

higher in CA when compared with CD ( P=0.013) and SG (P=0.037). GPR43 expression was significantly higher in the ileum compared with CA ( P<0.001), CT (P<0.001), CD (P= 0.001), and SG (P<0.001). This expression pattern was observed in all 5 patients that donated biopsies from 3 or more locations ( figure 2). Discussion and conclusion: This study in which biopsies from different intestinal locations were obtained within the same subject allowed us to profile SCFA receptor expression throughout the human ileum and colon. Gene expression of GPR41 and GPR43 was significantly higher in the ileum when compared with all parts of the colon. In addition, expression of GPR41 was significantly higher in the proximal part of the colon when compared with the distal colon. These findings might suggest that prebiotic fibers fermented in the ileum or proximal colon are most suitable for exerting beneficial health effects through GPR41 and GPR43 activation and downstream signaling in the human gastrointestinal tract.