Mehmet Hakan Ozdener

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.

Volatile biomarkers from human melanoma cells.

Dogs can identify, by olfaction, melanoma on the skin of patients or melanoma samples hidden on healthy subjects, suggesting that volatile organic compounds (VOCs) from melanoma differ from those of normal skin. Studies employing gas chromatography-mass spectrometry (GC-MS) and gas sensors reported that melanoma-related VOCs differed from VOCs from normal skin sources. However, the identities of the VOCs that discriminate melanoma from normal skin were either unknown or likely derived from exogenous sources. We employed solid-phase micro-extraction, GC-MS and single-stranded DNA-coated nanotube (DNACNT) sensors to examine VOCs from melanoma and normal melanocytes. GC-MS revealed dozens of VOCs, but further analyses focused on compounds most likely of endogenous origin. Several compounds differed between cancer and normal cells, e.g., isoamyl alcohol was higher in melanoma cells than in normal melanocytes but isovaleric acid was lower in melanoma cells. These two compounds share the same precursor, viz., leucine. Melanoma cells produce dimethyldi- and trisulfide, compounds not detected in VOCs from normal melanocytes. Furthermore, analyses of the total volatile metabolome from both melanoma cells and normal melanocytes by DNACNT sensors, coupled with the GC-MS results, demonstrate clear differences between these cell systems. Consequently, monitoring of melanoma VOCs has potential as a useful screening methodology.

Chemosensory loss: functional consequences of the world trade center disaster.

Background Individuals involved in rescue, recovery, demolition, and cleanup at the World Trade Center (WTC) site were exposed to a complex mixture of airborne smoke, dust, combustion gases, acid mists, and metal fumes. Such exposures have the potential to impair nasal chemosensory (olfactory and trigeminal) function. Objective The goal of this study was to evaluate the prevalence of chemosensory dysfunction and nasal inflammation among these individuals. Methods We studied 102 individuals who worked or volunteered at the WTC site in the days and weeks during and after 11 September 2001 (9/11) and a comparison group with no WTC exposure matched to each participant on age, sex, and job title. Participants were comprehensively evaluated for chemosensory function and nasal inflammation in a single session. Individual exposure history was obtained from self-reported questionnaires. Results The prevalence of olfactory and trigeminal nerve sensitivity loss was significantly greater in the WTC-exposed group relative to the comparison group [prevalence ratios (95% confidence intervals) = 1.96 (1.2–3.3) and 3.28 (2.7–3.9) for odor and irritation thresholds, respectively]. Among the WTC responders, however, individuals caught in the dust cloud from the collapse on 9/11 exhibited the most profound trigeminal loss. Analysis of the nasal lavage samples supported the clinical findings of chronic nasal inflammation among the WTC-exposed cohort. Conclusions The prevalence of significant chemosensory impairment in the WTC-exposed group more than 2 years after their exposure raises concerns for these individuals when the ability to detect airborne odors or irritants is a critical safety factor. Relevance to clinical practice This outcome highlights the need for chemosensory evaluations among individuals with exposure to acute high or chronic levels of airborne pollutants.

Characterization of Human Fungiform Papillae Cells in Culture

The ability to maintain human fungiform papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Here we describe a method for enzymatically isolating human cells from fungiform papillae obtained by biopsy and maintaining them in culture for more than 7 passages (7 months) without loss of viability and while retaining many of the functional properties of acutely isolated taste cells. Cells in these cultures exhibited increases in intracellular calcium when stimulated with perceptually appropriate concentrations of several taste stimuli, indicating that at least some of the native signaling pathways were present. This system can provide a useful model for molecular studies of the proliferation, differentiation, and physiological function of human fungiform papillae cells.

Primary Culture of Mammalian Taste Epithelium

Establishment of primary and immortalized cultures of many cell types has facilitated efforts to understand the signals involved in proliferation and differentiation and yielded tools to rapidly assay new molecules targeting specific receptor pathways. Taste cells are specialized sensory epithelial cells which reside within taste buds on the lingual epithelium. Only recently have successful culturing protocols been developed which maintain essential molecular and functional characteristics. These protocols provide a tractable tool to examine the molecular, regenerative, and functional properties of these unique sensory cells within a controlled environment. The method involves an enzymatic isolation procedure and standardized culture conditions, and may be applied to either dissected rodent tissue or human fungiform papillae obtained by biopsy. Human fungiform cells can be maintained in culture for more than seven passages, without loss of viability and with retention of the molecular and biochemical properties of acutely isolated taste cells. Cultured primary human fungiform papillae cells also exhibit functional responses to taste stimuli indicating the presence of taste receptors and at least some relevant signaling pathways. While the loss of the three-dimensional structure of the intact taste bud must be taken into consideration in interpreting results obtained with these cells, this culture protocol provides a useful model for molecular studies of the proliferation, differentiation, and physiological function of mammalian taste receptor cells.

ERK1/2 activation in human taste bud cells regulates fatty acid signaling and gustatory perception of fat in mice and humans.

Obesity is a major public health problem. An in‐depth knowledge of the molecular mechanisms of orosensory detection of dietary lipids may help fight it. Humans and rodents can detect fatty acids via lipido‐receptors, such as CD36 and GPR120. We studied the implication of the MAPK pathways, in particular, ERK1/2, in the gustatory detection of fatty acids. Linoleic acid, a dietary fatty acid, induced via CD36 the phosphorylation of MEK1/2‐ERK1/2ETS‐like transcription factor‐1 cascade, which requires Fyn‐Src kinase and lipid rafts in human taste bud cells (TBCs). ERK1/2 cascade was activated by Ca2+ signaling via opening of the calcium‐homeostasis modulator‐1 (CALHM1) channel. Furthermore, fatty acid–evoked Ca2+ signaling and ERK1/2 phosphorylation were decreased in both human TBCs after small interfering RNA knockdown of CALHM1 channel and in TBCs from Calhm1‐/‐ mice. Targeted knockdown of ERK1/2 by small interfering RNA or PD0325901 (MEK1/2 inhibitor) in the tongue and genetic ablation of Erk1 or Calhm1 genes impaired preference for dietary fat in mice. Lingual inhibition of ERK1/2 in healthy volunteers also decreased orogustatory sensitivity for linoleic acid. Our data demonstrate that ERK1/2‐MAPK cascade is regulated by the opening of CALHM1 Ca2+ channel in TBCs to modulate orogustatory detection of dietary lipids in mice and humans.—Subramaniam, S., Ozdener, M. H., Abdoul‐Azize, S., Saito, K., Malik, B., Maquart, G., Hashimoto, T., Marambaud, P., Aribi, M., Tordoff, M.G., Besnard, P., Khan, N.A. ERK1/2 activation in human taste bud cells regulates fatty acid signaling and gustatory perception of fat in mice and humans. FASEBJ. 30, 3489–3500 (2016). www.fasebj.org

Primary Culture of the Human Olfactory Neuroepithelium

The central cell type involved in the initial perception of odors and transduction of the sensory signal are the olfactory receptor neurons (ORNs) located in the olfactory neuroepithelium of the nasal cavities. The olfactory epithelium is a unique system similar to the neuroepithelium of the embryonic neural tube, in which new neurons are continually generated throughout adult life. Olfactory neurons are derived from precursor cells that lie adjacent to the basal lamina of the olfactory epithelium; these precursor cells divide several times and their progeny differentiate into mature sensory neurons throughout life. Thus, the human olfactory epithelium has the potential to be used as a tool to examine certain human disorders resulting from abnormal development of the nervous system. This chapter presents methods for primary culture of human ORNs, which have been used successfully by multiple investigators. The protocol provides a consistent, heterogeneous cell population, which demonstrates functional responses to odorant mixtures and exhibits a complex neuronal phenotype, encompassing receptors and signaling pathways pertinent to both olfaction and other aspects of CNS function. These cultured neural cells exhibit neurotransmitter pathways important in a number of neuropsychiatric disorders, and the ability to culture cells from living human subjects provides a tool for assessing cellular neuropathology at the individual patient level.

Mechanisms of chloride uptake in frog olfactory receptor neurons

Odorant stimulation of olfactory receptor neurons (ORNs) leads to the activation of a Ca2+ permeable cyclic nucleotide-gated (CNG) channel followed by opening of an excitatory Ca2+-activated Cl− channel, which carries about 70% of the odorant-induced receptor current. This requires ORNs to have a [Cl−]i above the electrochemical equilibrium to render this anionic current excitatory. In mammalian ORNs, the Na+-K+-2Cl− co-transporter 1 (NKCC1) has been characterized as the principal mechanism by which these neurons actively accumulate Cl−. To determine if NKCC activity is needed in amphibian olfactory transduction, and to characterize its cellular location, we used the suction pipette technique to record from Rana pipiens ORNs. Application of bumetanide, an NKCC blocker, produced a 50% decrease of the odorant-induced current. Similar effects were observed when [Cl−]i was decreased by bathing ORNs in low Cl− solution. Both manipulations reduced only the Cl− component of the current. Application of bumetanide only to the ORN cell body and not to the cilia decreased the current by again about 50%. The results show that NKCC is required for amphibian olfactory transduction, and suggest that the co-transporter is located basolaterally at the cell body although its presence at the cilia could not be discarded.

Assessment of smoking status based on cotinine levels in nasal lavage fluid

Cotinine is a principal metabolite of nicotine with a substantially longer half-life, and cotinine levels in saliva, urine or serum are widely used to validate self-reported smoking status. The nasal cavity and olfactory system are directly exposed to tobacco smoke in smokers and in non-smokers who live with or work around smokers. However, despite the potential for a direct impact of tobacco smoke on the nasal epithelium and olfactory neurons, no prior studies have assessed cotinine levels in nasal mucus. We sought to determine whether cotinine levels in nasal lavage fluid (NLF) would provide a reasonable estimate of smoke exposure. We assayed cotinine using a competitive immunoassay in NLF from 23 smokers, 10 non-smokers exposed to tobacco smoke (ETS) and 60 non-smokers who did not report smoke exposure. NLF cotinine levels were significantly higher in smokers than in non-smokers, regardless of their exposure to ambient tobacco smoke. Cotinine levels in this small group of exposed non-smokers were not significantly different than those of non-exposed non-smokers. A cutoff of 1 ng/ml provided a sensitivity of 91% and a specificity of 99% for smoking status in this sample. Data were consistent with self-reported smoking status, and a cutoff of 1.0 ng/ml NLF cotinine may be used to classify smoking status. While saliva is the most easily obtained body fluid, NLF can be used to provide an objective and precise indication of smoking status and more directly reflects smoke exposure in the nasal and olfactory mucosa.

Arginyl dipeptides increase the frequency of NaCl-elicited responses via epithelial sodium channel alpha and delta subunits in cultured human fungiform taste papillae cells

Salty taste is one of the five basic tastes and is often elicited by NaCl. Because excess sodium intake is associated with many health problems, it could be useful to have salt taste enhancers that are not sodium based. In this study, the regulation of NaCl-induced responses was investigated in cultured human fungiform taste papillae (HBO) cells with five arginyl dipeptides: Ala-Arg (AR), Arg-Ala (RA), Arg-Pro (RP), Arg-Glu (RE), and Glu-Arg (ER); and two non-arginyl dipeptides: Asp-Asp (DD) and Glu-Asp (ED). AR, RA, and RP significantly increased the number of cell responses to NaCl, whereas no effect was observed with RE, ER, DD, or ED. We also found no effects with alanine, arginine, or a mixture of both amino acids. Pharmacological studies showed that AR significantly increased responses of amiloride-sensitive but not amiloride-insensitive cells. In studies using small interfering RNAs (siRNAs), responses to AR were significantly decreased in cells transfected with siRNAs against epithelial sodium channel ENaCα or ENaCδ compared to untransfected cells. AR dramatically increased NaCl-elicited responses in cells transfected with NHE1 siRNA but not in those transfected with ENaCα or ENaCδ siRNAs. Altogether, AR increased responses of amiloride-sensitive cells required ENaCα and ENaCδ.