Global, neuronal or β cell-specific deletion of inceptor improves glucose homeostasis in male mice with diet-induced obesity

Abstract

Insulin resistance is an early complication of diet-induced obesity (DIO)¹, potentially leading to hyperglycaemia and hyperinsulinaemia, accompanied by adaptive β cell hypertrophy and development of type 2 diabetes². Insulin not only signals via the insulin receptor (INSR), but also promotes β cell survival, growth and function via the insulin-like growth factor 1 receptor (IGF1R)^3-6. We recently identified the insulin inhibitory receptor (inceptor) as the key mediator of IGF1R and INSR desensitization⁷. But, although β cell-specific loss of inceptor improves β cell function in lean mice⁷, it warrants clarification whether inceptor signal inhibition also improves glycaemia under conditions of obesity. We assessed the glucometabolic effects of targeted inceptor deletion in either the brain or the pancreatic β cells under conditions of DIO in male mice. In the present study, we show that global and neuronal deletion of inceptor, as well as its adult-onset deletion in the β cells, improves glucose homeostasis by enhancing β cell health and function. Moreover, we demonstrate that inceptor-mediated improvement in glucose control does not depend on inceptor function in agouti-related protein-expressing or pro-opiomelanocortin neurons. Our data demonstrate that inceptor inhibition improves glucose homeostasis in mice with DIO, hence corroborating that inceptor is a crucial regulator of INSR and IGF1R signalling.

Fig. 1. Whole-body inceptor deletion improves glucose metabolism in male mice with DIO.

a, Inceptor immunoreactivity in whole brain, pituitary and pancreas of 10-week-old male C57BL/6J inceptor Iir^+/+ (WT) and Iir^−/− (KO) mice. Scale bars, 1 mm for brain, 200 µm for pituitary and 20 µm for pancreas. b,c, Food intake (b) in 28-week-old and body weight (c) in 10- to 30-week-old male C57BL/6J WT and KO mice (n = 8 for each genotype). d,e, Intraperitoneal glucose tolerance (d) and area under the curve (AUC) (e) in 19-week-old male C57BL/6J WT and KO mice (n = 7 WT, n = 8 KO). f,g, Intraperitoneal insulin tolerance (f) and AUC (g) in 23-week-old male C57BL/6J WT and KO mice (n = 8 for each genotype). h,i, Fasting levels of plasma insulin (n = 8 for each genotype) in 19-week-old male C57BL/6J WT and KO mice (n = 8 for each genotype) (h) and levels of HbA1c in 27-week-old male C57BL/6J WT and KO mice (n = 8 for each genotype) (i). j, Glucose-stimulated insulin secretion in 27-week-old male C57BL/6J WT and KO mice (n = 8 for each genotype). k,l, Western blots of liver protein of 30-week-old male C57BL/6J WT and KO mice treated with a single dose of saline or 1 mU per kg of insulin (n = 4 for each group) (k) and densitometric quantification (l). m,n, Pancreatic α cell (m) and β cell (n) mass in 30-week-old male C57BL/6J WT and KO mice (n = 5 for each genotype). o,p, Fasting plasma levels of glucagon (o) and blood glucose (p) in 19-week-old male C57BL/6J WT and KO mice (n = 8 for each genotype). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Data are presented as mean ± s.e.m. Data in b, e, g–i and l–p were analysed by two-sided, two-tailed Student’s t-test. Data in c, d, f and j were analysed by two-way ANOVA with Bonferroni’s post hoc, multiple-comparison test for the different time points. P values for group differences are: P = 0.0049 (c), P = 0.0082 (d), P = 0.010 (e), P = 0.0098 (i), P = 0.0021 (j), P = 0.002 (WT) and P = 0.0016 (KO) (l) and P = 0.0232 (o). P values for the multiple-comparison tests are provided in Source data. Corr., corrected; vhcl, vehicle. Source data

Fig. 2. Central inceptor immunoreactivity is restricted to neurons, including those regulating energy and glucose metabolism.

a,b, Representative image (a) and quantification (b) of inceptor immunoreactivity in hypothalamic nuclei of 18- to 24-week-old male C57BL/6J WT mice (n = 30 mice for ARC and DMH, n = 28 mice for VMH, n = 19 mice for PVN). Scale bar, 200 μm. c, Inceptor colocalization with NeuN, Pomc–green fluorescent protein (GFP), Npy–GFP, Aldhl1–GFP, Gfap and Iba1 in the hypothalamus of 28-week-old male C57BL/6J WT mice. Scale bars, 50 μm. d, Inceptor immunoreactivity in 28-week-old chow- versus HFD-fed male C57BL/6J WT mice (n = 5 for all regions except PVN: n = 3). e, Hypothalamic inceptor messenger RNA expression in 10-week-old male C57BL/6J WT mice fed with either chow or HFD (n = 4 per group). f, HOMA-IR in 14-week-old C57BL/6J WT mice fed with either chow or HFD (n = 8). ^*P < 0.05, Data are presented as mean ± s.e.m. Data in b and e were analysed by one-way ANOVA, data in d were analysed by two-way ANOVA and data in f were analysed using the Kruskal–Wallace test. P values for group differences are: P < 0.0001 (ARC versus VMH), P < 0.0001 (ARC versus DMH), P = 0.0046 (ARC versus PVN), P = 0.0014 (VMH versus DMH), P < 0.0001 (VMH versus PNV), P < 0.0001 (DMH versus PVN) (b), P = 0.0156 (d), P = 00137 (e), P = 0.0217 (chow versus HFD 14 d) and P = 0.0232 (chow versus HFD 28 d) (f). A more detailed statistical report is provided in Source data. Rel., relative. Source data

Fig. 3. Neuronal loss of inceptor improves glucose metabolism without affecting body weight in male mice with DIO.

a, Inceptor immunoreactivity in the hypothalamus of 11-week-old male C57BL/6J Nestin Cre^+/−Iir^wt/wt (WT) and Nestin Cre^+/−Iir^flx/flx (KO) mice. Scale bars, 200 μm. b–f, Body weight (b), cumulative food intake (c), as well as fat mass (d) and lean tissue mass (e) and fasting plasma levels of blood glucose (f), in 26-week-old male C57BL/6J WT and KO mice (n = 8 for each genotype) on an HFD. g,h, Intraperitoneal glucose tolerance (g) and AUC (h) in 27-week-old male C57BL/6J WT and KO mice (n = 8 for each genotype). i,j, Intraperitoneal insulin tolerance (i) and AUC (j) in 28-week-old male C57BL/6J mice on an HFD (n = 6 WT, n = 5 KO). k,l, Fasting levels of insulin in 27-week-old and 32-week-old C57BL/6J WT and KO mice (n = 8 for each group) (k) and fasting levels of glucagon in 27-week-old and 32-week-old C57BL/6J WT (n = 6) and KO mice (n = 5) (l). m,n, Pancreatic α cell (m) and β cell (n) mass in 36-week-old C57BL/6J WT and KO mice (n = 6 WT, n = 5 KO). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Data are presented as mean ± s.e.m. Data in d, e, h, j, k, l, m and n were analysed by two-sided, two-tailed Student’s t-test, data in f were analysed using the Mann–Whitney U-test and data in b, c, g and i were analysed by two-way ANOVA with Bonferroni’s post hoc comparison for individual time points. P values for group differences are P < 0.0169 (e), P = 0.0123 (g) and P = 0.0074 (h). A more detailed statistical report and the P values for the multiple-comparison test (g) are provided in Source data. Source data

Fig. 4. DIO male mice with adult-onset, β cell-specific inceptor deletion show improved glucose metabolism.

a, Inceptor immunoreactivity in the pancreas of male 26-week-old Ins1Cre^ERT−/−Iir^flx/flx (WT) and Ins1Cre^ERT+/−Iir^flx/flx (KO) C57BL/6J mice. Scale bars, 20 μm. b–e, Body weight (b), as well as lean (c) and fat (d) tissue mass and fasting glucose (e) in 30-week-old male C57BL/6J WT and KO mice (n = 8 for each genotype). Tam, Tamoxifen. f,g, Intraperitoneal glucose tolerance (f) and AUC (g) in 18-week-old male C57BL/6J WT and KO mice on an HFD (n = 8 for each genotype). h,i, Intraperitoneal insulin tolerance (h) and AUC (i) in 18-week-old male C57BL/6J WT and KO mice on an HFD (n = 10 for each genotype). j,k, Fasting levels of plasma insulin (n = 10 for each genotype) (j) and glucagon (k) (n = 10 for each genotype) in 22-week-old male C57BL/6J WT and KO mice. l,m, Pancreatic α cell mass (l; n = 5 each genotype) and β cell mass (m; n = 6 WT, n = 5 KO) in 26-week-old male C57BL/6J WT and KO mice. n, Whole-blood HbA1c in 18-week-old male C57BL/6J WT and KO mice (n = 10 mice per genotype). ^*P < 0.05, ^**P < 0.01. Data are presented as mean ± s.e.m. Data in c, d, e, g, l, j, k, l, m and n were analysed by two-sided, two-tailed Student’s t-test, data in b, f and h were analysed by two-way ANOVA with Bonferroni’s post hoc comparison for individual time points and data in n were analysed using Mann–Whitney U-test. P values for group differences are P < 0.0426 (f), P = 0.0322 (g), P = 0.0382 (h), P = 0.0379 (i), P = 0.0142 (m) and P = 0.002 (n). A more detailed statistical report and the P value for the multiple-comparison test (f) are provided in Source data. Source data

Extended Data Fig. 1. Proteome analysis of liver and muscle of whole body inceptor KO vs. WT mice.

Principle component analysis (PCA) of all tissue samples (a). Clustering of quadriceps muscle (left), liver (middle) and hypothalamus (right) samples as well as z-scored protein intensities. Bar beneath the clustering indicates genotype (WT: white, KO: red) (n = 3 each genotype for liver, n = 3 WT, n = 2 KO for muscle) (b). Volcano plots of the same organs as in (b) ProteinGroups only found in one condition are indicated in black (c) (n = 3 each genotype for liver, n = 3 WT, n = 2 KO for muscle). Source data

Extended Data Fig. 2. Proteome analysis of liver, muscle, and hypothalamus of neuronal inceptor KO vs. WT mice.

Principle component analysis (PCA) of all tissue samples (a). Clustering of quadriceps muscle (left) and liver (right) samples as well as z-scored protein intensities. Bar beneath the clustering indicates genotype (WT: white, KO: red) (n = 3 each genotype) (b). Volcano plots of the same organs as in (b) ProteinGroups only found in one condition are indicated in black (c) (n = 3 each genotype. Source data

Extended Data Fig. 3. AgRP or POMC neurons do not mediate neuronal effects of inceptor on glucose homeostasis.

Inceptor immunoreactivity in the hypothalamus of 20 week old C57BL/6J Agrp-Cre^+/− Iir^wt/wt (WT) and Agrp-Cre^+/− Iir^flx/flx mice (KO). Scale bar is 100 µm (a). b–e, Body weight (b), as well as lean (c) and fat (d) tissue mass and fasting glucose (e) in 30 week old C57BL/6J WT and KO mice (n = 9 WT, n = 10 KO). f,g, Intraperitoneal glucose tolerance (f) and area under the curve (AUC) (g) in 30 week old male C57BL/6J WT and KO mice on a HFD (n = 8 WT, n = 9 KO). h,i, Intraperitoneal insulin tolerance (h) and AUC (i) in 31 week old male mice on a HFD (n = 8 WT, n = 9 KO). j, Inceptor immunoreactivity in the hypothalamus of 20 week old C57BL/6J Pomc-Cre^+/− Iir^wt/wt (WT) and Pomc-Cre^+/− Iir^flx/flx (KO) mice. Scale bar is 50 µm. k–n, Body weight (k), as well as lean (l) and fat (m) tissue mass and fasting glucose (n) in 30 week old male C57BL/6J WT and KO mice on a HFD (n = 9 each genotype). o,p, Intraperitoneal glucose tolerance (o) and area under the curve (AUC) (p) in 30 week old male C57BL/6J WT and KO mice on a HFD (n = 9 each genotype). q,r, Intraperitoneal insulin tolerance (q) and area under the curve (AUC) (r) in 31 week old C57BL/6J WT and KO mice (n = 9 each genotype). Asterisks indicate ** p < 0.01. Data present mean ± SEM. Data in panel C, E, G, I, L, M, N, P, R were analyzed by students two-sided, two-tailed test. Data in panel D were analyzed using the Mann-Whitney Test. Data in panel B, F, H, K, O, Q were analyzed by two-way ANOVA with Bonferroni post-hoc comparison for individual time points. P-value for group differences is p < 0.00377 (E). Corr., corrected. Source data

Extended Data Fig. 4. Proteome analysis of liver and muscle of adult-onset β-cell-specific inceptor KO vs. WT mice.

Principle component analysis (PCA) of all tissue samples (a). Clustering of quadriceps muscle (left) and liver (right) samples as well as z-scored protein intensities. Bar beneath the clustering indicates genotype (WT: white, KO: red) (n = 4 KO, n = 5 WT for muscle, n = 5 each genotype for liver) (b). Volcano plots of the same organs as in (b) ProteinGroups only found in one condition are indicated in black (c) (n = 5 each genotype). Source data