Acta Diabetol (2004) 41:194–199 DOI 10.1007/s00592-004-0165-8 ORIGINAL
R.A. Kowluru • S. Chakrabarti • S. Chen
Re-institution of good metabolic control in diabetic rats and activation of caspase-3 and nuclear transcriptional factor (NF-kB) in the retina
Received: 16 March 2004 / Accepted in revised form: 21 October 2004
Abstract Hyperglycemia is one of the major underlying factors in the development of retinopathy in diabetes. Retinal microvascular cells undergo accelerated apoptosis before other histopathological changes are detectable in diabetes. We examined the effect of re-institution of good metabolic control (GC) on the activation of retinal apoptosis executor enzyme, caspase-3, and nuclear transcriptional factor NF-kB. In streptozotocin diabetic rats, two or six months of poor metabolic control (PC) with glycated hemoglobin >11.0% was followed by seven additional months of GC (glycated hemoglobin <5.5%). Caspase-3
activity in retina was measured by the cleavage of its substrate, the expression of active 17 kD subunit, and cleavage of poly(ADP ribosyl) polymerase. NF-kB activation was determined by electrophoretic shift assay and by western blots for P65 subunit. Caspase-3 activity in diabetic rats kept in PC for 13 months was 175% that in normal rats. Re-institution of GC after two months of PC partially normalized the hyperglycemia-induced activation of caspase3 (to 140% of normal values) while re-institution of GC after six months of PC had no significant effect on the activation of caspase-3 NF-kB activity was 2.5-fold higher in diabetic rats kept in PC than in normal rats. Re-institution of GC after 2 months of PC partially reversed this increase (X-fold over normal), but GC after 6 months of PC had no effect. Initiation of GC soon after induction of diabetes in rats prevented activation of retinal caspase-3 and NF-kB. These results suggest that the process of activation of apoptosis execution enzyme and NF-kB in retina that starts before appearance of histopathological changes is not easily reversed by re-institution of GC. Characterization of the abnormalities responsible for the resistance of retinopathy to halt after re-institution of GC will help identify potential therapies for inhibition of progression of diabetic retinopathy. Key words Apoptosis Retinopathy
R.A. Kowluru () Kresge Eye Institute Wayne State University Detroit, MI 48201, USA E-mail: [email protected] S. Chakrabarti • S. Chen Department of Pathology University of Western Ontario London, Ontario, Canada
Introduction Hyperglycemia-induced metabolic disorders initiate a sequence of events leading to the development of retinopathy in diabetes . However, which of those abnormalities are critical to the etiology of the retinopathy remains to be established. Good metabolic control is beneficial in preventing retinopathy in diabetes [2–4], and the duration of
R.A. Kowluru et al.: Reversal of hyperglycemia and retinal caspase-3
hyperglycemia before initiation of normal glycemia plays a major role in the outcome of good metabolic control [2, 5, 6]. Re-institution of normal glycemia after a period of poor metabolic control does not produce immediate benefits on the progression of retinopathy [2–4]. We have shown that nitrative modifications of retinal proteins that occur early in the course of development of diabetic retinopathy in rats are not easily reversed by re-institution of normal glycemia . Furthermore, using another animal model of diabetic retinopathy (experimentally galactosemic rats), we have shown that retinal dysmetabolism continues to progress after galactose feeding is terminated in rats . In diabetes, retinal capillary cells, Muller cells and ganglions are lost before other histopathological changes are detectable, and apoptosis has been implicated as one of the mechanisms of this accelerated cell death [8–10]. Apoptosis execution enzyme, caspase-3, and nuclear transcriptional factor, NF-kB, are activated in rat retina when the duration of diabetes is such that the capillary cell apoptosis and histopathological changes are detectable [11–13]. While activation of caspase-3 in retina is not seen for six to eight months after induction of diabetes in rats , increased oxidative stress, nitric oxide levels and activation of NF-kB are considered early events in the pathogenesis of diabetic retinopathy [6, 13, 14]. The present study investigated the effect of re-institution of normal glycemia in diabetic rats on activation of caspase-3 and NF-kB in retina. Since the preexisting damage at the time of intervention is considered a primary factor in determining the outcome of a therapy , we have also determined the effect of duration of hyperglycemia before initiation of normal glycemia on these abnormalities by allowing the rats to remain in hyperglycemic state for two or six months before initiation of normal glycemia for seven additional months.
Materials and methods Rats (Wistar, male, 200 g) were randomly assigned to normal (n=8) or diabetic (n=28) groups, and diabetes was induced with streptozotocin (55 mg/kg body weight). Diabetic rats were further divided into four groups of 7 animals each according to intended degree and duration of good metabolic control: – PC group. Rats remained in poor metabolic control for 13 months. – GC group. Rats maintained in good metabolic control for the entire duration of 13 months. – PC2→GC group. Rats were in poor metabolic control for two months followed by good metabolic control for seven additional months. – PC6→GC group. Rats were in poor metabolic control for six months, followed by good metabolic control for seven additional months.
195 Diabetic rats received insulin (NPH) injections: the rats in which poor metabolic control was intended received a single injection of insulin (1–2 units) four to five times a week to prevent ketosis and weight loss, and the rats in which good metabolic control was intended received insulin twice daily (8–10 units total) to maintain a steady gain in body weight and urine glucose values below 150 mg/24 hours . Rats were housed in metabolism cages: 24-hour urine samples were tested for glycosuria daily with Keto-Diastix (Bayer Pharmaceuticals, West Haven, USA), and three to four times every week using quantitative methods (Glucose Kit, 510-A, Sigma Chemicals, St. Louis, USA). Blood glucose was measured once a week (Glucometer Elite, Bayer), and glycated hemoglobin (GHb) was measured using kit 442-B (Sigma Chemicals) every two months. The entire rat colony received powdered diet (Purina 5001, St. Louis, USA), and the food consumption and body weights were measured two to three times every week. These experiments conformed to the Association for Research in Vision and Ophthalmology’s Resolution on Treatment of Animals in Research (National Institutes of Health). At the end of the desired duration of metabolic control, the animals were sacrificed; eyes from each animal were used to gently isolate the fresh retina using a microspatula under a dissecting microscope. Activation of caspase-3 in the retina was measured by three independent methods: cleavage of fluorogenic substrate specific for caspase-3, and detection of active subunit of caspase-3 (17 kD) and 85 kD subunit of poly(ADP-ribose) polymerase (PARP, Santa Cruz Biotechnology, Santa Cruz, USA) . Caspase-3 activity was measured in the supernatant using 15–20 µg protein/tube and 25 µM fluorogenic substrate N-acetyl-Asp-GluVal-Asp-7-amino-4-trifluoromethyl coumarin (DEVD-AFC, Biomol Research (National Institutes of Health) Laboratories, Plymouth Meeting, USA). Increased expression of the active subunit of caspase-3 (17 kD) was detected by western blot analysis using polyclonal antibody against caspase-3. To ensure equal loading among lanes, values were normalized to the expression of “housekeeping” protein β-actin . Furthermore, the formation of 85 kD fragment of PARP was determined by western blot analysis using a polyclonal antibody against PARP. The membranes were developed using ECL-Plus western blotting detection kit . Activation of NF-kB was demonstrated by two independent methods: electrophoretic mobility shift assay and western blot . Nuclear protein (5 µg) obtained from rat retina was incubated with 100 000 cpm of 32P-labeled consensus oligonucleotides (5-AGT TGA GGG GAC TTT CCC AGG C-3, 3-TCA ACT CCC CTG AAA GGG TCC G-5). The specificity of binding was further confirmed by incubation with 100-fold excess of unlabeled oligonucleotides . NF-kB activation was determined also by performing western blots to measure the expression of p65 subunit using antibody against NF-kB (p65; 1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, USA). To ensure equal loading among lanes, values were normalized to the expression of β-actin. Tissue protein was measured in triplicate using bicinchoninic acid kit (Sigma Chemicals), and bovine serum albumin was used as a standard. Data were analyzed statistically using the nonparametric Kruskal-Wallis test followed by Mann-Whitney test for multiple group comparisons. Similar conclusions were reached also by using ANOVA with Fisher or Tukey.
R.A. Kowluru et al.: Reversal of hyperglycemia and retinal caspase-3
Results We studied the pathogenesis of diabetic retinopathy in streptozotocin diabetic rats subjected to different programs of metabolic control (Table 1). Diabetic rats who had poor metabolic control for the entire study duration (13 months; PC group) had glycated hemoglobin values >11%, compared to values <5% in the normal control rats. Diabetic rats with good metabolic control (GC group) had glycated hemoglobin values similar to those of normal control rats (p>0.05 vs. normal). Glycated hemoglobin values before initiation of good metabolic control in both PC2→GC and PC6→GC groups were >11%, and decreased to less than 5.5% two months after initiation of good metabolic control. These values remained between 4.5% and 6% for the entire 7 months of good metabolic control (Table 1). Twenty four-hour urine volumes were three to four times higher when the diabetic rats were in poor metabolic control, and were similar in rats in normal control and GC groups (<30 ml in normal and >90 ml in PC rats). Caspase-3 activity, as determined by cleavage of the substrate, was increased 75% in diabetic PC group (poor metabolic control for 13 months) compared to normal rats (Fig. 1). When diabetic rats were maintained in good metabolic control for 13 months (GC group), caspase-3 activity was similar to that of normal rats. Seven months of good metabolic control following two months of poor metabolic control (PC2→GC group) was beneficial in reversing the elevation of caspase-3 activity. However, re-institution of good metabolic control for 7 months after 6 months of poor control (PC6→GC) failed to block the increase in caspase-3 activity. Expression of the active 17 kDa subunit of caspase-3 was increased by 160% in PC group compared to control rats (Fig. 2a), while that of the 85 kDa subunit of PARP was increased by 93% (Fig. 2b). Good metabolic control (GC group) helped maintained the expression levels of the 17 kDa caspase-3 subunit near normal. Re-institution of good metabolic control for 7 months (PC2→GC group) had beneficial effects on expression of both proteins,
whereas only 2 months of good metabolic control (PC6→GC) did not. NF-kB in retina was activated by 13 months of poor metabolic control in diabetic rats compared to normal rats (Fig. 3). Densitometric quantification of the shifted band on the electrophoretic mobility assay revealed a 2.7-fold increase of NF-kB in PC group (p<0.05), which was mostly restored by good metabolic control (GC group). Reinstitution of good metabolic control after 6 months of poor control (PC6→GC) failed to block NF-kB activation.
Discussion Apoptosis has been implicated as one of the mechanisms in the accelerated death of retinal capillary cells [8, 9]. We have shown that apoptosis execution enzyme, caspase-3,
Fig. 1 Caspase-3 activity in normal and diabetic rats, and the effects of metabolic control. Caspase-3 activity was measured using fluorogenic substrate DEVD-AFC. The values are means and SD for seven rats each in normal and GC groups, and six rats each in PC, PC2→GC and PC6→GC groups. *p<0.05 vs. normal; **p<0.05 vs. PC group
Table 1 Metabolic control in normal and diabetic rats subjected to different programs of metabolic control. Values of body weight and glycated hemoglobin are expressed as mean (SD). Body weight represents the mean value during the entire duration of the intended metabolic control
*p<0.05 vs. normal; **p<0.05 vs. PC group; *** Data published in 
85 kD band absorbance (arbitrary units)
17 kD band absorbance (arbitrary units)
R.A. Kowluru et al.: Reversal of hyperglycemia and retinal caspase-3
Fig. 2a, b Western blot analyses for 17 kD subunit of caspase-3 and 85 kD subunit of PARP in rat retina samples. To ensure equal loading among the lanes, values were normalized for the expression of β-actin. The values are mean and SD for six rats in normal, PC and PC6→GC groups, and five rats each in GC and PC2→GC groups. Values for normal rats were arbitrarily set to 100 units. *p<0.05 vs. normal; **p<0.05 vs. PC group
Fig. 3a, b Activation of NF-kB in diabetic rat retina and reversal by good metabolic control shown on mobility shift assay. a Shift in electrophoretic migration for one representative rat in each group. Specificity of the assay was demonstrated by incubation with p65 antibody or 100-fold excess unlabeled probe (right panel). *, supershifted band. b The intensity of P50/65 complex was quantified by densitometry, and the bars represent mean and SD for 4 rats per group, analyzed in duplicate. *p<0.05 vs. normal; **p<0.05 vs. PC group
Band Intensity (arbitrary units)
and NF-kB are activated in the rat retina when the duration of diabetes is such that the capillary cell death and histopathological changes are detectable [11, 13]. Although the activation of NF-kB in retina can be seen early in the pathogenesis of diabetic retinopathy , activation of caspase-3 is observed only after 6–8 months of diabetes in rats, a duration when capillary cell apoptosis is detectable [8, 9,
11]. Here we provide evidence that even a short duration of hyperglycemia followed by good metabolic control is sufficient to activate caspase-3, and activation of caspase-3 and NF-kB continues for some time (at least for seven months in rats) after cessation of hyperglycemia. Previous studies have revealed that the preexisting damage at the time of intervention of hyperglycemia is a pri-
mary factor in determining the outcome of a therapy to inhibit retinopathy, and instituting tight metabolic control in diabetes does not necessarily immediately benefit the progression of retinopathy [2–5]. Our recent study with diabetic rats demonstrated that if good metabolic control is initiated soon after the induction of diabetes, oxidative stress and nitrative stress are not elevated in the retina; however, if hyperglycemia is maintained for even two months before instituting good metabolic control, these abnormalities are partially reversed, while the abnormalities are not reversed if hyperglycemia is maintained for six months before reinstituting good metabolic control . Here we show that once even a short duration of hyperglycemia (two months) activates NF-kB and caspase-3 in the retina, termination of hyperglycemia does not completely reverse these abnormalities. This suggests that some of the signaling pathways involving NF-kB initiate early in the development of retinopathy (duration of diabetes in rats, two months or less), and continue to progress for at least seven months after re-institution of good metabolic control. Since apoptosis of pericytes and endothelial cells in diabetes is seen before other histopathological changes are detectable, the clinically silent initial phase of diabetic retinopathy appears to consist of irreversible cellular events with late structural consequences [8, 9]. Caspases have been reported to play a pivotal role in the apoptotic cascade, and the processing of pro-caspase-3 to its active form is regarded as a point of no return in the death-signaling cascade . Only partial reversal of caspase-3 activation after two months of hyperglycemia followed by seven months of good metabolic control in rats is unexpected because at two months of diabetes caspase-3 activation is not detectable in rat retina [11, 12]. This implies that the process involved in activation of caspase-3 starts early in the pathogenesis of retinopathy in diabetes. Thus, it is likely that the oxidative nitrative modifications of retinal caspase-3 are initiated early in the development of diabetic retinopathy, they continue for a time even after termination of hyperglycemia, and once caspase-3 is compromised, re-institution of good metabolic control fails to have beneficial effects on its activation. Activation of NF-kB is considered a key signaling pathway by which high glucose concentration induces apoptosis in endothelial cells , and in retinal pericytes activated NF-kB triggers a proapoptotic program in diabetes . Activation of NF-kB modulates the expression of iNOS . The results presented here show that NF-kB, once activated early in the pathogenesis of diabetic retinopathy, can be inhibited only partially by early reinstitution of good metabolic control, but is not reversed if poor metabolic control is maintained for seven months after induction of hyperglycemia. A study with diabetic dogs has clearly shown that good metabolic control can prevent development of acellular capillaries and microaneurysyms in the retinal microvessels only
R.A. Kowluru et al.: Reversal of hyperglycemia and retinal caspase-3
if it is initiated within a few weeks after the onset of diabetes, but if good control is initiated after a longer duration of poor glycemic control, retinal histopathology has a tendency to persist . Our results show that if the rats are allowed to maintain good metabolic control soon after induction of diabetes, retinal caspase-3 and NF-kB are not significantly activated. This further strengthens the possibility that the activation of NF-kB and caspase-3 (and possibly their signaling pathways) are initiating the sequence of events that are not easily reversible, and are contributing to the progression of retinopathy after re-institution of good metabolic control. Development of retinal histopathology in diabetic rats is associated with the activation of caspase-3 and NF-kB, and antioxidants that inhibit the development of retinopathy inhibit the activation of both caspase-3 and NF-kB [11–13, 17]. Diabetic retinopathy continues to progress for some time after correction of hyperglycemia [2–4], and the results presented here show that caspase-3 and NF-kB remain active in retina even after termination of hyperglycemia, suggesting the importance of these abnormalities in the failure of retinopathy to halt. The rats in PC2→GC group were sacrificed nine months after initiation of the experiment (two months of PC and seven months of GC) as compared to 13 months in the other four groups. The partial reversal of the activation of retinal caspase-3 and NF-kB by good metabolic control in this group, however, cannot be accounted by the differences in the duration of diabetes since the activations of caspase-3 and NF-kB are not different at 8 and 14 months of diabetes in rats [11, 13]. Thus, our study shows that the process of activation of the enzyme involved in the execution of apoptosis and of nuclear transcriptional factor occur early in the development of retinopathy in diabetes and re-institution of good metabolic control after a short duration of hyperglycemia in diabetic rats does not reverse the activation of caspase3 and NF-kB. The duration of poor metabolic control before initiation of good metabolic control influences the reversal of theses abnormalities, thus strengthening the importance of early metabolic control in diabetic patients. Acknowledgements Technical assistance of Prashant Koppolu, Saiyeda Noor Abbas, and Xiaohua Zhou is sincerely appreciated. This work was supported in part by grants from Juvenile Diabetes Research Foundation, the Thomas Foundation, and Research to Prevent Blindness.
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