Complements in non-insulin-dependent diabetes mellitus with complications

Diabetes Research and Cl#~ical Practice, 5 (1988) 233-238

233

Elsevier DRC 00223

Brief Report

Complements in non-insulin-dependent diabetes mellitus with complications Yasuo M o r i m o t o , Hiroshi Taniguchi, Yuki Yamashiro, Kazushige Ejiri, Shigeaki Baba and Yasufumi Arimoto a Second Department of Internal Medichw. Kobe University School of Medichw. Kobe, Japan and aArhnoto Clinic, Akashi. Japan

(Received 5 January 1988, revision received 15 February 1988, accepted 8 March 1988)

Key words: Complications;Complements;C3d: Non-insulin-dependentdiabetes mellitus

Summary A relation of the complement system to the development of complications in non-insulin-dependent diabetes mellitus (NIDDM) was evaluated by measuring some components of the complement system. CH50, C3, C4 and C3bINA were significantly elevated in subjects with NIDDM as compared with healthy non-diabetic controls. However, CH50 and C3 did not differ between diabetics with and without complications. C4 was higher in diabetics with retinopathy as well as with retinopathy and neuropathy than in diabetics without these complications. ACH50,/~lHgl and C3d were similar in subjects with NIDDM and non-diabetics, and not associated with complications of NIDDM. C3d/C3 in NIDDM without complications was lower than in healthy subjects, but did not significantly differ between the types of complications. These results suggest that the high level of complements in NIDDM might be due to enhanced production of complements and the development of diabetic complications would be related to the elevated level of complements.

Introduction It is, well known that diabetic microangiopathy is affected by diabetic control and duration [1,2]. Genetic factors are also suggested to play a role in the development of microangiopathy, but this is not largely accepted now [3]. In some observations cirAddress for correspondence: Dr. Y. Morimoto, Second Department of InternalMedicine,KobeUniversitySchoolof Medicine, 5-1, Kusunoki-cho7 chome,Chuo-ku, Kobe 650, Japan.

culating immune complexes increase in diabetes, especially with microangiopathy [4,5]. The deposition of immune complexes is considered to affect the pathogenesis of diabetic microangiopathy [6]. However, the process toward tissue injury after the deposition of immune complexes in the tissue has not been clarified in terms of the development of microangiopathy [7]. A low C4 concentration is reported in about 25% of the cases of insulin-dependent diabetes mellitus (IDDM) [8,9]. As for noninsulin-dependent diabetes mellitus (NIDDM), the

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234 TABLE 1 CHARACTERISTICS OF SUBJECTS No. of subjects NIDDM with retinopathy (group R) NIDDM with neuropathy (group N) NIDDM with retinopathy and neuropathy (group B) NIDDM without complications (group F) Healthy subjects (group H)

5 7 7 31 27

complement system has not been investigated well. Therefore, in the present study, we examined the complement system in N I D D M in terms of complement components as well as C3d to clarify the underlying mechanism of the altered complement level.

Subjects and method We studied five groups of subjects consisting of five N I D D M patients with retinopathy (group R), seven with neuropathy (group N), seven with retinopathy and neuropathy (group B), 31 N I D D M without complications (group F), and 27 healthy nondiabetic subjects (group H) (Table 1). There was no significant difference in age, sex and duration among each group of N I D D M . However, the age in N I D D M was significantly higher than in group H. In our unpublished observations age did not correlate with CH50, ACH50, C3, C4, C3blNA, fllHgl and C3d. The eye fundus was examined by an ophthalmologist at the Department of Ophthalmology, Kobe University School of Medicine Hospital. Both background and proliferative retinopathies were included in group R. Neuropathy was considered to be present in case of impaired ankle and/or knee jerk reflex, disturbed sense of touch,

Age (years) Mean + SEM (range) 54.8 4- 7.1 (31-74) 60.6 4- 3.4 (48-76) 52.0 + 7.9 (38-79) 56.2 + 1.9 (37-78) 30.8 + 1.4 (21-46)

Male/ Female 1/4 3/4 3/4 20/11

Duration (years) Mean 4- SEM (range) 7.4 + 2.3 (2-16) 8.9 .4- 2.7 (0-17) 15.1 + 3.0 (6-31) 7.0 4- 1.8 (0-51)

13/14

pain and vibration, or abnormal function disclosed by cardiac beat-to-beat variation and/or Schellong's test [10]. CH50 was obtained through hemolytic assays modified from Mayer's method [11]. ACH50 was determined using the method described by Amano et al. [12]. C3, C4, C3blNA and fllHgl were measured by single radial immunodiffusion. C3d was assayed by single immunoelectrophoresis using the corresponding specific antibody [13,14]. Antibodies against C3 and C4 were purchased from Hoechst (F.R.G.) and antibody against C3d from Dako (Denmark). The results are expressed as mean 4- SEM. Statistical analysis was performed using Student's ttest and the chi-square test.

Results The titer of CH50 in group F was 47.7 + 14.8 U/ml. It was significantly increased compared with that in group H, 37.6 + 5.1 U/ml, but no significant difference in CH50 was observed between the groups of N I D D M , i.e., 51.8 ± 5.2, 51.4 ± 9.8 and 57.7 + 15.1 U/ml in groups R, N and B respectively (Fig. 1, upper panel). The titer of ACH50 was 16.3 + 1.5, 17.5 + 4.5, 17.4 + 2.0, 18.3 + 3.8 and 17.4

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Fig. 1. CH50 and ACH50 in healthy subjects (H), frank diabetics (F), and diabetics with retinopathy (R), neuropathy (N) or retinopathy plus neuropathy (B).

Fig. 2. C4 and C3 in healthy subjects (H), frank diabetics (F), and diabetics with retinopathy (R), neuropathy (N) or retinopathy plus neuropathy (B).

+ 1.8 U/ml in groups H, F, R, N and B respectively (Fig. 1, lower panel). There was no significant difference between these groups. The serum C4 concentration was significantly higher in N I D D M than in group H, i.e., 28.2 + 7.4 mg/dl. In particular it was significantly increased in group R (58.9 4- 17.7 mg/dl) and group B (54.2 + 15.8 mg/dl) as compared with group F (41.3 + 11.8 mg/dl) (Fig. 2, upper panel). The serum C3 concentration was significantly

higher in groups F (80.5 • 16.5 mg/dl), N (83.6 14.4 mg/dl) and B (82.3 + 13.8 mg/dl) than in group H (70.5 + 13.5 mg/dl), but there was no significant difference between these groups of N I D D M (Fig. 2, lower panel). C3d levels were 34.2 ± 9.8, 30.0 + 10.5, 33.8 + 9.2, 37.0 + 12.4 and 31.4 + 15.0 mg/dl in groups H, F, R, N and B respectively. These values did not differ (Fig. 3, upper panel). C3d/C3, a ratio of destruction of C3, was 0.5 + 0.16, 0.39 -4- 0.16, 0.43

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+ 0.11, 0.44 + 0.12 and 0.39 4- 0.14 in groups H, F, R, N and B respectively. In group F this ratio was significantly smaller than in group H, but there was no significant difference in the ratio between the groups of N I D D M (Fig. 3, lower panel). C3bINA was 93.5 + 21.3, 129.0 + 30.0, 127.4 + 40.7, 119.0 4- 29.4 and 132.6 4- 38.9% in groups H, F, R, N and B respectively. It was significantly elevated in N I D D M compared with group H, but did not differ between N I D D M groups (Fig. 4, upper panel). /~lHgl was 105.2 + 12.0, 113.3 + 23.9, 124.0 + 25.8, 102.3 + 21.6 and 121.3 4- 24.8% in groups H, F, R, N and B respectively. Elevation o f / ~ l H g l was detected in group B as compared with group H, but the values in groups F, R, N and B were no different from that in group H (Fig. 4, lower panel).

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Discussion Barnett and coworkers reported that a low serum C4 concentration was present in about 25% of I D D M and the prevalence of low serum C4 concentration is higher in patients with microangiopathy than in those without it [15]. Cooper et al. showed that the serum C4 concentration is significantly lower in I D D M than in N I D D M , but in both types of diabetes no difference is present between diabetes with and without microangiopathy [16]. As for neuropathy, the C4 concentration does not vary between I D D M patients with this complication and non-diabetic controls [17]. However, a low C4 concentration was reported in those with microangiopathy compared with non-diabetic subjects [15]. Thus, there have been no consistent views

237 on the relationship of complement to diabetic complications, since few studies on complements have been documented, in particular with regard to NIDDM. Some reports showed no reduction of C4 in NIDDM [8,16]. In our present study on NIDDM, CH50 and serum C3 and C4 concentrations were elevated compared with the non-diabetic subjects. This possibly indicates an accelerated production of the complements, which may lead to activation of the complement system. The concomitant increase of C3bINA may reflect a homeostatic suppression of this potential complement activation, though /~lHgl did not show a similar tendency. However, C3d, the breakdown product of C3, did not show any increase in the subjects with NIDDM in comparison with non-diabetics. The C3d concentration is affected by both classical and alternative pathways and partially dependent upon the amount of C3 [14], so that C3d/C3, an indicator for the activation of both pathways, was examined and not shown to be high in NIDDM. This suggested that the activation of complement is not enhanced in NIDDM. The C4 concentration was significantly higher in patients with diabetic retinopathy irrespective of the concomitant presence of neuropathy than in non-diabetic subjects as well as those with frank NIDDM, but no difference was found between diabetics with retinopathy and neuropathy. These findings contrast with those of Barnett et al. [15]. It may be due to the different types of diabetes studied, i.e., NIDDM vs. IDDM. No difference in CH50, ACH50 and C3 values in the present investigation was apparent between diabetics with and without complications. This together with the elevation of C4 suggests that the classical rather than the alternative pathway of the complement system is associated with the development of diabetic complications, especially retinopathy. In summary, the high level of C3 and C4 in NIDDM may derive rather from the increased production of complements and/or delayed degradation than from their simple activation. The more detailed process of involvement of the complement system in NIDDM and the development of its complications remain to be clarified.

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238 16 Cooper, M., Duff, R., Buchanan, R., McPherson, J. and Jerurns, G. (1986) Low serum C4 concentrations and microangiopathy in type 1 and type 2 diabetes. Br. Med. J. 292, 801.

17 Gilbey, S.G., Guy, R.J., Jones, H., Vergani, D. and Watkins, P.J. (1986) Diabetes and autonomic neuropathy: an immunological association? Diabetic Med. 3. 241-245.