Abstract
The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.
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Data availability
All raw data for all figures are available at https://monash.figshare.com/articles/Treatment_of_type_2_diabetes_with_the_designer_cytokine_IC7Fc/9637040.
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Acknowledgements
This study was funded, in part, by the National Health & Medical Research Council of Australia (project grant 526606 and APP1156511 to M.A.F. and S.R.-J.; project grant APP1039502 to M.A.F.; development grant APP1039502 to M.A.F., T.E.A. and M.A.C.; principal research fellowship 445302 to M.A.F., senior principal research fellowship (SPRF) APP1021168 to M.A.F. and SPRF APP1116936 to M.A.F. This project was also funded, in part, by a CASS Foundation grant awarded to T.L.A. and M.A.F. and CIHR foundation grant 164321 to D.J.D. The project was also supported in part by the Victorian Government’s OIS Program. We thank F. T. Wunderlich and C. M. Wunderlich for providing reagents to generate the ROSA26-IC7Fc mouse, as well as A. Nenci and J. Gonzales from the Monash Gene Targeting Facility. M.P. was supported by a research fellowship (DFG, PA-2459/1-1). We thank J. Scoble and L. Sparrow for their help in preparing PEGylated forms of IC7, J. Bentley for help with europium assays, X. Xiao and G. Lovrecz for mammalian cell line development and scale-up, L. Pontes-Braz for protein purification and the Burnet ImmunoMonitoring Facility for conducting the human PBMC experiments. The work of S.R.-J. was funded by the Deutsche Forschungsgemeinschaft (DFG), Bonn (grant no.: SFB841, project C1; grant no.: SFB877, project A1), and by the Cluster of Excellence ‘Inflammation at Interfaces’.
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M.F. and T.L.A. conducted experiments and performed analyses described in all figures. P.G. and K.I.W. performed YAP1 experiments in Fig. 1. D.C.H., H.K., E.E., C.Y., C.E., S.R., R.S.L., M.J.K., N.A.M., L.M.T., J.S. and E.T.K. conducted experiments and performed analyses for GLP-1RA plus IC7Fc experiments. T.E.A. supplied IC7A, IC7B and IC7Fc and provided the europium binding methodology. L.C. performed western blot analyses. A.E.B. and G.J.C. conducted euglycaemic clamp experiments. G.M.K. and C.R.B. conducted tracer determined OGTT experiments. L.O. and T.J.B. conducted GSIS experiments. E.S., D.J.K. and R.L.Y. performed surgeries, experiments and analyses in GLP-1 secretion from human patient samples. L.L.B. and D.J.D. conducted experiments and performed analyses in GLP-1R knockout mice. M.A.C. co-ordinated non-human primate studies. M.P. generated the ROSA26-IC7Fc mouse. P.J.M. co-ordinated lipidomic experiments. P.A.B. conducted bone density experiments in IC7FcAlb-cre mice. J.G., C.G. and S.R.-J. made native IC7. G.K., T.E.A., T.L.A. and M.A.F. conceived and synthesized mIC7A, mIC7B and IC7Fc. M.A.F. and S.R.-J. conceived the project. M.A.F., T.L.A. and M.F. wrote the manuscript. M.A.F. co-ordinated all aspects of this work.
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M.A.F. and S.R.-J. are the inventors of IC7Fc and hold patents for this molecule (US 60/920,822; WO/2008/119110 A1). M.A.F. is the founder and CSO of Kinomedica Pty Ltd. G.K., T.L.A. and S.R.-J. have financial interests in Kinomedica Pty Ltd.
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Extended data figures and tables
Extended Data Fig. 1 IC7 can bind and signal in vitro.
a, Sequence of IC7Fc. Blue shading denote IC7 site 3 loop sequence; magenta shading denote a spacer sequence from the BssHII restriction site; grey shading denote the Fc fragment sequence; blue residues denote mutated amino acids in the Fc fragment. b, HepG2 cells were stimulated with IL-6 (left) or IC7 (right) in the absence or presence of soluble gp130 (sgp130) and low or high doses of soluble IL-6R (sIL-6R). c, Ability of IC7 to compete with europium-labelled IL-6 to bind to sgp130 and sIL-6R. d, Ability of IC7 to activate gp130 signalling and induce haptoglobin secretion. n = 3 technical replicates per condition in c and d. e, pSTAT3 treated with IC7 (lanes 2–4) and PEGylated mIC7A (30 kDa; lanes 5–7) and mIC7B (40 kDa; lanes 8–10). f, Mice were fed a HFD for 8 weeks and treated daily with IC7, mIC7A, mIC7B (1 mg kg−1) or vehicle over 8 days. The effects on glucose tolerance in an intraperitoneal glucose tolerance test on day 5 of the intervention are shown (n = 7 for all groups, except n = 6 for vehicle). Left, change in blood glucose. Right, incremental area under the curve (iAUC). P value determined by one-way ANOVA with Dunnett’s multiple comparison test. g–i, Modified and unmodified IC7 were assessed for their ability to compete with europium-labelled IL-6 to bind to the sIL-6R and sgp130 (g, i) and to induce haptoglobin secretion in HepG2 cells (h). j–l, C57BL/6 mice were fed a HFD for 4 weeks and injected intraperitoneally with a single dose of vehicle or IC7Fc in the fed state (mice were killed 30 and 120 min after injection), and STAT3 phosphorylation was analysed in liver (j), epididymal fat (k) and quadriceps muscles (l). Blots represent n = 6 mice per group. m, Pharmacokinetic profile over 72 h in plasma of mice after a single intraperitoneal dose (1 mg kg−1) of IC7 or IC7Fc. Mice were killed after 2, 6, 24, 48 or 72 h (n = 10 for IC7 and n = 25 for IC7Fc). Data are mean ± s.e.m. (except in d, data are mean ± s.d.).
Extended Data Fig. 2 Effect of IC7Fc on DIO in mice.
a, Mice received a single escalating dose of IC7Fc (low: 0.1 mg kg−1; high: 1 mg kg−1; n = 5–25) or an equal volume of vehicle (n = 5), administered either intraperitoneally (IP) or subcutaneously (SC). b, c, Liver AMPK activity (b) and mRNA expression of SREBP1c and Dgat1 (c) in DIO mice injected daily for 7 days with vehicle (n = 6) or PEGylated (PEG) IC7 (1 mg kg−1, n = 13). d–f, Sixteen-day intervention study in DIO mice fed a HFD for 8 weeks and injected every other day with vehicle (n = 7) or IC7Fc (1 mg kg−1, n = 8) for 16 days. Body composition was analysed as follows: change in total mass (top) and as percentage of starting weight (bottom) of total body mass (d), fat mass (e), and lean mass (f) during intervention. g, h, Chronically treated mice (n = 10 per treatment) were monitored in metabolic cages at 30 °C for 48 h. Oxygen consumption (VO2) recorded over 48 h during 13 days and 15 days of intervention (g) and average VO2 (h) were determined. i–l, Raw data of absolute weight changes (i), liver triacylglycerol (TAG; left) diacylglycerol (DAG; right) (j), gastrocnemius Yap1 mRNA (k) and the ratio of YAP1(Ser112) to YAP1 protein (l) during the paired-feeding study (related to Fig. 1j–l). m, Quantification of total YAP1 levels in C2C12 myotubes treated with vehicle or IC7Fc (1 ng ml−1) for 24 h after knockdown of YAP1 (siYAP) or siRNA control (ON-TARGETplus, OTP); n = 3. n, Quantification of YAP1 protein levels in liver tissue of chronically treated mice. n = 7 (Veh), n = 8 (Veh PF, IC7Fc) in j–l, n. Data are mean ± s.e.m. P values determined by: two-way ANOVA with Sidak’s multiple comparison test (a, d–f), multiple t-test using the Holm–Sidak method (b, c, h, k, m), or ordinary one-way ANOVA with Tukey’s multiple comparison test (g, j, l).
Extended Data Fig. 3 IC7Fc lowers glucose via increased insulin secretion.
a–g, Acute metabolic studies in 10–11-week-old C57BL/6 mice. a, Study design. b–d, Chow-fed mice received a single intraperitoneal dose of vehicle (n = 13) or IC7Fc (n = 14) in the fed state and blood glucose was monitored over 6 h (b). Change in plasma insulin (c) and C-peptide (d) 2 h after injection (n = 7 per treatment). e–g, DIO mice were fasted for 6 h and injected intraperitoneally with vehicle or IC7Fc 30 min before an OGTT. Mice were killed at −30, 0, 15 and 30 min, respectively. Changes in plasma blood glucose (e), insulin (f) and C-peptide (g) were determined. n = 16–25 (e), n = 15–22 (f), n = 15–23 mice (g); for details, see Source Data. h–m, Acute metabolic studies in the genetically obese leptin receptor-deficient (Leprdb/db) mouse model. h, Study design. i, Blood glucose levels in fed 7-week-old mice; n = 16 (db/+), n = 15 (db/db). j, Blood glucose levels after a single intraperitoneal dose of vehicle or IC7Fc (1 mg kg−1) in control mice (db/+) at the age of 7 weeks (left), 13 weeks (middle) and 17 weeks (right); n = 8 (per treatment). k, Body composition analysed as weight changes in total body mass (left), fat mass (middle) and lean mass (right) 10 days after a single injection in db/+ mice (n = 8). l, Change in fasting plasma insulin in db/+ mice 17 days after treatment (n = 7 per group). m, Change in fasting blood glucose in db/db (n = 8 each treatment) and db/+ mice with treated with vehicle (n = 9) or IC7Fc (n = 6), 17 days after injection. n–p, Acute studies in chemically induced model of diabetes. Six-to-seven-week-old male mice were injected intraperitoneally with either placebo (CON) or STZ (55 mg kg−1) over five consecutive days to induce diabetes (n = 20 per group). n, Study design. o, Fasting blood glucose and insulin levels in control and STZ mice 6, 10 and 18 days after induction. Left, STZ mice with fasting blood glucose below 15 mM (highlighted in blue) were excluded from the study. Right, corresponding fasting insulin in control and STZ mice (n = 17) 10 days after induction. p, Change in plasma insulin over 42 h; n = 10 CON (all groups), n = 9 STZ (Veh) and 8 STZ (IC7Fc). Data are mean ± s.e.m. P values were determined by: two-way ANOVA with Sidak’s multiple comparison test (b, e–g, j; IC7Fc versus Veh at indicated time points); two-tailed unpaired Mann–Whitney U-test (c), Student’s two-tailed unpaired t-test (d, i, k–m, o); ordinary two-way ANOVA with Tukey’s multiple comparison test for main treatment effect (p). ****P < 0.0001 for STZ (Veh) versus CON (Veh), STZ (Veh) versus CON (IC7Fc), STZ (IC7Fc) versus CON (Veh), STZ (IC7Fc) versus CON (IC7Fc); §P = 0.0062 for STZ (Veh) versus STZ (IC7Fc); ‡P = 0.0104 for CON (Veh) versus CON (IC7Fc).
Extended Data Fig. 4 IC7Fc suppresses glucose production and increases glucose disposal.
a–d, Stable isotope tracer studies in DIO mice treated with vehicle or IC7Fc (1 mg kg−1) 30 min before an OGTT using d-[6,6′-2H2]glucose (n = 8 per group). Blood glucose (a, left) and iAUC (a, right), proportion of blood glucose from hepatic glucose production (b), the ratio of exogenous (tracer) and endogenous (hepatic) glucose (that is, tracer enrichment) (c), and d-[6,6′-2H2]glucose (d) during the OGTT. e, GIR during an euglycaemic–hyperinsulinaemic clamp in vehicle- or IC7Fc-injected mice (n = 7 each group). f–n, Euglycaemic clamp (EC) studies in DIO and chow-fed mice with intravenous bolus injections of vehicle or IC7Fc in the presence and absence of constant octreotide infusion (7.5 μg kg−1 min−1). f, Study design. g, Experimental protocol. h, Insulin levels in the basal period of the clamp before and after octreotide or vehicle infusion (n = 12). i–n, Euglycaemic clamp experiments in 14–16-week-old chow-fed mice; n = 5 (Veh), n = 9 (IC7Fc), n = 4 (Oct Veh), n = 8 (Oct IC7Fc). i, j, Blood glucose levels (i) and GIR (j) over time (left) and before and after clamp (right). k, l, Change in HGP (k) and glucose turnover (R; l) during the euglycaemic clamp. Data are normalized and expressed as percentage of basal levels. m, Glucose uptake (Rg’) in BAT; n = 3 (Veh), n = 6 (IC7Fc), n = 4 (Oct Veh), n = 7 (Oct IC7Fc). n, Insulin levels throughout the clamps. Data are mean ± s.e.m. P values were determined by: two-way ANOVA with Sidak’s multiple comparison test (a–d, g, i (right; between basal and clamp); k, l, n (between groups at time points)); multiple comparison test using the Holm–Sidak method (Veh versus Oct −30 and 0 min in h); two-way ANOVA with Tukey’s multiple comparison test (main treatment effect in i (left) and j (left)); ordinary one-way ANOVA with Tukey’s multiple comparisons test (i (right; between groups)); nonparametric Kruskal–Wallis test and Dunn’s multiple comparison test (m).
Extended Data Fig. 5 Effect of IC7Fc on insulin secretion and hormonal milieu.
a, GSIS in pancreatic islets isolated from 10-week-old normal chow-fed mice. Stimulation with saline (control) or IC7Fc (30 or 100 ng ml−1), in the presence of 2 or 20 mM glucose. n = 3 replicates in three independent experiments. b–d, DIO mice intraperitoneally injected daily with vehicle or IC7Fc (1 mg kg−1) over 8 days in week 8 during high-fat feeding (n = 10 mice per group). Mice were killed in the fed state 2 h after the last intraperitoneal injection. b, c, Pancreatic islets were isolated from DIO mice that had been injected daily. Insulin and glucagon content (b) and active GLP-1 (c) were determined in isolated islets from five mice per group; b, left, three to four technical replicates; right, two independent experiments; c, two technical replicates except one Veh mouse. d, Blood glucose, insulin, C-peptide, glucagon, total and active GIP (1–42); n = 10 (Veh), n = 10 (IC7Fc), except n = 9 (Veh) for insulin and active GIP, n = 8 (Veh) for C-peptide. P values determined by: multiple two-tailed t-test (a); ordinary one-way ANOVA followed by Sidak’s multiple comparisons test (b (left)); Student’s two-tailed unpaired t-test (b (right), c and d); or two-tailed unpaired Mann–Whitney U-test (d, glucagon).
Extended Data Fig. 6 Generation and phenotype of mice overexpressing IC7Fc.
a, Schematic describing the generation of ROSA26-IC7Fc mice. The panel shows (1) ROSA26 wild-type locus; (2) ROSA26-IC7Fc targeting vector; and (3) ROSA26 locus after homologous recombination. b–d, Eight-week-old male mice that overexpress liver-specific IC7Fc (IC7FcAlb-cre) and littermate controls (IC7FcWT) were killed in the fed state. IC7Fc expression was determined by western blots in different tissues (b) and circulating IC7Fc levels in plasma were detected by ELISA (c); n = 4 (WT), n = 9 (Alb). d, STAT3 phosphorylation in liver tissues from IC7FcAlb-cre mice. e–i, Eight-week-old IC7FcAlb-cre and control (IC7FcWT) mice fed a chow diet until 19 weeks of age. e, Growth curve as change in total body mass (left), fat mass (middle) and lean mass (right) of chow-fed IC7FcAlb-cre mice (n = 9) and littermate controls (n = 5). f, Fasting blood glucose (left) and insulin levels (right) in 8-week-old mice; left, n = 10 and 12, and right, n = 5 and 9 for WT and Alb mice, respectively. g, Glucose levels during an OGTT shown as time course (left) and expressed as iAUC (right) in fasted mice; n = 5 (WT), n = 8 (Alb). h, i, Plasma levels of NEFA and glucagon in 19-week-old transgenic mice; n = 5 and 9 (h) and n = 8 and 19 (i) for WT and Alb mice, respectively. j–t, Differences between 7-week-old male IC7FcMCK-cre mice (MCK, n = 8)—which predominantly overexpress muscle-specific IC7Fc (mice were kept at 30 °C holding temperature)—and littermate control mice (WT, n = 5), before the start of diet intervention. j, Total body mass, fat mass and lean mass. k, Fasting blood glucose. l, Glucose levels during an OGTT shown as time course (left) and expressed as iAUC (right). m, Fasting insulin levels after a 4-week HFD in IC7FcMCK-cre mice (n = 6) and littermate controls (n = 5). n–t, Eight-week-old IC7FcMCK-cre mice (n = 5) and littermate controls (n = 7) fed a HFD for 6 weeks. Total body mass (n), fat mass (o) and lean mass (p). Fasting blood glucose levels (q, left), insulin levels (q, right), glucose during an OGTT (r) and plasma insulin (s) after a HFD. t, Difference in weight of tibialis anterior (TA) of mice at cull at 14 weeks of age. u, Normal chow-fed 26-week-old male IC7FcMCK-cre and littermate mice were kept at 22 °C holding temperature (n = 4 per genotype) and femurs were analysed as follows: Percentage of BV/TV (left) and quantification of trabecular number (right). Data are mean ± s.e.m. P values determined by: Student’s two-tailed unpaired t-test (c, f (right), g–m, q (right), r, t, u); two-tailed unpaired Mann–Whitney U-test (f (left) and q (left)); two-way ANOVA with Sidak’s multiple comparisons test (e, g, l, n–p, r, s).
Extended Data Fig. 7 IC7Fc reverses some of the unwanted side effects of GLP-1RA treatment.
Effects of a single injection of vehicle, IC7Fc (1 mg kg−1), dulaglutide (GLP-1RA; 0.5 mg kg−1) or a combination of the latter (IC7Fc + GLP-1RA) on body mass, energy homeostasis and metabolism in DIO mice; n = 10 mice per treatment unless specified otherwise. a, Study design. b, Percentage change in fat mass (left) and lean mass (right) 5–7 days after injection (n = 8 per group, except n = 7 for GLP-1RA). c, Glycaemia in fed mice shown as mean blood glucose (left) and as stratified data points (right) in mice after acute injection over time. d, Circulating plasma glucagon at cull 6 h after injection. e–h, Oxygen consumption (VO2, e) carbon dioxide production (VCO2, f), respiratory exchange ratio (RER, g) and total energy expenditure (TEE, h). i, Physical activity of mice analysed as percentage of time spent voluntary walking and presented as average of a 12-h dark or light phase and 24-h recording period. j, Physical activity as average value of measurements made within the 8 h of the first dark phase flanking 07:00. Data are mean ± s.e.m. P values determined by ordinary one-way ANOVA with Dunnett’s multiple comparisons test (b, d); two-way ANOVA with Dunnett’s multiple comparison test (c); or multiple two-tailed t-tests (e–j).
Extended Data Fig. 8 Safety profile of IC7Fc.
a–g, Acute study in non-human primates (cynomolgus macaques); n = 3 unless specified otherwise. a, Study design. b–m, Markers of inflammation in blood samples 24 h and 7 days after treatment were analysed as follows: changes in C-reactive protein (CRP, b), creatine kinase (CK, c, n = 2 for 24 h Veh), in alanine aminotransferase (ALT, d, n = 2 for 24 h Veh), aspartate aminotransferase (AST, e, n = 2 for 24 h Veh), white blood cell count (WCC, f), lymphocytes (g), monocytes (h), neutrophils (i), platelets (j), cholesterol (k), urea (l) and gamma-glutamyltransferase (GGT, m). o–x, Human PBMCs were cultured for 48 h with medium, IC7Fc (3 μg ml−1) or PHA (5 μg ml−1); n = 9. Supernatant was analysed as follows: TNF (n), IL-1β (o), IL-6 (p), MCP-1, q), IL-2 (r), IL-4 (s), IL-8 (t), IL-17α (u), hepatocyte growth factor (HGF, v), nerve growth factor (NGF, w) and leptin (x). P values were determined by: two-way ANOVA (b, f–m) or mixed effects model (c–e), with Sidak’s multiple comparison test for differences between treatment at time points, and Tukey’s multiple comparison test to detect differences in-between time points for one condition; Student’s two-tailed unpaired t-test (q, s–x); or non-parametric two-tailed unpaired Mann–Whitney U-test (all other panels) comparing IC7Fc and medium only. See Source Data for PHA.
Extended Data Fig. 9 Graphical summary of the metabolic actions of IC7Fc.
IC7Fc has several beneficial actions on metabolic processes.
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Findeisen, M., Allen, T.L., Henstridge, D.C. et al. Treatment of type 2 diabetes with the designer cytokine IC7Fc. Nature 574, 63–68 (2019). https://doi.org/10.1038/s41586-019-1601-9
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DOI: https://doi.org/10.1038/s41586-019-1601-9
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