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A Commentary on 10 Years of Aldose Reductase Inhibition for Limited Joint Mobility in Diabetes
A Commentary on 10 Years of Aldose Reductase Inhibition for Limited Joint Mobility in Diabetes R. Philip Eaton W. L. Sibbitt, Jr. V. O. Shah R. I. Dorin P. G. Zager J. M. Bicknell
ABSTRACT This investigation examines the clinical response to long-term treatment of the diabetic syndrome of limited joint mobility (LJM) using an aldose reductase inhibitor (ARI) in comparison to historical controls, and proposes a potential role of aldose reductase (AR) genotype and expression in the clinical response to ARI. Clinical parameters, including quantitative hand movement and electromyogram, were followed over a decade of continuous ARI treatment with sorbinil (400 mg/ day) in two patients with insulin-dependent diabetes mellitus (IDDM) and severe compromising LJM, and compared to the published 10-year prospective investigation of untreated IDDM diabetic patients with LJM. Both
subjects were homozygous for the Z-2 AR allele (A-C)23 that has been linked with microvascular complications of DM. Cellular AR mRNA/b-actin ratios for both treated patients while on ARI therapy were approximately one-half the value observed in untreated patients with the complications of nephropathy or neuropathy. This is the longest reported experience of ARI intervention for any diabetic complication, documenting sustained correction of LJM, lack of side effects, and a potential molecular basis for the therapeutic response. ( Journal of Diabetes and Its Complications 12;1:34–38, 1998.) 1998 Elsevier Science Inc.
INTRODUCTION
ductase inhibitor (ARI). Since that time, McCance et al. have reported a 10-year prospective study of diabetic patients with LJM who demonstrated no resolution of their untreated LJM, providing an historical control for our ARI treated patients.2 An additional control regarding the natural history of LJM has been reported during treatment with an ineffective inhibitor representing a different chemical class, demonstrating no improvement over a 6-month span in 13 patients.3 It has now been 10 years that our patients have been continuously treated with sorbinil, and it may be valuable to report the continued alleviation of LJM in these ARI-treated subjects over this decade of experimental intervention to compare with the reported un-
I
n 1985, we reported three patients with the diabetic syndrome of limited joint mobility (LJM) who responded dramatically to several months of therapy with sorbinil,1 a hydantoin aldose re-
Department of Medicine (R.P.E., W.L.S., V.O.S., R.I.D., P.G.Z.) and Department of Neurology (J.M.B.), University of New Mexico School of Medicine, and Veterans Administration Medical Center, Albuquerque, New Mexico, USA Reprint requests to be sent to: Dr. R. Philip Eaton, Department of Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131. Journal of Diabetes and Its Complications 12:34–38 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
1056-8727/98/$19.00 PII S1056-8727(97)00049-4
J Diab Comp 1998; 12:34–38
LONG-TERM ARI FOR LIMITED JOINT MOBILITY
treated natural history. Moreover, with the advances in understanding of the heterogeneity of aldose reductase (AR)-genotype4 and expression 5–8 that have been linked to the development of diabetic complications, it is possible to hypothesize that a specific AR genotype or regulated expression may have influenced the dramatic clinical response to ARI observed in the 10 years of our patient management. In this context, we report our experience with ARI intervention for LJM conducted in two insulin-dependent diabetes mellitus (IDDM) patients examined over a 10-year period. We also characterize their AR-mRNA and genotype in order to suggest a potential role of these patient variables in determining the clinical response to ARI treatment. METHODS Clinical Evaluation. Patients were maintained on sorbinil (400mg/day) for a period of 10 years. Compliance with medication was determined by monthly pill count and demonstration of reduced serum uric acid levels, which is a known effect of AR inhibition in humans. Quantitative hand motion and nerve conduction velocity were assessed as previously described.1 Peripheral Blood Mononuclear Cell Aldose Reductase and b-actin mRNA. Peripheral Blood Mononuclear Cells (PBMC) were isolated by Ficoll-Hypaque gradient centrifugation. Genomic DNA and total cellular RNA were isolated using RNA-DNA STAT 60 (Teltest ‘‘B’’, Inc., Frenchwood, TX). RNase protection assay was performed using 10 mg of total cellular RNA hybridized with single-strand, antisense cRNA probes derived from the 222-nt Sau3A1-EcoRI fragment from the 39-untranslated region of the human AR cDNA9 and a 129 nt KpnI-EcoRI probe from the human b-actin cDNA (Ambion, Woodward, TX). Methods for probe preparation, hybridization, and determination of molar ratios were as previously described.10 Comparative AR/b-actin values were determined in control and diabetic populations as previously reported.7,8 AR Genotype. A 138 bp fragment of the hAR gene containing the variable dinucleotide (A-C) repeat was amplified by PCR using previously described primers and PCR conditions.4 The PCR product was analyzed by direct sequencing, using the Sequenase PCR kit (United States Biochemical Corp., Cleveland, OH) and confirmed by sequencing of eight or more individual subclones of the PCR product after intermediate subcloning in the TA cloning vector (Invitrogen, San Diego, CA). RESULTS Clinical Course. The clinical description of both patients with IDDM and initial 12-month response to ARI has been previously reported.1 Both subjects experi-
35
enced a sustained clinical improvement in LJM symptoms and functional capacity following initiation of ARI treatment. As shown in Table 1, the initial and dramatic clinical improvement in grip strength and hand motion were sustained over the decade of ARI treatment, with no deterioration toward pre-therapy values. Neither patient experienced any recognizable symptoms or complications of ARI treatment. Microvascular complications were stable during longitudinal evaluation, with no progression of pre-existing retinopathy in patient 1 and no clinical or EMG evidence of neuropathy (see Table 1) or nephropathy in either patient. AR Genotype and mRNA Expression. Both patients were homozygous for the Z-2 allele, characterized by the (A-C)23 dinucleotide repeat, of the AR gene, and this is the same allele implicated in diabetic retinopathy by Ko et al.4 AR mRNA levels in PBMC were assessed while the patients were receiving ARI therapy.6 The molar ratios for AR/b-actin, 0.045 and 0.046, respectively, were decreased compared to AR/b-actin ratios in diabetics with nephropathy (0.088 6 0.010, mean 6 SE, N 5 19) or retinopathic complications (0.079 6 0.01, mean 6 SE, N 5 20).6,7 In the absence of pre-ARI levels, it is not possible to state that the lower AR values in the two ARI-treated subjects relative to that observed in both nephropathy and retinopathy groups represent a lowering in value due to ARI treatment. Similarly, we cannot establish other than by coexisting events, that this normalization of AR levels caused their clinical improvement in LJM with ARI treatment. CONCLUSIONS Neither the pathophysiology nor effective therapy has been determined for the diabetic complication of limited joint mobility. Sometimes termed the stiff hand syndrome, this readily diagnosed problem has been a therapeutic challenge since first described by Jung et al. in 1971.11 The cumulative prevalence of LJM in adult IDDM from examination of 1028 patients is reported to be 49%,11–18 with an incidence of 41% over a 10-year prospective study.2 Both increased age2,11–13,18 and the duration of diabetes 2,11,13–18 correlate with LJM, as well as increased frequencies of the complications of retinopathy,12,14–17 and neuropathy.11,15,17 The pathogenesis of LJM is uncertain, however, changes in the hydration state of collagen matrix, affecting tensile properties of the connective tissue,19,20 have been implicated. The metabolic accumulation of polyol as a consequence of aldose reductase action on glucose substrate may generate the osmotic changes central to altered tissue hydration.21 A second hypothesis is that fibroblast growth and infiltration of the synovial tissues contribute to development of contractures of the tendon sheath.22,23 This synovial tissue is com-
36
EATON ET AL.
J Diab Comp 1998; 12:34–38
TABLE 1. CLINICAL RESPONSE TO ALDOSE REDUCTASE INHIBITOR INTERVENTION OVER 10 YEARS 1983 (Pre-Rx)
1984 (Post-Rx)
1993 (Post-Rx)
Grip strength (mm Hg)(left/right) Patient 1 100/200 240/240 180/235 Patient 2 110/120 145/130 NE* Range of motion in metacarpo-phalangeal joints (degrees) (flex/extension) Patient 1 85/0 90/20 90/5 Patient 2 80/10 100/0 NE Range of motion in interphalangeal joints (degrees) (flex/extension) Patient 1 90/10 90/0 110/0 Patient 2 90/-10 95/5 NE Ulnar nerve distal latencies (ms) (motor/sensory) Patient 1 3.3/2.8 NE 2.7/2.0 Patient 2 2.4/2.5 NE NE Median nerve distal latencies (ms) (motor/sensory) Patient 1 4.5/3.4 NE 4.1/2.9 Patient 2 3.4/2.7 NE NE
1995 (Post-Rx)
Normal
190/200 200/190
180/180 (F) 240/240 (M)
87/25 100/25
100/20 100/20
120/0 100/10
100/20 100/20
2.8/2.5 2.5/2.6
<4.5/<2.7 <4.5/<2.7
4.6/3.4 3.6/2.7
<4.5/<2.7 <4.5/$2.7
NE, not examined.
posed of fibroblast and macrophage-like cells with AR sensitive growth behavior.24 Cytopathic effects leading to secondary inflammatory changes in the tendon sheath21 or local increases in transforming growth factor b (TGF-b) may also play a role in pathogenesis.25 Whatever the mechanism, the improvement in LJM symptoms was sustained over a decade-long period of ARI treatment with no untoward consequences and complete patient satisfaction with normalized hand function. Because the severity of impaired hand function in these two patients prevented them from pursuing their professional activities of employment, this sustained amelioration of the problem with AR inhibition is important to document. This response stands in contrast with the historical controls of McCance et al.2 and of Rosenbloom et al.3 demonstrating no expectation of improvement in LJM with time. Thus, the clinical response observed in our patients was likely the result of ARI treatment rather than spontaneous remission occurring as part of the natural history of the diabetic LJM syndrome. Although a role for the AR enzyme in the pathogenesis of diabetic complications was proposed more than 20 years ago,26 the results of clinical trials of ARI remain equivocal. However, a great deal of progress in basic scientific research concerning aldose reductase has begun to emerge in recent years, including determination of the sequence9,27 and crystal structure28 of the AR gene and cognate protein, definition of physiological and molecular determinants of gene expression,29,30 and examination of the role of AR gene expression in polyoldependent and diabetic complications in transgenic animal models.31,32 In particular, the rapid development and tissue distribution of microvascular disease in
transgenic mice that overexpress the human AR gene support a potential role for modulation of AR gene expression in diabetic complications in humans.31,32 Consistent with this possibility, molecular epidemiologic studies have identified an association between the level of AR activity or expression in peripheral blood elements, including erythrocytes5 and mononuclear cells6–8 and the development of microvascular diabetic complications. In addition, a higher incidence of diabetic complications has been observed in patients who, like ours, are homozygous for the Z-2 (A-C)23 allele of the AR gene.4 In this context, the normal PBMC AR mRNA levels observed during ARI treatment in our patients with LJM syndrome may reflect a polyoldependent effect of sorbinil to decrease AR mRNA levels, or alternatively, intrinsic levels of AR expression. Though no human studies of ARI exposure influencing AR gene expression have been reported, normal Sprague-Dawley rats given 9 weeks of sorbinil treatment have shown a reduction in AR mRNA in the renal cortex and medulla.10 Such data are consistent with the hypothesis that AR gene expression is regulated, at least in part, by tissue polyol content. In contrast, exposure of normal or diabetic rats to only 3 or fewer weeks of treatment with either sorbinil or a nonhydantoin ARI, statil, have failed to demonstrate an effect on AR message concentration.33,34 Clearly, further human studies are needed to evaluate the effects of sorbinil on AR mRNA and protein expression and to examine the relationship between AR levels in peripheral blood elements and target tissues of human diabetic complications. Both LJM patients were homozygous for the Z-2 (A-C)23 allele. Based upon the reported allelic frequency
J Diab Comp 1998; 12:34–38
of 0.217,4 this genetic composition should occur with an expected frequency of one in 25 persons in the population. Although allelic frequencies and relationship to AR expression need to be assessed further in defined populations, the linkage disequilibrium previously reported suggests that individuals with this genotype may demonstrate increased susceptibility to polyol-dependent complications. While no basic or clinical investigation has disproven a significant role for AR in diabetic complications, the broad base of scientific enthusiasm for the hypothesis has waned for lack of concrete success in either therapeutic response or irrefutable establishment of pathophysiology. The reasons for the dramatic response in our patients are not clear. Several possibilities include (1) this hydantoin-based inhibitor contains the necessary biological and pharmacokinetic behavior for tissue specific AR inhibition; (2) the LJM syndrome is more responsive to AR inhibition than microvascular complications of DM; (3) the early presentation and intervention in the natural history of diabetic stiff-hand syndrome is critical to the therapeutic response; (4) genetic variability in AR genotype or expression may influence the response to ARI; and (5) this drug may be effective via a non-AR mechanism. Thus, the apparent outcome in these two patients suggests that a possible beneficial role for ARI in genetically specific diabetic patients merits further investigation. ACKNOWLEDGEMENT This study was supported in part by General Clinical Research Center grant 5M01-RR-997 and Department of Veterans Affairs. The sorbinil (CP45,634) was provided by Pfizer Central Research, Groton, CT. We thank K. Kilpatrick, J. K. Griffith, and M. Jones for expert technical assistance and M. Eckhouse for secretarial support.
REFERENCES
LONG-TERM ARI FOR LIMITED JOINT MOBILITY
37
Increased levels of aldose reductase and glyoxalase-I in mononuclear cells from IDDM patients. J Clin Endocrinol Metab 81:488–492, 1996. 7. Shah VO, Dorin RI, Brown M, Sun Y, Zager PG: Aldose reductase (AR) gene expression in peripheral blood mononuclear cells (PBMC) is increased in patients with diabetic nephropathy (DN); role of leukocyte aldose reductase in development of diabetic nephropathy [Abstract]. J Am Soc Nephrol 6:455, 1995. 8. Das A, Shah VO, Dorin RI, Zager PG: Increase in aldose reductase gene expression in patients with diabetic retinopathy [Abstract]. Invest Ophthalmol Vis Sci 3:S105, 1996. 9. Bohren KM, Bullock B, Wermuth B, Gabbbay KH: The aldo-keto reductase superfamily. cDNAs and deduced amino acid sequences of human aldehyde and aldose reductases. J Biol Chem 264:9547–9551, 1989. 10. Dorin RI, Shah VO, Kaplan DL, Vela BS, Zager PG: Regulation of aldose reductase gene expression in renal cortex and medulla of rats. Diabetologia 38:46–54, 1995. 11. Jung Y, Hohmann TC, Gerneth JA, et al.: Diabetic hand syndrome. Metabolism 20:1008–1015, 1971. 12. Chammas M, Bousquet P, Renard E, et al.: Dupuytren’s disease, carpal tunnel syndrome, trigger finger, and diabetes mellitus type 2 diabetes mellitus: Various entities and associated significance. J Hand Surg 20:109–114, 1995. 13. Arkkila PE, Kantola IM, Viikari JS: Limited joint mobility in type 1 diabetic patients: correlation to other diabetic complications. J Int Med 236:215–223, 1994. 14. Pal B, Anderson J, Dick WC, et al.: Limitation of joint mobility and shoulder capsulitis in insulin- and noninsulin-dependent diabetes mellitus. Br J Rheum 25:147– 151, 1986. 15. Starkman HS, Gleason RE, Rand LI, et al.: Limited joint mobility (LJM) of the hand in patients with diabetes mellitus: Relation to chronic complications. Ann Rheum Dis 45:130–135, 1986. 16. Mengistu M, Aabdulkadir J: Limited finger joint mobility in insulin-dependent Ethiopian diabetics. Diabetic Med 2:387–389, 1985.
1. Eaton RP, Sibbitt WL Jr, Harsh A: The effect of an aldose reductase inhibition agent on limited joint mobility in diabetic patients. JAMA 253:1437–1440, 1985.
17. Beacom R, Gillespie EL, Middleton D, et al.: Limited joint mobility in insulin-dependent diabetes: Relationship to retinopathy, peripheral nerve function and HLA status. Q J Med 56:337–344, 1985.
2. McCance DR, Crowe G, Quinn MJ, et al.: Incidence of microvascular complications in type 1 diabetic subjects with limited joint mobility: A 10-year prospective study. Diabetic Med 10:807–810, 1993.
18. Renard E, Jacques D, Chammas M, et al.: Increased prevalence of soft tissue hand lesions in type 1 and type 2 diabetes mellitus: Various entities and associated significance. Diabetes Metab 20:513–521, 1994.
3. Rosenbloom AL, Buithieu M, Jelliffee KA, and et al.: Effect of an aldose reductase inhibiting agent on limited joint mobility in IDDM. Diabetes Care 15:588–589, 1992.
19. Nomura S, Hiltner A, Lando JB, et al.: Interaction of water with native collagen. Bipolymers 16:231–246, 1977.
4. Ko BC, Lam KS, Wat NM, Chung SS: An (A-C) dinucleotide repeat polymorphic marker at the 59 end of the aldose reductase gene is associated with early-onset diabetic retinopathy in NIDDM patients. Diabetes 44:727– 732, 1995.
20. Bazin S, LeLous M, Duance VC, et al.: Biochemistry and histology of the connective tissue of Dupuytren’s disease lesions. Eur J Clin Invest 10–16, 1980. 21. Eaton RP, Qualls C, Bicknell J, et al.: Structure-function relationships within peripheral nerves in diabetic neuropathy: the hydration hypothesis. Diabetologia 39:439– 446, 1996.
5. Nishimura C, Saito T, Ito T, Omori Y, Tanimoto T: High levels of erythrocyte aldose reductase and diabetic retinopathy in NIDDM patients. Diabetologia 37:328–330, 1994.
22. Hamlin CR, Kohn RP, Luschin JH: Apparent accelerated aging of human collagen in diabetes mellitus. Diabetes 24:902, 1975.
6. Ratliff DM, Vanderjagt DF, Eaton RP, Vanderjagt DL:
23. Buckingham BAA, Uitto J, Sandborg C, et al.: Sclero-
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J Diab Comp 1998; 12:34–38
derma-like changes in insulin-dependent diabetes mellitus: Clinical and biochemical studies. Diabetes Care 7:163– 169, 1984.
osmoregulation of aldose reductase mRNA, protein, and sorbitol in renal medulla. Am J Physiol 258:F154–F161, 1990.
24. Sibbitt WL Jr, Mills RG, Bigler CF, Eaton RP, et al.: Glucose inhibition of human fibroblast proliferation and response to growth factors is prevented by inhibitors of aldose reductase. Mech Aging Dev 47:265–274, 1989.
30. Smardo FL Jr, Burg MB, Garcia-Perez A: Kidney aldose reductase gene transcription is osmotically regulated. Am J Physiol 262:C776–C782, 1992.
25. Border WA, Ruoslahti E: Transforming growth factor-b in disease: The dark side of tissue repair. J Clin Invest 90:1–7, 1992. 26. Gabbay KH: The sorbitol pathway and the complications of diabetes. N Engl J Med 288:831–836, 1973. 27. Graham A, Brown L, Hedge PJ, Gammack AJ, Markham AF: Structure of the human aldose reductase gene. J Biol Chem 266:6872–6877, 1991. 28. Wilson DK, Bohren KM, Gabbay KH, Quiocho FA: An unlikely substrate site in the 1.65 A structure of the human aldose reductase holoenzyme implicated in diabetic complications. Science 257:81–84, 1992. 29. Cowley BD Jr, Ferraris JD, Carper D, Burg MB: In vivo
31. Yamaoka T, Nishimura C, Yamashita K, et al.: Acute onset of diabetic pathological changes in transgenic mice with human aldose reductase cDNA. Diabetologia 38:255– 261, 1995. 32. Yagihashi S, Yamagishi S, Wada R, et al.: Galactosemic neuropathy in transgenic mice for human aldose reductase. Diabetes 45:56–59, 1996. 33. Ghahary A, Chakrabarti S, Sima AAF, et al.: Effect of insulin and statil on aldose reductase expression in diabetic rats. Diabetes 40:1391–1396, 1991. 34. Bondy C, Cowley BD Jr, Lightman SL, et al.: Feedback inhibition of aldose reductase gene expression in rat renal medulla. Galactitol accumulation reduces enzyme mRNA levels and depletes cellular inositol content. J Clin Invest 86:1103–1108. 1990.
ABSTRACT This investigation examines the clinical response to long-term treatment of the diabetic syndrome of limited joint mobility (LJM) using an aldose reductase inhibitor (ARI) in comparison to historical controls, and proposes a potential role of aldose reductase (AR) genotype and expression in the clinical response to ARI. Clinical parameters, including quantitative hand movement and electromyogram, were followed over a decade of continuous ARI treatment with sorbinil (400 mg/ day) in two patients with insulin-dependent diabetes mellitus (IDDM) and severe compromising LJM, and compared to the published 10-year prospective investigation of untreated IDDM diabetic patients with LJM. Both
subjects were homozygous for the Z-2 AR allele (A-C)23 that has been linked with microvascular complications of DM. Cellular AR mRNA/b-actin ratios for both treated patients while on ARI therapy were approximately one-half the value observed in untreated patients with the complications of nephropathy or neuropathy. This is the longest reported experience of ARI intervention for any diabetic complication, documenting sustained correction of LJM, lack of side effects, and a potential molecular basis for the therapeutic response. ( Journal of Diabetes and Its Complications 12;1:34–38, 1998.) 1998 Elsevier Science Inc.
INTRODUCTION
ductase inhibitor (ARI). Since that time, McCance et al. have reported a 10-year prospective study of diabetic patients with LJM who demonstrated no resolution of their untreated LJM, providing an historical control for our ARI treated patients.2 An additional control regarding the natural history of LJM has been reported during treatment with an ineffective inhibitor representing a different chemical class, demonstrating no improvement over a 6-month span in 13 patients.3 It has now been 10 years that our patients have been continuously treated with sorbinil, and it may be valuable to report the continued alleviation of LJM in these ARI-treated subjects over this decade of experimental intervention to compare with the reported un-
I
n 1985, we reported three patients with the diabetic syndrome of limited joint mobility (LJM) who responded dramatically to several months of therapy with sorbinil,1 a hydantoin aldose re-
Department of Medicine (R.P.E., W.L.S., V.O.S., R.I.D., P.G.Z.) and Department of Neurology (J.M.B.), University of New Mexico School of Medicine, and Veterans Administration Medical Center, Albuquerque, New Mexico, USA Reprint requests to be sent to: Dr. R. Philip Eaton, Department of Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131. Journal of Diabetes and Its Complications 12:34–38 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
1056-8727/98/$19.00 PII S1056-8727(97)00049-4
J Diab Comp 1998; 12:34–38
LONG-TERM ARI FOR LIMITED JOINT MOBILITY
treated natural history. Moreover, with the advances in understanding of the heterogeneity of aldose reductase (AR)-genotype4 and expression 5–8 that have been linked to the development of diabetic complications, it is possible to hypothesize that a specific AR genotype or regulated expression may have influenced the dramatic clinical response to ARI observed in the 10 years of our patient management. In this context, we report our experience with ARI intervention for LJM conducted in two insulin-dependent diabetes mellitus (IDDM) patients examined over a 10-year period. We also characterize their AR-mRNA and genotype in order to suggest a potential role of these patient variables in determining the clinical response to ARI treatment. METHODS Clinical Evaluation. Patients were maintained on sorbinil (400mg/day) for a period of 10 years. Compliance with medication was determined by monthly pill count and demonstration of reduced serum uric acid levels, which is a known effect of AR inhibition in humans. Quantitative hand motion and nerve conduction velocity were assessed as previously described.1 Peripheral Blood Mononuclear Cell Aldose Reductase and b-actin mRNA. Peripheral Blood Mononuclear Cells (PBMC) were isolated by Ficoll-Hypaque gradient centrifugation. Genomic DNA and total cellular RNA were isolated using RNA-DNA STAT 60 (Teltest ‘‘B’’, Inc., Frenchwood, TX). RNase protection assay was performed using 10 mg of total cellular RNA hybridized with single-strand, antisense cRNA probes derived from the 222-nt Sau3A1-EcoRI fragment from the 39-untranslated region of the human AR cDNA9 and a 129 nt KpnI-EcoRI probe from the human b-actin cDNA (Ambion, Woodward, TX). Methods for probe preparation, hybridization, and determination of molar ratios were as previously described.10 Comparative AR/b-actin values were determined in control and diabetic populations as previously reported.7,8 AR Genotype. A 138 bp fragment of the hAR gene containing the variable dinucleotide (A-C) repeat was amplified by PCR using previously described primers and PCR conditions.4 The PCR product was analyzed by direct sequencing, using the Sequenase PCR kit (United States Biochemical Corp., Cleveland, OH) and confirmed by sequencing of eight or more individual subclones of the PCR product after intermediate subcloning in the TA cloning vector (Invitrogen, San Diego, CA). RESULTS Clinical Course. The clinical description of both patients with IDDM and initial 12-month response to ARI has been previously reported.1 Both subjects experi-
35
enced a sustained clinical improvement in LJM symptoms and functional capacity following initiation of ARI treatment. As shown in Table 1, the initial and dramatic clinical improvement in grip strength and hand motion were sustained over the decade of ARI treatment, with no deterioration toward pre-therapy values. Neither patient experienced any recognizable symptoms or complications of ARI treatment. Microvascular complications were stable during longitudinal evaluation, with no progression of pre-existing retinopathy in patient 1 and no clinical or EMG evidence of neuropathy (see Table 1) or nephropathy in either patient. AR Genotype and mRNA Expression. Both patients were homozygous for the Z-2 allele, characterized by the (A-C)23 dinucleotide repeat, of the AR gene, and this is the same allele implicated in diabetic retinopathy by Ko et al.4 AR mRNA levels in PBMC were assessed while the patients were receiving ARI therapy.6 The molar ratios for AR/b-actin, 0.045 and 0.046, respectively, were decreased compared to AR/b-actin ratios in diabetics with nephropathy (0.088 6 0.010, mean 6 SE, N 5 19) or retinopathic complications (0.079 6 0.01, mean 6 SE, N 5 20).6,7 In the absence of pre-ARI levels, it is not possible to state that the lower AR values in the two ARI-treated subjects relative to that observed in both nephropathy and retinopathy groups represent a lowering in value due to ARI treatment. Similarly, we cannot establish other than by coexisting events, that this normalization of AR levels caused their clinical improvement in LJM with ARI treatment. CONCLUSIONS Neither the pathophysiology nor effective therapy has been determined for the diabetic complication of limited joint mobility. Sometimes termed the stiff hand syndrome, this readily diagnosed problem has been a therapeutic challenge since first described by Jung et al. in 1971.11 The cumulative prevalence of LJM in adult IDDM from examination of 1028 patients is reported to be 49%,11–18 with an incidence of 41% over a 10-year prospective study.2 Both increased age2,11–13,18 and the duration of diabetes 2,11,13–18 correlate with LJM, as well as increased frequencies of the complications of retinopathy,12,14–17 and neuropathy.11,15,17 The pathogenesis of LJM is uncertain, however, changes in the hydration state of collagen matrix, affecting tensile properties of the connective tissue,19,20 have been implicated. The metabolic accumulation of polyol as a consequence of aldose reductase action on glucose substrate may generate the osmotic changes central to altered tissue hydration.21 A second hypothesis is that fibroblast growth and infiltration of the synovial tissues contribute to development of contractures of the tendon sheath.22,23 This synovial tissue is com-
36
EATON ET AL.
J Diab Comp 1998; 12:34–38
TABLE 1. CLINICAL RESPONSE TO ALDOSE REDUCTASE INHIBITOR INTERVENTION OVER 10 YEARS 1983 (Pre-Rx)
1984 (Post-Rx)
1993 (Post-Rx)
Grip strength (mm Hg)(left/right) Patient 1 100/200 240/240 180/235 Patient 2 110/120 145/130 NE* Range of motion in metacarpo-phalangeal joints (degrees) (flex/extension) Patient 1 85/0 90/20 90/5 Patient 2 80/10 100/0 NE Range of motion in interphalangeal joints (degrees) (flex/extension) Patient 1 90/10 90/0 110/0 Patient 2 90/-10 95/5 NE Ulnar nerve distal latencies (ms) (motor/sensory) Patient 1 3.3/2.8 NE 2.7/2.0 Patient 2 2.4/2.5 NE NE Median nerve distal latencies (ms) (motor/sensory) Patient 1 4.5/3.4 NE 4.1/2.9 Patient 2 3.4/2.7 NE NE
1995 (Post-Rx)
Normal
190/200 200/190
180/180 (F) 240/240 (M)
87/25 100/25
100/20 100/20
120/0 100/10
100/20 100/20
2.8/2.5 2.5/2.6
<4.5/<2.7 <4.5/<2.7
4.6/3.4 3.6/2.7
<4.5/<2.7 <4.5/$2.7
NE, not examined.
posed of fibroblast and macrophage-like cells with AR sensitive growth behavior.24 Cytopathic effects leading to secondary inflammatory changes in the tendon sheath21 or local increases in transforming growth factor b (TGF-b) may also play a role in pathogenesis.25 Whatever the mechanism, the improvement in LJM symptoms was sustained over a decade-long period of ARI treatment with no untoward consequences and complete patient satisfaction with normalized hand function. Because the severity of impaired hand function in these two patients prevented them from pursuing their professional activities of employment, this sustained amelioration of the problem with AR inhibition is important to document. This response stands in contrast with the historical controls of McCance et al.2 and of Rosenbloom et al.3 demonstrating no expectation of improvement in LJM with time. Thus, the clinical response observed in our patients was likely the result of ARI treatment rather than spontaneous remission occurring as part of the natural history of the diabetic LJM syndrome. Although a role for the AR enzyme in the pathogenesis of diabetic complications was proposed more than 20 years ago,26 the results of clinical trials of ARI remain equivocal. However, a great deal of progress in basic scientific research concerning aldose reductase has begun to emerge in recent years, including determination of the sequence9,27 and crystal structure28 of the AR gene and cognate protein, definition of physiological and molecular determinants of gene expression,29,30 and examination of the role of AR gene expression in polyoldependent and diabetic complications in transgenic animal models.31,32 In particular, the rapid development and tissue distribution of microvascular disease in
transgenic mice that overexpress the human AR gene support a potential role for modulation of AR gene expression in diabetic complications in humans.31,32 Consistent with this possibility, molecular epidemiologic studies have identified an association between the level of AR activity or expression in peripheral blood elements, including erythrocytes5 and mononuclear cells6–8 and the development of microvascular diabetic complications. In addition, a higher incidence of diabetic complications has been observed in patients who, like ours, are homozygous for the Z-2 (A-C)23 allele of the AR gene.4 In this context, the normal PBMC AR mRNA levels observed during ARI treatment in our patients with LJM syndrome may reflect a polyoldependent effect of sorbinil to decrease AR mRNA levels, or alternatively, intrinsic levels of AR expression. Though no human studies of ARI exposure influencing AR gene expression have been reported, normal Sprague-Dawley rats given 9 weeks of sorbinil treatment have shown a reduction in AR mRNA in the renal cortex and medulla.10 Such data are consistent with the hypothesis that AR gene expression is regulated, at least in part, by tissue polyol content. In contrast, exposure of normal or diabetic rats to only 3 or fewer weeks of treatment with either sorbinil or a nonhydantoin ARI, statil, have failed to demonstrate an effect on AR message concentration.33,34 Clearly, further human studies are needed to evaluate the effects of sorbinil on AR mRNA and protein expression and to examine the relationship between AR levels in peripheral blood elements and target tissues of human diabetic complications. Both LJM patients were homozygous for the Z-2 (A-C)23 allele. Based upon the reported allelic frequency
J Diab Comp 1998; 12:34–38
of 0.217,4 this genetic composition should occur with an expected frequency of one in 25 persons in the population. Although allelic frequencies and relationship to AR expression need to be assessed further in defined populations, the linkage disequilibrium previously reported suggests that individuals with this genotype may demonstrate increased susceptibility to polyol-dependent complications. While no basic or clinical investigation has disproven a significant role for AR in diabetic complications, the broad base of scientific enthusiasm for the hypothesis has waned for lack of concrete success in either therapeutic response or irrefutable establishment of pathophysiology. The reasons for the dramatic response in our patients are not clear. Several possibilities include (1) this hydantoin-based inhibitor contains the necessary biological and pharmacokinetic behavior for tissue specific AR inhibition; (2) the LJM syndrome is more responsive to AR inhibition than microvascular complications of DM; (3) the early presentation and intervention in the natural history of diabetic stiff-hand syndrome is critical to the therapeutic response; (4) genetic variability in AR genotype or expression may influence the response to ARI; and (5) this drug may be effective via a non-AR mechanism. Thus, the apparent outcome in these two patients suggests that a possible beneficial role for ARI in genetically specific diabetic patients merits further investigation. ACKNOWLEDGEMENT This study was supported in part by General Clinical Research Center grant 5M01-RR-997 and Department of Veterans Affairs. The sorbinil (CP45,634) was provided by Pfizer Central Research, Groton, CT. We thank K. Kilpatrick, J. K. Griffith, and M. Jones for expert technical assistance and M. Eckhouse for secretarial support.
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