Diabetes reduces β-cell mitochondria and induces distinct morphological abnormalities, which are reproducible by high glucose in vitro with attendant dysfunction

Abstract

We investigated the impact of a diabetic state with hyperglycemia on morphometry of β cell mitochondria and modifying influence of a K (+) -ATP channel opener and we related in vivo findings with glucose effects in vitro. For in vivo experiments islets from syngeneic rats were transplanted under the kidney capsule to neonatally streptozotocin-diabetic or non-diabetic recipients. Diabetic recipients received vehicle, or tifenazoxide (NN414), intragastrically for 9 weeks. Non-diabetic rats received vehicle. Transplants were excised 7 d after cessation of treatment (wash-out) and prepared for electron microscopy. Morphological parameters were measured from approx. 25,000 mitochondria. Rat islets were cultured in vitro for 2-3 weeks at 27 or 11 (control) mmol/l glucose. Transplants to diabetic rats displayed decreased numbers of mitochondria (-31%, p < 0.05), increased mitochondrial volume and increased mitochondrial outer surface area, p < 0.001. Diabetes increased variability in mitochondrial size with frequent appearance of mega-mitochondria. Tifenazoxide partly normalized diabetes-induced effects, and mega-mitochondria disappeared. Long-term culture of islets at 27 mmol/l glucose reproduced the in vivo morphological abnormalities. High-glucose culture was also associated with reduced ATP and ADP contents, reduced oxygen consumption, reduced signaling by MitoTracker Red and reduction of mitochondrial proteins (complexes I-IV), OPA 1 and glucose-induced insulin release. We conclude that (1) a long-term diabetic state leads to a reduced number of mitochondria and to distinct morphological abnormalities which are replicated by high glucose in vitro; (2) the morphological abnormalities are coupled to dysfunction; (3) K (+) -ATP channel openers may have potential to partly reverse glucose-induced effects.

Figure 1. In vivo study design.

Figure 2. Blood glucose (A) and body weight (B) of transplanted rats that were non-diabetic and vehicle-treated, diabetic and vehicle-treated or diabetic and tifenazoxide-treated. There were eight rats in each group. Treatment was removed after 9 weeks i.e., after day 63.

Figure 3. Quantitative morphology of primary (upper panel) and secondary (lower panel) mitochondrial parameters. Data were collected from 22 sections per islet graft. There were eight transplants per group. Totally 528 EM sections were evaluated (176 from each treatment group i.e., approx. 25,000 mitochondria altogether). *p < 0.05 vs. mitochondria from transplants to non-diabetic animals, ^†p < 0.05 vs. mitochondria from transplants to vehicle-treated diabetic animals. NN414 = tifenazoxide.

Figure 4. Electron microscopy showing representative sections of β-cells from transplanted islets to non-diabetic, vehicle-treated (A), diabetic, vehicle-treated (B and D) and diabetic, tifenazoxide-treated (C) rats. (E) shows an example of a likely fusion event in a section from a diabetic vehicle-treated rat. In (B) black arrows = mitochondria. In (C) black arrows = mature granule and white arrows = immature granule.

Figure 5. Frequency distribution of average mitochondrial volume (A) and average mitochondrial outer surface area (B).

Figure 6. Quantitative morphology of primary (upper panel) and secondary (lower panel) mitochondrial parameters of islets cultured in vitro. Data were collected from 22 sections per rat. Data from 2 weeks (n = 2) and 3 weeks (n = 1) culture in 11 or 27 mmol/l glucose were pooled since quantitative morphology was similar after 2 and 3 weeks culture. *p < 0.05 vs. mitochondria from islets cultured in 11 mmol/l glucose.

Figure 7. Effects of 3 weeks culture in vitro in 11 or 27 mmol/l glucose on sub-units of mitochondrial complexes I-V (A and quantified in B), OPA 1 (A and quantified in C) and on ATP and ADP levels (D). Immunoblotting was performed on islets cultured either at 27 or 11 mmol/l glucose. In (A), (B) and (C) mean ± SEM of four individual experiments expressed as percentage of results in islets cultured in 11 mmol/l glucose (100%). *p < 0.05 or less vs. 11 mmol/l glucose culture. ATP and ADP levels: Mean ± SEM of four individual experiments. *p < 0.001 or less vs. 11 mmol/l glucose culture.