GLP-1 Improves Adipocyte Insulin Sensitivity Following Induction of Endoplasmic Reticulum Stress

Yaojing Jiang, Zhihong Wang, Bo Ma, Linling Fan, Na Yi, Bin Lu, Qinghua Wang, Rui Liu, Yaojing Jiang, Zhihong Wang, Bo Ma, Linling Fan, Na Yi, Bin Lu, Qinghua Wang, Rui Liu

Abstract

Glucagon-like peptide 1 (GLP-1) improves insulin resistance of adipose tissue in obese humans. However, the mechanism of this effect is unclear. Perturbation of endoplasmic reticulum (ER) homeostasis impairs insulin signaling. We hypothesized that GLP-1 could directly improve insulin signaling in ER-stressed adipocytes. Here, we examined the effects of GLP-1 on ER stress response in fat cells in an obese and insulin-resistant murine model. We found that GLP-1 analog liraglutide reduced ER stress related gene expression in visceral fat cells accompanied by improved systemic insulin tolerance. Consistently, GLP-1 decreased CHOP expression and increased insulin stimulated AKT phosphorylation (p-AKT) in thapsigargin, a ER stress inducer, treated white fat cells differentiated from visceral stromal vascular fraction. We further found blocking CHOP expression increased insulin stimulated p-AKT in ER-stressed fat cells. Of note, we found mTOR signaling pathway contributed to the expression of ATF4 and subsequently the CHOP expression in ER stress response, while GLP-1 inhibited mTOR activity as exemplified by elevated autophagosome formation and increased LC3II/LC3I ratio. These findings suggest that GLP-1 directly modulates the ER stress response partially via inhibiting mTOR signaling pathway, leading to increased insulin sensitivity in adipocytes.

Keywords: adipocyte; endoplasmic reticulum stress; glucagon-like peptide-1; insulin sensitivity; mTOR.

Figures

FIGURE 1
FIGURE 1
GLP-1 reduced the expression of makers of ER stress in primary rat adipocytes subjected to ER stress and improves insulin sensitivity of ob/ob mice. ob/ob mice (6 weeks old) were given once-daily i.p. injection of PBS or liraglutide for 2 weeks. Blood glucose level in PBS- and liraglutide-treated mice were determined during IPITT (A) and IPGTT (B). Glucose responsiveness of the corresponding experimental groups was shown as a measurement of glucose area under the curve (AUC) of the IPGTT or IPITT graph. Data represent means ± SE of 6 mice per group. (C) Photomicrographs of representative adipose CHOP staining (arrows point to CHOP-positive nuclei) in PBS- and liraglutide-treated mice. Magnification, 200×. The numbers of CHOP-positive adipocyte were calculated in a total of 21 microscopical sections from three mice per group. Data represent means ± SE. (D) Fat pad from two rats was pooled for SVF isolation. Adipocytes differentiatied from SVF were fasted for 6 h in low-glucose DMEM media with 0.2% BSA (fasting media) and then were exposed to either vehicle alone or 5 μM thapsigargin (Tg) in fasting media for 16 h in the absence or presence of 50 nM GLP-1. The cells were then stimulated with insulin (100 nM) in fasting media for 10 min. Two wells of cell extracts were pooled together and were analyzed by immunoblotting for phospho-AKT Ser47 (p-AKT), total AKT, CHOP, and GAPDH (loading control). Results are means ± SE of four independent experiments. ∗P < 0.05 and ∗∗P < 0.01.
FIGURE 2
FIGURE 2
GLP-1 attenuated insulin resistance (IR) and regulated both PERK and IRE1/XBP-1 arm of the UPR in mouse adipose cell line. 3T3-L1 preadipocytes were induced to differentiate into mature adipocytes. (A) The differentiated adipocytes were fasted for 6 h in low-glucose DMEM media with 0.2% BSA (fasting media) and then were treated with vehicle alone or thapsigargin (Tg) in the absence or presence of 50 nM GLP-1 in fasting media for 16 h. The cells were then stimulated with insulin (100 nM) in fasting media for 10 min. (B–D) The differentiated adipocytes were fasted for 16 h in fasting media and then were treated with vehicle alone or thapsigargin (Tg) in the absence or presence of 50 nM GLP-1 in fating media for 9 h (B–D) or for 2–4 h (E). Three wells of cell extracts were pooled together and were analyzed by immunoblotting for (A) phospho-AKT Ser47 (p-AKT), total AKT and CHOP, (E) ATF-4, P(Ser51)-eIF2a and P(Thr980)-PERK, or by quantitative PCR for (B,C) total and spliced XBP-1 and (D) BIP. GAPDH or b-tublin was used as internal control. Data represent means ± SE of four (A) or three (B–E) independent experiments. ∗P < 0.05 and ∗∗P < 0.01.
FIGURE 3
FIGURE 3
GLP-1 affected insulin signaling in ER-stressed adipocytes via ATF4. The adipocytes were transfected with non-specific (siRNA-np) or ATF4 specific siRNA fragments (ATF4i-32, 33, 34) to inhibit transcription of ATF4. Total cell extracts were then analyzed by quantitative PCR for ATF4 (A) and immunoblotting for CHOP (B). Data represent means ± SE of images from the plot B and plot C. ∗P < 0.05. (C) 24 h after transfection with siRNA-np or ATF4i, cells were fasted for 6 h in low-glucose DMEM media with 0.2% BSA (fasting media) and then were treated with vehicle alone or thapsigargin (Tg) in the absence or presence of 50 nM GLP-1 in fasting media for 16 h. The cells were then stimulated with insulin (100 nM) in fasting media for 10 min. Three wells of cell extracts were pooled together and were analyzed by immunoblotting for phospho-AKT Ser47 (p-AKT), CHOP, and b-tublin (loading control). Data represent means ± SE of three independent experiments. ∗P < 0.05 and ∗∗P < 0.01.
FIGURE 4
FIGURE 4
GLP-1 promoted the autophagy process activated by ER stress. The differentiated adipocytes fasted for 16 h in low-glucose DMEM media with 0.2% BSA (fasting media) and then were treated with vehicle alone or thapsigargin (Tg) in the absence or presence of GLP-1 for 4 h. (A) Three wells of cell extracts were pooled together and were then analyzed by immunoblotting for LC3. Conversion of LC3-I to LC3-II is qualified by calculating the intensities of Western blotting. Data represent means ± SE of three independent experiments. (B) Four wells of cells were pooled together for electron microscopic analysis. Panels d to f were high magnification images of panels a to c, respectively. Typical autophagosomes (arrows) are indicated. The numbers of autophagosomes in adipocytes were calculated in a total of 15 electron microscopical sections. Data represent means ± SE. ∗P < 0.05 and ∗∗P < 0.01.
FIGURE 5
FIGURE 5
GLP-1 affects ATF4 translation induction in adipocytes following ER stress via preventing mTOR signaling pathway. (A) The differentiated adipocytes were fasted for 16 h in low-glucose DMEM media with 0.2% BSA (fasting media) and then were exposed to vehicle alone or thapsigargin (Tg) in the absence or presence of GLP-1 in fasting media for 0.5–3.0 h as indicated. Three wells of cell extracts were pooled together and were analyzed by immunoblotting for P(2448)-mTOR, mTOR, and b-tublin (loading control). Data represent means ± SE of four independent experiments. ∗P < 0.05 and ∗∗P < 0.01. Data for replication see Supplementary Figure 5. (B) The differentiated adipocytes were fasted for 16 h in fasting media and then were exposed to vehicle alone or thapsigargin (Tg) in the absence or presence of rapamycin (Rm) in fasting media for 0.5–3.0 h as indicated. Three wells of cell extracts were pooled together and were analyzed by immunoblotting for ATF-4, P(Ser51)-eIF2a, P(Thr980)-PERK, LC3 and b-tublin (loading control). Data represent means ± SE of three independent experiments. ∗P < 0.05. (C,D) The differentiated adipocytes were fasted for 6 h in fasting media and then were exposed to vehicle alone or thapsigargin (Tg) in the absence or presence of rapamycin (Rm) in fasting media for 16 h. Three wells of cell extracts were pooled together and were analyzed by immunoblotting for CHOP, phospho-AKT Ser47 (p-AKT) and b-tublin (loading control). Data represent means ± SE of three independent experiments. ∗P < 0.05 and ∗∗P < 0.01. (E) Model depicting the proposed mechanism of effects of GLP-1 on ER stress in adipocyte.

References

    1. B’chir W., Maurin A. C., Carraro V., Averous J., Jousse C., Muranishi Y., et al. (2013). The eIF2alpha/ATF4 pathway is essential for stress-induced autophagy gene expression. Nucleic Acids Res. 41 7683–7699. 10.1093/nar/gkt563
    1. Bergman R. N., Mittelman S. D. (1998). Central role of the adipocyte in insulin resistance. J. Basic. Clin. Physiol. Pharmacol. 9 205–221. 10.1515/JBCPP.1998.9.2-4.205
    1. Boyce M., Bryant K. F., Jousse C., Long K., Harding H. P., Scheuner D., et al. (2005). A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science 307 935–939. 10.1126/science.1101902
    1. Cabou C., Vachoux C., Campistron G., Drucker D. J., Burcelin R. (2011). Brain GLP-1 signaling regulates femoral artery blood flow and insulin sensitivity through hypothalamic PKC-delta. Diabetes Metab. Res. Rev. 60 2245–2256. 10.2337/db11-0464
    1. Drucker D. J. (2006). The biology of incretin hormones. Cell Metab. 3 153–165. 10.1016/j.cmet.2006.01.004
    1. Gastaldelli A., Gaggini M., Daniele G., Ciociaro D., Cersosimo E., Tripathy D., et al. (2016). Exenatide improves both hepatic and adipose tissue insulin resistance: a dynamic positron emission tomography study. Hepatology 64 2028–2037. 10.1002/hep.28827
    1. Hirosumi J., Tuncman G., Chang L., Gorgun C. Z., Uysal K. T., Maeda K., et al. (2002). A central role for JNK in obesity and insulin resistance. Nature 420 333–336. 10.1038/nature01137
    1. Idris I., Patiag D., Gray S., Donnelly R. (2002). Exendin-4 increases insulin sensitivity via a PI-3-kinase-dependent mechanism: contrasting effects of GLP-1. Biochem. Pharmacol. 63 993–996. 10.1016/S0006-2952(01)00924-8
    1. Kim J., Kundu M., Viollet B., Guan K. L. (2011). AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 13 132–141. 10.1038/ncb2152
    1. Kumar M., Hunag Y., Glinka Y., Prud’homme G. J., Wang Q. (2007). Gene therapy of diabetes using a novel GLP-1/IgG1-Fc fusion construct normalizes glucose levels in db/db mice. Gene Ther. 14 162–172. 10.1038/sj.gt.3302836
    1. Lai E., Bikopoulos G., Wheeler M. B., Rozakis-Adcock M., Volchuk A. (2008). Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic beta-cells. Am. J. Physiol. Endocrinol. Metab. 294 E540–E550. 10.1152/ajpendo.00478.2007
    1. Lee Y. S., Park M. S., Choung J. S., Kim S. S., Oh H. H., Choi C. S., et al. (2012). Glucagon-like peptide-1 inhibits adipose tissue macrophage infiltration and inflammation in an obese mouse model of diabetes. Diabetologia 55 2456–2468. 10.1007/s00125-012-2592-3
    1. Liu R., Ding X., Wang Y., Wang M., Peng Y. (2013). Glucagon-like peptide-1 upregulates visfatin expression in 3T3-L1 adipocytes. Horm. Metab. Res. 45 646–651. 10.1055/s-0033-1343472
    1. Liu R., Li N., Lin Y., Wang M., Peng Y., Lewi K., et al. (2016). Glucagon like peptide-1 promotes adipocyte differentiation via the Wnt4 mediated sequestering of Beta-Catenin. PLoS One 11:e0160212. 10.1371/journal.pone.0160212
    1. Liu R., Ma D., Li Y., Hu R., Peng Y., Wang Q. (2014). The anorexic effect of Ex4/Fc through GLP-1 receptor activation in high-fat diet fed mice. Acta Biochim. Biophys. Sin. 46 675–681. 10.1093/abbs/gmu044
    1. Maris M., Overbergh L., Gysemans C., Waget A., Cardozo A. K., Verdrengh E., et al. (2012). Deletion of C/EBP homologous protein (Chop) in C57Bl/6 mice dissociates obesity from insulin resistance. Diabetologia 55 1167–1178. 10.1007/s00125-011-2427-7
    1. Oyadomari S., Mori M. (2004). Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 11 381–389. 10.1038/sj.cdd.4401373
    1. Ozawa K., Miyazaki M., Matsuhisa M., Takano K., Nakatani Y., Hatazaki M., et al. (2005). The endoplasmic reticulum chaperone improves insulin resistance in type 2 diabetes. Diabetes Metab. Res. Rev. 54 657–663.
    1. Ozcan U., Yilmaz E., Ozcan L., Furuhashi M., Vaillancourt E., Smith R. O., et al. (2006). Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science 313 1137–1140. 10.1126/science.1128294
    1. Palakurthi S. S., Aktas H., Grubissich L. M., Mortensen R. M., Halperin J. A. (2001). Anticancer effects of thiazolidinediones are independent of peroxisome proliferator-activated receptor gamma and mediated by inhibition of translation initiation. Cancer Res. 61 6213–6218.
    1. Rhodes C. J. (2005). Type 2 diabetes-a matter of beta-cell life and death? Science 2005 380–384.
    1. Rzymski T., Milani M., Pike L., Buffa F., Mellor H. R., Winchester L., et al. (2010). Regulation of autophagy by ATF4 in response to severe hypoxia. Oncogene 29 4424–4435. 10.1038/onc.2010.191
    1. Salvado L., Palomer X., Barroso E., Vazquez-Carrera M. (2015). Targeting endoplasmic reticulum stress in insulin resistance. Trends Endocrinol. Metab. 26 438–448. 10.1016/j.tem.2015.05.007
    1. Senft D., Ronai Z. A. (2015). UPR, autophagy, and mitochondria crosstalk underlies the ER stress response. Trends Biochem. Sci. 40 141–148. 10.1016/j.tibs.2015.01.002
    1. Shang L., Wang X. (2011). AMPK and mTOR coordinate the regulation of Ulk1 and mammalian autophagy initiation. Autophagy 7 924–926. 10.4161/auto.7.8.15860
    1. Sheng R., Liu X. Q., Zhang L. S., Gao B., Han R., Wu Y. Q., et al. (2012). Autophagy regulates endoplasmic reticulum stress in ischemic preconditioning. Autophagy 8 310–325. 10.4161/auto.18673
    1. Shoelson S. E., Lee J., Goldfine A. B. (2006). Inflammation and insulin resistance. J. Clin. Invest. 116 1793–1801. 10.1172/JCI29069
    1. Silva A. M., Wang D., Komar A. A., Castilho B. A., Williams B. R. (2007). Salicylates trigger protein synthesis inhibition in a protein kinase R-like endoplasmic reticulum kinase-dependent manner. J. Biol. Chem. 282 10164–10171. 10.1074/jbc.M609996200
    1. Soltani N., Kumar M., Glinka Y., Prud’homme G. J., Wang Q. (2007). In vivo expression of GLP-1/IgG-Fc fusion protein enhances beta-cell mass and protects against streptozotocin-induced diabetes. Gene Ther. 14 981–988. 10.1038/sj.gt.3302944
    1. Soltani N., Qiu H., Aleksic M., Glinka Y., Zhao F., Liu R., et al. (2011). GABA exerts protective and regenerative effects on islet beta cells and reverses diabetes. Proc. Natl. Acad. Sci. U.S.A. 108 11692–11697. 10.1073/pnas.1102715108
    1. Vendrell J., El Bekay R., Peral B., Garcia-Fuentes E., Megia A., Macias-Gonzalez M., et al. (2011). Study of the potential association of adipose tissue GLP-1 receptor with obesity and insulin resistance. Endocrinology 152 4072–4079. 10.1210/en.2011-1070
    1. Wang Q., Chen K., Liu R., Zhao F., Gupta S., Zhang N., et al. (2010). Novel GLP-1 fusion chimera as potent long acting GLP-1 receptor agonist. PLoS One 5:e12734. 10.1371/journal.pone.0012734
    1. Wu J., Kaufman R. J. (2006). From acute ER stress to physiological roles of the unfolded protein response. Cell Death Differ. 13 374–384. 10.1038/sj.cdd.4401840
    1. Xue R., Lynes M. D., Dreyfuss J. M., Shamsi F., Schulz T. J., Zhang H., et al. (2015). Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes. Nat. Med. 21 760–768. 10.1038/nm.3881
    1. Yin J., Gu L., Wang Y., Fan N., Ma Y., Peng Y. (2015). Rapamycin improves palmitate-induced ER stress/NF κ B pathways associated with stimulating autophagy in adipocytes. Mediators Inflamm. 2015:272313. 10.1155/2015/272313
    1. Zhang N., Cao M. M., Liu H., Xie G. Y., Li Y. B. (2015). Autophagy regulates insulin resistance following endoplasmic reticulum stress in diabetes. J. Physiol. Biochem. 71 319–327. 10.1007/s13105-015-0384-1

Source: PubMed

Sponsorzy i współpracownicy

Warunki medyczne

Interwencje lekowe

3
Subskrybuj