Oro-gustatory perception of dietary lipids and calcium signaling in taste bud cells are altered in nutritionally obesity-prone Psammomys obesus

Abstract

Since the increasing prevalence of obesity is one of the major health problems of the modern era, understanding the mechanisms of oro-gustatory detection of dietary fat is critical for the prevention and treatment of obesity. We have conducted the present study on Psammomys obesus, the rodent desert gerbil which is a unique polygenic natural animal model of obesity. Our results show that obese animals exhibit a strong preference for lipid solutions in a two-bottle test. Interestingly, the expression of CD36, a lipido-receptor, in taste buds cells (TBC), isolated from circumvallate papillae, was decreased at mRNA level, but remained unaltered at protein level, in obese animals. We further studied the effects of linoleic acid (LA), a long-chain fatty acid, on the increases in free intracellular calcium (Ca(2+)) concentrations, [Ca(2+)]i, in the TBC of P. obesus. LA induced increases in [Ca(2+)]i, largely via CD36, from intracellular pool, followed by the opening of store-operated Ca(2+) (SOC) channels in the TBC of these animals. The action of this fatty acid on the increases in [Ca(2+)]i was higher in obese animals than that in controls. However, the release of Ca(2+) from intracellular stores, studied also by employing thapsigargin, was lower in TBC of obese animals than control rodents. In this study, we show, for the first time, that increased lipid intake and altered Ca(2+) signaling in TBC are associated with obesity in Psammomys obesus.

Figure 1. Expression of and CD36 and α-gustducin.

The CD36-positive TBC were isolated from CVP of P. obesus as described in Materials and Methods. Relative mRNA expression of CD36 (Fig. 1A) and α-gustducin (Fig. 1B) in CD36-positive TBC cells was assessed. p<0.001 shows the significant differences between TBC of obese and control animals. NS = insignificant differences. The CD36 and β-actin proteins were also detected by western blots in CD36-positive TBC (Fig. 1C) and intestinal cells (Fig. 1D) of control and obese animals. For experimental details, see Materials and Methods.

Figure 2. Effects of LA on the increases in [Ca²⁺]i in CD36-positive TBC.

The CD36-positive TBC were isolated from CVP of P. obesus as described in Materials and Methods. Ca²⁺ imaging studies were performed on CD36-positive TBC in calcium-containing media. The changes in intracellular Ca²⁺ (F₃₄₀/F₃₈₀) were monitored under the Nikon microscope (TiU) by using S-fluor 40x oil immersion objectives, as described in Materials and Methods. Colored time-lapse changes show the kinetics of the rise in [Ca²⁺]i, following addition of linoleic acid (LA), in a CD36-positive taste bud cell, freshly isolated from CVP of control (A) and obese (B) animals. (C) represents the single traces of observations and the arrow indicates when the test molecule (LA, 20 µM) was added into the cuvette without interruptions in the recording.

Figure 3. LA-induced increases in [Ca²⁺]i via CD36 in TBC.

The CD36-positive TBC cells were incubated with or without SSO (50 µM) for 20 min and then treated with LA at 20 µM. Ca²⁺ imaging studies were performed on CD36-positive TBC in Ca²⁺-containing media. The changes in intracellular Ca²⁺ (F₃₄₀/F₃₈₀) were monitored under the Nikon microscope (TiU) by using S-fluor 40x oil immersion objectives, as described in Materials and Methods. The arrows indicate when the test molecule (LA, 20 µM) was added into the cuvette without interruptions in the recording in TBC of control (A) and obese (B) animals. (C) represents histograms of the changes in [Ca²⁺]i.

Figure 4. Implication of Ca²⁺ influx in LA-induced increases in [Ca²⁺]i in CD36-positive TBC.

The CD36-positive TBC were isolated from CVP of P. obesus as described in Materials and Methods. Ca²⁺ imaging studies were performed in Ca²⁺-free (0% Ca²⁺) or Ca²⁺-containing (100% Ca²⁺) media. The changes in intracellular Ca²⁺ (F₃₄₀/F₃₈₀) were monitored under the Nikon microscope (TiU) by using S-fluor 40× oil immersion objectives. (A) represents the experiments performed in Ca²⁺-containing (100% Ca²⁺) and Ca²⁺-free medium (0% Ca²⁺). (B) represents the Δ increases in [Ca²⁺]i in the presence of a SOC channel blocker (ABP at 5 µM) in Ca²⁺-containing (100% Ca²⁺) medium. In both the experimental conditions, LA was used at 20 µM.

Figure 5. Effects of TG on the increases in [Ca²⁺]i in CD36-positive TBC.

The CD36-positive TBC were isolated from CVP of P. obesus as described in Materials and Methods. Ca²⁺ imaging studies were performed on CD36-positive TBC in Ca²⁺-containing (100% Ca²⁺) media. The changes in intracellular Ca²⁺ (F₃₄₀/F₃₈₀) were monitored under the Nikon microscope (TiU) by using S-fluor 40x oil immersion objectives. Colored time-lapse changes show the kinetics of the rise in [Ca²⁺]i, induced by TG at 5 µM, in a CD36-positive taste bud cell freshly isolated from CVP of control (A) and obese (B) animals. (C) represents the single traces of observations and the arrow indicates when the test molecule (TG, 5 µM) was added into the cuvette without interruptions in the recording.

Figure 6. Implication of Ca²⁺ influx in TG-induced increases in [Ca²⁺]i in CD36-positive TBC.

The CD36-positive TBC were isolated from CVP of P. obesus as described in Materials and Methods. The changes in intracellular Ca²⁺ (F₃₄₀/F₃₈₀) were monitored under the Nikon microscope (TiU) by using S-fluor 40x oil immersion objectives. (A) represents the histograms of the experiments performed in Ca²⁺-containing (100% Ca²⁺) and Ca²⁺-free (0% Ca²⁺) media with TG (5 µM). (B) represents the Δ increases in [Ca²⁺]i evoked by TG (5 µM) in the presence or absence of ABP at 5 µM in Ca²⁺-containing (100% Ca²⁺) medium. (C) represents the changes in intracellular Ca²⁺ (F₃₄₀/F₃₈₀) evoked by ionomycin at 500 nM in Ca²⁺-containing (100% Ca²⁺) medium.