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Department of Medicine, Huddinge University Hospital, and Department of Surgery, Danderyd Hospital (E.N.), Karolinska Institute, SE-141 86 Stockholm, Sweden
Address all correspondence and requests for reprints to: Dr. Peter Arner, Department of Medicine, M63, Huddinge University Hospital, SE-141 86 Stockholm, Sweden. E-mail: . peter.arner{at}medhs.ki.se
Abstract
Polymorphism in the calpain-10 gene is linked to type 2 diabetes, insulin resistance, and decreased thermogenesis. In view of the role of ß-adrenoceptors in thermogenesis we investigated the relationship between ß1-, ß2-, and ß3-adrenoceptor-stimulated lipolysis in abdominal sc fat cells and 3 different previously described single nucleotide polymorphisms (SNPs) in the calpain-10 gene (SNP-19, SNP-43, and SNP-63). The study sample comprised 240 healthy subjects. A strong association between lipolytic ß3-receptor function in adipocytes and the SNP-19, which is a deletion/insertion (1/2) was observed in overweight subjects (body mass index, >25 kg/m2), but not in lean ones. No association was found between any of the polymorphisms and lipolytic function of either ß1- or ß2-receptors. Carriers of 1/1 in SNP-19 had 30-fold decreased lipolytic sensitivity of ß3-adrenoceptors in comparison to 1/2 or 2/2 carriers (P = 0.0019, by ANOVA). This was found in both genders and was not influenced by SNP–43 or SNP–63 in the calpain-10 gene or by the Trp64Arg polymorphism in the ß3-adrenoceptor gene. In conclusion, a deletion/insertion polymorphism in the calpain-10 gene (SNP-19) is associated with reduced ß3-adrenoceptor function in obesity. This could be of importance for regulating thermogenesis in overweight subjects.
THE GENETIC COMPONENT of type 2 diabetes remains to be established. Recently, however, results of positional cloning suggested that the calpain-10 gene could be an important type 2 diabetes gene (1). Independent association studies confirmed a relationship between the calpain-10 gene polymorphism and type 2 diabetes (2). Furthermore, the latter study showed that variation in the calpain-10 gene in subjects of British ancestry is associated with development of type 2 diabetes in a more obese group with an earlier age at onset.
The biological role of calpain-10 is unknown (see Ref. 3 for a review), it being an atypical member of the calpain family. These proteins are intracellular nonlysomal proteases that are believed to play a role in calcium-regulated signaling pathways; whether calpain-10 has similar or other effects has not been elucidated. Calpain-10 is expressed at the mRNA and protein levels in a variety of tissues, and a number of different mRNA isoforms have been observed. It is therefore possible that calpain-10 has tissue specific effects (3, 4).
Interestingly, Baier and colleagues (5) found that the calpain-10 gene polymorphism was associated with insulin resistance and reduced resting energy expenditure. The latter suggests that calpain-10 could be involved in the regulation of thermogenesis. We examined this question by comparing calpain-10 polymorphisms with ß-adrenoceptor function, bearing in mind the important role of the sympathetic system in stimulating thermogenesis through different ß-adrenoceptor subtypes (6). As a model we used ß-adrenergic receptor-mediated lipolysis in human sc fat cells. These cells are relatively easy to obtain, and independent in vivo studies have shown that lipolysis in human sc abdominal adipose tissue can be activated by selective stimulation of each of the three known ß-adrenoceptor subtypes (7, 8). In an ongoing project designed to characterize lipolysis regulation in abdominal sc fat cells in healthy subjects with a large interindividual variation in body mass index (BMI) we genotyped 240 subjects for the previously described single nucleotide polymorphisms (SNPs) in the calpain-10 gene, termed SNP-19, SNP-43, and SNP-63 (1). A strong association between ß3-adrenoceptor lipolytic function and SNP-19 was found in overweight subjects.
Subjects and Methods
We investigated 148 overweight subjects (34 men and 114 women) as well as 92 lean subjects (28 men and 64 women) of Scandinavian origin who were consecutively recruited to study adrenergic regulation of lipolysis in sc fat cells. The subjects were either healthy volunteers or obese otherwise healthy subjects who were referred to our obesity unit for treatment of overweight. None of the subjects was either sedentary or involved in sporting activities. None had undertaken a slimming diet during the 6 months before the study. Overweight was defined using WHO criteria (BMI, >25 mg/m2). The BMI of all the subjects ranged from 18–53 kg/m2. None of the subjects was taking regular medication, including anticontraceptives for the women. The subjects’ ages ranged from 19–72 yr. Fifteen women were menopausal. It has recently been shown that menopause does not influence lipolysis regulation in human sc fat cells (9). Adipose tissue was also obtained from 5 subjects during elective surgery because of nonmalignant disorders and was used for methodological experiments.
The subjects came to the laboratory in the morning after an overnight fast. Height and weight were determined, and a venous blood sample was obtained for determinations of plasma glucose and insulin by the hospital’s routine chemistry laboratory. An sc fat biopsy (
1–2 g) was then obtained under local anesthesia from the umbilical region (10). The study was approved by the ethics committee of Huddinge Hospital. The procedure was initially explained to each subject, and his or her consent was obtained.
Fat cell lipolysis experiments
The methods used to perform and analyze adipocyte cellularity and lipolysis have been described in detail previously (11). In brief, isolated fat cells were prepared, and their average size, volume, and weight were determined. Diluted fat cell suspensions (2%, v/v) were incubated in duplicate for 2 h at 37 C in a Krebs-Henseleit phosphate buffer (pH 7.4) containing bovine albumin (20 g/liter), glucose (1 mg/ml), and ascorbic acid (0.1 mg/ml). At the end of the incubation, an aliquot of the medium was removed for glycerol analysis (lipolysis index), and the amount of glycerol release was related to the number of incubated fat cells.
The following agents were added (at concentrations of 10-10–10-4 mol/liter, depending on the type) to the incubation medium at the start of the experiment: dobutamine (a selective ß1-adrenoceptor agonist; n = 232), terbutaline (a selective ß2-adrenoceptor agonist; n = 231), and CGP 12177 (a selective ß3-adrenoceptor agonist; n = 172). The basal condition was defined as that with no agonist present. Because of the lack of tissue it was not always possible to perform complete lipolysis experiments. In methodological lipolysis experiments isoprenaline (nonselective ß-adrenoceptor agonist) and CGP 20712 (highly selective ß1-adrenoceptor blocker) were used.
As human sc fat cells have functional spare ß-adrenergic receptors (12), it is possible to use classical pharmacological methods to evaluate receptor function (13). The individual concentration-response curves were linearized by log-logit transformation and analyzed for pD2 (negative logarithm of half-maximum effective concentration). Furthermore, responsiveness (glycerol release at maximum effective drug concentration) was determined from the original curves. Changes in pD2 reflect receptor events (binding and coupling to effector), whereas changes in responsiveness reflect receptor events as well as more distal and postreceptor-related events (13). Although the ß1-, ß2-, and ß3-adrenoceptor agonists used might be less selective in other systems, we have previously demonstrated the selectivity of dobutamine, terbutaline, and CGP 12177 on ß-adrenoceptor-mediated lipolysis in isolated human fat cells (14). A plateau of response was reached at the highest concentrations in all cases and with all drugs. Thus, it was possible to get an accurate measure of pD2 and responsiveness of the different drugs in each of the subjects investigated.
Genotyping
DNA was prepared from venous blood and used for genotyping of the three SNPs in the calpain-10 gene. We used previously defined nomenclature to define the SNPs (1). SNP-19, which is a deletion/insertion variant (1/2), was genotyped by PCR using the forward primer 5'-GTT TGG TTC TCT TCA GCG TGG AG-3' and the reverse primer 5'-CAT GAA CCC TGG CAG GGT CTA AG-3'. SNP-43 (G/A) was genotyped using the forward primer 5'-GCT GGC TGG TGA CAT CAG TGC and the reverse primer 5'-ACC AAG TCA AGG CTT AGC CTC ACC TTC ATA-3' and subsequent digestion with enzyme NdeI. SNP-63 (G/T) was genotyped using the forward primer 5'-AAG GGG GGC CAG GGC CTG ACG GGG GTG GCG-3' and the reverse primer 5'-AGC ACT CCC AGC TCC TGA TC-3' and digestion using the enzyme HhaI. Previously genotyped DNA (1) was used to confirm the accuracy of determining the three polymorphisms.
Statistics
Values are the mean ± SE. ANOVA, analysis of covariance (using sex and age as cofactors), paired t test, and 
2 analysis were performed. P 
0.05 was considered statistically significant.
Results
Genotype frequency
Results of genotyping of SNP-19, SNP-43, and SNP-63 in the calpain-10 gene in all material are shown in Table 1
. SNP-19 and SNP-43 were common. SNP-63 was less common; the uncommon allele occurred in 12.5% of the subjects. All genotypes were in Hardy-Weinberg equilibrium, and there was no significant difference in genotype distribution either between lean and overweight or between genders (values not shown). The frequency distribution of the three polymorphisms was similar, as reported for other Caucasian populations (1, 2).
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It is well established that body weight may influence most of the measured parameters. Therefore, the subjects were divided into a lean and an overweight group. The phenotypic effects of the three polymorphisms in the calpain-10 gene were investigated. Neither SNP-43 nor SNP-63 had any significant effect on the measured parameters of age, BMI, plasma glucose, plasma insulin, fat cell volume, and lipolysis (values not shown). Likewise, SNP-19 did not influence age, BMI, plasma glucose and insulin, or fat cell volume (Table 2). However, some marked phenotypic effects of the latter polymorphism on lipolysis were observed (Table 3 and Fig. 1). In the overweight group, subjects with 1/1 (i.e. deletion/deletion) had 1.2 log unit lower pD2 for CGP 12177 than those with 2/2 (i.e. insertion/insertion). This corresponds to a 30-fold difference in ß3-adrenoceptor sensitivity. Heterozygote subjects had similar pD2 as 2/2 subjects. This genotype effect was highly significant and remained after adjustment for age together with gender and BMI (P = 0.004). There was no genotype effect on the responsiveness (maximal lipolytic effect) of CGP 12177. For terbutaline and dobutamine neither responsiveness nor pD2 was influenced by SNP-19. Likewise, the basal rate of lipolysis was not influenced by the polymorphism. Thus, in overweight subjects, carriers of 1/1 in SNP-19 had a marked and selective decrease in lipolytic ß3-adrenoceptor sensitivity. The mean concentration- response curves for terbutaline, dobutamine, and CGP 12177 are shown for illustrative purposes in Fig. 2. It is apparent that the CGP 12177 curve for 1/1 subjects was markedly shifted to the right compared with that for 2/2 subjects. Corresponding curves for terbutaline and dobutamine were superimposed in 2/2 and 1/2 subjects. The CGP 12177 curve for 1/2 subjects (not displayed in the figure) was superimposed over the 2/2 curve.
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was 0.00190. By correction for multiple comparison, i.e. P value multiplied by (n - 1), the corrected value was 0.032, which is still statistically significant. It should be noted that lipolysis values for CGP 12177 were lower than those for dobutamine and terbutaline. This is due to the fact that the latter two agents are full agonists, whereas CGP 12177 is a partial agonist (14).
Subgroup analysis
To study the interaction with gender and other genotypes, a subgroup analysis of the pD2 for CGP 12177 was performed on the group of overweight subjects (Table 4). Similar results were found regardless of subgroup analysis, namely that the pD2 for CCP 12177 was 1–1.5 log units lower in 1/1 carriers of SNP-19 than in the other two groups. This was true for men and women and for carriers who had an identical background for the SNP-43 and SNP-63 polymorphisms in the calpain-10 gene. We choose the most common genotypes for these polymorphisms, which were G/A in SNP-43 (n = 49) and G/G in SNP-63 (n = 95). We also investigated the influence of the Trp64Arg polymorphism in the ß3-adrenoceptor gene, because it influences the pD2 for CGP 12177 in lipolysis experiments (15). Ninety-eight of the overweight subjects had been previously genotyped for this polymorphism (see Ref. 15 for details) and were used for analysis. Interestingly in both the common form (Trp64Trp; n = 86) and the rare form (Trp64Arg; n = 12) of the ß3-adrenoceptor gene polymorphism, the pD2 for CGP 12177 was much lower in 1/1 subjects than in either 1/2 or 2/2 subjects with regard to SNP-19.
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It has been questioned whether CGP 12177 exerts its agonist effects solely through ß3-adrenoceptors (16). In certain cells it can also interact with a fraction of ß1-adrenoceptors that are in alternative confirmation (17). However, the latter does not seem to be true for lipolysis in human fat cells. We previously demonstrated the selectivity of CGP 12177 as a ß3-agonist on human fat cell lipolysis in a detailed pharmacological study (18). This was confirmed in an extensive pharmacological study of lipolysis in isolated human fat cells performed by those who had challenged the idea that CGP 12177 is not solely mediating agonist effects through ß3-receptors (19). To again reevaluate the selectivity of the CGP compound, we performed experiments designed by Konkar et al. (20) in such a way so as to address this issue. Isolated fat cells from five subjects were incubated either with isoprenaline (nonselective ß-adrenoceptor agonist) or CGP 12177 in the absence or presence of 10-6 mmol/liter of a highly selective ß1-adrenergic receptor antagonist (CGP 20712). Glycerol release was measured. As observed in Fig. 2 the ß1-blocker shifted the isoprenaline curve markedly to the right, whereas the CGP 12177 concentration-response curves with or without the ß1-blocker were almost superimposed. The individual experiments were subjected to statistical evaluation of pD2 values by paired t test. The mean pD2 values for isoprenaline in the absence and presence of CGP 20712 were 9.6 ± 0.3 and 8.4 ± 0.4, respectively (P = 0.008). In other words, there was a 30-fold decrease in isoprenaline sensitivity induced by CGP 20712. Corresponding values for CGP 12177 were 8.3 ± 0.7 and 7.9 ± 0.7 (P = 0.52). This means that the ß1-blocker was not able to induce a significant shift in CGP 12177 sensitivity (although it had this effect on isoprenaline sensitivity in the same subjects). Thus, although minor ß1-adrenergic receptor-mediated effects of CGP 12177 on lipolysis in human fat cells cannot be excluded, it is evident that the quantitatively important effects are mediated through ß3-adrenoceptors.
Discussion
This study provides for the first time some evidence that the calpain-10 gene might be directly involved in the regulation of thermogenesis. In overweight subjects we observed a marked effect SNP-19 on adipocyte ß3-adrenoceptor function. Homozygous carriers of deletion (1/1) had a 30-fold decrease in the agonist lipolytic sensitivity of this receptor compared with 1/2 or 2/2 carriers, who did were not different from each other in this respect. The genotype effect was specific for the ß3-adrenoceptor subtype, as no variation was seen in either ß1- or ß2-adrenoceptor lipolytic function.
The role of the ß3-adrenoceptor in thermogenesis of animal models is well established as reviewed previously (21). The importance in humans is less well documented. For example, short-term iv infusions of the nonselective ß-adrenoceptor agonist isoprenaline together with ß1/ß2-adrenergic receptor blockers failed to stimulate thermogenesis (22). However, long-term treatment of healthy volunteers with a selective ß3-adrenoceptor agonist under placebo-controlled conditions clearly stimulated thermogenesis (23). It is also possible that ß3-adrenoceptors stimulate thermogenesis through their lipolytic action in adipose tissue by increased fatty acid output. Although the ß3-adrenergic receptor is less lipolytic in sc adipose tissue than are ß1- and ß2-subtypes, the former has a marked lipolytic effect in visceral fat (14). Unfortunately, it is not possible to study the visceral fat depot in this type of clinical study.
The molecular mechanisms responsible for the interaction between calpain-10 and the ß3-adrenoceptor are currently unknown. Unfortunately, no good tools, such as potent agonists, selective radioligands, or selective antibodies, are available for mechanistic ß3-adrenoceptor studies in man. Even the use of CGP 12177 has been criticized because it is believed that this ligand, in addition to its ß3-adrenoceptor properties, can also stimulate ß1-adrenoceptors under certain circumstances (17). However, present and previous (18, 19) pharmacological experiments suggest that, unlike in many other cell types, CGP 12177 exerts its important agonist action solely through ß3-adrenoceptors in human fat cells at least when lipolysis is concerned. We cannot exclude minor or ß1-receptor-mediated effects, but these are probably not important for the interpretation of the present data. There is an alternative ß3-agonist available for human studies of fat cell lipolysis termed CL 316,243. Unfortunately, this agent has a very low sensitivity and is therefore not suitable for clinical studies; the half-maximal effect occurs at 0.1 mmol/liter compared with 10 nmol/liter for CGP 12177, which is a 10,000-fold difference in sensitivity (24).
The present findings are not likely due to chance or to poor selection of study groups. First, the findings remained significant when corrected for multiple comparisons. Second, the results held true whether various subgroups, such as men, women, and carriers of other SNPs (i.e. SNP-43 and SNP-63) of the calpain-10 gene, were analyzed or when the Trp64Arg polymorphism in the ß3-adrenceptor gene was considered. However, we could not demonstrate any effect of SNP-19 in lean subjects. This suggests that an important gene-environmental interaction may be directly linked to total body fat accumulation. The latter question cannot be answered until further studies of lean and obese subjects have been conducted. However, it is tempting to speculate that when overweight develops, the carriers of 1/1 in SNP-19 are prone to decreased thermogenesis due to impaired ß3-adrenoceptor function. There is also earlier evidence of an effect of body fat on the possible diabetic effects of the calpain-10 gene (2).
It is of interest to compare the present findings with those published by Baier et al. (5). In the former study low thermogenesis was associated with SNP-43 and not with SNP-19. The reason for this discrepancy is unclear, although it can be due to the fact that different ethnic groups were investigated, specifically Pima Indians in the earlier study and Scandinavians in our study. Evidence has indeed been presented that specific calpain-10 alleles associate with type 2 diabetes in different populations (1, 2). It is also possible that the present gene markers are in linkage dysequilibrium with other functional polymorphisms within or near the calpain-10 gene. It should be noted, though, that in some individuals SNP-19 is significantly associated with insulin sensitivity (25).
There was no apparent association between the calpain-10 polymorphism and in vivo insulin sensitivity, as judged by findings with plasma glucose and insulin. This might be due to the fact that we did not directly measure insulin sensitivity or that only healthy subjects (except for overweight) were investigated. In this respect it should be noted that the calpain-10 gene polymorphism does not seem to influence insulin secretion in nondiabetic subjects (26). There was also no association between calpain-10 gene polymorphism and overweight in our material.
In summary, this study shows that the calpain-10 gene polymorphism influences ß3-adrenoceptor action. A markedly reduced receptor lipolytic sensitivity is found in subjects homozygous for the deletion at SNP-19. This could suggest that calpain-10 is involved in the regulation of thermogenesis.
Acknowledgments
The excellent technical assistance of Britt-Marie Leijonhufvud, Katarina Hertel, Kerstin Wåhlin, Eva Sjölin, and Elisabeth Dungner is greatly appreciated. We thank Prof. L. Groop (Malmö, Sweden) for the donation of DNA previously genotyped for the three calpain-10 gene polymorphisms.
Footnotes
This work was supported by grants from the Swedish Medical Research Council, the Swedish Diabetes Association, the Novo Nordic Foundation, the Thuring Foundation, the Wiberg Foundation, the Bergvall Söderberg Foundation, and the Swedish Heart and Lung Foundation.
Abbreviations: BMI, Body mass index; SNP, single nucleotide polymorphism.
Received October 23, 2001.
Accepted April 8, 2202.
References
- and ß- adrenoceptors in sympathetically mediated thermogenesis. Am J Physiol 264:E11–E17
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