Int. J. Exp. Path. (2014), 95, 95–100
ORIGINAL ARTICLE
Effects of long-acting somatostatin analogues on redox systems in rat lens in experimental diabetes Sirilaksana Kunjara*, A. Leslie Greenbaum*, Milena Sochor*, Allan Flyvbjerg†, Henning Grønbaek† and Patricia McLean* *Division of Biosciences, Research Department of Cell and Developmental Biology, University College London, London, UK and † Department of Endocrinology and Internal Medicine & The Medical Research Laboratories, Clinical Institute, Aarhus University Hospital and Aarhus University, Aarhus C, Denmark
INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY doi: 10.1111/iep.12069
Received for publication: 26 November 2012 Accepted for publication: 26 November 2013 Correspondence: Patricia McLean Division of Biosciences Research Department of Cell and Developmental Biology University College London Gower Street London WC1E 6BT UK Tel.: +44 207 6796576 Fax: +44 207 6792091 E-mail: [email protected]
SUMMARY The effects of long-acting somatostatin analogues, angiopeptin (AGP) and Sandostatin (SMS), on the early decline in the lens content of glutathione (GSH), ATP and NADPH and increase in sorbitol were studied in STZ diabetic rats, and comparison was made with the effect of insulin. Three factors prompted this study: (i) the known increase in IGF-1 in ocular tissue in diabetes and antagonistic effect of somatostatins, (ii) the known effect of IGF-1 in increasing lens aldose reductase and (iii) the lack of effect of somatostatins on diabetic hyperglycaemia, the latter enabling a differentiation to be made between effects of hyperglycaemia per se and site(s) of IGF-1/somatostatins. All four metabolites studied showed a significant restoration towards the normal control level after 7 days of treatment with AGP and SMS, and AGP was more effective on levels of GSH and ATP. A significant correlation was found between GSH and ATP across all groups at 7 days treatment. The redox state changes in diabetes include both NADP+/NADPH and NAD+/NADH in the conversion of glucose to sorbitol and via sorbitol dehydrogenase to fructose with a linked decrease in ATP formation via NAD+/NADH regulation of the glycolytic pathway. The interlinked network of change includes the requirement for ATP in the synthesis of GSH. The present study points to possible loci of action of somatostatins in improving metabolic parameters in the diabetic rat lens via effects on aldose reductase and/or glucose transport at GLUT 3. Keywords angiopeptin, ATP, experimental diabetes (STZ), glutathione, insulin, rat lens, redox systems, sandostatin
Cataract formation is a major cause of blindness in ageing and in diabetes mellitus (see Resnikoff et al. 2004). Extensive studies have been made of the early biochemical changes involved in cataract formation using as model lenses from streptozotocin or alloxan diabetic rats, many of these centring upon early changes in the metabolite profile. Changes in the integration of pathways linked to glucose metabolism, redox systems resulting from hyperglycaemia, the role of the NADPH-dependent aldo-keto reductase, the rate-limiting enzyme of the polyol pathway, and the inhibition of this enzyme by Sorbinil have been discussed (Gabbay 1973; Kinoshita et al. 1979; Varma 1980; Gonzalez et al.1983, 1986; McLean et al. 1985; Brownlee 2005; Lu
2009). In parallel, measures to correct the redox state changes by the use of compounds such as pyruvate and ascorbate have been studied (Zhao et al. 2000; Hedge & Varma 2004). Glutathione was a focal point of the present study, because as reviewed by Lu (2009), glutathione has diverse functions including antioxidant defence, maintenance of thiol groups and cell proliferation. Alterations in the regulation of glutathione synthesis in ageing and disease, including diabetes and insulin treatment, have been reviewed by Lu (2009) with emphasis on the regulation of the ratelimiting enzyme glutamate cysteine ligase and the coordinated change in glutathione synthase. At present, there does not appear to be information on changes in these enzymes
© 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology
95
96
S. Kunjara et al.
in the rat lens in diabetes although the decrease in lens glutathione is well established. Recently, attention has been drawn to the role of growth factors such as fibroblast growth factors and insulin-like growth factor 1 and to the effect of a somatostatin analogue, octreotide, on lens epithelial cell proliferation (BaldysiakFigiel et al. 2005; Kampmeier et al. 2006). IGF-1 has been shown to regulate lens cell differentiation and proliferation and specifically binds to bovine lens epithelial cells (Palmade et al. 1994; Klok et al. 1998). It has also been shown that insulin and IGF-1 affect the protein composition of the lens fibre cells with possible consequences for cataract formation (Civil et al. 2000). In this context, it is significant that high levels of IGF 1 were found in the vitreous in diabetic patients (Boehm et al. 2001) and that Octreotide has been shown to have anti-proliferative effects on endothelial cells and to reduce the progression of diabetic retinopathy (Boehm et al. 2001; Boehm 2007). Thus, the use of somatostatin receptor ligands has been proposed for use in obesity and diabetic complications including cataract formation and retinopathy (Civil et al. 2000; Boehm & Lustig 2002; Cervia et al. 2008). The present study measures the effects of somatostatin analogues on early changes in glutathione, NADPH and ATP content of the rat lens in experimental diabetes, metabolites central to the maintenance of normal lens structure and function. .
Materials and methods Animals and reagents Male Wistar rats (Taconic, Eiby, Denmark) with an initial mean body weight of 230 g were used. Rats were housed, three per cage, in a room with 12:12 h (06.00–1800 h) artificial light cycle, temperature 21 2 °C and humidity 55 2%. The animals had free access to standard rat chow (Altromin, Lage, Germany) and tap water throughout the experiment. The blood glucose of the control rats was 6.7 0.2 mM. Diabetes was induced by a single i.v. injection of STZ (55 mg/kg body weight) in acidic 0.154 M NaCl (pH 4.0) following 12 h of food deprivation. Eighteen hours after the administration of STZ, and daily thereafter, the animals were weighed, urinalysis performed for glucose and ketones using Neostix (Ames Limited, Stoke Poges, Slough, UK) and tail-vein blood glucose determined by Haemoglucotest 1–44 and Reflolux II reflectance meter (Boehringer-Mannheim, Germany). Only animals with blood glucose levels above 11 mM and without ketonuria were included in the study. The STZ-treated rats remained diabetic over the entire 7-day period of the experiment. The rats were randomly distributed among 8 groups matched for body weight, each containing seven animals, with treatment duration of 7 days as follows: two groups of non-diabetic rats; two groups of diabetic rats, injected with saline s.c. twice daily; two groups of diabetic rats treated with insulin (s.c.) daily with a very long-acting, heat-treated ultralente insulin (Novo-Nordisk, Bagsvaerd, Denmark); one
group of diabetic rats given daily AGP s.c.; and one group of diabetic rats given daily doses of SMS s.c. Insulin treatment was initiated 18 h after the administration of STZ and was given in an initial dose of 4–6 U, followed by 1–3 U thereafter, depending on blood glucose values. AGP and SMS treatments were initiated 2–4 h after injection with STZ. AGP was given s.c. at a dose of 100 lg (in 0.5 ml 0.154 M NaCl) twice daily and SMS 100 lg s.c. (in 0.5 ml acidic 0.154 M NaCl) (pH 5.5) twice daily. The control and diabetic rats were treated with placebo (0.5 ml 0.154 M NaCl) twice daily. After completion of the treatment period, the rats were anaesthetized with sodium barbital (50 mg/kg body weight i.p.), and the lens removed, snapfrozen in liquid nitrogen. They were stored at 80 °C until required for the measurement of total glutathione, ATP, NADPH and sorbitol.
Ethical approval The principles of laboratory care were in accordance with the Home Office Animal (Scientific Procedures) Act 1986 requirements.
Metabolite measurements Metabolites were measured in neutralised perchloric acid extracts of rapidly frozen lens tissue. Total glutathione was assayed by the DNTB / glutathione reductase recycling method (Akerboom & Sies 1981). Other metabolites were measured according to the methods described by Bergmeyer (1962), and the resultant oxidation/reduction in NAD+/ NADH or NADP+/NADPH was recorded spectrophotometrically. In outline, the lens content of ATP was linked to glucose 6-phosphate formation via the addition of glucose and purified hexokinase followed by the addition of glucose 6phosphate dehydrogenase and NADP+, and the formation of 6-phosphogluconate and NADPH. The following are linked reactions: ATP+glucose+hexokinase?G6P+ADP G6P+G6P dehydrogenase+NADP+? 6-phosphogluconate+NADPH. Sorbitol was determined using sorbitol dehydrogenase and the conversion of NAD+ to NADH. The equilibrium of sorbitol dehydrogenase in favour of the conversion of fructose+NADH to sorbitol. The equilibrium was shifted in favour of the oxidation of sorbitol was achieved by using a pH of approximately 9.0–9.5. Sorbitol+sorbitol dehydrogenase+NAD +fructose +NADH. NADPH was measured by the enzymic recycling method using the rate of oxygen uptake with an oxygen electrode as described by Greenbaum et al. (1965). Purified enzymes, substrates and cofactors were obtained from Roche Diagnostics GmbH, Roche Applied Sciences, 68298 Mannheim, Germany. The results are expressed as the mean SEM and Fisher’s P test was used for statistical analysis of significance. International Journal of Experimental Pathology, 2014, 95, 95–100
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lens glutathione content showed the most significant fall of 70% relative to the control value; SMS and AGP partially prevented this decline; angiopeptin had a more marked effect (Figure 1b); and insulin treatment ab initio prevented the fall in glutathione. The ATP content of the lens is significantly decreased by 30% in diabetes; here again, AGP has the more marked effect in preventing the loss of ATP, and in contrast, the SMS treatment had no significant effect (Figure 1c). The lens content of NADPH showed a significant fall of 30% in untreated diabetes; only SMS and insulin treatment raised the NADPH towards normal levels contrasting with AGP that had no significant effect after 7 days of treatment (Figure 1d).
Results The changes in glutathione, NADPH and ATP 7 days after induction of diabetes with STZ and the effects of treatment with insulin, SMS and AGP on these metabolites in rat lens are shown in Figure 1a–d as content/g lens together with statistical differences between the groups; no significant differences were observed in the lens content of these metabolites over the period of the first 4 days of treatment. The effect of somatostatins on the lens content of sorbitol in diabetes is shown in Figure 1a, and SMS and AGP had similar effects in significantly lowering the sorbitol content by 30% and 34% respectively. Among the metabolites measured, the (a)
(b)
(c)
(d)
Figure 1 Effect of long-acting somatostatin analogues (Sandostatin, SMS and angiopeptin, AGP) and insulin on the sorbitol (a), glutathione (b), ATP (c) and NADPH (d) content of diabetic rat lens. The columns represent the mean values and the vertical bars the SEM of not
ORIGINAL ARTICLE
Effects of long-acting somatostatin analogues on redox systems in rat lens in experimental diabetes Sirilaksana Kunjara*, A. Leslie Greenbaum*, Milena Sochor*, Allan Flyvbjerg†, Henning Grønbaek† and Patricia McLean* *Division of Biosciences, Research Department of Cell and Developmental Biology, University College London, London, UK and † Department of Endocrinology and Internal Medicine & The Medical Research Laboratories, Clinical Institute, Aarhus University Hospital and Aarhus University, Aarhus C, Denmark
INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY doi: 10.1111/iep.12069
Received for publication: 26 November 2012 Accepted for publication: 26 November 2013 Correspondence: Patricia McLean Division of Biosciences Research Department of Cell and Developmental Biology University College London Gower Street London WC1E 6BT UK Tel.: +44 207 6796576 Fax: +44 207 6792091 E-mail: [email protected]
SUMMARY The effects of long-acting somatostatin analogues, angiopeptin (AGP) and Sandostatin (SMS), on the early decline in the lens content of glutathione (GSH), ATP and NADPH and increase in sorbitol were studied in STZ diabetic rats, and comparison was made with the effect of insulin. Three factors prompted this study: (i) the known increase in IGF-1 in ocular tissue in diabetes and antagonistic effect of somatostatins, (ii) the known effect of IGF-1 in increasing lens aldose reductase and (iii) the lack of effect of somatostatins on diabetic hyperglycaemia, the latter enabling a differentiation to be made between effects of hyperglycaemia per se and site(s) of IGF-1/somatostatins. All four metabolites studied showed a significant restoration towards the normal control level after 7 days of treatment with AGP and SMS, and AGP was more effective on levels of GSH and ATP. A significant correlation was found between GSH and ATP across all groups at 7 days treatment. The redox state changes in diabetes include both NADP+/NADPH and NAD+/NADH in the conversion of glucose to sorbitol and via sorbitol dehydrogenase to fructose with a linked decrease in ATP formation via NAD+/NADH regulation of the glycolytic pathway. The interlinked network of change includes the requirement for ATP in the synthesis of GSH. The present study points to possible loci of action of somatostatins in improving metabolic parameters in the diabetic rat lens via effects on aldose reductase and/or glucose transport at GLUT 3. Keywords angiopeptin, ATP, experimental diabetes (STZ), glutathione, insulin, rat lens, redox systems, sandostatin
Cataract formation is a major cause of blindness in ageing and in diabetes mellitus (see Resnikoff et al. 2004). Extensive studies have been made of the early biochemical changes involved in cataract formation using as model lenses from streptozotocin or alloxan diabetic rats, many of these centring upon early changes in the metabolite profile. Changes in the integration of pathways linked to glucose metabolism, redox systems resulting from hyperglycaemia, the role of the NADPH-dependent aldo-keto reductase, the rate-limiting enzyme of the polyol pathway, and the inhibition of this enzyme by Sorbinil have been discussed (Gabbay 1973; Kinoshita et al. 1979; Varma 1980; Gonzalez et al.1983, 1986; McLean et al. 1985; Brownlee 2005; Lu
2009). In parallel, measures to correct the redox state changes by the use of compounds such as pyruvate and ascorbate have been studied (Zhao et al. 2000; Hedge & Varma 2004). Glutathione was a focal point of the present study, because as reviewed by Lu (2009), glutathione has diverse functions including antioxidant defence, maintenance of thiol groups and cell proliferation. Alterations in the regulation of glutathione synthesis in ageing and disease, including diabetes and insulin treatment, have been reviewed by Lu (2009) with emphasis on the regulation of the ratelimiting enzyme glutamate cysteine ligase and the coordinated change in glutathione synthase. At present, there does not appear to be information on changes in these enzymes
© 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology
95
96
S. Kunjara et al.
in the rat lens in diabetes although the decrease in lens glutathione is well established. Recently, attention has been drawn to the role of growth factors such as fibroblast growth factors and insulin-like growth factor 1 and to the effect of a somatostatin analogue, octreotide, on lens epithelial cell proliferation (BaldysiakFigiel et al. 2005; Kampmeier et al. 2006). IGF-1 has been shown to regulate lens cell differentiation and proliferation and specifically binds to bovine lens epithelial cells (Palmade et al. 1994; Klok et al. 1998). It has also been shown that insulin and IGF-1 affect the protein composition of the lens fibre cells with possible consequences for cataract formation (Civil et al. 2000). In this context, it is significant that high levels of IGF 1 were found in the vitreous in diabetic patients (Boehm et al. 2001) and that Octreotide has been shown to have anti-proliferative effects on endothelial cells and to reduce the progression of diabetic retinopathy (Boehm et al. 2001; Boehm 2007). Thus, the use of somatostatin receptor ligands has been proposed for use in obesity and diabetic complications including cataract formation and retinopathy (Civil et al. 2000; Boehm & Lustig 2002; Cervia et al. 2008). The present study measures the effects of somatostatin analogues on early changes in glutathione, NADPH and ATP content of the rat lens in experimental diabetes, metabolites central to the maintenance of normal lens structure and function. .
Materials and methods Animals and reagents Male Wistar rats (Taconic, Eiby, Denmark) with an initial mean body weight of 230 g were used. Rats were housed, three per cage, in a room with 12:12 h (06.00–1800 h) artificial light cycle, temperature 21 2 °C and humidity 55 2%. The animals had free access to standard rat chow (Altromin, Lage, Germany) and tap water throughout the experiment. The blood glucose of the control rats was 6.7 0.2 mM. Diabetes was induced by a single i.v. injection of STZ (55 mg/kg body weight) in acidic 0.154 M NaCl (pH 4.0) following 12 h of food deprivation. Eighteen hours after the administration of STZ, and daily thereafter, the animals were weighed, urinalysis performed for glucose and ketones using Neostix (Ames Limited, Stoke Poges, Slough, UK) and tail-vein blood glucose determined by Haemoglucotest 1–44 and Reflolux II reflectance meter (Boehringer-Mannheim, Germany). Only animals with blood glucose levels above 11 mM and without ketonuria were included in the study. The STZ-treated rats remained diabetic over the entire 7-day period of the experiment. The rats were randomly distributed among 8 groups matched for body weight, each containing seven animals, with treatment duration of 7 days as follows: two groups of non-diabetic rats; two groups of diabetic rats, injected with saline s.c. twice daily; two groups of diabetic rats treated with insulin (s.c.) daily with a very long-acting, heat-treated ultralente insulin (Novo-Nordisk, Bagsvaerd, Denmark); one
group of diabetic rats given daily AGP s.c.; and one group of diabetic rats given daily doses of SMS s.c. Insulin treatment was initiated 18 h after the administration of STZ and was given in an initial dose of 4–6 U, followed by 1–3 U thereafter, depending on blood glucose values. AGP and SMS treatments were initiated 2–4 h after injection with STZ. AGP was given s.c. at a dose of 100 lg (in 0.5 ml 0.154 M NaCl) twice daily and SMS 100 lg s.c. (in 0.5 ml acidic 0.154 M NaCl) (pH 5.5) twice daily. The control and diabetic rats were treated with placebo (0.5 ml 0.154 M NaCl) twice daily. After completion of the treatment period, the rats were anaesthetized with sodium barbital (50 mg/kg body weight i.p.), and the lens removed, snapfrozen in liquid nitrogen. They were stored at 80 °C until required for the measurement of total glutathione, ATP, NADPH and sorbitol.
Ethical approval The principles of laboratory care were in accordance with the Home Office Animal (Scientific Procedures) Act 1986 requirements.
Metabolite measurements Metabolites were measured in neutralised perchloric acid extracts of rapidly frozen lens tissue. Total glutathione was assayed by the DNTB / glutathione reductase recycling method (Akerboom & Sies 1981). Other metabolites were measured according to the methods described by Bergmeyer (1962), and the resultant oxidation/reduction in NAD+/ NADH or NADP+/NADPH was recorded spectrophotometrically. In outline, the lens content of ATP was linked to glucose 6-phosphate formation via the addition of glucose and purified hexokinase followed by the addition of glucose 6phosphate dehydrogenase and NADP+, and the formation of 6-phosphogluconate and NADPH. The following are linked reactions: ATP+glucose+hexokinase?G6P+ADP G6P+G6P dehydrogenase+NADP+? 6-phosphogluconate+NADPH. Sorbitol was determined using sorbitol dehydrogenase and the conversion of NAD+ to NADH. The equilibrium of sorbitol dehydrogenase in favour of the conversion of fructose+NADH to sorbitol. The equilibrium was shifted in favour of the oxidation of sorbitol was achieved by using a pH of approximately 9.0–9.5. Sorbitol+sorbitol dehydrogenase+NAD +fructose +NADH. NADPH was measured by the enzymic recycling method using the rate of oxygen uptake with an oxygen electrode as described by Greenbaum et al. (1965). Purified enzymes, substrates and cofactors were obtained from Roche Diagnostics GmbH, Roche Applied Sciences, 68298 Mannheim, Germany. The results are expressed as the mean SEM and Fisher’s P test was used for statistical analysis of significance. International Journal of Experimental Pathology, 2014, 95, 95–100
Somatostatins, lens redox status in diabetes
97
lens glutathione content showed the most significant fall of 70% relative to the control value; SMS and AGP partially prevented this decline; angiopeptin had a more marked effect (Figure 1b); and insulin treatment ab initio prevented the fall in glutathione. The ATP content of the lens is significantly decreased by 30% in diabetes; here again, AGP has the more marked effect in preventing the loss of ATP, and in contrast, the SMS treatment had no significant effect (Figure 1c). The lens content of NADPH showed a significant fall of 30% in untreated diabetes; only SMS and insulin treatment raised the NADPH towards normal levels contrasting with AGP that had no significant effect after 7 days of treatment (Figure 1d).
Results The changes in glutathione, NADPH and ATP 7 days after induction of diabetes with STZ and the effects of treatment with insulin, SMS and AGP on these metabolites in rat lens are shown in Figure 1a–d as content/g lens together with statistical differences between the groups; no significant differences were observed in the lens content of these metabolites over the period of the first 4 days of treatment. The effect of somatostatins on the lens content of sorbitol in diabetes is shown in Figure 1a, and SMS and AGP had similar effects in significantly lowering the sorbitol content by 30% and 34% respectively. Among the metabolites measured, the (a)
(b)
(c)
(d)
Figure 1 Effect of long-acting somatostatin analogues (Sandostatin, SMS and angiopeptin, AGP) and insulin on the sorbitol (a), glutathione (b), ATP (c) and NADPH (d) content of diabetic rat lens. The columns represent the mean values and the vertical bars the SEM of not
