Adiponectin expression in adipose tissue is reduced in first-degree relatives of type 2 diabetic patients

A. S. Lihn1, T. Østergård2, B. Nyholm2, S. B. Pedersen1, B. Richelsen1, and O. Schmitz2

1 Department of Endocrinology and Metabolism C, Aarhus Amtssygehus, Aarhus University Hospital and Faculty of Health Sciences, Aarhus University and 2 Department of Endocrinology and Diabetes, Aarhus Kommunehospital, Aarhus University Hospital, DK-8000 Aarhus C, Denmark


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adiponectin is suggested to be an important mediator of insulin resistance. Therefore, we investigated the association between adiponectin and insulin sensitivity in 22 healthy first-degree relatives (FDR) to type 2 diabetic patients and 13 matched control subjects. Subcutaneous adipose tissue biopsies were taken before and after a hyperinsulinemic euglycemic clamp. FDR subjects were insulin resistant, as indicated by a reduced M value (4.44 vs. 6.09 mg · kg-1 · min-1, P < 0.05). Adiponectin mRNA expression was 45% lower in adipose tissue from FDR compared with controls (P < 0.01), whereas serum adiponectin was similar in the two groups (6.4 vs. 6.6 µg/ml, not significant). Insulin infusion reduced circulating levels of adiponectin moderately (11-13%) but significantly in both groups (P < 0.05). In the control group, adiponectin mRNA levels were negatively correlated with fasting insulin (P < 0.05) and positively correlated with insulin sensitivity (P < 0.05). In contrast, these associations were not found in the FDR group. In conclusion, FDR have reduced adiponectin mRNA in subcutaneous adipose tissue but normal levels of circulating adiponectin. Adiponectin mRNA levels are positively correlated with insulin sensitivity in control subjects but not in FDR. These findings indicate dysregulation of adiponectin gene expression in FDR.

30-kilodalton adipose complement-related protein; gene expression; genetic predisposition to disease; insulin; first-degree relatives


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EVIDENCE IS ACCUMULATING that molecules produced by adipose tissue are important for the development of obesity-related health complications such as insulin resistance and atherosclerosis (9, 15, 26). In response to metabolic signals, the adipose tissue produces and releases various biologically active proteins, including tumor necrosis factor-alpha (TNF-alpha ), interleukin-6, interleukin-8, leptin, plasminogen activator inhibitor-1, and adiponectin (1, 5). Excess amounts of adipose tissue, as seen in obesity, and lack of adipose tissue in lipodystrophic states might cause dysregulated production of these substances.

The present study focuses on the adipocyte-specific secretory protein adiponectin, also known as adipocyte complement-related protein of 30 kDa (21), AdipoQ (12), adipose most abundant gene transcript 1 (14), and gelatin-binding protein 28 (16). Adiponectin is a polypeptide of 244 amino acids, and it has some structural homology with collagen, complement factor C1q, and TNF-alpha (14, 22). Interestingly, adiponectin is abundantly present in plasma (3). In healthy nonobese subjects, the plasma level of adiponectin is 4-14 µg/ml, but lower levels are seen in relation to obesity, type 2 diabetes, and coronary artery disease (CAD; see Refs. 3, 10, 23). Weight reduction elevates the plasma levels in both diabetic and nondiabetic subjects (10, 25).

The physiological role of adiponectin is yet to be determined, but protective effects against the development of atherosclerosis (18, 19) and a causative role in the development of insulin resistance (10, 13) have been proposed. Recent studies in both monkeys and humans have shown that plasma adiponectin concentration is more closely related to insulin sensitivity than to body mass index (BMI) and percent body fat (11, 23). Further indications of antidiabetic effects of adiponectin were found in rodent studies. Injection of adiponectin in both wild-type and diabetic mice models decreased hepatic glucose output, resulting in reduced serum glucose levels without an increase in insulin levels (4). This finding was supported by a study of Yamauchi et al. (24) demonstrating amelioration of hyperglycemia and hyperinsulinemia by adiponectin infusion to lipoatrophic mice, which were insulin resistant and had nondetectable plasma adiponectin levels.

To gain further insight into the pathogenesis of type 2 diabetes, we explored the relationship between a prediabetic state and adiponectin. The circulating levels of adiponectin and adiponectin gene expression in adipose tissue were determined in a group of healthy first-degree relatives of type 2 diabetic patients (FDR) and in a group of age, sex, and BMI-matched healthy control subjects. FDR are individuals at increased risk of developing type 2 diabetes but still without major abnormalities in glucose metabolism. Adipose tissue adiponectin gene expression and serum adiponectin levels were examined during baseline and hyperinsulinemic conditions.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. Thirteen control subjects without any family history of diabetes and 22 FDR were included in this study (Table 1). All results presented in text, Tables 1 and 2, and Figs. 1 and 2 are based on these 13 control and 22 FDR subjects. Among the 22 FDR, 5 had two FDRs, 12 had one parent and two or more second-degree relatives, and 5 had only one parent with type 2 diabetes. The subjects of the two groups compared were from unrelated families, and the groups were matched for age, sex, and BMI. All of the subjects were Caucasian, healthy, not taking any medication, and exhibited a normal oral glucose tolerance test (75 g glucose) according to World Health Organization criteria. Instruction was given to abstain from strenuous exercise for at least 3 days before participation in the study. The study was approved by the local Ethics Committee, and all subjects provided written informed consent to participate.

                              
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Table 1.   Clinical characteristics of the study population


                              
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Table 2.   Correlations between BMI, insulin sensitivity, and adiponectin



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Fig. 1.   Adipose tissue adiponectin mRNA expression (A) and serum adiponectin concentration (B) in control subjects (open bars, n = 13) and first-degree relatives (FDR; filled bars, n = 22) at baseline and during hyperinsulinemic euglycemic clamp. Adiponectin mRNA levels are expressed as relative to beta -actin mRNA levels. Mean values ± SE are shown. *P < 0.05, **P < 0.01, and ***P < 0.001.



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Fig. 2.   Correlation between adiponectin mRNA expression and M value in control subjects (open circle , n = 13, r = 0.62, P < 0.05) and FDR (, n = 22, r = -0.04, not significant). Adiponectin mRNA levels are expressed as relative to beta -actin mRNA levels.

Experimental design. All subjects underwent a hyperinsulinemic euglycemic clamp, and subcutaneous fat biopsies were taken from the abdominal region before and after the clamp.

Measurements of body composition. BMI was calculated as total body weight (TBW, kg) divided by squared height (m2). In the supine position, waist-to-hip ratio (WHR) was measured as the smallest circumference around the waist divided by the largest circumference around the hips. Body composition was evaluated by dual-energy X-ray absorptiometry scanning with a QDR-2000 densitometer (Hologic, Waltham, MA), and fat mass (FM, kg) and fat mass percent (FM%) were calculated.

Hyperinsulinemic euglycemic clamp. In the morning, after an overnight fast, two intravenous cannulas were inserted, one in a dorsal hand vein and the other in an antecubital vein. The hand vein was kept heated (55°C) and used for sampling of arterialized blood, whereas the antecubital vein was used for infusions. After 90 min, insulin infusion at a rate of 0.6 mU · kg-1 · min-1 was started and maintained for 150 min. Plasma glucose concentration was kept at ~5 mmol/l by adjusting the infusion rate of 20% glucose. The last 30 min of the clamp (time 120-150 min) were defined as the hyperinsulinemic steady-state period. During this period, the amount of glucose (mg · kg TBW-1 · min-1) required to maintain euglycemia was taken as an index of insulin action (M value). Plasma glucose was determined every 5-10 min during the clamp, and blood samples for determination of serum insulin were taken at -90, -30, -15, 0, 60, 120, 135, and 150 min.

Fat biopsies. At -90 min (baseline) and at 150 min (during hyperinsulinemia), adipose tissue biopsies were taken from the subcutaneous abdominal region (periumbilically), as previously described (20). Using local anesthesia (5 mg/ml lidocaine), the biopsies were taken by needle aspiration (liposuction). The adipose tissue was washed thoroughly with isotonic saline and then frozen in liquid nitrogen for later RNA extraction.

Analytical techniques. Immediately after sampling, plasma glucose was measured in duplicate (Beckman Instruments, Palo Alto, CA). Serum insulin was determined by a two-site ELISA (Dako Diagnostics, Cambridgeshire, UK; see Ref. 2). The intra-assay coefficient of variation was 2.0% (n = 75) at a serum level of 200 pmol/l. Serum adiponectin was measured by RIA (Linco Research), and the intra-assay coefficient of variation was 5.0% (n = 12).

Isolation of RNA. Total RNA was isolated using Tri-Zol reagent (GIBCO-BRL Life Technologies, Roskilde, Denmark). RNA was quantified by measuring absorbency at 260 and 280 nm, and the integrity of the RNA was checked by visual inspection of 18S and 28S rRNA on an agarose gel.

Real-time RT-PCR measurement of adiponectin mRNA. RNA was reverse transcribed with RT and random hexamer primers at 23°C for 10 min, 42°C for 60 min, and 95°C for 10 min according to the manufacturers' instructions (GeneAmp RNA PCR Kit from Perkin-Elmer Cetus, Norwalk, CT). Next, PCR-mastermix containing the specific primers, Hot Star Taq DNA polymerase and SYBR-Green PCR buffer, was added. All samples were determined as duplicates.

The adiponectin primers amplified a product of 301 bp as follows: 5'-CATGACCAGGAAACCACGACT and 5'-TGAATGCTGAGCGGTAT. As a housekeeping gene, beta -actin was amplified using the following primers: 5'-ACGGGGTCACCCACACTGTGC and 5'-CTAGAAGCATTTG-CGGTGGACGAT-G. Real-time quantization of adiponectin to beta -actin mRNA was performed using a SYBR-Green PCR assay and an iCycler PCR machine (Bio-Rad Laboratories, Hercules, CA), as previously described (6). In brief, adiponectin mRNA and beta -actin mRNA were amplified in separate tubes at 95°C for 10 min and thereafter repeating cycles comprised of 95°C for 30 s, 57°C for 30 s, and extension at 72°C for 60 s. During the extension, a step increase in fluorescence was measured in real time. Data were obtained as threshold cycle (CT) values. CT was defined as the cycle number at which the fluorescence reached 10 times the SD of the baseline fluorescence. Relative gene expression was calculated using the formula 1/2(CT adiponectin - CT beta -actin), essentially as described in the User Bulletin 2, 1997 from Perkin-Elmer (Perkin-Elmer Cetus).

Statistical analysis. The distribution of adiponectin mRNA and serum adiponectin data was not normal; therefore, nonparametric analysis was performed. Mann-Whitney's test was used to test the statistical significance of differences between control and FDR values. For comparison of mean values before and after the hyperinsulinemic clamp, Wilcoxon Signed Ranks test was used. Correlation between variables was tested by Pearsons's correlation coefficient after logarithmic transformation of data, which were not normally distributed. Values are presented as means ± SE, and a P value <0.05 was considered statistically significant. For analyses, the SPSS statistical package was used (SPSS, Chicago, IL).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Clinical characteristics. Although the FDR and the controls were matched for BMI, the FM turned out to be higher in the FDR group compared with the control group (21.7 ± 1.2 vs. 16.9 ± 1.6 kg, P < 0.05; Table 1). There was no difference in WHR between FDR and controls.

After an overnight fast, subject plasma glucose level was higher in the FDR group than in the control group (5.3 ± 0.1 vs. 5.0 ± 0.1 mmol/l, P < 0.05). Fasting plasma insulin levels tended to be higher among the FDR, but this difference was not statistically significant. The amount of glucose required to maintain euglycemia during the clamp, the M value, was significantly lower in FDR compared with control subjects (4.44 ± 0.40 vs. 6.09 ± 0.61 mg · kg-1 · min-1, P < 0.05; Table 1).

Adiponectin mRNA expression in adipose tissue. At baseline, the expression of adiponectin mRNA in subcutaneous, abdominal adipose tissue was significantly lower in FDR compared with control subjects (13.2 ± 2.6 vs. 24.1 ± 4.5, P < 0.01; Fig. 1A). During the hyperinsulinemic clamp, the adiponectin mRNA expression tended to decline in the control group (24.1 ± 4.5 to 17.5 ± 3.8, P = 0.20), but insulin did not affect adiponectin mRNA expression among FDR [13.2 ± 2.6 vs. 16.4 ± 2.2, not significant (NS); Fig. 1A].

Circulating levels of adiponectin. Mean fasting serum adiponectin concentration for all subjects in this study was 6.5 ± 0.5 µg/ml. Unlike adiponectin mRNA expression, we found no difference between circulating adiponectin concentrations in FDR compared with controls in the basal state [6.6 ± 1.0 vs. 6.4 ± 0.5 µg/ml (NS); Table 1]. Hyperinsulinemia reduced serum adiponectin levels by 11-13% in control subjects (6.6 ± 1.0 vs. 5.9 ± 9.3 µg/ml, P < 0.05) and in FDR (6.4 ± 0.5 vs. 5.6 ± 0.5 µg/ml, P < 0.001; Fig. 1B).

Gender differences. Serum adiponectin concentrations were significantly lower in men than in women when all subjects were analyzed together (5.3 ± 0.5 vs. 7.7 ± 0.7 µg/ml, n = 35, P < 0.05). Adiponectin mRNA levels were 29% lower in men compared with women, but the gender difference was not statistically significant [14.4 ± 2.6 vs. 20.3 ± 4.2 (NS); data not shown].

Correlations. In the control group, subcutaneous adipose tissue adiponectin mRNA levels were positively correlated with insulin sensitivity determined by the M value (r = 0.62, P < 0.05; Fig. 2), negatively correlated with the fasting insulin level (r = -0.65, P < 0.05), and tended to be negatively correlated with BMI (r = -0.38, P = 0.20). In contrast, no such associations were found in the FDR group (Table 2). Concerning serum adiponectin, it was only found to be negatively correlated with BMI in the control group (r = -0.58, P < 0.05), not in the FDR group [r = 0.13 (NS); Table 2], and no correlations were found between serum adiponectin and M value or fasting insulin in either the control group or the FDR group (Table 2).

There was no correlation between adiponectin mRNA expression and circulating levels of adiponectin in the two groups separately, nor in the two groups together [r = 0.21 (NS); data not shown]. Furthermore, the basal plasma level of free fatty acids was not correlated to either adiponectin mRNA or to serum adiponectin in any of the study groups (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A role for adiponectin in insulin resistance has been suggested on the basis of an association between low levels of serum adiponectin and insulin resistance found in both epidemiological (10, 13, 23) and experimental (11) studies. Furthermore, levels of circulating adiponectin have been investigated in normal subjects, in type 2 diabetic patients with and without CAD, and in different ethnic groups (3, 10, 23). However, this is the first study in which serum adiponectin and adiponectin gene expression have been investigated in FDRs of type 2 diabetic patients.

FDR are subjects at increased risk of developing type 2 diabetes, and, as shown earlier (17), the FDR subjects in this study were characterized by reduced insulin sensitivity. Accordingly, we found substantially reduced levels of adiponectin mRNA in subcutaneous adipose tissue from FDR compared with control subjects. In contrast to these findings, the circulating levels of adiponectin did not differ between the two groups compared. This indicates that serum adiponectin levels are controlled both transcriptionally and posttranscriptionally, which is in agreement with the fact that, in the present study, no clear association was found between adiponectin mRNA and serum adiponectin.

In comparison with our findings, Weyer et al. (23) found statistically significant higher plasma adiponectin levels in a group of 23 Caucasians compared with a group of 121 Pima Indians, the latter established to be an ethnic group with high propensity for obesity and type 2 diabetes. Furthermore, Lindsay et al. (13) recently showed in a nested case-control study with Pima Indians that higher plasma levels of adiponectin protected against later development of type 2 diabetes. In both of these studies, adiponectin mRNA levels were, however, not determined. The reduced level of adipose tissue adiponectin mRNA found in FDR in this study could suggest that altered adiponectin gene expression plays a pathogenic role in the development of insulin resistance in FDR. However, it has been shown that insulin in vitro is able to suppress adiponectin gene expression (7). Thus, if this is the case, then the difference we found in adipose tissue adiponectin gene expression is more likely the consequence, rather than the cause, of insulin resistance.

In vitro studies of insulin effects on adiponectin mRNA expression and production in adipocytes have shown conflicting results. The first studies showed stimulating effects of insulin on adiponectin mRNA in 3T3-L1 adipocytes (21) and in human visceral adipose tissue explants (8), but recently Fasshauer et al. (7) showed a time- and dose-dependent decrease in adiponectin mRNA levels by incubating 3T3-L1 adipocytes with insulin. Significant effects were found already after 4 h of incubation with insulin. In the present study, we show that insulin, in vivo, reproducibly reduces serum adiponectin levels within 3 h in both groups, but only modestly (11-13%). During the hyperinsulinemic-euglycemic clamp, adiponectin mRNA levels among control subjects were also decreased, albeit not significantly. The FDR group had lower levels of adiponectin mRNA at baseline, and these levels were not reduced further by the acute insulin challenge. These results suggest that, in insulin-sensitive control subjects, insulin affects both adiponectin gene expression and circulating levels of adiponectin in vivo. In contrast, insulin did not have effects on adipose tissue adiponectin mRNA in the FDR group but reduced their levels of serum adiponectin. During the clamp, an effect of concomitant fasting on adiponectin mRNA and serum adiponectin levels cannot be excluded. However, from preliminary studies from our laboratory on extended fasting, we found no effect of fasting on adiponectin (data not shown). Taken together, these findings could implicate that the effect of insulin on serum adiponectin is more related to changes in adiponectin degradation in the body than to changes in adiponectin production.

In several studies, correlations between obesity measurements, insulin resistance, and plasma adiponectin have been investigated. The major findings in these studies are that BMI and plasma adiponectin are negatively correlated (3, 10, 11, 13, 23) and that plasma adiponectin is positively correlated with insulin sensitivity (11, 23). In the control group, we found similar correlations particularly between adiponectin gene expression and BMI (negative), M value (positive), and plasma insulin (negative). For serum adiponectin, the tendencies were the same in the control group. However, all correlations did not reach statistical significance, which may be because of the relatively narrow range of BMI (21.6-29.6 kg/m2) and the relatively small number of control subjects (n = 13). Surprisingly, none of these correlations was observed in the FDR group, although the number of subjects was higher (n = 22). We presently have no explanation why well-described correlations between adiponectin and BMI/insulin sensitivity were only found in the control group and not among the FDR, but this finding might indicate a dysregulated adiponectin production in FDR subjects. Possibly, some kind of tonic inhibition of adipose tissue adiponectin gene expression or a change in the level of transcription factors regulating the gene expression of adiponectin may be present in the FDR, obscuring the conventional associations observed in insulin-sensitive subjects.

As mentioned above, we found no correlation between subcutaneous adipose tissue adiponectin gene expression and serum adiponectin in the two groups. This lack of association has been observed by others (11) and could indicate that adipose tissue depots other than the subcutaneous abdominal depot may be of importance for determining the serum level of adiponectin. However, in preliminary studies, we have found lower adiponectin mRNA levels in omental adipose tissue compared with subcutaneous abdominal adipose tissue, suggesting that this depot might be of less importance. Differences in the level of adiponectin gene expression and production in various subcutaneous adipose tissue depots, such as abdominal vs. femoral and gluteal depots, could be another explanation for the lack of association between circulating levels and gene expression. On the other hand, other factors, such as posttranscriptional modification or degradation, might be more important for the regulation of serum adiponectin levels than regulation of gene transcription in adipocytes.

Thus the present study, together with other recent studies (3, 11), raises important questions concerning which factors are regulating adipose tissue adiponectin production in addition to questions concerning the relationship between gene expression and adiponectin secretion. To get a clearer picture of the physiological role of adiponectin in various metabolic states, these questions should be addressed in future studies.

In conclusion, FDRs of type 2 diabetic patients have substantially reduced levels of adipose tissue adiponectin mRNA compared with control subjects, but they have equal levels of circulating adiponectin. Circulating levels of adiponectin are reduced moderately by insulin infusion in both FDR and control subjects, possibly because of an effect of insulin on adiponectin degradation. In control subjects, adiponectin mRNA expression is correlated negatively with BMI and positively with insulin sensitivity, although no such correlations were found in the FDR group. These findings indicate that, in FDR, adipose tissue adiponectin gene expression is dysregulated. Whether this dysregulation is a consequence or the cause of the insulin-resistant state cannot be determined from the present study.


    ACKNOWLEDGEMENTS

We appreciate the expert technical assistance of Lenette Pedersen, Dorte Phillip, Lene Trudsø, and Annette Mengel.


    FOOTNOTES

The study was supported by the Danish Research Council, the Danish Diabetes Association, the Novo Nordic Foundation, the Institute of Experimental Clinical Research at Aarhus University, and the Aarhus University-Novo Nordic Center for Research in Growth and Regeneration.

Address for reprint requests and other correspondence: A. S. Lihn, Dept. of Endocrinology and Metabolism, Aarhus Amtssygehus, Tage Hansensgade 2, 8000 Aarhus C, Denmark (E-mail: lihn{at}dadlnet.dk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published October 8, 2002;10.1152/ajpendo.00358.2002

Received 13 August 2002; accepted in final form 1 October 2002.


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DISCUSSION
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