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Non-invasive Cell Type Selective in Vivo Monitoring of Insulin Resistance Dynamics

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Non-invasive Cell Type Selective in Vivo Monitoring of Insulin Resistance Dynamics

Meike Paschen et al. Sci Rep.

Abstract

Insulin resistance contributes to the development of cardio-vascular disease and diabetes. An important but unresolved task is to study the dynamics of insulin resistance in selective cell types of insulin target tissues in vivo. Here we present a novel technique to monitor insulin resistance dynamics non-invasively and longitudinally in vivo in a cell type-specific manner, exemplified by the pancreatic β-cell situated within the micro-organ the islet of Langerhans. We utilize the anterior chamber of the eye (ACE) as a transplantation site and the cornea as a natural body-window to study the development and reversibility of insulin resistance. Engrafted islets in the ACE that express a FoxO1-GFP-based biosensor in their β-cells, report on insulin resistance measured by fluorescence microscopy at single-cell resolution in the living mouse. This technique allows monitoring of cell type specific insulin sensitivity/resistance in real-time in the context of whole body insulin resistance during progression and intervention of disease.

Conflict of interest statement

P.-O. Berggren is cofounder of Biocrine AB, S.J. is employed by Biocrine AB, I.B.L. and B.L. are consultants for Biocrine AB.

Figures

Figure 1
Figure 1. Cell-type selective analysis of insulin resistance in the islet of Langerhans.
(a) Schematic illustration of β-cell insulin resistance biosensor (βIRB). β-cell specific expression is ensured by the rat-insulin 1 promoter (RIP1). FoxO1 was mutated to FoxO1(H215R) to avoid DNA-binding and coupled to GFP for its detection. 3Tomato was introduced as reference signal for identification of transduced cells. An IRES-element ensures stoichiometric expression of the two fluorescent indicators under the same promoter. (b) MIN6 cells and pancreatic islets are transduced with βIRB-encoding adenoviruses. Upon excitation with 488 nm, the fluorescent indicator GFP is identified by laser-scanning fluorescent confocal microscopy. FoxO1(H215R)GFP is cytoplasmic under normal conditions and shuttles into the nucleus at insulin resistance. c) For the analysis of in vitro and in vivo imaging data, three regions of interest (nucleus, cytoplasm and background) were placed for every cell. The obtained intensities were used for calculation of a ratio. The ratios were categorized into two groups: 1) Ratios < 1, where less FoxO1(H215R)GFP is present in the nucleus than in the cytoplasm (=insulin sensitive) and 2) Ratios ≥ 1, where equal/more FoxO1(H215R)GFP is present in the nucleus compared to the cytoplasm ( = insulin resistant). The percentage of insulin resistant cells was calculated for each animal and for each experimental condition.
Figure 2
Figure 2. In vitro characterization of βIRB in MIN6 cells.
Transduced MIN6 cells under (a) normal culture conditions or (b) after treatment with the Akt-inhibitor (n = 50). (c) β-cell insulin resistance in transduced MIN6 cells after Akti-1/2 treatment in a dose-dependent matter (n = 3 experiments). (d) β-cell insulin resistance in transduced MIN6 cells treated with the Akt2 inhibitor Akti-2 (n = 3 experiments). (e) β-cell insulin resistance in transduced MIN6 cells treated with IGF-1R inhibitor (IGF1R1) and insulin receptor tyrosine kinase inhibitor HNMPA(AM)3 (HNMPA) (n = 3 experiments). Data are shown as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars, 5 μm.
Figure 3
Figure 3. In vitro characterization of βIRB in pancreatic islets.
(a) Representative single focal plane of an isolated islet, transduced with βIRB-encoding adenovirus and immuno-stained for insulin (n = 10). (b) Representative maximum projection of an isolated mouse islet transduced with βIRB imaged before and after incubation with Akti-1/2 (n = 3). Change in nuclear localization of FoxO1(H215R)GFP is shown in blow-ups and indicated by an arrow. (c) Representative maximum projection of an isolated mouse islet transduced with βIRB and imaged after treatment with HNMPA(AM)3. (n = 3) (d) Representative maximum projection of an isolated mouse islet transduced with βIRB and imaged after treatment with IGF-1R inhibitor (IGF1R1) (n = 3). (e) Representative maximum projection of isolated mouse islets transduced with βIRB and imaged after treatment with HNMPA(AM)3 or IGF-1R inhibitor (IGF1R1). (f) Representative maximum projection of mouse islets transduced with the biosensor and treated with palmitate for 48 h and 96 h and 48 h after transfer to RPMI after treatment (re-treatment). Nuclear FoxO1(H215R)GFP-positive cells are indicated by arrows (n = 4). (g) Effect of palmitate treatment and medium re-treatment on subcellular distribution of FoxO1(H215R)GFP (n = 3). (h) Representative single focal planes of human islets transduced with the biosensor under normal culture conditions (n = 5). (i) Representative single focal planes of human islets transduced with the biosensor after treatment with palmitate (n = 5). (j) Representative single focal planes of human islets transduced with the biosensor under normal culture conditions or after treatment with palmitate (n = 5). (k) Effect of palmitate treatment for 144 h on subcellular distribution of FoxO1(H215R)GFP in human islets (n = 5). Data are shown as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars, 30 μm.
Figure 4
Figure 4. In vivo characterization of the insulin resistance biosensor βIRB.
(a) Whole-body insulin resistance in ob-control and ob/ob mice from 1 to 12 months of age obtained by intraperitoneal insulin tolerance tests (IPITT24) and depicted as average area under the curve (AUC) of the IPITT (n = 7). **p < 0.01, ***p < 0.001. (b) Whole-body insulin resistance in ob-control and ob/ob mice at an age of 3 and 10 months obtained by IPITT (n = 7). ***p < 0.001: 3 months old ob-control vs. 3 months old ob/ob mice; +++p < 0.001: 3 months vs. 10 months old ob/ob mice; #p < 0.05: 10 months old ob-control vs. 10 months old ob/ob mice. (c) Representative single focal planes of engrafted islets in ob-control and ob/ob mice at 3 and 10 months of age. Scale bars, 30 μm. (d) In vivo measurement of β-cell insulin resistance in 3 and 10 months old ob/ob and ob-control mice. (n = 13) ***p < 0.001: 3 months old ob-control vs. 3 months old ob/ob mice; ##p < 0.01: 3 months vs. 10 months old ob/ob mice. (e) Representative single focal planes of FoxO-immunostained islets of 3 and 10 months old ob-control and ob/ob mice. Scale bars, 30 μm. (f) Subcellular distribution of endogenous FoxO1 in in situ islets obtained from the pancreas. (n = 4) ***p < 0.001: 3 months old ob-control vs. 3 months old ob/ob mice; ##p < 0.01: 3 months vs. 10 months old ob/ob. (g) Ratio of phosphoFoxO1 Ser256 (pFoxO1) and FoxO1 expression in 3 and 10 months old ob/ob and ob-control mice obtained by Western blot analysis. (n = 3) **p < 0.01: 10 months old ob-control vs. 10 months ob/ob mice; ***p < 0.001: 3 months old ob-control vs. 3 months ob/ob mice; ##p < 0.01: 3 months vs. 10 months old ob/ob mice. Data are shown as mean ± SEM. See also Supplementary Figs S2, S3 and S4.
Figure 5
Figure 5. Technical and optical characteristics of the different objectives used for in vitro and in vivo imaging.
(a) Schematic illustration of a test bead (Invitrogen, Focal Check™ fluorescence microscope test slide #1, F36909, Lot 627811) with a green fluorescent shell and a non-fluorescent core that were used to study the effect of different objectives on the core/mantle fluorescence ratio in an ideal system. (b) Mean ratios obtained when imaging test beads using different objectives, ***p < 0.001. (c) Equation for axial resolution, d, for point scan confocal microscopy where K is a scalar correction factor where for an ideal very small pinhole Kpinhole = 0.67, λ is the emission wavelength, n is the refractive index of medium (in our case 1.33 for water) and NA the numerical aperture was used to calculate the axial-resolution that can theoretically be reached with each objective (d). Data are shown as mean ± SEM, ***p < 0.001.

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