Chronic fractalkine administration improves glucose tolerance and pancreatic endocrine function - PubMed (original) (raw)

. 2018 Apr 2;128(4):1458-1470.

doi: 10.1172/JCI94330. Epub 2018 Mar 5.

Jong Bae Seo 1 2, Gautam K Bandyopadhyay 1, Pingping Li 1 3, Joshua Wollam 1, Heekyung Chung 1, Seung-Ryoung Jung 2, Anne Murphy 4, Maria Wilson 5, Ron de Jong 5, Sanjay Patel 5, Deepika Balakrishna 5, James Bilakovics 5, Andrea Fanjul 5, Artur Plonowski 5, Duk-Su Koh 2, Christopher J Larson 5 6, Jerrold M Olefsky 1, Yun Sok Lee 1 7

Affiliations

Chronic fractalkine administration improves glucose tolerance and pancreatic endocrine function

Matthew Riopel et al. J Clin Invest. 2018.

Abstract

We have previously reported that the fractalkine (FKN)/CX3CR1 system represents a novel regulatory mechanism for insulin secretion and β cell function. Here, we demonstrate that chronic administration of a long-acting form of FKN, FKN-Fc, can exert durable effects to improve glucose tolerance with increased glucose-stimulated insulin secretion and decreased β cell apoptosis in obese rodent models. Unexpectedly, chronic FKN-Fc administration also led to decreased α cell glucagon secretion. In islet cells, FKN inhibited ATP-sensitive potassium channel conductance by an ERK-dependent mechanism, which triggered β cell action potential (AP) firing and decreased α cell AP amplitude. This results in increased glucose-stimulated insulin secretion and decreased glucagon secretion. Beyond its islet effects, FKN-Fc also exerted peripheral effects to enhance hepatic insulin sensitivity due to inhibition of glucagon action. In hepatocytes, FKN treatment reduced glucagon-stimulated cAMP production and CREB phosphorylation in a pertussis toxin-sensitive manner. Together, these results raise the possibility of use of FKN-based therapy to improve type 2 diabetes by increasing both insulin secretion and insulin sensitivity.

Keywords: Diabetes; Endocrinology; Gluconeogenesis; Insulin; Metabolism.

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Conflict of interest statement

Conflict of interest: HC is employed by Samumed LLC. MW, RDJ, SP, DB, JB, AF, and AP are employed by Takeda California Inc. YSL and JMO have received research funding from Takeda California Inc. FKN and CX3CR1 molecules and related methods of use reported in this study are covered in patent 9764001 invented by JMO and YSL.

Figures

Figure 1

Figure 1. Chronic administration of a chimeric FKN-Fc fusion protein exerts durable effects to improve glucose tolerance with increased insulin and decreased glucagon secretion in HFD/obese mice.

(A) Intraperitoneal glucose tolerance tests (IPGTTs) in NCD WT mice at day 0. n = 8 for both groups. (B) GTTs in NCD WT mice at day 5. A single injection of 10 mg/kg FKN-Fc or vehicle was given to NCD WT mice at day 0 and, at day 5, glucose tolerance (left) and plasma insulin levels (right) were measured with (FKN-Fc 10 mg/kg ×2) or without (vehicle and FKN-Fc 10 mg/kg ×1) acute FKN-Fc administration. n = 8 for each group. (C) GTTs were performed in HFD WT mice at 0 (left), 2 (middle) or 5 (right panel) days after a single FKN-Fc injection (day 0). n = 8 for both groups. (D) Fasting plasma glucagon levels in NCD and HFD (16 week) WT mice before and 10 min after 30 mg/kg FKN-Fc injection. Mean ± SEM. n = 8 for each group. (E–I) Effects of chronic FKN-Fc administration in HFD mice. Body weight (E; n = 20 WT mice), daily food intake (F; n = 5 WT mice), glucose tolerance (G, n = 8 WT mice; H, n = 8 CX3CR1 KO mice) and serum insulin (I, n = 8 WT mice) levels were measured during or after 8 weeks of FKN-Fc treatment. V, vehicle; F, FKN-Fc. For statistical analysis, 2-way ANOVA with post-hoc t tests between the individual groups (A–C and E–H), 1-way ANOVA (D) or 2-tailed unpaired t test (I) was performed. In all panels, values are mean ± SEM and the symbols indicate statistical analysis: *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle controls or lane 1; #P < 0.05 versus lane 4. See also Supplemental Figure 1.

Figure 2

Figure 2. Chronic FKN-Fc administration enhances GSIS and decreases apoptosis in the islets of obese mice.

(A) Static GSIS in primary mouse islets. (B) Relative apoptotic activity in primary mouse islet cells. Pal, palmitate. (C–E) Chronic FKN-Fc administration improves GSIS and decreases apoptosis in islets of HFD WT mice. 10 week HFD mice were treated with vehicle or FKN-Fc for an additional 8 weeks. Islets were isolated and similar sized islets were picked under the microscope and subjected to in vitro GSIS (C), quantitative RT-PCR (Q-PCR) (D) and caspase-3/7 activity assays (E). (F–G) Chronic FKN-Fc administration decreases β cell apoptosis in ob/ob mice. 8 week-old ob/ob mice were ip injected with vehicle or 30 mg/kg FKN-Fc every other day for 7 weeks. β cell apoptosis and apoptoic gene expression was assessed by immunohistochemistry (IHC) analyses using anti-insulin and anti-active (cleaved) caspase-3 antibodies (F) and Q-PCR (G), respectively. n = 4. (H and I) Morphometric analyses of HFD mouse islets. 10 week HFD mice were treated with FKN-Fc every other day for 8 weeks. A whole pancreas was harvested from each mouse, weighed and then fixed for IHC analyses. β Cell mass (H) and islet number per unit pancreatic area (I) were measured after staining with anti-insulin antibody, as described in Methods. Images are obtained at ×20 magnification. AU, arbitrary unit. For statistical analysis, 2-tailed paired t test (C, E, F, and H) or 1-way ANOVA (A, B, D, and G) was performed. In all graph panels, values are mean ± SEM and the symbols indicate statistical analysis: *P < 0.05 versus lane 1; **P < 0.01 versus lane 1; #P < 0.05 versus lane 2; ##P < 0.01 versus lane 2. See also Supplemental Figure 2.

Figure 3

Figure 3. Chronic FKN-Fc administration exerts tissue-specific effects to improve hepatic insulin sensitivity in HFD/obese insulin resistant mice.

(A–D) FKN-Fc treatment improves hepatic insulin sensitivity in obese mice. 10 week HFD mice were treated with vehicle or 30 mg/kg FKN-Fc for 8 weeks and subjected to ITTs (A) or hyperinsulinemic euglycemic clamp studies (B–D). Final FKN-Fc dosing was given 6 hours before the tests, at the start of fasting. Glucose infusion rate (GIR; B), hepatic glucose production (HGP; C) and basal (GDR; D) and insulin-stimulated glucose disposal rates (IS-GDR; D) were calculated as described previously (53). n = 8 for ITTs and n = 4 for clamp studies. In panel A, ITT was performed 2 times in separate cohorts of mice and a representative figure is shown. (E) Gluconeogenic activity in NCD mouse hepatocytes. n = 4. (F) Intracellular cAMP levels 15 min after hormonal treatment(s) in NCD mouse hepatocytes. n = 4. (G) CREB and Akt phosphorylation 30 min after hormonal stimulation. (H) Gluconeogenic activity in pertussis toxin (PTX)-treated (30 min pretreatment) NCD mouse hepatocytes. n = 4. (I) Gluconeogenic activity in HFD mouse hepatocytes. n = 4. (J) Intracellular cAMP levels in HFD mouse hepatocytes 15 min after hormonal treatment(s). n = 4. For statistical analysis, ANOVA with post-hoc t tests between the individual groups (A), 2-tailed unpaired t test (B) or 1-way ANOVA (C, E, F, H–J) was performed. In all graph panels, values are mean ± SEM and the symbols indicate statistical analysis: *P < 0.05 versus lane 1; **P < 0.01 versus lane 1; ***P < 0.001 versus lane 1; #P < 0.05 versus lane 2; ##P < 0.01 versus lane 2. See also Supplemental Figure 3. See complete unedited blots in the supplemental material.

Figure 4

Figure 4. Electrophysiology studies in β cells.

(A) KATP channel current in Min6 cells. U0216 (200 nM) was pretreated 1 hour before the measurements. (B) Dose-dependent inhibition of KATP channel activity by FKN in the presence or absence of 200 nM U0126 or 10 nM PD98059. n = 3, 12, 3, 4, 5, 8 and 4 for lane 1–7, respectively. **P < 0.01; ***P < 0.001; 2-tailed unpaired t test. (C) ATP/ADP ratio in Min6 cells with or without FKN (100 ng/ml) treatment for 60 min. (D) Oxygen consumption rate in Min6 cells. Oligo, oligomycin (ATP synthase inhibitor); FCCP, trifluoromethoxy carbonylcyanide phenylhydrazone (a protonophoric uncoupler). (E and F) KATP channel current in primary WT (n = 5; E) and CX3CR1 KO (n = 4; F) β cells in intact islets. FKN effects were seen relatively slower, probably due to surrounding cells in the intact islet. (G) Plasma membrane potential (Vm) and AP firing in low glucose conditions in Min6 cells. A representative figure is presented (left). Bar graphs (right) represent average electrical activities 30 seconds after FKN application. (H) Vm and AP firing in high glucose conditions in Min6 cells. Min6 cells were incubated in 2 mM glucose for 16 h and challenged by high glucose. 5 minutes after, FKN was applied. A representative figure from 7 independent experiments (left). n = 8 (17 mM glucose) and n = 7 (17 mM glucose + FKN condition). *P < 0.05. (I) VDCC currents activated by voltage steps (insets). A representative recording from four measurements. (J) Time course measurement of VDCC currents at +20 mV. n = 4. Throughout the figure, values in bar graphs represent mean ± SEM. See also Supplemental Figure 4.

Figure 5

Figure 5. Electrophysiology studies in α cells.

(A) Glucagon secretion in primary mouse islets. Islets were incubated in 1 mM glucose medium for 16 hours and washed. Islet glucagon (Gcg) release was measured after incubation with insulin (100 nM) or FKN (100 or 500 ng/ml) for 1 hour. Mean ± SEM; n = 5 for control and 6 for other groups. *P < 0.05 vs. lane 1; **P < 0.01 vs. lane 1; #P < 0.05 vs. lane 2; 1-way ANOVA. (B) Glucagon secretion in αTC1 cells. αTC1 cells were incubated in 1 mM glucose medium for 16 hours and washed. Glucagon release was measured after incubation of the cells with FKN for 1 hour. Mean ± SEM; n = 3 (lane 4) or 7 (lane 1–3). *P < 0.05 vs. lane 1; #P = 0.051 vs. lane 1; 1-way ANOVA. (C) IHC analysis of CX3CR1 expression in primary WT mouse islets. Images were obtained at ×20 magnification. (D) Flow cytometry analysis of CX3CR1 expression in primary α and β cells in dispersed islet cells. Right lower box in the graph is for glucagon+/insulin– subset. Left upper box is for the glucagon–/insulin+ subset. (E) KATP currents in the presence or absence of FKN (100 ng/ml) in αTC1 cells. (F) KATP currents in the presence or absence of FKN (100 ng/ml) in primary α cells. (G) Membrane potential (Vm) and AP firing before and after FKN (100 ng/ml) treatment in αTC1 cells. Cells were incubated in 0.5 mM glucose medium, and depolarization of membrane potential (ΔVm), average AP peak, and AP frequency upon FKN treatment were analyzed. Ctl, control (before FKN treatment). Mean ± SEM; n = 7. **P < 0.01; 2-tailed unpaired t test. (H) Intracellular Ca2+ levels in the presence (n = 47) or absence (n = 31) of FKN. Lowering extracellular glucose concentration from 10 to 0.5 mM triggered Ca2+ rise in αTC1 cells. ***P < 0.01; 1-way ANOVA. See also Supplemental Figure 5.

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