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. 2013 Apr 11;153(2):413-25.
doi: 10.1016/j.cell.2013.03.001.

The fractalkine/CX3CR1 system regulates β cell function and insulin secretion

Yun Sok Lee  1 , Hidetaka Morinaga, Jane J Kim, William Lagakos, Susan Taylor, Malik Keshwani, Guy Perkins, Hui Dong, Ayse G Kayali, Ian R Sweet, Jerrold Olefsky
Affiliations

The fractalkine/CX3CR1 system regulates β cell function and insulin secretion

Yun Sok Lee et al. Cell. .

Abstract

Here, we demonstrate that the fractalkine (FKN)/CX3CR1 system represents a regulatory mechanism for pancreatic islet β cell function and insulin secretion. CX3CR1 knockout (KO) mice exhibited a marked defect in glucose and GLP1-stimulated insulin secretion, and this defect was also observed in vitro in isolated islets from CX3CR1 KO mice. In vivo administration of FKN improved glucose tolerance with an increase in insulin secretion. In vitro treatment of islets with FKN increased intracellular Ca(2+) and potentiated insulin secretion in both mouse and human islets. The KO islets exhibited reduced expression of a set of genes necessary for the fully functional, differentiated β cell state, whereas treatment of wild-type (WT) islets with FKN led to increased expression of these genes. Lastly, expression of FKN in islets was decreased by aging and high-fat diet/obesity, suggesting that decreased FKN/CX3CR1 signaling could be a mechanism underlying β cell dysfunction in type 2 diabetes.

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Figure 1
Figure 1
CX3CR1 KO mice exhibit normal body weight, food intake, fat and liver mass, and inflammatory and metabolic gene expression in adipose tissue. (A) Body weight change on HFD. Mean+/-SEM, n=20 for both WT and KO. (B) Cumulative food intake on HFD. Food intake was measured from 5 different cages per group, and 4 mice were housed in each cage. Mean+/-SEM. (C) Liver mass. Mean+/-SEM, n=8 per group. N.S., not significant. NCD, normal chow diet. (D) Epididymal fat mass. Mean+/-SEM, n=8 per group. *, P<0.05. (E) Immunohistochemistry analysis of epididymal adipose tissue using anti-F4/80 antibody. Representative figures were presented from the analyses of 5 different mice per group. (F) mRNA expression of inflammatory and metabolic genes in epididymal adipose tissue on NCD and HFD. Mean+/-SEM, n=5 per group. N, normal chow diet; H, high fat diet. AU, arbitrary unit.
Figure 2
Figure 2
CX3CR1 KO mice exhibit impaired glucose tolerance due to reduced insulin secretion. (A-D) CX3CR1 KO mice manifest impaired glucose tolerance with normal insulin sensitivity either on NCD (n=11) or HFD (n=12). Mean+/-SEM. (A) Oral glucose tolerance test. ##, P<0.01 WT-NCD vs KO-NCD; ###, P<0.001 WT-NCD vs KO-NCD; ***, P<0.001 WT-HFD vs KO-HFD. AUC, area under the curve. (B) Insulin tolerance test. *, P<0.05 WT-HFD vs KO-HFD; **, P<0.01 WT-HFD vs KO-HFD; N.S., not significant. (C) Plasma insulin (left), C-Peptide (middle), and GLP1 (right) levels of NCD mice during OGTT in panel A. #, P<0.05; ##, P<0.01. (D) Plasma insulin, C-peptide, and GLP1 levels of HFD mice during OGTT in panel A. *, P<0.05; **, P<0.01. (E) Intravenous glucose tolerance test (left) and plasma insulin level during IVGTT (right). Mean+/-SEM, n=4 per group. (F-G) Plasma insulin and C-Peptide levels of NCD (F) or HFD (G) mice during arginine tolerance test. Mean+/-SEM, n=8 per group. #, P<0.05; ##, P<0.01; *, P<0.05; **, 0.01. (H) Fractalkine neutralization reduces insulin secretion and causes glucose intolerance in WT mice. FKN neutralizing antibody was injected IP to WT mice, and 30 min later the mice were given oral gavage of glucose (2g/kg) for GTT. Mean+/-SEM, n=10 per group. Left, plasma insulin level during GTT; right, GTT. See also Figure S1.
Figure 3
Figure 3
CX3CR1 KO islets display reduced insulin secretion with lower expression of genes involved in beta cell function and communication. (A) Static GSIS test using primary mouse islets from WT and CX3CR1 KO mice fed NCD. Mean+/-SEM, n=6 per group. *, P<0.05. (B) Static GSIS test using primary mouse islets from WT and CX3CR1 KO mice fed HFD for 10 weeks. Mean+/-SEM, n=6 per group. (C) Static GSIS in the presence or absence of arginine (Arg; 10mM) using primary mouse islets. (D) Knockdown of CX3CR1 decreases GSIS in Min6 cells. Min6 cells were transfected with scrambled siRNA (Scrb) or 2 different CX3CR1 siRNAs (CX-1 and CX-2). 48h after transfection, GSIS was measured in the presence or absence of 100ng/ml FKN (left panel), or quantitative realtime RT-PCR was performed for CX3CR1 expression (right panel). **, P<0.01; ***, P<0.001. (E) Perifusion experiment using islets from WT and CX3CR1 KO mice on NCD. Mean+/-SEM, n=5 per group. *, P<0.05; **, P<0.01. (F-G) STZ-treated mice transplanted with CX3CR1 KO islets are more glucose intolerant than mice transplanted with WT islets. Plasma glucose (F) and insulin (G) levels during GTTs. Mean+/-SEM, n=6 per group. n=5 per group. (H) mRNA level of genes involved in beta cell function and communication in islets from WT or CX3CR1 KO mice either on chow and HFD. mRNA level of each gene was normalized to 18S rRNA level in the same sample. Mean+/-SEM, n=6 per group. *, P<0.05 WT-NCD vs KO-NCD or WT-NCT vs WT-HFD; **, P<0.01 WT-NCD vs KO-NCD or WT-NCT vs WT-HFD; #, P<0.05 WT-HFD vs KO-HFD. See also Figure S2.
Figure 4
Figure 4
CX3CR1 KO mice exhibit increased beta cell mass and insulin content. (A) Immunohistochemistry analysis of WT and CX3CR1 KO islets using anti-insulin (green) and anti-glucagon (red) antibodies. (B) Beta cell mass of WT and CX3CR1 KO mice on NCD. Mean+/-SEM, n=8 per group. *, P<0.05. (C) Pancreatic insulin content. Mean+/-SEM, n=10 per group. (D) Relative islet cell size of WT and CX3CR1 KO mice. Relative islet cell size was calculated by dividing beta cell area by nuclei number. AU, arbitrary unit. Mean+/-SEM, n=8 per group. (E-F) Ultramicroscopic analysis of WT and CX3CR1 KO beta cells. (E) Ultramicroscopic pictures of WT and CX3CR1 KO mouse islets on NCD. (F) Mitochondrial length (upper left) and width (upper right), mitochondrial number per given area (lower left), and cristae abundance (lower right) was calculated using ImageJ software. Mean+/-SEM. 10 different EM pictures of WT and KO islets, and at least 2 mitochondria located nearest to the center of each EM picture were analyzed for the morphometry. AU, arbitrary unit; N.S., not significant. (G) Vascular density in the islets of WT and CX3CR1 KO mice on NCD was analyzed by immunohistochemistry. Pancreatic sections were co-stained with anti-CD34 (endothelial cell; green) and anti-insulin (beta cell; red) antibodies, and the intensity of CD34-positive signals in the insulin-positive area was measured and graphed in the right panel. Mean+/-SEM, n=6 (WT) and 8 (KO). (H) Immunohistochemistry analysis of mouse islets using anti-CX3CR1 (red, on the left), anti-FKN (red, on the right), or anti-insulin (green) antibodies. (I) Immunohistochemistry of human islet using anti-CX3CR1 (green) and anti-insulin (red), or anti-glucagon (red) antibodies. See also Figure S3.
Figure 5
Figure 5
FKN enhances insulin secretion and improves glucose tolerance in mice in a CX3CR1-dependnet manner. (A-C) WT and CX3CR1 KO mice (on NCD) were injected IP with glucose (2 g/kg) + FKN (40 μg/kg) or glucose + vehicle solution, and blood glucose and insulin levels were measured at the indicated time points. Mean+/-SEM, n=6 (vehicle) or 7 (FKN). (A) Glucose tolerance test in WT mice. *, P<0.05; **, P<0.01. (B) Glucose tolerance test in CX3CR1 KO mice. (C) Plasma insulin level during the GTTs. (D) In vitro static GSIS studies using WT and KO islets in the presence or absence of mouse FKN (100 ng/ml). n=6 per group. (E) Perifusion experiment using primary mouse islets in response to the indicated levels of glucose, GLP1 (100 nM) and FKN (100 ng/ml). ***, P<0.001. (F) Oxygen consumption rate in islets during the perifusion experiment in panel E. (G) In vitro static GSIS studies using human islets in the presence or absence of human FKN (100 or 400 ng/ml). n=4. #, P=0.076; *, P<0.05 compared with lane 2. See also Figure S4.
Figure 6
Figure 6
FKN stimulates insulin secretion by increasing intracellular calcium levels in a CX3CR1- and MEK-dependent manner. (A) GSIS test using primary mouse islets with or without pertussis toxin (PTX; 250 ng/ml), Wortmannin (Wort; 10 μM), or PD98059 (50 μM), in the presence or absence of FKN (100 ng/ml). Mean+/-SEM, n=6 per group. (B) GSIS test using Min6 cells with or without FKN (100 ng/ml) or U0126 (10 μM). Mean+/-SEM. (C-I) Intracellular calcium level in Min6 mouse beta cells in the presence or absence of glucose (C, with glucose; D, without glucose), nimodipine (10 μM) (E), control antibody (F), anti-CX3CR1 neutralizing antibody (G), PTX (250 ng/ml) (H), or U0126 (10 μM) (I). Mean+/-SEM. (J) Intracellular cAMP level. Min6 cells were pre-incubated with isobutylmethylxanthine for 30 min, and then treated with GLP1 (100 nM), forskolin (100 μM), or FKN (100 ng/ml) at low (2.8 mM) or high (16.7 mM) glucose conditions for 30 min. Mean+/-SEM. (K) PKA enzymatic activity was measured in Min6 cells incubated in a low (2.8 mM) or high (16.7 mM) glucose condition for 15 min in the presence or absence of GLP1 (100 nM) or FKN (100 ng/ml). Mean+/-SEM. (L) FKN stimulates expression of genes involved in beta cell function. Primary mouse islets were incubated for 7 days with or without FKN (100 ng/ml), and mRNA expression of PDX-1, NeuroD, Glut2, and HIF-1α was measured by quantitative realtime RT-PCR. Mean+/-SEM. *, P<0.05. (M) ICER-1 mRNA expression in WT and CX3CR1 KO islets. Mean+/-SEM. (N) Palmitate-induced ICER-1 expression is suppressed by FKN in Min6 cells. Mean+/-SEM. (O) GSIS by Min6 cells treated with palmitate (Pal; 0.4 mM) for 48 h in the presence or absence of FKN (100 ng/ml). (P) FKN represses binding of ICER-1 to NeuroD promoter. Min6 cells were incubated in serum free media in the presence or absence of palmitate (100 μM) and/or FKN (100ng/ml). After 48h, the cells were fixed and subjected to chromatin immunoprecipitation with anti-ICER-1 antibody. (Q) Suppressive effect of FKN on ICER-1 expression is abolished by MEK inhibitor (U0126). Min6 cells were incubated with palmitate (Pal; 0.4 mM) in the presence or absence of FKN (100 ng/ml) or U0126 (10 μM). 48 h later, cells were harvested and subjected to quantitative realtime RT-PCR. See also Figures S5, S6 and S7.
Figure 7
Figure 7
FKN expression is decreased by aging and HFD in islets. (A) FKN mRNA expression in 7 week old (7w)-NCD (N), 24 week old (24w)-NCD, or 24 week old-HFD mice. *, P<0.05 24w-NCD vs 24w-HFD. FKN mRNA level was normalized to 18S rRNA level in each sample. Mean+/-SEM, n=6 per group. (B) FKN mRNA expression in 7 week old (7w)-NCD (N; n=5), 1 year old (1y)-NCD (n=8), or 1 year old-HFD (H; n=6) mice. Mean+/-SEM. *, P<0.05 7w-NCD vs 1y-NCD or 7w-NCD vs 1y-HFD. (C) FKN protein expression levels were measured in aliquots of the samples used in panel B. FKN protein levels were normalized by total protein concentration. Mean+/-SEM. *, P<0.05 7w-NCD vs 1y-NCD; **, P<0.01 7w-NCD vs 1y-HFD; #, P<0.05 1y-NCD vs 1y-HFD. (D) FKN mRNA expression is decreased in in vitro expanded human beta cells. mRNA levels of FKN and CX3CR1 were analyzed by Affymetrix GeneChip microarrays (Kayali et al., 2007; Kutlu et al., 2009) in freshly isolated human islets (Islet) or in vitro expanded islets (Exp) as described in experimental procedures. Mean+/-SEM.
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References

    1. Aoyama T, Inokuchi S, Brenner DA, Seki E. CX3CL1-CX3CR1 interaction prevents carbon tetrachloride-induced liver inflammation and fibrosis in mice. Hepatology. 2010;52:1390–1400. - PMC - PubMed
    1. Boehme SA, Lio FM, Maciejewski-Lenoir D, Bacon KB, Conlon PJ. The chemokine fractalkine inhibits Fas-mediated cell death of brain microglia. J Immunol. 2000;165:397–403. - PubMed
    1. Calabrese A, Zhang M, Serre-Beinier V, Caton D, Mas C, Satin LS, Meda P. Connexin 36 controls synchronization of Ca2+ oscillations and insulin secretion in MIN6 cells. Diabetes. 2003;52:417–424. - PubMed
    1. Cardona AE, Pioro EP, Sasse ME, Kostenko V, Cardona SM, Dijkstra IM, Huang D, Kidd G, Dombrowski S, Dutta R, et al. Control of microglial neurotoxicity by the fractalkine receptor. Nature neuroscience. 2006;9:917–924. - PubMed
    1. Carvalho CP, Barbosa HC, Britan A, Santos-Silva JC, Boschero AC, Meda P, Collares-Buzato CB. Beta cell coupling and connexin expression change during the functional maturation of rat pancreatic islets. Diabetologia. 2010;53:1428–1437. - PubMed

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