Histopathological nerve and skeletal muscle changes in rats subjected to persistent insulin-induced hypoglycemia

Abstract

New insulin analogues with a longer duration of action and a flatter pharmacodynamic profile are developed to improve convenience and safety for diabetic patients. During the nonclinical development of such analogues, safety studies must be conducted in nondiabetic rats, which consequently are rendered chronically hypoglycemic. A rat comparator model using human insulin would be valuable, as it would enable differentiation between effects related to either persistent insulin-induced hypoglycemia (IIH) or a new analogue per se. Such a model could alleviate the need for an in-study-comparator and thereby reduce the number of animals used during development. Thus, the aims of the present study were i) to develop a preclinical animal model of persistent hypoglycemia in rats using human insulin infusion for four weeks and ii) to investigate histopathological changes in sciatic nerves and quadriceps femoris muscle tissue, as little is known about the response to persistent hypoglycemia in these tissues. Histopathologic changes in insulin-infused animals included axonal degeneration and myofibre degeneration. To our knowledge, this is the first study to show that persistent IIH provokes peripheral nerve and skeletal myofiber degeneration within the same animals. This suggests that the model can serve as a nonclinical comparator model during development of long-acting insulin analogues.

Chronic Hyperinsulinaemic Hypoglycaemia in Rats Is Accompanied by Increased Body Weight, Hyperleptinaemia, and Decreased Neuronal Glucose Transporter Levels in the Brain

“…The animals were randomised into four main groups stratified for body weight and sex (Table 1). To allow for infusion (see Study Design), the animals received surgical implantation of a vascular catheter inserted into the caudal vena cava through the right femoral vein as previously described [21]. Postsurgery animals were single-housed and allowed at least seven days of recovery before start of insulin infusion.…”

“…Consequently, the animals were closely monitored for clinical signs of hypoglycaemia, and doses were lowered, if the maximum tolerable dose was considered to be exceeded. Doses were chosen based on results from a previous 4-week HI-infusion study [21]. Females received lower doses than males as they are known to possess higher insulin sensitivity [25–27].…”

“…Plasma was sampled for glucose profiling to monitor the decrease in blood glucose levels following the start of HI-infusion in the morning of Day 1 and on Day 25 and Day 53 (at 0.25, 0.5, 6, 12, or 24 h, relating to the start of infusion at 0 h on day 1); time points were determined based on the approximate half-life of HI in the rat of approximately 0.25 h [28], as well as on results from a previous study using the same animal model [21], and to confirm a persistent 24 h decrease as would be expected from continuous infusion. As this was the main purpose, only 2 animals/sex/group were sampled at each time point, to minimise stress (induced by handling and/or sampling) in the animals.…”

“…In addition, blood samples for plasma glucose quantification were obtained at a single time point from all the animals on day 8 and before termination of the animals on Day 29, Day R2, and Day R29. Plasma glucose level was quantified as described previously [21]. On Day 1 and 53, blood was drawn also for plasma HI quantification at the same time points as for plasma glucose profiling measurements.…”

“…Composition of buffered HI stock formulation, buffered vehicle, and dilution medium was as described previously [21]. For the infusion, external syringe infusion pumps and a vascular access harness connected to a tether kit were used as described previously [21]. …”