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Departments of Endocrinology and Metabolism (M.O., D.G., G.O., I.C.O.), Hydroclimatology (M.T.), and Biochemistry (T.O.), Gulhane School of Medicine, Etlik-Ankara 06018, Turkey; and Bayindir Medical Center (N.B.), Sogutozu-Ankara 06018, Turkey
Address all correspondence and requests for reprints to: Metin Ozata, M.D., Department of Endocrinology and Metabolism, Gulhane School of Medicine, Etlik-Ankara 06018, Turkey. E-mail: metinozata{at}hotmail.com
Abstract
Acylation-stimulating protein is an adipocyte-derived protein that has recently been suggested to play an important role in the regulation of triglyceride storage. To date, little information has been reported with regard to fasting acylation-stimulating protein levels and its relation to metabolic control, leptin, and/or lipids in subjects with diabetes mellitus. We therefore evaluated fasting acylation-stimulating protein, leptin, and lipid levels before and 4 months after improving glycemic control with sulfonylurea treatment in a group of poorly controlled obese women with type 2 diabetes and in age- and body mass index-matched nondiabetic obese women.
Fasting plasma acylation-stimulating protein (49.67 ± 19.73 vs. 48.49 ± 20.70 nmol/liter) and leptin concentrations (33.7 ± 23.2 vs. 26.2 ± 10.6 ng/ml) were not significantly different between the groups. Improvement of glycemic control produced parallel falls in fasting blood glucose and hemoglobin A1c. Plasma leptin concentrations were also significantly reduced (33.69 ± 23.2 vs. 22.73 ± 11.26 ng/ml; P = 0.036), whereas fasting acylation-stimulating protein concentrations were significantly increased after treatment (48.49 ± 20.70 vs. 72,82 ± 29,72 nmol/liter; P = 0.004). Nevertheless, lipids and apolipoprotein B did not significantly improve. We could not find any correlation between elevated acylation-stimulating protein levels and changes in body mass index, glucose, insulin, hemoglobin A1c, leptin, or lipid levels. Similarly, the decrement in circulating leptin levels observed after treatment did not correlate with changes in the levels of glucose, insulin, hemoglobin A1c, or any lipid parameters.
We conclude that improved glycemic control increases fasting acylation-stimulating protein and decreases leptin concentrations, but not corrects critical lipid abnormalities in type 2 obese diabetic subjects. Moreover, altered plasma acylation-stimulating protein levels are not associated with changes in body mass index or lipid, leptin, insulin, or glucose levels. Thus, our findings suggest that improved glycemic control or insulin resistance is not responsible for abnormal fatty acid trapping, and failure of lipids to improve after treatment in our patients is consistent with the acylation-stimulating protein resistance concept.
RECENT STUDIES have demonstrated a dual
role for adipocytes both as the primary site of energy storage and as a
source of numerous hormones or cytokines, such as leptin, TNF
, and
IL-6, which regulate adipocyte functions as well as body weight or
glucose homeostasis (1, 2). Leptin, the product of the
obese gene, is mainly produced by adipocytes, and its circulating
levels reflect the amount of energy stored in adipose tissue (3, 4). Although body weight accounts for approximately 50–60% of
leptin’s variability, other factors, such as gender, age, hormones
(mainly insulin), and cytokine levels also contribute to the regulation
of circulating leptin levels (5). In addition to leptin’s
role as a hormonal regulator of body weight and energy expenditure, it
is now being considered whether leptin plays a regulatory role in
various physiological states, such as lipid metabolism, hemopoiesis,
insulin action, ovarian function, reproduction, immune function, and
angiogenesis (6). Human congenital leptin deficiency is
also shown to be associated with multiple hormonal defects
(7).
Acylation-stimulating protein (ASP) is an adipocyte- derived
protein that has recently been recognized to play an important role in
the regulation of lipoprotein metabolism and triglyceride (TG) storage
(8). ASP is generated by the interaction of factor B and
adipsin with the third component of complement (C3), all three of which
are synthesized and secreted by human adipocytes (9, 10).
The product, C3a, is a nonglycosylated 77-amino acid N-terminal
fragment of the -chain of C3. The terminal arginine is then rapidly
removed by carboxypeptidase N to produce C3adesArg, also known as
ASP.
ASP increases the rate of fatty acid uptake of adipocytes by increasing TG synthesis, which is achieved by increasing specific membrane glucose transport (11, 12) and by increasing the activity of diacylglycerol acyltransferase, which is the final enzyme involved in the synthesis of a triglyceride molecule (13). It has been shown that ASP also influences fatty acid release from adipocytes (14). Therefore, ASP plays a role in determining fatty acid balance in the adipocyte. The actions of insulin and ASP on all of these processes are independent and additive (15).
In vivo studies in humans have demonstrated that the production and release of ASP by adipocytes markedly increase in the second half of the postprandial period (16). Furthermore, the increase in ASP production during this period correlates with maximal TG clearance and fatty acid uptake by adipocytes. On the other hand, Charlesworth et al. (17) showed that plasma ASP levels do not increase after an oral fat load. Moreover, obese subjects have elevated plasma ASP levels (18, 19), but their plasma ASP decreases with prolonged fasting and weight loss (20). Thus, plasma ASP levels correlate with expansion and contraction of adipose tissue mass, resembling those changes in leptin. Presumably, the ASP pathway may be involved in the communication between dietary fat intake and fat storage in adipocytes.
ASP stimulates glucose uptake in various cell types, including adipocytes (12), fibroblasts (11), and myotubes (21). ASP also facilitates glucose transport via increased translocation of glucose transporters (GLUT1, GLUT3, and GLUT4) to the cell surface (11, 12). In muscle, ASP appears to enhance the effect of insulin on glucose transport (21), which suggests that ASP may play an independent role in the regulation of insulin-stimulated glucose uptake. It has also been demonstrated that the ip injection of ASP into C57BL/6 mice reduced glucose excursions in response to oral fat load (22). Several studies reported elevated fasting plasma ASP concentrations in individuals with obesity (18, 19) and coronary artery disease (23). However, obese Pima Indians do not have elevated plasma ASP (24). A recent study in Pima Indians has also demonstrated that insulin action and insulinemia are closely related to the fasting complement C3, but not the ASP, concentration (25). However, there are no data regarding either the interaction between fasting ASP concentrations and leptin in diabetes mellitus or the effect of glycemic control on this circulating peptide. The aims of this study were 1) to determine fasting plasma ASP and leptin levels in type 2 diabetic individuals and 2) to evaluate the influence of glycemic control on plasma ASP, leptin, and lipid levels.
Subjects and Methods
Patients
Twenty-four poorly controlled, obese, female patients [mean ± SD age and body mass index (BMI) were 50.7 ± 7.9 yr and 32.5 ± 3.8 kg/m2, respectively] with type 2 diabetes mellitus and without major diabetic complication (retinopathy, neuropathy, or nephropathy) were enrolled in the study. The duration of diabetes after diagnosis was 12.6 ± 3.6 months. Diabetes was diagnosed using WHO criteria (26). All patients were being treated with diet alone and were investigated before the initiation of additional treatment (sulfonylurea). Evaluation with standard physical examination, chest x-ray, baseline electrocardiogram, exercise electrocardiogram, two-dimensional echocardiography, and routine clinical laboratory tests, including liver and kidney function tests and 24-h urinary protein measurements, was performed in each patient. None of the patients had hypertension, nephropathy, depression, coronary heart disease, heart failure, renal failure, autoimmune disease, or acute infection. Diabetic retinopathy was excluded by funduscopic examination and fluoroangiography.
Twenty-two age- and BMI-matched obese women (mean ± SD age and BMI were 54.7 ± 6.1 yr and 33.02 ± 2.5 kg/m2, respectively) were chosen as a control group. They underwent routine physical and laboratory evaluations to ensure that none had diabetes mellitus; hypertension; hyperlipidemia; psychiatric, metabolic, hepatic, renal, or autoimmune disease; or acute infection. None of the healthy subjects had a family history of hypertension, autoimmune disease, or diabetes. All subjects gave informed consent for participating in the study. The study was approved by the local ethics committee of Gulhane School of Medicine. Subjects in both groups reported that their weight had been stable for at least 3 months before the start of the study. All venous blood and urine samples were collected at 0800 h after an overnight fast.
All subjects were studied on an out-patient basis before treatment (baseline) and 4 months after initiation of treatment. Patients were treated with sulfonylurea (glybenclamide) once or twice daily each for 4 months, and none was taking any other medications. Dosage adjustment was made every 2 wk on the basis of blood glucose monitoring (first months) followed by monthly visits, aiming for a fasting blood glucose and hemoglobin A1c (HbA1c) levels within the acceptable range (27). Blood samples were drawn at baseline and 4 months after initiating treatment for assessing fasting blood glucose, HbA1c, insulin, apolipoprotein AI (ApoAI), ApoB, total cholesterol, triglyceride, very low density lipoprotein (VLDL) and high density lipoprotein (HDL) cholesterol, leptin, and ASP.
Biochemical assays
Baseline blood samples were drawn for determination of plasma ASP and insulin concentrations using prechilled syringes and prechilled glass tubes (EDTA) for insulin and ASP. All blood samples were drawn after an overnight fast, immediately centrifuged at 4 C, and stored at -80 C until assay. Fasting blood glucose was measured by a glucose oxidase-peroxidase calorimetric method using a Tecnicon Dax-48 system analyzer (Miles, Inc., Tarrytown, NY). Glycosylated HbA1c was measured by a latex immunoagglutination inhibition method, using a DCA 2000 analyzer (Bayer Corp., Elkhart, IN; normal range, 4.2–6.5%). Microalbuminuria was detected by an immunoturbidimetric method (Micro ALBUMIN, Beckman Inst., Galway, Ireland) in 24-h urine specimens. Fasting insulin was measured by RIA (insulin reagent, Diagnostics Products Corp., Biaobac, NJ; normal range: <30 mIU/ml). ApoAI and ApoB were analyzed by the immunonephelometric method using reagents from Beckman Coulter, Inc. (Galway, Ireland). Serum cholesterol was measured by the cholesterol oxidase-peroxidase enzymatic method, TG by the glycerol-3-phosphate oxidase-peroxidase enzymatic method, and HDL cholesterol by the direct enzymatic method. Serum low density lipoprotein (LDL) cholesterol was calculated using Friedewald’s formula. Plasma leptin levels were measured in duplicate by immunoradiometric assay (human leptin IRMA, DSL-23100, Diagnostics Systems Laboratories, Inc., Webster, TX). The assay sensitivity was 0.10 ng/ml. The intraassay coefficient of variation at 13.50 ng/ml was 4.9% (n = 12). Plasma ASP concentrations were measured by RIA (Biotrak, Amersham Pharmacia Biotech, Little Chalfont, UK), which used a rabbit anti-C3a des-Arg (ASP) and avoided interference with the precursor (C3) by a selective precipitation step (normal range, 20–500 ng/ml; sensitivity, <40 ng/ml; coefficient of variation, 3–9%).
Statistical analysis
All results are given as the mean ± SD. According to the distribution of the data, either paired t test or Wilcoxon signed ranks test was used to compare related samples data, and either unpaired t test or Mann-Whitney U test was used to compare independent samples data. Correlations between various parameters were determined by Spearman Rho correlation analysis. P < 0.05 was considered statistically significant.
Results
Clinical and laboratory features of the untreated patient and
control groups are shown in Table 1
. As
expected, pretreatment levels of fasting blood glucose,
HbA1c, and insulin were significantly higher in
the patient group than in the controls. However, fasting ASP
(49,67 ± 19,73 vs. 48,49 ± 20,70 nmol/liter;
P = 0.6) and leptin concentrations (33.7 ± 23.2
vs. 26.2 ± 10.6 ng/ml; P = 0.5) were
not significantly different between the two groups. Plasma lipid
concentrations in diabetic patients were higher than those in the
controls, but only plasma TG levels were significantly higher than
those in the control group (2.29 ± 1.16 vs. 1.29
± 0.65 mmol/liter; P = 0.001). BMI, waist and hip
circumference, and waist to hip ratio were not significantly different
between patient and control groups.
|
. Treatment resulted in a statistically
significant decrease in systolic blood pressure, BMI, and waist and
hip circumference associated with parallel falls in fasting blood
glucose and HbA1c. HbA1c
and fasting blood glucose levels were reduced to close to and within
the normal range. Plasma leptin concentrations were also significantly
reduced after treatment (33.69 ± 23.2 vs. 22.73
± 11.26 ng/ml; P = 0.036; Fig. 1). However, fasting ASP concentrations
were significantly increased after treatment (48.49 ± 20.70
vs. 72.82 ± 29.72 nmol/liter; P =
0.004; Fig. 2). Of interest, a marked
increase in ASP (29.2%) and a decrease in leptin (15.9%) were noted.
These changes are comparable to or smaller than changes in glucose
(59.7%) and HbA1c (36.1%). However, changes in
ASP were not correlated to changes in leptin, BMI, insulin, glucose, or
lipid levels. Although these results fall short of statistical
significance, decreases in ApoAI, ApoB, LDL cholesterol, VLDL, TG, and
HDL cholesterol levels was noted after treatment, and total cholesterol
levels were reduced significantly.
|
|
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, changes in lipids were subtle and did not
correlate to those in ASP. No significant correlation was found between fasting ASP and BMI (r = 0.22; P = NS), waist circumference (r = 0.19; P = NS), hip circumference (r = 0.30; P = NS), waist to hip ratio (r = 0.04; P = NS), fasting blood glucose (r = -0.030; P = NS), insulin (r = 0.21; P = NS), ApoAI (r = 0.26; P = NS), ApoB (r = 0.124; P = NS), TG (r = 0.29; P = NS), total cholesterol (r = 0.32; P = NS), VLDL (r = 0.15; P = NS), LDL (r = 0.21; P = NS), HDL (r = 0.24; P = NS), systolic blood pressure (r = 0.10; P = NS), or diastolic blood pressure (r = 0.01; P = NS) in the patient group before treatment. Fasting plasma ASP concentrations were also not correlated to those of leptin (r = 0.20; P = NS) in diabetic subjects. After having improved glycemic control, no correlation between fasting ASP and TG, total cholesterol, ApoAI, ApoB, VLDL, LDL, BMI, waist to hip ratio, insulin, or leptin could be found (data not shown). As expected, plasma leptin levels were correlated to BMI in the patient group before treatment (r = 0.62; P = 0.001) as well as in the control group (r = 0.56; P = 0.001). However, plasma leptin levels were not correlated to insulin, fasting plasma glucose, or any lipid parameters in either group.
Discussion
It is of considerable interest that fasting ASP levels are remarkably increased by improving glycemic control. As alterations in fasting ASP levels are not correlated to those in HbA1c, fasting blood glucose, insulin, or BMI, factors other than HbA1c, fasting blood glucose, insulin, and BMI may have a role in elevated ASP levels. In support of this view, previous studies have emphasized the existence of a large interindividual variability in ASP levels in both obese diabetic subjects and nondiabetic obese controls (18) suggesting that either the regulation of ASP is not tight or there are many factors contributing to ASP homeostasis (9). Moreover, Weyer et al. (25) reported no association between fasting ASP and insulin action or insulinemia in nondiabetic Pima Indians.
It has been shown that total ASP production correlates positively to fatty acid incorporation into adipose tissue (16). With regard to data from both mice and humans, elevated plasma ASP levels might be due to either impaired or enhanced adipocyte fatty acid trapping, and there is no direct way to clarify which prevails (28). As suggested by Sniderman et al. (28), plasma ApoB levels may help distinguish between those two mechanisms. ApoB levels are elevated in the presence of impaired trapping, leading to increased fatty acid flux to the liver. However, ApoB levels are normal in the latter setting, as fatty acid flux is expected to be normal. As ApoB levels did not change significantly after treatment, it is unlikely that a change in fatty acid trapping is the reason for increased ASP levels. Thus, we can propose that insulin or insulin resistance is not responsible for abnormal fatty acid trapping, as improving insulin/glucose did not correct critical lipid abnormalities.
TNF, a peptide secreted from adipocytes, mediates a number of
effects on adipocytes, including decreases in lipoprotein lipase
activity, glucose transport, and adipsin secretion (29).
TNF production increases as adipocytes enlarge and, in turn, impairs
the secretion of adipsin, which is an enzyme involved in ASP production
(30). Previous studies have shown that sulfonylurea
treatment inhibits TNF production in type 2 diabetic subjects
(31, 32). Thus, it is possible that inhibition of TNF
after sulfonylurea treatment may lead to an increase in adipsin
secretion, and consequently, this may lead to increased ASP
generation.
Sulfonylureas may also cause alterations in ASP production and lead to increases in ASP generation. Recent studies have demonstrated that sulfonylurea receptor 1 is expressed in human adipose tissue and other endocrine organs and responds to glibenclamide (33, 34, 35, 36). This receptor mediates physiological responses such as lipogenesis and lipolysis in adipocytes (34). Sulfonylureas were found to stimulate the glycosyl phosphatidylinositol-specific PLC activity and tyrosine phosphorylation of caveolin-1 through direct interaction with their respective enzymes (37), indicating their effects on lipid synthesis (38). Thus, sulfonylurea treatment might cause alteration in ASP production, probably as a result of a primary drug effect, although further studies are needed to reach such a conclusion. Alternatively, one reasonable explanation is that sulfonylurea treatment increases insulin sensitivity, and then insulin increases C3 and consequently ASP. It is known that insulin enhances the production of ASP (14, 39).
One might also argue that an elevated ASP concentration may result from increased chylomicron-activated ASP generation (39). Recent studies have suggested that transthyretin may transfer, from chylomicrons to adipocytes, a factor that stimulates C3 and ASP production (40). However, we did not find any association between fasting ASP and lipid levels. Improving glycemic control did not significantly improve lipid or ApoB levels. Moreover, changes in ASP are not correlated with changes in lipid levels. Thus, it is unlikely that increased chylomicron- or transthyretin-activated ASP production is the reason for observed increase in ASP levels. Similarly, Maslowska et al. (18) found no correlation between fasting ASP and TG or ApoB levels in obese patients. However, Cianflone et al. (23) found significant positive correlations between fasting ASP and TG, VLDL, or ApoB in patients with coronary artery disease. Thus, the selection of the study population or sample size may explain this discrepancy. Although our finding suggests that circulating ASP is not associated with plasma lipids, it does not exclude the role of ASP in the regulation of fatty acid uptake by adipocytes, as the action of ASP is not dependent only on the existing ASP concentration, but also on the sensitivity of target tissues to ASP as indicated by Sniderman et al. (15). Thus, ASP resistance is as real as insulin resistance, and failure of lipids to improve in our patients is consistent with that concept (15).
Interestingly, the decrease in leptin and the increase in ASP were of equal magnitude, suggesting a cross-talk between the two hormones. Animal studies also suggest a relation between ASP and leptin. Markedly reduced adipsin expression has been previously shown in ob/ob and db/db mice (41). Thus, defects in leptin signaling may influence ASP production. On the other hand, mice lacking ASP have marked alterations in plasma leptin levels (42). However, changes in leptin were not correlated to changes in ASP. The lack of association between these two hormones may be due to their inherently different regulatory pathways. Although the underlying mechanisms are not clear, sulfonylurea treatment or glycemic control may have different impacts on each hormone, which, in turn, leads to an increase in one and a decrease in the other. Alternatively, the selection of a specific study population or sample size may be responsible for the lack of association.
Our finding demonstrates that improved glycemic control decreases plasma leptin levels. Leptin, the product of the obese gene, is mainly produced by adipocytes, and its circulating levels correlate significantly to BMI or fat mass (3, 4). As BMI was decreased after treatment, it is most likely that this is responsible for leptin reduction after treatment. In support of our finding, Halle et al. (43) reported that weight loss in obese subjects with type 2 diabetes mellitus leads to a reduction of serum leptin. However, Haffner et al. (44) reported elevated leptin levels after sulfonylurea treatment in type 2 diabetes, as their patients had gained weight after treatment. On the other hand, we could not find any correlation between changes in leptin and changes in glucose, HbA1c, or insulin, and there is no significant difference in leptin levels between type 2 diabetic subjects and controls. Sinha et al. (45) and Malstrom et al. (46) also found no independent effect of type 2 diabetes per se on leptin concentrations. Recent studies suggest a complex interaction between leptin and insulin or insulin resistance (6). On the other hand, it was shown that insulin does not stimulate leptin secretion acutely; however, a long-term effect of insulin on leptin secretion has been demonstrated (47). The recent literature is controversial with regard to the relationship between insulin resistance and leptin. A number of studies have reported associations between insulin resistance and leptin in normal glucose-tolerant subjects (48, 49), but one study did not (50). Other studies reported no associations between insulin resistance and leptin in subjects with diabetes (51, 52).
We conclude that improved glycemic control increases fasting ASP and decreases leptin concentrations, but does not correct critical lipid abnormalities in type 2 obese diabetic subjects. Moreover, altered plasma ASP levels are not associated with changes in BMI, lipids, leptin, insulin, or glucose levels. Thus, our findings suggest that improved glycemic control or insulin resistance is not responsible for abnormal fatty acid trapping, and failure of lipids to improve after treatment in our patients is consistent with the ASP resistance concept.
Acknowledgments
We are grateful to Dr. Allan D. Sniderman (McGill University Health Center, Montréal, Canada) for his critical review of the manuscript.
Footnotes
This work was supported by the Research Center of Gulhane School of Medicine.
Abbreviations: Apo, Apolipoprotein; ASP, acylation-stimulating protein; BMI, body mass index; C3, third component of complement; HbA1c, hemoglobin A1c; HDL, high density lipoprotein; LDL, low density lipoprotein; TG, triglyceride; VLDL, very low density lipoprotein.
Received December 20, 2000.
Accepted April 3, 2001.
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