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Increased leptin concentrations and lack of gender difference in Type 2 diabetic patients with nephropathy
Diabetes Research and Clinical Practice 64 (2004) 93–98
Increased leptin concentrations and lack of gender difference in Type 2 diabetic patients with nephropathy W.B. Chan, R.C.W. Ma, N.N. Chan∗ , M.C.Y. Ng, Z.S.K. Lee, C.W.K. Lai, P.C.Y. Tong, W.Y. So, J.C.N. Chan Department of Medicine and Therapeutics, The Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, China Accepted 28 October 2003
Abstract Leptin plays an important role in the regulation of body weight and energy balance. Women have higher circulating leptin level than men. In this study, we examined serum leptin concentrations in Type 2 diabetic men and women with or without nephropathy. Fasting plasma glucose (FPG), lipid profile, and serum leptin concentrations were measured in 34 Type 2 diabetic patients with nephropathy (DMN), 12 normoalbuminuric Type 2 diabetic subjects (DM) and 34 non-diabetic control subjects, all matched for age and body mass index (BMI). Result: Patients with diabetic nephropathy had lower high-density lipoprotein cholesterol and higher triglyceride, FPG, urinary albumin/creatinine ratio (ACR) and serum creatinine than the other two groups. There was a significant trend in serum leptin concentrations (P < 0.001, analysis of variance ANOVA) across the three groups with the main difference being detected between DMN and control subjects (DMN: 17.5 ± 16.8 ng/ml, DM: 14.6 ± 10.5 ng/ml and control: 9.1 ± 7.1 ng/ml). Women had higher serum leptin concentration than men in the control group (12.5 ± 7.3 ng/ml versus 4.2 ± 2.0 ng/ml, P = 0.001) and in the DM group (18.9 ± 11 ng/ml versus 8.6 ± 5.9 ng/ml, P = 0.07) whereas this gender difference was not observed in the DMN group (18.6 ± 17.0 ng/ml versus 16.8 ± 17.0 ng/ml, P = 0.754). On multivariate analysis, ACR (β = 0.411, P < 0.001) and BMI (β = 0.240, P = 0.002) were independently associated with serum leptin concentrations (R2 = 0.194, F = 22.1, P < 0.001) in the whole group. In the DMN group, ACR (β = 0.370, P = 0.016) was the only independent determinant of serum leptin concentrations (R2 = 0.159, F = 11.4, P = 0.016). Serum leptin concentrations were higher in Type 2 diabetic patients with nephropathy than normoalbuminuric diabetic patients and controls. Diabetic men with nephropathy had proportionally higher serum leptin such that the gender difference in leptin observed in non-nephropathic individuals was abolished. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Leptin; Diabetic nephropathy; Chinese; Gender
Serum leptin, encoded by the ob gene, is a 167amino acid hormone produced by adipoctyes [1,2]. Circulating leptin relays information on body adipos∗ Corresponding author. Tel.: +86-852-26323845; fax: +86-852-26375396. E-mail address: [email protected] (N.N. Chan).
ity and energy intake to the hypothalamus [3] and plays a key adipostatic role by suppressing appetite, enhancing energy expenditure and reducing fat deposition [4]. Administration of leptin to ob/ob mice was associated with reduction in body weight [2]. However, the majority of obese subjects have increased serum leptin concentrations, suggesting some degree of lep-
0168-8227/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2003.10.023
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W.B. Chan et al. / Diabetes Research and Clinical Practice 64 (2004) 93–98
tin resistance [5,6]. Although factors such as gender, hormones and cytokine concentrations can influence serum leptin concentrations, body weight, in particular, subcutaneous body fat, accounted for most of its inter-individual variations [7]. Yet, despite the central role of obesity in insulin resistance syndrome and Type 2 diabetes [8], several studies have shown that diabetes per se had no effect on serum leptin concentrations [9–12]. On the other hand, animal data have suggested that leptin might have direct effects on renal growth [13]. In this study, we aimed to explore the relationship between serum leptin concentration and diabetic nephropathy, as well as other determinants of leptin in both diabetic and non-diabetic subjects, matched for age and body mass index (BMI). 1. Patients and methods 1.1. Patients The study was carried out at the Diabetes Mellitus and Endocrine Centre at The Prince of Wales Hospital. Thirty-four Chinese Type 2 diabetic patients with nephropathy (DMN) (serum creatinine: 120–450 mol/l and spot urinary albumin/creatinine ratio (ACR) ≥ 25 mg/mmol), and 12 newly diagnosed Type 2 diabetic patients (DM) with normoalbuminuria (spot urinary ACR <3.5 mg/mmol and serum creatinine <120 mol/l) and 34 age and BMI-matched Chinese non-diabetic subjects, were recruited. All subjects were aged 50–80 years. Subjects in the DMN group were treated with insulin and multiple anti-hypertensive drugs. The distribution in drug usage was comparable between men and women in this group. All subjects gave informed written consent and the study was approved by The Chinese University of Hong Kong Clinical Research Ethics Committee. 1.2. Measurements Physical measurements including body weight and height were taken for calculation of BMI. Blood pressures were measured in both arms after the patients had rested for at least 5 min with a mercury sphygmomanometer. The Korotkoff sound V was taken as the diastolic blood pressure. The mean of two readings measured 1 min apart was used. Funduscopy was per-
formed through dilated pupils. Blood samples for determination of serum leptin, creatinine, glucose, lipid profile and glycated haemoglobin (HbA1c ) were obtained between 8.30 and 9.30 a.m. after an overnight fast of at least 8 h. Early morning urine samples were collected for measurement of albumin/creatinine ratio. All medications including insulin injection were omitted on the study day. 1.3. Laboratory assays Serum leptin concentration was measured by an ELISA kit manufactured by Diagnostic Systems Laboratories. The intra-assay precision was 1.5–6.2% CV and inter-assay precision was 4.2–5.3% CV. Plasma glucose (hexokinase method), total cholesterol (TC; enzymatic method), triglycerides (TG; enzymatic method without glycerol blanking), high-density lipoprotein cholesterol (HDL-C; dextran sulphateMgCl2 precipitation) were measured on a Hitachi 911 automated analyser (Boehringer Mannheim, Mannheim, Germany) using reagent kits supplied by the manufacturer of the analyser. Low-density lipoprotein cholesterol (LDL-C) was calculated by the Friedewald’s equation [14] for TG < 4.5 mmol/l. The precision performance of these assays was within the manufacturer’s specifications. Glycated haemoglobin (HbA1c ) was measured by an automated ion-exchange chromatographic method (Bio-Rad Laboratory, Hercules, CA, USA) and the reference range was 5.1–6.4%. Inter-assay coefficients of variation (CV) were better than 3.1% for HbA1c values below 6.5%. Urinary creatinine was measured using the Jaffe’s kinetic method and albumin was determined by immunoturbidimetry. The inter-assay precision CV was 12.0 and 2.3% for urinary albumin concentrations of 8.0 and 68.8 mg/l, respectively. The lowest detection limit was 3.0 mg/l. Serum electrolytes (direct ion-selective electrodes), urea (enzymatic) and creatinine (Jaffe’s kinetic method) were measured on a Dimension AR system (Dade Behring, Deerfield, IL, USA). The reagent kits were supplied by the manufacturer and the precision performance of these methods was within the manufacturer’s specifications. 1.4. Statistical analysis Statistical analysis was carried out using SPSS (Statistical Package for Social Science) 8.0. Skewed
W.B. Chan et al. / Diabetes Research and Clinical Practice 64 (2004) 93–98
data including ACR, serum creatinine and triglycerides were logarithmically transformed. All data are expressed as mean ± S.D. and geometric mean (×/÷) antilog S.D. as appropriate. One way analysis of variance (ANOVA) was used for between-group comparisons with adjustment for multiple comparisons using Bonferroni’s equation. Spearman correlation and linear regression analyses were performed to test for associations. Stepwise forward multiple regression analysis was used to identify the independent predictors for serum leptin concentrations in all subjects and in patients with DMN. A P-value <0.05 (two-tailed) was considered significant.
2. Results All three groups of subjects had similar age, blood pressure, TC and LDL-C but patients with DMN had higher urinary ACR, serum creatinine, HbA1c , FPG, TG and lower HDL-C than normoalbuminuric diabetic and control subjects. None of the newly-diagnosed Type 2 diabetic patients had retinopathy. Amongst the 34 patients with diabetic nephropathy, seven had no clinical evidence of retinopathy, five had mild background retinopathy and the remaining 22 had advanced
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retinopathy with previous laser treatment. There was a trend of rising serum leptin concentration across the three groups (P < 0.01) with significant difference between the DMN and control groups (P < 0.01). Serum leptin concentrations were similar between the newly diagnosed diabetic and control subjects (Table 1). In the control group, serum leptin concentration was higher in women then men (12.5 ± 7.3 ng/ml versus 4.2 ± 2.0 ng/ml, P = 0.001). A similar trend was observed in the DM group but fell short of significance (18.9 ± 11 ng/ml versus 8.6 ± 5.9 ng/ml, P = 0.071). In the DMN group, there was no difference in serum leptin concentration between women and men (18.6 ± 17.0 ng/ml versus 16.8 ± 17.0 ng/ml, P = 0.754, Fig. 1). The interaction between gender and group using ANOVA model was of borderline significance (P = 0.052) after adjustment for BMI and ACR. There was no relationship between serum leptin concentration and FPG (R = 0.104, P = 0.408), HbA1c (R = 0.207, P = 0.219) or serum creatinine (r = 0.006, P = 0.972) in the diabetic nephropathy group. Fig. 2 shows the relationship between leptin, ACR and serum creatinine in the DMN group. Using stepwise regression analysis, in the whole group, ACR (β = 0.411, P < 0.001) and BMI (β = 0.240, P = 0.002) were independently associated with serum
Table 1 Clinical and biochemical parameters including serum leptin concentrations in Type 2 diabetic patients with or without nephropathy and non-diabetic control subjects matched for age and BMI
Age (years) Male SBP (mmHg) DBP (mmHg) Body mass index (kg/m2 ) Fasting glucose (mmol/l) HbA1c (%) Serum creatinine (mol/) Total cholesterol (mmol/l) Triglyceride (mmol/l) HDL-cholesterol (mmol/l) LDL-cholesterol (mmol/l) Urinary ACR (mg/mmol) Leptin (ng/ml)
Diabetic patients with nephropathy (n = 34)
Normoalbuminuric diabetic patients (n = 12)
Non-diabetic control subjects (n = 34)
62.1 ± 6.6 21 (62%) 147 ± 21 82 ± 10 24.4 ± 2.8 7.7 ± 2.2∗ 7.7 ± 1.5∗ 213 ×/÷ 1.5∗ 6.1 ± 1.2 1.9 ×/÷ 1.7∗ 1.1 ± 0.3∗ 3.9 ± 0.9 135 ×/÷ 3.5∗ 17.5 ± 16.8†
62.5 ± 10.7 5 (42%) 145 ± 22 81 ± 10 24.8 ± 4.2 5.9 ± 1.4 6.4 ± 0.7 76 ×/÷ 1.2 5.7 ± 1.0 1.4 ×/÷ 1.7 1.3 ± 0.5 3.7 ± 1.0 2.1 ×/÷ 2.8 14.6 ± 10.5
65.9 ± 6.1 14 (41%) 141 ± 17 80 ± 10 24.6 ± 3.1 5.0 ± 0.7 – 77 ×/÷ 1.2 5.8 ± 1.0 1.3 ×/÷ 1.5 1.4 ± 0.4 3.8 ± 0.9 1.5 ×/÷ 3.2 9.1 ± 7.1
Data are expressed as mean ± S.D. or Geometric mean ×/÷ antilog S.D. Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; HbA1c , glycated haemoglobin; ACR, albumin/creatinine ratio; LDL, low-density lipoprotein; HDL, high-density lipoprotein. ∗ P < 0.05 between diabetic nephropathy group and the other two groups. † P < 0.05 between diabetic nephropathy group and control subjects.
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Fig. 1. Serum leptin concentrations in male and female Type 2 diabetic patients with or without nephropathy and non-diabetic control subjects.
leptin concentration (R2 = 0.194, F = 11.7, P < 0.001) while in the DMN group, ACR (β = 0.370, P = 0.016) was the only determinant of serum leptin concentration (R2 = 0.159, F = 11.4, P = 0.016). 3. Discussion
Fig. 2. Regression line (95% confidence intervals) showing the relationships between serum leptin concentrations and spot urinary albumin/creatinine ratio (ACR, top panel: R2 = 0.159, P = 0.016) and serum creatinine (bottom panel: R2 < 0.001, P = 0.972) in 34 Type 2 diabetic patients with nephropathy and impaired renal function.
Our study showed that normoalbuminuric Type 2 diabetic patients had similar serum leptin concentrations as age and body mass index-matched control subjects. These findings were consistent with previous studies reporting the lack of effect of diabetes on serum leptin concentrations [10–12]. It is unclear how changes in plasma glucose concentrations may affect the release or the metabolism of leptin at a physiological level. However, based on previous findings of the lack of a positive correlation between plasma glucose (or long-term glycaemic control) and leptin concentrations [10–12], our results simply extend this observation to Chinese diabetic patients. Similarly, we did not find a relationship between serum leptin concentration and plasma glucose concentrations. Despite the relatively low BMI of 24–25 kg/m2 of our subjects, we confirmed the independent relationship between BMI and serum leptin concentrations in the whole group. As in Type 1 [15] and Type 2 diabetic patients [10,11], we also found that women had higher serum leptin concentrations than men in both normoalbuminuric diabetic subjects and non-diabetic controls. In contrast, there was a markedly elevated serum leptin concentration in diabetic patients with nephropathy.
W.B. Chan et al. / Diabetes Research and Clinical Practice 64 (2004) 93–98
A gender difference in serum leptin concentration has been described in both rodents and human subjects. Frederich et al. [16] found that female normal and transgenic mice had higher serum leptin concentrations than male mice for any amount of total body fat content. In humans, leptin secretion from omental tissue in vitro was higher in samples obtained from women than men and was independent of BMI [17]. Other corroborative evidence include the findings of disproportionately high serum leptin concentrations in women than men, at any level of body fat measurement, in Type 1 [15] and Type 2 diabetic [11] as well as non-diabetic populations [10,11]. Of note, subcutaneous fat has been shown to express more leptin mRNA than intra-abdominal fat [18]. Since female tend to accumulate more subcutaneous and less abdominal fat than men, this may account for the gender-difference in serum leptin concentrations. Leptin is primarily degraded by kidneys [19]. Normal kidneys do not excrete leptin but in children with nephrotic syndrome, increased urinary loss of leptin has been reported [20]. In a study involving 10 subjects in each group, Type 2 diabetic patients with micro- and macro-albuminuria [9] had higher serum leptin concentrations than control subjects matched for age, sex and body fat. In this small study, patients were overweight (mean BMI 25.2 and 26.8 kg/m2 for micro- and macro-albuminuric patients, respectively) and had impaired renal function (mean serum creatinine 163 mol/l for macro-albuminuric patients). Using a larger sample size, we have now extended this finding to Type 2 diabetic patients with more normal weight and moderately impaired renal function. Intriguingly, we found that the well-established gender difference in serum leptin concentration [10–12] was abolished amongst diabetic patients with nephropathy. It remains to be determined whether the increased serum leptin concentrations in patients with diabetic nephropathy was due to increased production, reduced clearance or increased leptin resistance. However, we were unable to demonstrate a relationship between serum leptin and creatinine concentrations in our subjects. Furthermore, on multivariate analysis, 14% of the variance of serum leptin concentrations was explained by albuminuria and BMI in the whole group. In patients with diabetic nephropathy, albuminuria was the only significant predictor explaining 11% of the variance of serum leptin concentrations.
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There is in vitro and in vivo evidence from an animal study that leptin had direct effects on renal growth through stimulation of proliferation and expression of transforming growth factor-beta (TGF-) in renal glomerular endothelial cells of rats [13]. Direct evidence that leptin contributes to the development of nephropathy is lacking in humans although several lines of evidence are suggestive. In obesity-related hypertension, leptin has been shown to have a pathophysiological role in the elevation of blood pressure in part through its activating effects on sympathetic nervous and angiotensin systems [21,22]. Type 2 diabetic patients with high serum leptin concentrations have an increased risk for developing end-stage renal failure [23]. In Pima Indians, urine leptin concentrations positively correlated with urinary albumin/creatinine ratios and inversely correlated with glomerular filtration rates [24]. Increased serum leptin concentrations also correlated with increased urine albumin excretion in women with type 1 diabetes [25]. Correlations are important for trying to understanding pathophysiological mechanisms and the real role of leptin in the development of diabetic nephropathy. Thus, the above observations indicate that leptin may play a key role in the pathogenesis and progression of diabetic glomerulopathy. The results of our study are consistent with these observations although our study was not designed to explore the aetiological role of leptin in diabetic nephropathy. Some of the limitations of this study include the small sample size, a single rather than multiple measurements of serum leptin which are known to follow a circadian pattern as well as the lack of detailed measurements of body fat and its distribution. Although there might be potential confounding effects of medications on leptin concentrations in patients with nephropathy, the usage of drugs were similar between men and women. Furthermore, none of these patients were receiving steroids which might enhance leptin secretion [26]. Despite these limitations, we were able to demonstrate the independent relationship between albuminuria and leptin especially in patients with diabetic nephropathy in whom the gender-difference in leptin was also abolished. Given the potential effects of leptin on renal growth in animal studies, these findings lend support to further testing of the aetiological role of leptin in diabetic nephropathy in human.
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In summary, serum leptin concentrations were elevated in patients with diabetic nephropathy. Urinary ACR was an important determinant of serum leptin concentration in Type 2 diabetic patients with nephropathy. Compared to normoalbuminuric diabetic counterparts, men with diabetic nephropathy had higher serum leptin concentrations proportionally than women with diabetic nephropathy, thus abolishing the well-established gender difference in leptin concentrations. Whether Type 2 diabetic men with high serum leptin concentrations have a more rapid progression of diabetic nephropathy than diabetic men with low serum leptin concentrations in the Chinese population requires further investigations.
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[12]
[13]
[14]
[15]
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Increased leptin concentrations and lack of gender difference in Type 2 diabetic patients with nephropathy W.B. Chan, R.C.W. Ma, N.N. Chan∗ , M.C.Y. Ng, Z.S.K. Lee, C.W.K. Lai, P.C.Y. Tong, W.Y. So, J.C.N. Chan Department of Medicine and Therapeutics, The Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, China Accepted 28 October 2003
Abstract Leptin plays an important role in the regulation of body weight and energy balance. Women have higher circulating leptin level than men. In this study, we examined serum leptin concentrations in Type 2 diabetic men and women with or without nephropathy. Fasting plasma glucose (FPG), lipid profile, and serum leptin concentrations were measured in 34 Type 2 diabetic patients with nephropathy (DMN), 12 normoalbuminuric Type 2 diabetic subjects (DM) and 34 non-diabetic control subjects, all matched for age and body mass index (BMI). Result: Patients with diabetic nephropathy had lower high-density lipoprotein cholesterol and higher triglyceride, FPG, urinary albumin/creatinine ratio (ACR) and serum creatinine than the other two groups. There was a significant trend in serum leptin concentrations (P < 0.001, analysis of variance ANOVA) across the three groups with the main difference being detected between DMN and control subjects (DMN: 17.5 ± 16.8 ng/ml, DM: 14.6 ± 10.5 ng/ml and control: 9.1 ± 7.1 ng/ml). Women had higher serum leptin concentration than men in the control group (12.5 ± 7.3 ng/ml versus 4.2 ± 2.0 ng/ml, P = 0.001) and in the DM group (18.9 ± 11 ng/ml versus 8.6 ± 5.9 ng/ml, P = 0.07) whereas this gender difference was not observed in the DMN group (18.6 ± 17.0 ng/ml versus 16.8 ± 17.0 ng/ml, P = 0.754). On multivariate analysis, ACR (β = 0.411, P < 0.001) and BMI (β = 0.240, P = 0.002) were independently associated with serum leptin concentrations (R2 = 0.194, F = 22.1, P < 0.001) in the whole group. In the DMN group, ACR (β = 0.370, P = 0.016) was the only independent determinant of serum leptin concentrations (R2 = 0.159, F = 11.4, P = 0.016). Serum leptin concentrations were higher in Type 2 diabetic patients with nephropathy than normoalbuminuric diabetic patients and controls. Diabetic men with nephropathy had proportionally higher serum leptin such that the gender difference in leptin observed in non-nephropathic individuals was abolished. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Leptin; Diabetic nephropathy; Chinese; Gender
Serum leptin, encoded by the ob gene, is a 167amino acid hormone produced by adipoctyes [1,2]. Circulating leptin relays information on body adipos∗ Corresponding author. Tel.: +86-852-26323845; fax: +86-852-26375396. E-mail address: [email protected] (N.N. Chan).
ity and energy intake to the hypothalamus [3] and plays a key adipostatic role by suppressing appetite, enhancing energy expenditure and reducing fat deposition [4]. Administration of leptin to ob/ob mice was associated with reduction in body weight [2]. However, the majority of obese subjects have increased serum leptin concentrations, suggesting some degree of lep-
0168-8227/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2003.10.023
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tin resistance [5,6]. Although factors such as gender, hormones and cytokine concentrations can influence serum leptin concentrations, body weight, in particular, subcutaneous body fat, accounted for most of its inter-individual variations [7]. Yet, despite the central role of obesity in insulin resistance syndrome and Type 2 diabetes [8], several studies have shown that diabetes per se had no effect on serum leptin concentrations [9–12]. On the other hand, animal data have suggested that leptin might have direct effects on renal growth [13]. In this study, we aimed to explore the relationship between serum leptin concentration and diabetic nephropathy, as well as other determinants of leptin in both diabetic and non-diabetic subjects, matched for age and body mass index (BMI). 1. Patients and methods 1.1. Patients The study was carried out at the Diabetes Mellitus and Endocrine Centre at The Prince of Wales Hospital. Thirty-four Chinese Type 2 diabetic patients with nephropathy (DMN) (serum creatinine: 120–450 mol/l and spot urinary albumin/creatinine ratio (ACR) ≥ 25 mg/mmol), and 12 newly diagnosed Type 2 diabetic patients (DM) with normoalbuminuria (spot urinary ACR <3.5 mg/mmol and serum creatinine <120 mol/l) and 34 age and BMI-matched Chinese non-diabetic subjects, were recruited. All subjects were aged 50–80 years. Subjects in the DMN group were treated with insulin and multiple anti-hypertensive drugs. The distribution in drug usage was comparable between men and women in this group. All subjects gave informed written consent and the study was approved by The Chinese University of Hong Kong Clinical Research Ethics Committee. 1.2. Measurements Physical measurements including body weight and height were taken for calculation of BMI. Blood pressures were measured in both arms after the patients had rested for at least 5 min with a mercury sphygmomanometer. The Korotkoff sound V was taken as the diastolic blood pressure. The mean of two readings measured 1 min apart was used. Funduscopy was per-
formed through dilated pupils. Blood samples for determination of serum leptin, creatinine, glucose, lipid profile and glycated haemoglobin (HbA1c ) were obtained between 8.30 and 9.30 a.m. after an overnight fast of at least 8 h. Early morning urine samples were collected for measurement of albumin/creatinine ratio. All medications including insulin injection were omitted on the study day. 1.3. Laboratory assays Serum leptin concentration was measured by an ELISA kit manufactured by Diagnostic Systems Laboratories. The intra-assay precision was 1.5–6.2% CV and inter-assay precision was 4.2–5.3% CV. Plasma glucose (hexokinase method), total cholesterol (TC; enzymatic method), triglycerides (TG; enzymatic method without glycerol blanking), high-density lipoprotein cholesterol (HDL-C; dextran sulphateMgCl2 precipitation) were measured on a Hitachi 911 automated analyser (Boehringer Mannheim, Mannheim, Germany) using reagent kits supplied by the manufacturer of the analyser. Low-density lipoprotein cholesterol (LDL-C) was calculated by the Friedewald’s equation [14] for TG < 4.5 mmol/l. The precision performance of these assays was within the manufacturer’s specifications. Glycated haemoglobin (HbA1c ) was measured by an automated ion-exchange chromatographic method (Bio-Rad Laboratory, Hercules, CA, USA) and the reference range was 5.1–6.4%. Inter-assay coefficients of variation (CV) were better than 3.1% for HbA1c values below 6.5%. Urinary creatinine was measured using the Jaffe’s kinetic method and albumin was determined by immunoturbidimetry. The inter-assay precision CV was 12.0 and 2.3% for urinary albumin concentrations of 8.0 and 68.8 mg/l, respectively. The lowest detection limit was 3.0 mg/l. Serum electrolytes (direct ion-selective electrodes), urea (enzymatic) and creatinine (Jaffe’s kinetic method) were measured on a Dimension AR system (Dade Behring, Deerfield, IL, USA). The reagent kits were supplied by the manufacturer and the precision performance of these methods was within the manufacturer’s specifications. 1.4. Statistical analysis Statistical analysis was carried out using SPSS (Statistical Package for Social Science) 8.0. Skewed
W.B. Chan et al. / Diabetes Research and Clinical Practice 64 (2004) 93–98
data including ACR, serum creatinine and triglycerides were logarithmically transformed. All data are expressed as mean ± S.D. and geometric mean (×/÷) antilog S.D. as appropriate. One way analysis of variance (ANOVA) was used for between-group comparisons with adjustment for multiple comparisons using Bonferroni’s equation. Spearman correlation and linear regression analyses were performed to test for associations. Stepwise forward multiple regression analysis was used to identify the independent predictors for serum leptin concentrations in all subjects and in patients with DMN. A P-value <0.05 (two-tailed) was considered significant.
2. Results All three groups of subjects had similar age, blood pressure, TC and LDL-C but patients with DMN had higher urinary ACR, serum creatinine, HbA1c , FPG, TG and lower HDL-C than normoalbuminuric diabetic and control subjects. None of the newly-diagnosed Type 2 diabetic patients had retinopathy. Amongst the 34 patients with diabetic nephropathy, seven had no clinical evidence of retinopathy, five had mild background retinopathy and the remaining 22 had advanced
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retinopathy with previous laser treatment. There was a trend of rising serum leptin concentration across the three groups (P < 0.01) with significant difference between the DMN and control groups (P < 0.01). Serum leptin concentrations were similar between the newly diagnosed diabetic and control subjects (Table 1). In the control group, serum leptin concentration was higher in women then men (12.5 ± 7.3 ng/ml versus 4.2 ± 2.0 ng/ml, P = 0.001). A similar trend was observed in the DM group but fell short of significance (18.9 ± 11 ng/ml versus 8.6 ± 5.9 ng/ml, P = 0.071). In the DMN group, there was no difference in serum leptin concentration between women and men (18.6 ± 17.0 ng/ml versus 16.8 ± 17.0 ng/ml, P = 0.754, Fig. 1). The interaction between gender and group using ANOVA model was of borderline significance (P = 0.052) after adjustment for BMI and ACR. There was no relationship between serum leptin concentration and FPG (R = 0.104, P = 0.408), HbA1c (R = 0.207, P = 0.219) or serum creatinine (r = 0.006, P = 0.972) in the diabetic nephropathy group. Fig. 2 shows the relationship between leptin, ACR and serum creatinine in the DMN group. Using stepwise regression analysis, in the whole group, ACR (β = 0.411, P < 0.001) and BMI (β = 0.240, P = 0.002) were independently associated with serum
Table 1 Clinical and biochemical parameters including serum leptin concentrations in Type 2 diabetic patients with or without nephropathy and non-diabetic control subjects matched for age and BMI
Age (years) Male SBP (mmHg) DBP (mmHg) Body mass index (kg/m2 ) Fasting glucose (mmol/l) HbA1c (%) Serum creatinine (mol/) Total cholesterol (mmol/l) Triglyceride (mmol/l) HDL-cholesterol (mmol/l) LDL-cholesterol (mmol/l) Urinary ACR (mg/mmol) Leptin (ng/ml)
Diabetic patients with nephropathy (n = 34)
Normoalbuminuric diabetic patients (n = 12)
Non-diabetic control subjects (n = 34)
62.1 ± 6.6 21 (62%) 147 ± 21 82 ± 10 24.4 ± 2.8 7.7 ± 2.2∗ 7.7 ± 1.5∗ 213 ×/÷ 1.5∗ 6.1 ± 1.2 1.9 ×/÷ 1.7∗ 1.1 ± 0.3∗ 3.9 ± 0.9 135 ×/÷ 3.5∗ 17.5 ± 16.8†
62.5 ± 10.7 5 (42%) 145 ± 22 81 ± 10 24.8 ± 4.2 5.9 ± 1.4 6.4 ± 0.7 76 ×/÷ 1.2 5.7 ± 1.0 1.4 ×/÷ 1.7 1.3 ± 0.5 3.7 ± 1.0 2.1 ×/÷ 2.8 14.6 ± 10.5
65.9 ± 6.1 14 (41%) 141 ± 17 80 ± 10 24.6 ± 3.1 5.0 ± 0.7 – 77 ×/÷ 1.2 5.8 ± 1.0 1.3 ×/÷ 1.5 1.4 ± 0.4 3.8 ± 0.9 1.5 ×/÷ 3.2 9.1 ± 7.1
Data are expressed as mean ± S.D. or Geometric mean ×/÷ antilog S.D. Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; HbA1c , glycated haemoglobin; ACR, albumin/creatinine ratio; LDL, low-density lipoprotein; HDL, high-density lipoprotein. ∗ P < 0.05 between diabetic nephropathy group and the other two groups. † P < 0.05 between diabetic nephropathy group and control subjects.
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Fig. 1. Serum leptin concentrations in male and female Type 2 diabetic patients with or without nephropathy and non-diabetic control subjects.
leptin concentration (R2 = 0.194, F = 11.7, P < 0.001) while in the DMN group, ACR (β = 0.370, P = 0.016) was the only determinant of serum leptin concentration (R2 = 0.159, F = 11.4, P = 0.016). 3. Discussion
Fig. 2. Regression line (95% confidence intervals) showing the relationships between serum leptin concentrations and spot urinary albumin/creatinine ratio (ACR, top panel: R2 = 0.159, P = 0.016) and serum creatinine (bottom panel: R2 < 0.001, P = 0.972) in 34 Type 2 diabetic patients with nephropathy and impaired renal function.
Our study showed that normoalbuminuric Type 2 diabetic patients had similar serum leptin concentrations as age and body mass index-matched control subjects. These findings were consistent with previous studies reporting the lack of effect of diabetes on serum leptin concentrations [10–12]. It is unclear how changes in plasma glucose concentrations may affect the release or the metabolism of leptin at a physiological level. However, based on previous findings of the lack of a positive correlation between plasma glucose (or long-term glycaemic control) and leptin concentrations [10–12], our results simply extend this observation to Chinese diabetic patients. Similarly, we did not find a relationship between serum leptin concentration and plasma glucose concentrations. Despite the relatively low BMI of 24–25 kg/m2 of our subjects, we confirmed the independent relationship between BMI and serum leptin concentrations in the whole group. As in Type 1 [15] and Type 2 diabetic patients [10,11], we also found that women had higher serum leptin concentrations than men in both normoalbuminuric diabetic subjects and non-diabetic controls. In contrast, there was a markedly elevated serum leptin concentration in diabetic patients with nephropathy.
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A gender difference in serum leptin concentration has been described in both rodents and human subjects. Frederich et al. [16] found that female normal and transgenic mice had higher serum leptin concentrations than male mice for any amount of total body fat content. In humans, leptin secretion from omental tissue in vitro was higher in samples obtained from women than men and was independent of BMI [17]. Other corroborative evidence include the findings of disproportionately high serum leptin concentrations in women than men, at any level of body fat measurement, in Type 1 [15] and Type 2 diabetic [11] as well as non-diabetic populations [10,11]. Of note, subcutaneous fat has been shown to express more leptin mRNA than intra-abdominal fat [18]. Since female tend to accumulate more subcutaneous and less abdominal fat than men, this may account for the gender-difference in serum leptin concentrations. Leptin is primarily degraded by kidneys [19]. Normal kidneys do not excrete leptin but in children with nephrotic syndrome, increased urinary loss of leptin has been reported [20]. In a study involving 10 subjects in each group, Type 2 diabetic patients with micro- and macro-albuminuria [9] had higher serum leptin concentrations than control subjects matched for age, sex and body fat. In this small study, patients were overweight (mean BMI 25.2 and 26.8 kg/m2 for micro- and macro-albuminuric patients, respectively) and had impaired renal function (mean serum creatinine 163 mol/l for macro-albuminuric patients). Using a larger sample size, we have now extended this finding to Type 2 diabetic patients with more normal weight and moderately impaired renal function. Intriguingly, we found that the well-established gender difference in serum leptin concentration [10–12] was abolished amongst diabetic patients with nephropathy. It remains to be determined whether the increased serum leptin concentrations in patients with diabetic nephropathy was due to increased production, reduced clearance or increased leptin resistance. However, we were unable to demonstrate a relationship between serum leptin and creatinine concentrations in our subjects. Furthermore, on multivariate analysis, 14% of the variance of serum leptin concentrations was explained by albuminuria and BMI in the whole group. In patients with diabetic nephropathy, albuminuria was the only significant predictor explaining 11% of the variance of serum leptin concentrations.
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There is in vitro and in vivo evidence from an animal study that leptin had direct effects on renal growth through stimulation of proliferation and expression of transforming growth factor-beta (TGF-) in renal glomerular endothelial cells of rats [13]. Direct evidence that leptin contributes to the development of nephropathy is lacking in humans although several lines of evidence are suggestive. In obesity-related hypertension, leptin has been shown to have a pathophysiological role in the elevation of blood pressure in part through its activating effects on sympathetic nervous and angiotensin systems [21,22]. Type 2 diabetic patients with high serum leptin concentrations have an increased risk for developing end-stage renal failure [23]. In Pima Indians, urine leptin concentrations positively correlated with urinary albumin/creatinine ratios and inversely correlated with glomerular filtration rates [24]. Increased serum leptin concentrations also correlated with increased urine albumin excretion in women with type 1 diabetes [25]. Correlations are important for trying to understanding pathophysiological mechanisms and the real role of leptin in the development of diabetic nephropathy. Thus, the above observations indicate that leptin may play a key role in the pathogenesis and progression of diabetic glomerulopathy. The results of our study are consistent with these observations although our study was not designed to explore the aetiological role of leptin in diabetic nephropathy. Some of the limitations of this study include the small sample size, a single rather than multiple measurements of serum leptin which are known to follow a circadian pattern as well as the lack of detailed measurements of body fat and its distribution. Although there might be potential confounding effects of medications on leptin concentrations in patients with nephropathy, the usage of drugs were similar between men and women. Furthermore, none of these patients were receiving steroids which might enhance leptin secretion [26]. Despite these limitations, we were able to demonstrate the independent relationship between albuminuria and leptin especially in patients with diabetic nephropathy in whom the gender-difference in leptin was also abolished. Given the potential effects of leptin on renal growth in animal studies, these findings lend support to further testing of the aetiological role of leptin in diabetic nephropathy in human.
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In summary, serum leptin concentrations were elevated in patients with diabetic nephropathy. Urinary ACR was an important determinant of serum leptin concentration in Type 2 diabetic patients with nephropathy. Compared to normoalbuminuric diabetic counterparts, men with diabetic nephropathy had higher serum leptin concentrations proportionally than women with diabetic nephropathy, thus abolishing the well-established gender difference in leptin concentrations. Whether Type 2 diabetic men with high serum leptin concentrations have a more rapid progression of diabetic nephropathy than diabetic men with low serum leptin concentrations in the Chinese population requires further investigations.
[11]
[12]
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