Effects of Aerobic Exercise on Microalbuminuria and Enzymuria in Type 2 Diabetic Patients

Abstract

Increased urinary albumin excretion is a strong predictor for the development of overt diabetic nephropathy and overall cardiovascular morbidity and mortality in patients with type 2 diabetes. In a previous study, regular aerobic physical activity in overweight/obese patients with type 2 diabetes mellitus was found to have significant beneficial effects on glycemic control, insulin resistance, cardiovascular risk factors, and oxidative stress. The aim of the present study was to investigate the effects of aerobic exercise in the same cohort of type 2 diabetic patients on urinary albumin excretion, serum levels and urinary excretion of enzymes, tubular damage, and metabolic control markers in type 2 diabetic patients. Changes from baseline to 3 and 6 months of aerobic exercise were assessed for urinary albumin excretion, serum activities, and urinary excretion of N-acetyl-β-D-glucosaminidase (NAGA), plasma cell glycoprotein 1 (PC-1) and aminopeptidase N (APN), as well as their association with insulin resistance, cardiovascular risk factors, and oxidative stress parameters in 30 male type 2 diabetic patients (aged 54.8 ± 7.3 years, with a mean BMI of 30.8 ± 3.0 kg/m²). Microalbuminuria was found in six (20%) diabetic patients at baseline, three of them (10%) after three months, and only one patient (3.33%) at the end of the study period. A significant correlation was found for urinary albumin excretion at baseline both with sulfhydryl-groups and catalase, but not for urinary albumin excretion with MDA and glutathione. The prevalence of microalbuminuria tended to decrease after six months of aerobic exercise in type 2 diabetic patients, independently of any improvement in insulin resistance and oxidative stress parameters. Neither between-group nor within-group changes were found for urinary PC-1, APN, and NAGA activity. Serum NAGA was significantly increased (p < 0.05) over the control level in diabetic patients at baseline, but it decreased to the normal level after six months of exercise. This study has shown that a six-month aerobic exercise, without any change in the medication, tended to decrease microalbuminuria without changing enzymuria. However, further studies are needed not only to confirm those findings, but to elucidate potential mechanisms that would clarify the beneficial effects of exercise.

Keywords:

• aerobic exercise
• diabetes mellitus type 2
• microalbuminuria
• NAGA
• PC-1
• APN

INTRODUCTION

Regular exercise is a powerful non-pharmacological tool for the prevention and treatment of diabetes mellitus type 2, as a sedentary lifestyle is one of its key contributing factors. Unfortunately, up to one-third of adults with diabetes are completely sedentary.

Increased urinary albumin excretion is a strong predictor for the development of glomerular dysfunction, overt diabetic nephropathy, and overall cardiovascular morbidity and mortality in patients with diabetes.[1] Its association with insulin resistance and related cardiovascular risk factors strongly supports the hypothesis that patients with increased urinary albumin excretion might benefit from specific treatments of insulin resistance, including regular exercise.

Urinary N-acetyl-beta-D-glucosaminidase (NAGA) originates in renal proximal tubular cells and positively correlates with microalbuminuria and glycated hemoglobin. Those findings clearly suggest that urinary NAGA might be a valuable parameter to evaluate tubular damage and metabolic control, in diabetic patients as well. Because physical activity had no effect on urinary NAGA excretion in healthy people and adolescents,[2],[3] with no available data for type 2 diabetic patients, one of the objectives was to determine if it affected NAGA in type 2 diabetic patients enrolled in the present study.

Plasma cell glycoprotein 1 (PC-1) is a class II transmembrane glycoprotein that is implicated in the pathogenesis of insulin resistance in obesity, diabetes, and uremia.[4] The therapeutic modification of PC-1 expression, as demonstrated for a three-month metformin treatment,[5] is meant to be of a great benefit for insulin-resistant type 2 diabetic patients.

Aminopeptidase N (APN), an exopeptidase with a wide substrate specificity, is widely expressed in numerous human cells and tissues.[6–8] Its urinary excretion is meant to be a marker of the damage of a brush border of the proximal tubule.

In a previous study, regular aerobic physical activity in overweight/obese patients with type 2 diabetes mellitus was found to have significant beneficial effects on glycemic control, insulin resistance, cardiovascular risk factors, and oxidative stress.[9] The aim of the present study was to investigate the effects of aerobic exercise, in the same cohort of type 2 diabetic patients, on urinary albumin excretion, serum levels, and urinary excretion of enzymes, tubular damage, and metabolic control markers in type 2 diabetic patients.

SUBJECTS AND METHODS

Subjects

A total of 30 male, previously sedentary, type 2 diabetic patients, identified from outpatient clinics, aged 54 ± 7.32 years and of the mean BMI 30.8 ± 3.04 kg/m², underwent regular aerobic exercise for six months. Diabetes mellitus type 2 was defined according to the revised National Diabetes Data Group/World Health Organization criteria and controlled by diet and, if necessary, oral hypoglycemic agents. Previously prescribed therapy was maintained, and participants were instructed not to change their usual diet habits during the study period. The control group consisted of 30 healthy blood bank donors of the same sex, age (48 ± 6.37 years), and BMI (29.6 ± 2.1 kg/m²), with no personal history of or first degree relatives with diabetes and receiving no medications. Informed written consent was obtained from all subjects before participation.

Baseline Assessments

A detailed medical evaluation was performed at baseline. It included an exercise stress test, as well as neurological and ophthalmological examinations, in order to assess cardiorespiratory fitness and help identify, if present, underlying or undiagnosed diabetic complications. A structured interview was used to assess the current level of physical activity and express it as a physical activity index (PAI).[10] Systolic (SBP) and diastolic blood pressure (DBP) were measured from the left arm in seated position; after 5 min rest, three measurements were taken, and the average value recorded. BMI was calculated as weight (kg) / height (m²). All outcome measures and procedures, carried out at baseline, were repeated after 3 and 6 months of exercise.

Biochemical Analyses

Urine and blood samples were taken for biochemical analyses after an overnight fast of 12 hours. Urinary albumin excretion, fasting glycemia, glycated hemoglobin, and lipid profile parameters were measured on OLYMPUS AU400^e® Chemistry Immuno Analyser.

Urinary albumin excretion was measured by immunoturbidimetric method, expressed as albumin-to-creatinine ratio (ACR), and microalbuminuria was defined as urinary albumin excretion of 2.5–25 mg/mmol creatinine. N-acetyl-β-D-glucosaminidase (NAGA) activity was determined by the spectrophotometric method[11] using N-acetyl-β-D-glucosaminide. Phosphodiesterase activity of plasma cell membrane glycoprotein 1 (PC-1) was measured by the hydrolysis of thymidine-5′-monophosphate p-nitrophenol ester (Sigma Chemical Co., St. Louis, Missouri, USA).[12] Aminopeptidase N (APN) activity was determined by the spectrophotometric method, using alanine-p-nitroaniline as a substrate.[8] Urinary enzyme activities were expressed as enzyme-to-creatinine ratios.

Fasting glycemia (FG) was determined enzymatically, fasting insulin was measured with radioimmunoassay (Inep, Zemun), and the homeostasis model assessment (HOMA) was calculated to estimate insulin resistance.[13] Glycated hemoglobin (HbA_1c) was measured by the immunoinhibition method using latex agglutination inhibition assay. Total cholesterol (TC), HDL cholesterol (HDL-C), and triglycerides (TG) were determined enzymatically, using cholesterol esterase, an immunoinhibition method with anti-human HDL antibodies, and coupled enzymatic reactions, respectively. The Friedewald formula[14] was used to calculate LDL cholesterol (LDL-C), except when triglycerides exceeded 3.96 mmol/L (in that case, data were treated as missing). The absolute ten-year probability of developing a fatal cardiovascular event was assessed using the Systematic Coronary Risk Evaluation (SCORE) model.[15]

The activity of catalase (CAT) was determined by the spectrophotometric method.[16] Reduced glutathione (GSH) was determined by the modification of the method of Ellman.[17] Sulfhydryl groups (SH-groups) were determined using Ellman reagent[18] as well. Plasma and erythrocyte malondialdehyde (MDA) were determined by modified thiobarbituric acid (TBA) methods.[19],[20]

INTERVENTION

Regular aerobic exercise of moderate frequency, intensity, and duration was performed under direct supervision and lasted for six months. It included at least 3–5 sessions per week that consisted of warm-up period, brisk walking, and cool-down period, of an average duration of 45–60 minutes and at a workload corresponding to 50–75% of maximal heart rate. Participants were instructed not to change their usual diet habits and prescribed medication during the study period.

Statistical Analysis

Data were analyzed using statistical software Jandel SigmaStat^® for Windows (version 2.0). Student's t-test, Mann-Whitney rank sum test, and Wilcoxon signed rank test were used as appropriate to test the differences of measured variables, and data were expressed as means ± SD, medians, and ranges. Regression analysis was used to correlate determined parameters. A p value of less than 0.05 was considered statistically significant.

RESULTS

Baseline Characteristics

The present study was carried out in a total of 60 subjects, including the diabetes group, that consisted of

• 30 previously sedentary male type 2 diabetic patients, aged 54.8 ± 7.3 years and with a mean BMI of 30.8 ± 3.0 kg/m², who received oral hypoglycemic agents and/or diet therapy, and

• the control group, which consisted of 30 healthy subjects, aged 48.0 ± 6.4 years and with a mean BMI of 29.6 ± 2.1 kg/m² (see Table 1)

  Table 1 Baseline characteristics of the study participants

  			Diabetes group (n = 30)
  		Control group (n = 30)	At baseline	After three months	After six months
  Age (years)	Mean ± SD	48.0 ± 6.4	54.8 ± 7.3
  	Median	47	55
  	Range	40–64	40–65
  BMI (kg/m^2)	Mean ± SD	29.6 ± 2.1	30.8 ± 3.0	29.3 ± 3.2	28.6 ± 3.3
  	Median	30.1	29.7	29.1	28.6^†
  	Range	25.0–34.6	25.4–39.4	23.7–38.7	23.1–37.2

  Abbreviations: BMI = body mass index, SD = standard deviation.

  *versus control group, p < 0.001.

  ^†versus control group, p < 0.05.

Urinary Albumin Excretion

Microalbuminuria was observed in six (20%) diabetic patients at baseline, three of them (10%) after three months, and only in one patient (3.33%) at the end of the study period. ACR (mg/mmol creatinine) in three patients who normalized microalbuminuria after three month exercise was in the first patient 2.92, 1.46, and 1.07; in the second patient 6.53, 2.38, and 1.96; in the third 3.19, 2.32, and 0.95; and at the baseline and after 3 and 6 months, respectively. ACR (mg/mmol creatinine) in two patients who normalized microalbuminuria after six-month exercise was in the first patient 14.1, 2.88, and 0.10 and in the second patient 7.67, 5.67, and 0.51 at the baseline and after three and six months, respectively. As expected, urinary albumin excretion (ACR) was in the normal range in healthy control subjects. However, neither between-group nor within-group differences in means and medians of ACR were significant during the whole exercise programme (see Table 2).

Table 2 Urinary albumin excretion (ACR) and urinary excretion of PC-1, APN, and NAGA

			Diabetes group (n = 30)
		Control group (n = 30)	At baseline	After three months	After six months
ACR (mg/mmol creatinine)	Mean ± SD	0.69 ± 0.59	1.87 ± 3.15	1.23 ± 1.81	1.02 ± 1.25
	Median	0.44	0.66	0.56	0.54
	Range	0.00–2.18	0.03–14.06	0.03–7.67	0.07–6.69
Urinary PC-1 (U/mmol creatinine)	Mean ± SD	0.09 ± 0.01	0.15 ± 0.02	0.09 ± 0.01	0.14 ± 0.01
	Median	0.06	0.09	0.09	0.13
	Range	0.01–0.35	0.01–0.86	0.01–0.26	0.01–0.38
Urinary APN (U/mmol creatinine)	Mean ± SD	0.093 ± 0.12	0.118 ± 0.1	0.091 ± 0.05	0.124 ± 0.09
	Median	0.057	0.093	0.073	0.100
	Range	0.008–0.579	0.009–0.361	0.024–0.218	0.011–0.295
Urinary NAGA (U/mmol creatinine)	Mean ± SD	0.420 ± 0.21	0.563 ± 0.35	0.452 ± 0.51	0.451 ± 0.29
	Median	0.422	0.435	0.330	0.395
	Range	0.000–0.961	0.181–1.459	0.081–2.676	0.084–1.314

Abbreviations: ACR = albumin-to-creatinine ratio, PC-1 = plasma cell glycoprotein 1, APN = aminopeptidase N, NAGA = N-acetyl-β-D-glucosaminidase, SD = standard deviation

Significant correlations were found for ACR and both SH-groups (p < 0.01, R = 0.69) and CAT (p < 0.05, R = 0.52) in the diabetes group at baseline. After three months of exercise, ACR significantly correlated both with age (p < 0.05, R = 0.37) and urinary APN excretion (p < 0.05, R = 0.44).

N-Acetyl-β-D-Glucosaminidase (NAGA)

NAGA activity in serum was initially increased (p < 0.05) in diabetics at baseline compared to control subjects (see Table 3). However, it decreased to the normal level after six months of exercise.

Table 3 Serum activities of PC-1, APN and NAGA

			Diabetes group (n = 30)
		Control group (n = 30)	Atbaseline	After three months	After six months
Serum PC-1 (U/L)	Mean ± SD	1.93 ± 1.13	1.92 ± 0.98	1.59 ± 0.6	1.63 ± 0.66
	Median	1.80	1.74	1.59	1.58
	Range	0.30–3.77	0.84–4.36	0.80–3.80	0.79–3.80
Serum APN (U/L)	Mean ± SD	11.00 ± 4.09	10.13 ± 2.9	9.94 ± 2.02	11.09 ± 2.44
	Median	10.01	10.16	9.51	11.41
	Range	5.48–19.65	4.59–18.71	4.59–13.86	6.01–16.24
Serum NAGA (U/L)	Mean ± SD	9.67 ± 3.15	12.37 ± 4.42	11.79 ± 3.89	10.49 ± 4.39
	Median	9.28	12.55	11.79	9.33
	Range	2.08–15.71	0.11–18.91	3.57–15.78	5.69–20.72

Abbreviations: PC-1 = plasma cell glycoprotein 1, APN = aminopeptidase N, NAGA = N-acetyl-β-D-glucosaminidase, SD = standard deviation.

*versus control group, p < 0.05.

Urinary NAGA excretion significantly correlated with age (p < 0.05, R = 0.44), FG (p < 0.05, R = 0.45), PAI (p < 0.001, R = 0.74), and SCORE (p < 0.05, R = 0.49) in the diabetes group at baseline, with age (p < 0.05, R = 0.44) after three months, and with HbA_1c (p < 0.05, R = 0.48) at the end of the follow-up period.

Plasma Cell Differentiation Antigen (PC-1)

Regular physical activity did not change significantly urinary excretion and serum PC-1 activities either between the control and diabetes groups, or within the diabetes group during the whole follow-up period (see Tables 2 and 3).

Urinary PC-1 excretion significantly correlated with HbA_1c (p < 0.05, R = 0.42) in the diabetes group at the beginning of the study, with urinary APN excretion (p < 0.05, R = 0.42) after three and six months, as well as with PAI (p < 0.05, R = 0.46) at the end of the follow-up period.

Aminopeptidase N (APN)

No significant differences either between groups or within the diabetes group were found for urinary excretion and serum APN activities during the whole study period (see Tables 2 and 3).

At baseline, urinary APN excretion significantly correlated with urinary NAGA excretion (p < 0.05, R = 0.48) in diabetic patients.

DISCUSSION

Microalbuminuria is present in up to one-third of patients with diabetes, in type 2 diabetics usually at diabetes diagnosis. It is an indicator of vascular disorders that belong to the metabolic syndrome, with the prevalence that progressively increases with the number of cardiovascular risk factors. It is a strong predictor not only of cardiovascular disorders and end-stage renal disease, but of all-cause mortality[21] as well.

Low development and progression, as well as high regression of microalbuminuria, that were demonstrated in type 2 diabetics under tight blood pressure and glycemic control strongly supported the hypothesis that regular non-strenuous physical activity might have a protective effect on the appearance of microalbuminuria in type 2 diabetics. Moreover, a significant improvement in microalbuminuria has already been demonstrated as a result of an intensive lifestyle intervention that included, not only an intense exercise, but also a dietary modification.[22] Just the same, urinary albumin excretion was not normalized until 24 months, and that late response of microalbuminuria was shown to be consistent with the concept that urinary albumin excretion integrated the degree of microvascular injury that resulted from hyperglycemia, hypertension, and dyslipidemia.

As far as the present study is concerned, in spite of the tight blood pressure and glycemic control, changes in microalbuminuria during the whole follow-up period have shown a slow response as well. However, it should be emphasized that the prevalence of microalbuminuria tended to decrease during the follow-up period, as it was observed in six (20%) diabetics at baseline, three of them (10%) after three months of exercise, and only one patient (3.33%) at the end of the study. The absence of any significant between-group or within-group differences in means and medians of ACR was probably due to the fact that the present study lasted only for six months, it was carried out in a small sample, patients were instructed not to change their usual diet habits, and exercise was not intense but of moderate frequency, intensity, and duration.

The relationship between insulin resistance and urinary albumin excretion in type 2 diabetic patients has been a matter of debate for a long period of time, both with positive and negative attitudes. The results of the present study have not shown any significant association between urinary albumin excretion with either HOMA or major cardiovascular risk factors, although previously cardiovascular risk factors were found to correlate with ACR.[23] High levels of oxidative stress parameters are known to be associated with premature vascular disorders in type 2 diabetic patients, so one of the aims was to investigate if they correlated with microalbuminuria in diabetics enrolled in the present study. However, a significant correlation was found for urinary albumin excretion both with sulphydryl-groups and catalase, but not for urinary albumin excretion with MDA and glutathione.

Conflicting results have been published on the role of NAGA as a valuable marker that reflected the risk of microalbuminuria (i.e., the tubular damage and metabolic control in type 2 diabetic patients). It has been demonstrated that NAGA began to rise in the third year of diabetes, maintained a plateau between three and ten years, and rapidly increased after the tenth year[24] of the duration of this disease. NAGA activity in serum was significantly over the control level in type 2 diabetic patients enrolled in the present study at baseline and decreased to the normal level after six months of exercise. Those findings clearly supported the hypothesis that regular aerobic physical activity of moderate frequency, intensity, and duration might be of a great benefit in slowing down an expected[11] increment of NAGA in type 2 diabetic patients.

In accord with previous findings,[25] urinary excretion of NAGA was in the normal range in diabetic patients at baseline, without any significant changes within the diabetes group during the follow-up period as well. In the present study, urinary excretion of NAGA significantly correlated not only with FG and HbA_1c, as expected,[26],[27] but also with age, physical activity index, and SCORE.

In conclusion, regular and moderate aerobic exercise has the potential to reduce microalbuminuria in overweight and obese type 2 diabetic patients. However, further studies are needed not only to confirm this finding, but to elucidate potential mechanisms that would clarify the beneficial effects of exercise in more details.

ACKNOWLEDGMENTS

This work was supported by a grant from the Ministry of Science and Environmental Protection of Serbia.