[1] 20w Protein kinase C beta inhibitor prevents diabetic peripheral neuropathy, but not histopathological abnormalities of retina in Spontaneously Diabetic Torii rat

[1] 153w Spontaneously Diabetic Torii (SDT) rat shows severe ocular complications such as tractional retinal detachment. In the present study, effect of protein kinase C beta (PKCβ) inhibitor JTT-010 was evaluated to clarify the involvement of PKCβ in complications of SDT rat. SDT rats were administered JTT-010 (10 or 50 mg/kg/day) for 48 weeks. SDT rats showed delayed oscillatory potentials in electroretinogram. Delayed motor nerve conduction velocity, decreased coefficients of variation of R-R intervals in electrocardiogram and thermal hypoalgesia were also observed. These functional disorders were prevented by administration of JTT-010. Abnormal retinal vascular was formed and the optic disc was protruded in SDT rat; however, JTT-010 did not prevent these hyperglycaemia-induced retinal abnormalities. These findings indicate that PKCβ is intimately involved in diabetic complications; however, it seems that other factor(s) are primary contributors to histopathological abnormalities in retina. Therefore, PKCβ inhibitors require concurrent administration of antihyperglycaemic drugs to achieve maximum effect on diabetic complications.

[1] 147w Although a strict control of blood glucose level can delay the onset and progression of diabetic microvascular complications [1], current oral antihyperglycaemic drugs have not shown sufficient efficacy. Activation of protein kinase C beta (PKCβ) through the de novo synthesis of diacylglycerol is observed under diabetic conditions, and administration of selective PKCβ inhibitor prevents development of diabetic complications in diabetic animal models [2,3]. However, diabetic patients often manifest more severe diabetic retinopathy (DR) than animal models such as streptozotocin (STZ)-induced diabetic rat. Therefore, the existing models are not sufficient for the study of DR. In the present study, we assess the pharmacological effect of a PKCβ inhibitor JTT-010 [2,4] on DR and diabetic peripheral neuropathy (DPN) using Spontaneously Diabetic Torii (SDT) rat, a severe model of diabetic complications particularly in eyes, such as tractional retinal detachment with fibrous proliferation and massive hemorrhage in the anterior chamber [5,6].

[1] 123w PKC activity of retina in normoglycaemic SDT rats was comparable with SD rats (8 weeks of age, figure 1a). At 24 weeks of age, SDT rats were hyperglycaemic and showed 1.5-fold higher retinal PKC activity than SD rats. Single oral administration of JTT-010 inhibited PKC activity of retina (ED 50 = 22.6 mg/kg, figure 1b). JTT-010 also inhibited PKC activity in other tissues associated with diabetic microangiopathy, such as sciatic nerve and glomeruli (ED 50 = 16.4 and 7.6 mg/kg respectively). At 68 weeks of age, SDT rats showed lower body weight, marked hyperglycaemia and hypoinsulinaemia compared with SD rats. Recruitment of insulin ameliorated body weight loss and hyperglycaemia. Administration of JTT-010 did not alter these parameters and food consumption (data not shown).

[2] 82w SDT rats showed a prolongation of the peak latencies of oscillatory potentials (OPs) in ERG at 32 weeks of age. Prolonged peak latencies were significantly reduced by JTT-010 administration (figure 1c). Tail MNCV of SDT rats was significantly delayed compared with normal rats (figure 1d). CV R-R was significantly lower in diabetic SDT rats (figure 1e), indicating disorder of autonomic nerve function. Marked thermal hypoalgesia was also observed in SDT rats (figure 1f). These nervous impairments were improved by administration of JTT-010.

[3] 77w At 68 weeks of age, SDT rat exhibited lens opacity as diabetic cataract (figure 2a-d). Fluorescein angiomicroscopy showed abnormal vascular formation, including venous dilation and meandering vascular networks with extensive leakage of fluorescein around the optic disc (figure 2e-h). Furthermore, the optic disc protrudes into the vitreous space. Histopathological examination showed slight thickening and distortion of the retina in SDT rats (figure 2i-l). These histopathological changes were completely prevented by insulin recruitment, but were not by JTT-010.

[1] 208w In the present study, we evaluated the effect of a PKCβ inhibitor on severe diabetic complications in SDT rat. Increased PKCβ activity is reported in tissues associated with microangiopathy in diabetic state [3,8]. JTT-010 ameliorated impaired MNCV and thermal hypoalgesia of SDT rat as previously reported using STZ rat [2]. Although the mechanism between hypoalgesia and PKCβ is unclear, these results are beneficial to treatment of DPN. PKCβ activation also contributes to nitric oxidedependent vascular and autonomic nerve dysfunction in diabetes, and abnormal variations in heart rate such as CV R-R were ameliorated by JTT-010. JTT-010 also clearly ameliorated the prolongation of OPs in ERG, a very early alteration among the diabetic eye complications indicating potential usefulness of PKCβ inhibitor on dysfunction of diabetic retina. The results signify that inhibiting tissue PKCβ activity is effective in preventing functional disorder of diabetic complications; however, the efficacy on histopathological changes in eyes, caused by sustained hyperglycaemia in SDT rat, is insufficient. Controlling blood glucose by insulin clearly prevented diabetic complications in SDT rat as we reported previously [7,9]. Therefore, factor(s) other than PKCβ may contribute to these retinal abnormalities in SDT rat. The mechanisms of these histopathological changes should be clarified and could become new targets of pharmacotherapy against DR.

[2] 134w In the clinical trials, ruboxistaurin, the first reported PKCβ-selective inhibitor demonstrated efficacy against diabetic macular oedema (DME); however, in the 30-month follow-up study, ruboxistaurin failed to delay progression of DME [10]. A clinical study of ruboxistaurin on nerve function and sensory symptoms showed that inhibition of PKCβ is beneficial to patients with symptomatic or less severe DPN [11]. Considering the results of clinical/pre-clinical trials and the results in the present study, it seems that administration of PKCβ inhibitor alone has limited effect on DR/DPN. Hence, it seems that PKCβ inhibitors require concurrent administration of antihyperglycaemic drugs to achieve its maximum effect on diabetic complications. Recently, treatment of epalrestat, an aldose reductase inhibitor, also showed benefit on MNCV and symptom score in diabetic patients, particularly in patients with good glycaemic control (HbA1c ≤ 7.0%) [12].

[3] 53w This study is the first report that evaluates DPN and pharmacotherapy of diabetic complications by PKCβ inhibitor using SDT rat. Considering the individual effects and limitations of PKCβ inhibitors on microangiopathy, PKCβ inhibitors offer a very promising therapeutic approach to diabetic complications and seem to be more effective in co-administration with antihyperglycaemic drugs.

[1] 131w Male SDT rats (CLEA Japan, Tokyo, Japan) already exhibiting hyperglycaemia (20 weeks of age, blood glucose level >14 mmol/l) were used in the study. Sprague-Dawley (SD) rats were used as control animal. All the experiments received prior approval from the committee for the humane care and use of animals of our laboratories. Animals were housed in a climatecontrolled room (temperature 23 ± 3 • C, humidity 55 ± 15%, 12 h lighting cycle) and allowed free access to diet and water. JTT-010, a selective PKCβ inhibitor, was administered to 20-week-old SDT rats for 48 weeks by food admixture (10 or 50 mg/kg/day). To compare the effect of PKCβ inhibitor with a hypoglycaemic agent, an insulin pellet was subcutaneously implanted into back and blood glucose level was controlled below 14 mmol/l [7].

[2] 73w Tissue PKC activity was measured 4 h after single oral administration of JTT-010 by its ability to transfer 32 P from [γ-32 P]-ATP into the PKC-specific substrate peptide [2]. Electroretinogram (ERG) was evaluated after 12 weeks administration of JTT-010 (32 weeks of age). Rats were adapted to darkness and anaesthetized with ketamine. A xenon lamp was flashed once to record the potential between the corneal contact lens electrode and the reference electrode [7].

[3] 109w After 12 weeks administration of JTT-010, motor nerve conduction velocity (MNCV) was measured under halothane anaesthesia. Tail nerve was stimulated electronically and muscle action potentials were recorded [2]. Antihypoalgesic response was evaluated by recording latency to withdrawal of the tail in response to noxious skin heating (tail-flick test) [2]. The basic limb lead method was performed using an electrocardiogram (ECG) amplifier (AC-601G, Nihon Kohden, Tokyo, Japan) and recorder (Acqknowledge, BIOPAC Systems, CA, USA) under mild ether anaesthesia. To calculate the coefficient of variance of R-R intervals (CV R-R ), standard deviation of R-R interval for 30 s just before cessation of anaesthesia was divided by average of R-R intervals.

[4] 56w At 68 weeks of age, cataracts were evaluated using a slit lamp (SL-14, Kowa, Aichi, Japan). Thereafter, rats were anaesthetized with sodium pentobarbital and FITC-dextran was perfused through the left ventricle. The retina from right eye was flat mounted onto glass slides for fluorescence-microscopic examination [7]. The left eye was used in histopathological examinations (Hematoxylin-Eosin stain).

[5] 28w Results are expressed as the mean ± s.e.m. Significant differences (p < 0.05) were determined using F-test followed by the unpaired t-test or ANOVA followed by Dunnett's test.