Diet-induced β-cell insulin resistance results in reversible loss of functional β-cell mass

Abstract

Although convincing in genetic models, the relevance of β-cell insulin resistance in diet-induced type 2 diabetes (T2DM) remains unclear. Exemplified by diabetes-prone, male, C57B1/6J mice being fed different combinations of Western-style diet, we show that β-cell insulin resistance occurs early during T2DM progression and is due to a combination of lipotoxicity and increased β-cell workload. Within 8 wk of being fed a high-fat, high-sucrose diet, mice became obese, developed impaired insulin and glucose tolerances, and displayed noncompensatory insulin release, due, at least in part, to reduced expression of syntaxin-1A. Through reporter islets transplanted to the anterior chamber of the eye, we demonstrated a concomitant loss of functional β-cell mass. When mice were changed from diabetogenic diet to normal chow diet, the diabetes phenotype was reversed, suggesting a remarkable plasticity of functional β-cell mass in the early phase of T2DM development. Our data reinforce the relevance of diet composition as an environmental factor determining different routes of diabetes progression in a given genetic background. Employing the in vivo reporter islet-monitoring approach will allow researchers to define key times in the dynamics of reversible loss of functional β-cell mass and, thus, to investigate the underlying, molecular mechanisms involved in the progression toward T2DM manifestation.-Paschen, M., Moede, T., Valladolid-Acebes, I., Leibiger, B., Moruzzi, N., Jacob, S., García-Prieto, C. F., Brismar, K., Leibiger, I. B., Berggren, P.-O. Diet-induced β-cell insulin resistance results in reversible loss of functional β-cell mass.

Keywords: imaging; biosensor; diabetes mellitus; diet intervention; fluorescence microscopy.

Figure 1

The combination of HFD and HSD, but not HFD and HFrD, leads to β-cell insulin resistance. A) Body weight of mice fed a control diet, HFHSD, or HFHFrD (n = 5–11). B) Glucose tolerance obtained by IPGTT and depicted as AUC of the IPGTT in mice fed a control diet, HFHSD, or HFHFrD (n = 5–10). C) Whole-body insulin tolerance obtained by IPITT and depicted as AUC of the IPITT in mice fed a control diet, HFHSD, or HFHFrD (n = 5–9). D) Liver insulin tolerance obtained by IPPTT and depicted as AUC of the IPPTT in mice fed a control diet, HFHSD, or HFHFrD (n = 5–8). E) Relative β-cell insulin resistance measured using the β-cell insulin resistance biosensor (βIRB) in islets transplanted into the ACE in mice fed a control diet, HFHSD, or HFHFrD (n = 4–14). *P < 0.05,**P < 0.01, ***P < 0.001. Data are expressed as means ± sem. See also Supplemental Fig. S1.

Figure 2

Verification of β-cell insulin resistance in HFHSD-fed mice. A) In vivo representative image of single-focal planes of engrafted islets transduced with βIRB in mice fed a control diet or HFHSD at 8 wk of diet treatment. In situ representative image of immunostaining of endogenous FoxO1 in situ in islets from pancreas sections from mice fed a control diet or HFHSD for 8 wk. Scale bars, 30 µm. B) Relative β-cell insulin resistance indicated by immunostaining of endogenous FoxO1 in situ in islets from pancreas sections from mice fed a control diet or HFHSD for 8 wk. C–E) Western blot quantification of pY-IR/IR, p-Akt/Akt, and p-FoxO1/FoxO1 in isolated islets from mice fed a control diet or HFHSD for 8 wk (n = 11–12). Representative Western blots for 3 control diet and HFHSD mice are shown. **P < 0.01, ***P < 0.001. Data are expressed as means ± sem. See also Supplemental Fig. S2.

Figure 3

The combination of lipotoxicity and high β-cell workload leads to β-cell insulin resistance. A) Body weight of mice fed a control diet, HFHSD, HSD, or HFD (n = 4–12). B) Glucose tolerance obtained by IPGTT and depicted as AUC of the IPGTT in mice fed a control diet, HFHSD, HSD, or HFD (n = 4–12). C) Whole-body insulin tolerance obtained by IPITT and depicted as AUC of the IPITT in mice fed a control diet, HFHSD, HSD, or HFD (n = 4–9). D) Liver insulin tolerance obtained by IPPTT and depicted as AUC of the IPPTT in mice fed a control diet, HFHSD, HSD, or HFD (n = 5–8). E) Relative β-cell insulin resistance measured by βIRB in islets transplanted into the ACE in mice fed a control diet, HFHSD, HSD, or HFD (n = 3–14). *P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as means ± sem. See also Supplemental Fig. S1.

Figure 4

Measurement of functional β-cell mass. A) Schematic illustration of βFLUOMETRI. Although all cells express Cerulean under the CMV promoter, all endocrine cells express EGFP under the rat β-cell-active glucokinase promoter fragment bGK-278, and only β cells express DsRed2 under the insulin promoter fragment RIP1-410. Cerulean under the CMV promoter was used for normalization of fluorescence changes in EGFP under bGK-278 and DsRed2 under RIP1-410. B) Schematic illustration of the metabolic response elements within RIP1-410 and bGK-278. Elements highlighted in green are glucose and insulin responsive; that in yellow is glucose responsive, and NFAT and CRE are Ca²⁺ responsive. C) Representative image of an islet expressing βFLUOMETRI (n = 6). Scale bars, 50 µm. D) Changes in EGFP, DsRed2, and Cerulean (CFP) fluorescence after glucose stimulation in islets in vitro (n = 6). E) Promoter activation after glucose stimulation in vitro. DsRed2 and EGFP fluorescence intensities were normalized to CFP fluorescence intensity (n = 6). F) Activation of RIP1-410 and bGK-278 after glucose and saline injection in vivo (n = 5). ***P < 0.001. Data are expressed as means ± sem. See also Supplemental Fig. S3.

Figure 5

βFLUOMETRI reveals loss of functional β-cell mass in response to HFHSD and the regaining of function upon recovery from the diet. A) Body weight of B6 mice before, during, and after feeding of a control diet or HFHSD (n = 5–10). B) Glucose tolerance obtained by IPGTT and depicted as the AUC of the IPGTT in B6 mice before, during, and after feeding of a control diet or HFHSD (n = 5–10). C) Whole-body insulin resistance obtained by IPITT and depicted as the AUC of the IPITT in B6 mice before, during, and after feeding of a control diet or HFHSD (n = 5–10). D) Relative β-cell insulin resistance indicated by βIRB in islets transplanted into the ACE in mice before, during, and after feeding of a control diet or HFHSD (n = 12–20). E) RIP1-410 promoter activation indicated by the βFLUOMETRI and obtained in vivo in transduced islets transplanted into the ACE of B6 mice before, during, and after feeding of a control diet or HFHSD (n = 5–10). F) bGK-278 promoter activation indicated by the βFLUOMETRI and obtained in vivo in islets transplanted into the ACE of B6 mice before, during, and after feeding of a control diet or HFHSD (n = 5–10). G) Proportion of glucose-responsive cells indicated by RIP1-410 of the βFLUOMETRI and obtained in vivo in islets transplanted into the ACE of B6 mice before, during, and after feeding of a control diet or HFHSD (n = 5–10). H) Proportion of glucose-responsive cells indicated by bGK-278 of the βFLUOMETRI and obtained in vivo in islets transplanted into the ACE of B6 mice before, during, and after feeding of a control diet or HFHSD (n = 5–10). *P < 0.05, **P < 0.01, ***P < 0.001. Data are expressed as means ± sem.

Figure 6

HFHSD leads to noncompensatory insulin secretion. A) Fasting blood glucose in mice fed a control diet or HFHSD. B) Fasting insulin in mice fed a control diet or HFHSD. C) Insulin secretion during an IPGTT in mice fed a control diet or HFHSD. D) Relative insulin secretion during the first 5 min of an IPGTT in mice fed a control diet or HFHSD. E) Fasting C-peptide results in mice fed a control diet or HFHSD. F) C-peptide secretion during an IPGTT in mice fed a control diet or HFHSD. G) Relative C-peptide secretion during the first 5 min of an IPGTT in mice fed a control diet or HFHSD. n = 5–6. *P < 0.05, **P < 0.01, ***P < 0.001 for control diet vs. HFHSD; ^#P < 0.01 for HFHSD at 8 wk of treatment vs. 0 wk of treatment. Data are expressed as means ± sem. See also Supplemental Fig. S4.

Figure 7

Impaired insulin secretion of HFHSD mice is due to a defect downstream of glucose-stimulated Ca²⁺ influx. A) Ca²⁺ excursions upon glucose stimulus, as indicated by the GCaMP3 Ca²⁺ biosensor, obtained in vivo in islets from Ins-Cre:GCaMP3-mice transplanted into the ACE of B6 mice at 8 wk of an HFHSD (n = 6–9). B) Amount of glucose-responsive cells indicated by the GCaMP3 Ca²⁺ biosensor obtained in vivo in islets from Ins-Cre:GCaMP3-mice transplanted to the ACE of B6 mice at 8 wk of an HFHSD (n = 6–9). C) Backscatter intensity of islets transplanted to the ACE of B6 mice at 8 wk of an HFHSD shown in arbitrary units (A.U.) (n = 10). D) Gene expression analysis of isolated islets from mice fed a control diet or HFHSD for 8 wk shown in A.U. (n = 6 mice). E) Western blot quantification of syntaxin-1A in isolated islets from mice fed a control diet or HFHSD for 8 wk (n = 11–12). Representative Western blot of 3 control diet and HFHSD mice is shown. *P < 0.05, ***P < 0.001. Data are expressed as means ± sem. See also Supplemental Figs. S5 and S6.