Therapeutic Effect of Exendin-4, a Long-Acting Analogue of Glucagon-Like Peptide-1 Receptor Agonist, on Nerve Regeneration after the Crush Nerve Injury

Glucagon-like peptide-1 (GLP-1) is glucose-dependent insulinotropic hormone secreted from enteroendocrine L cells. Its long-acting analogue, exendin-4, is equipotent to GLP-1 and is used to treat type 2 diabetes mellitus. In addition, exendin-4 has effects on the central and peripheral nervous system. In this study, we administered repeated intraperitoneal (i.p.) injections of exendin-4 to examine whether exendin-4 is able to facilitate the recovery after the crush nerve injury. Exendin-4 injection was started immediately after crush injury and was repeated every day for subsequent 14 days. Rats subjected to sciatic nerve crush exhibited marked functional loss, electrophysiological dysfunction, and atrophy of the tibialis anterior muscle (TA). All these changes, except for the atrophy of TA, were improved significantly by the administration of exendin-4. Functional, electrophysiological, and morphological parameters indicated significant enhancement of nerve regeneration 4 weeks after nerve crush. These results suggest that exendin-4 is feasible for clinical application to treat peripheral nerve injury

A novel regulatory function of sweet taste-sensing receptor in adipogenic differentiation of 3T3-L1 cells.

BACKGROUND: Sweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells. METHODOLOGY/PRINCIPAL FINDINGS: In undifferentiated preadipocytes, both T1R2 and T1R3 were expressed very weakly, whereas the expression of T1R3 but not T1R2 was markedly up-regulated upon induction of differentiation (by 83.0 and 3.8-fold, respectively at Day 6). The α subunits of Gs (Gαs) and G14 (Gα14) but not gustducin were expressed throughout the differentiation process. The addition of sucralose or saccharin during the first 48 hours of differentiation considerably reduced the expression of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer-binding protein α (C/EBPα at Day 2, the expression of aP2 at Day 4 and triglyceride accumulation at Day 6. These anti-adipogenic effects were attenuated by short hairpin RNA-mediated gene-silencing of T1R3. In addition, overexpression of the dominant-negative mutant of Gαs but not YM-254890, an inhibitor of Gα14, impeded the effects of sweeteners, suggesting a possible coupling of Gs with the putative sweet taste-sensing receptor. In agreement, sucralose and saccharin increased the cyclic AMP concentration in differentiating 3T3-L1 cells and also in HEK293 cells heterologously expressing T1R3. Furthermore, the anti-adipogenic effects of sweeteners were mimicked by Gs activation with cholera toxin but not by adenylate cyclase activation with forskolin, whereas small interfering RNA-mediated knockdown of Gαs had the opposite effects. CONCLUSIONS: 3T3-L1 cells express a functional sweet taste-sensing receptor presumably as a T1R3 homomer, which mediates the anti-adipogenic signal by a Gs-dependent but cAMP-independent mechanism

A model for signal transduction mechanism downstream of the sweet taste-sensing receptors in taste cells and 3T3-L1 cells.

<p>In taste cells (in the left side), T1R2+T1R3 heterodimeric sweet receptor activates PLCβ via gustducin (Ggust) or other G proteins, leading to [Ca²⁺]_c elevation and membrane depolarization. In 3T3-L1 cells (in the right side), T1R3 homomeric receptor may activates Gs, which mediates the anti-adipogenic signal by a cAMP-independent mechanism. PLCβ: phospholipase C-β; DAG: diacylglycerol; IP₃: inositol 1,4,5-trisphosphate; Px1: pannexin 1; AC: adenylate cyclase; PDE: cAMP phosphodiesterase.</p

Role for Gαs in adipogenesis.

<p>A. 3T3-L1 cells were differentiated for 48 hours in the absence (control) or the presence of cholera toxin (0.1 μg/ml) or forskolin (20 μM). Then the expression levels of PPARγ and C/EBPα were measured by immunoblotting. The representative immunoblot data (left panel) and the relative amounts of the proteins normalized with β-tubulin (right panel) are shown. Data are shown as the mean ± SE (n = 3)., P<0.05, P<0.01 (vs. control). Cont, control; CTX, cholera toxin; Fsk, forskolin. B. Undifferentiated 3T3-L1 cells were transfected with 20 nmoles of non-silencing or Gαs-targeting siRNAs by electroporation as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054500#s2" target="_blank">Materials and Methods</a>’. Cells were seeded on a 12-well plate, cultured to confluence and differentiated for 48 hours before the measurement of expression levels of PPARγ and C/EBPα by immunoblotting. The representative immunoblot data (left panel) and the relative amounts of the proteins normalized with β-tubulin (right panel) are shown. Data are shown as the mean ± SE (n = 3)., P<0.05, P<0.01 (vs. control).</p

Expression of T1Rs during differentiation of 3T3-L1 cells.

<p>A. The total RNAs prepared from 3T3-L1 cells at the indicated time points during differentiation were subjected to quantitative RT-PCR using mouse ribosomal protein S18 as an internal control as described in`Materials and Methods'. The mRNA levels of T1R2 and T1R3 are shown as the percentage of that of T1R3 at Day 6. Results are shown as the mean ± SE (n = 3–6). B. Immumoblot data for T1R3 and actin in undifferentiated (Day 0) and differentiated (Day 7) 3T3-L1 cells. C. Immunofluorescence staining images for T1R3 (<i>a</i> and <i>b</i>, red) and GLUT4 (<i>c</i> and <i>d</i>, green) in undifferentiated (Day 0, <i>a</i> and <i>c</i>) and differentiated (Day 7, <i>b</i> and <i>d</i>) 3T3-L1 cells. Nuclei were visualized with DAPI (blue). <i>e</i>, Subcellular distribution of T1R3 (red) in Day 7 cells. Arrowheads indicate peripheral localization of T1R3.</p