Insulin therapy for pre-hyperglycemic beta-cell endoplasmic reticulum crowding

Abstract

Insulin therapy improves β-cell function in early stages of diabetes by mechanisms that may exceed alleviation of glucotoxicity. In advance type 2 diabetes, hyperglycemia causes β-cell damage and ultimately β-cell loss. At such an advanced stage, therapeutic modalities are often inadequate. Growing evidence indicates that in early stages of type-2 diabetes and some types of monogenic diabetes linked with malfunctioning endoplasmic-reticulum (ER), the β-cell ER fails to process sufficient proinsulin once it becomes overloaded. These changes manifest with ER distention (ER-crowding) and deficiency of secretory granules. We hypothesize that insulin therapy may improves β-cell function by alleviating ER-crowding. To support this hypothesis, we investigated pre-diabetic β-cell changes in hProC(A7)Y-CpepGFP transgenic mice that develop prolonged pre-diabetes due to proinsulin dysmaturation and ER-crowding. We attenuated the β-cell ER proinsulin synthesis with a treat-to-target insulin therapy while avoiding hypoglycemia and weight gain. Alleviation of ER-crowding resulted in temporary improvement in proinsulin maturation, insulin secretion and glucose tolerance. Our observations suggest that alleviation of pre-diabetic ER-crowding using a treat-to-target insulin therapy may improve β-cell function and may prevent further metabolic deterioration.

Figure 1. Distribution of insulin and proinsulin in pancreatic islets of pre-diabetic mice.

Sections of paraffin embedded pancreata were immunostained with anti-insulin antibodies (red), anti-proinsulin antibodies (green) and anti-BIP (blue). The endogenous GFP fluorescence was quenched prior immunostaining. A. Endogenous insulin and proinsulin in pre-diabetic hProC(A7)Y-CpepGFP male compared to hProCpepGFP control. B. Endogenous insulin and proinsulin in pre-diabetic LepR^db/db male compare to LepR^db/+ control. C. β-cell BIP in pre-diabetic hProC(A7)Y-CpepGFP and LepR^db/db males. In genetically predisposed pre-diabetic mice, maturation of proinsulin to insulin is heterogeneously compromised, likely in β-cells more affected by the ER insult (white arrowheads). Red arrow denotes autofluorescence deriving from red blood cells.

Figure 2. Effect of a treat-to-target insulin therapy on insulin production.

Mice were treated with a treat-to-target insulin therapy for a month and tissues were harvested for mRNA and protein, before and after drug washout. A. Pharmacodynamic profiles of different insulin formulations following a single subcutaneous injection. B. Mean weight during therapy. C. Pre-treatment plasma C-peptide. D. Plasma C-peptide changes in insulin treated hProCpepGFP, insulin treated wild-type mice controls, and vehicle treated hProC(A7)Y-CpepGFP. E. Pre-proinsulin–II mRNA message normalized to β-actin, depicted as mean 2^−ΔCT ± SEM. F. BIP mRNA message. In pre-diabetic hProC(A7)Y-CpepGFP mice a treat-to-target insulin therapy suppressed proinsulin production, yet improved insulin secretion after therapy withdrawal.

Figure 3. Effect of a treat-to-target insulin therapy on ER crowding, proinsulin maturation and glucose tolerance.

Mice were treated with a treat-to-target insulin therapy for a month and tested with intraperitoneal glucose tolerance test. Pancreatic sections were imaged with a transmission electron microscope and pancreatic islets were collected 48 hours after therapy withdrawal. A. Electron microscope images of insulin producing β-cells in pre-diabetic hProC(A7)Y-CpepGFP mice (insulin Detemir and vehicle treated) compared to wild-type controls. B. Western blotting of islets lysate in reducing SDS-PAGE gel showing the steady state levels of endogenous proinsulin (proinsulin), endogenous insulin (insulin), GFP-labeled mutant proinsulin (hProC(A7)Y-CpepGFP), fully processed GFP labeled C-peptide (CpepGFP), BIP, and α-tubulin for internal control. The lower panel shows graphic representation of endogenous insulin/proinsulin ratio. Three samples were used from each group. Representative samples are from two different vehicle treated pre-diabetic hProC(A7)Y-CpepGFP mice (left panel) and two different insulin treated pre-diabetic hProC(A7)Y-CpepGFP mice (middle panel). hProCpepGFP transgenic mouse and transfected HEK293 cells were used as controls to identify the location of specific bands. C. Intraperitoneal glucose tolerance test 48 hours and a week after insulin withdrawal in the same mice. Alleviation of ER crowding increased endogenous insulin/proinsulin ratio in pre-diabetic hProC(A7)Y-CpepGFP mice and temporarily normalized glucose tolerance.