• Journal of Korean Medicine for Obesity Research
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• v.2 no.1
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• pp.43-52
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• 2002

Purpose & Methods: In order to study obese gene mutation rate in obese Korean patients and to investigate the effect at Chegamuiyiin-tang and electro-lipolysis-acupuncture on obesity treatment. the difference of the reaction to herbal dieting between patients with ${\beta}3$ adrenergic receptor mutation and the patients with wild type ${\beta}3$ adrenergic receptor is observed. Results: Chegamuiyiin-tang and electro-lipolysis-acupuncture treatment are effective on the treatment of obesity in weight reduction. body fat reduction and the circumferences of arm, abdomen, hip and thigh. In the comparison of ${\beta}3$ adrenergic receptor wild type and ${\beta}3$ adrenergic receptor mutation groups, body fat was more reduced with statistical significance, and as for BMI change and body weight change were higher in ${\beta}3$ adrenergic receptor mutation groups with no statistical significance. In the comparison of ${\beta}3$ adrenergic receptor wild type and ${\beta}3$ adrenergic receptor mutation groups among BMI under 25 patients change rate of body weight. BMI, body fact percentage, WHR and body circumference were higher in ${\beta}3$ adrenergic receptor mutation group than in ${\beta}3$ adrenergic receptor wild type group. Conclusion: These results imply that herbal dieting program combined with electro-lipolysis-acupuncture is more effective on reducing body weight and body fat in ${\beta}3$ adrenergic receptor mutation group than wild type group, and that the earlier the treatment is applied, the more effective it is.

• Biomolecules & Therapeutics
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• v.4 no.1
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• pp.103-109
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• 1996

To investigate the effect of the third intracellular loop (i3 loop) peptide of human $\beta$$_2$-adrenergic receptor on receptor agonist binding, we expressed third intracellular loop region of human $\beta$$_2$-adrenergic receptor as glutathione S-transferase fusion protein in E. coli. DNA fragment of the receptor gene which encodes amino acid 221-274 of human $\beta$$_2$-adrenergic receptor was amplified by polymerase chain reaction and subcloned into the bacterial fusion protein expression vector pGEX-CS and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein expressed in this study was purified to an apparent homogeneity by glutathione Sepharose CL-4B affinity chromatography. The purified i3 loop fusion proteins at a concentration of 10 $\mu\textrm{g}$/ι caused right shift of the isoproterenol competition curve of [$^3$H]Dihydroalprenolol binding to hamster lung $\beta$$_2$-adrenergic receptor indicating lowered affinity of isoproterenol to $\beta$$_2$-adrenergic receptor possibly due to the uncoupling of receptor and G protein in the presence of the fusion protein. The uncoupling of receptor and G protein suggests that i3 loop region plays a critical role on $\beta$$_2$-adrenergic receptor G protein coupling.

• Biomolecules & Therapeutics
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• v.1 no.2
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• pp.188-195
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• 1993

In order to understand the machanism of action and regulation of ${\beta}$-adrenergic receptor in terms of molecular level, the purification of receptor protein has a fundamental importance. Moreover, species differences among avian, amphibian and mammalian ${\beta}$-adrenergic receptors make it more important to purify mammalian ${\beta}$-adrenergic receptor. Because ${\beta}$-adrenergic receptor is an integral membrane protein, it must be solubilized from the membrane for the purification. The purpose of the present study was to solubilize and characterize the mammalian $\beta$-adrenergic receptor from guinea pig lung in quantities by more efficient and practical method eventually to purify receptor. Guinea pig lung membrane preparation was solubilized by sequential treatment of buffers containing low and high concentration of digitonin which are 0.2 and 1.2% respectively. About 50% of the total receptor pool was released by this double extraction procedure. The $\beta$-adrenoceptors in the digitonin extract were identified using the ${\beta}$-adrenergic antagonist, (-)-[$^3H$]-dihydroalprenolol ([$^3H$]DHA). The solubilized receptor retained all of the essential characteristics of membrane-bound receptor, namely saturability; stereoselectivity; high affinity to ${\beta}$-adrenergic drugs. For the measurement of soluble receptor activity, Sephadex G-50 chromatography method has been widely used. Inspite of its accuracy and wide acceptance, this technique employed troublesome column work which required long time to assay the activity of receptor. We employed another methods to measure receptor activity. When using 0.5% polyethylenimine pretreated GF/B glass fiber filter, filtration technique could be used to measure soluble receptor activity. This technique enabled us to reduce the total amount of time to assay by a factor of 4 as well as to detect soluble receptor. In the present study, we could establish more efficient and practical solubilization method of mammalian $\beta$-adrenergic receptor. The rapidity and high yield of this solubilization scheme, together with the favorable recovery of the receptor activity, are significant steps toward the ultimate purification of the mammalian $\beta$-adrenergic receptor. The result of this study together with more convenient purification method could provide large amount of purified receptor with ease for various research purposes.

• Biomolecules & Therapeutics
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• v.4 no.1
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• pp.97-102
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• 1996

To investigate whether human $\beta$$_2$-adrenergic receptor devoid of the C-terminal two transmembrane helices retain its ligand binding activity and specificity, 5'780-bp DNA fragment of the receptor gene which encodes amino acid 1-260 of human $\beta$$_2$-adrenergic receptor was subcloned into the bacterial fusion protein expression vector and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was expressed as a membrane bound form which was verified by SDS-PAGE and Western blot. The fusion protein expressed in this study specifically bound $\beta$-adrenergic receptor ligand [$^3$H] Dihydroalprenolol. In saturation ligand binding assay, the $K_{d}$ value was 7.6 nM which was similar to that of intact $\beta$$_2$-adrenergic receptor in normal animal tissue ( $K_{d}$=1~2 nM) and the $B_{max}$ value was 266 fmol/mg membrane protein. In competition binding assay, the order of binding affinity of various adrenergic receptor agonists to the fusion protein was isoproterenol》epinephrine norepinephrine, which was similar to that of intact receptor in normal animal tissue. These results suggest that N-terminal five transmembrane helices of the $\beta$$_2$-adrenergic receptor be sufficient to determine the ligand binding activity and specificity, irrespective of the presence or absence of the C-terminal two transmembrane helices.s.s.s.

• YAKHAK HOEJI
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• v.49 no.1
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• pp.44-50
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• 2005

The purpose of this study was to investigate the relationship between Trp64Arg polymorphism in the ${\beta}_3$-adrenergic receptor gene and complex phenotypes such as blood pressure, body compositions and bone parameters in young men about 20 years, and to collect the fundamental data in designing the exercise program. Eighty healthy young men including 41 controls and 39 athletes were recruited, Trp64Arg polymorphism in the ${\beta}_3$-adrenergic receptor gene was genotyped by PCR-RFLP method. By association study, there were no significance in genotype and allele frequencies of Trp64Arg polymorphism in the ${\beta}_3$-adrenergic receptor gene between controls and athletes, respectively (p>0.05). When the relationship between physiological parameters and Trp64Arg polymorphism in the ${\beta}_3$-adrenergic receptor gene was tested, this polymorphism was significantly associated with 3th lumber and left femoral neck Z-score values in controls (p<0.05), but these associations were not detected in athletic groups (p>0.05). It is likely that Trp64Arg polymorphism in the ${\beta}_3$-adrenergic receptor gene is a genetic marker for the bone mineral density index in young men, but environmental factors such as exercise modify the significant effect of this polymorphism. Thus, our results suggest that Trp64Arg polymorphism in the ${\beta}_3$-adrenergic receptor gene may be applicable as a predictive marker for osteoporosis in Korean young men, and regular exercise may prevent the disadventageous effect of this polymorphism for bone mineral density in male athletic group.

• Biomolecules & Therapeutics
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• v.2 no.4
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• pp.303-309
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• 1994

The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently through the use of specific antibodies. Two kinds of antibodies could be produced, one is from synthetic peptides and the other from proteins such as purified receptor. Anti-peptide antibodies gave some advantages; epitope is evident and also receptor purification in quantity is not prerequisite. It can be also applied to the study of receptor structure-activity relationship. The purpose of the present study was 1) to produce and characterize a polyclonal antibody against a synthetic $\beta$2-adrenergic receptor peptide(Phe-Gly-Asn-Phe-Trp-Cys-Phe-Trp-Thr-Ser-Ile-Asp-Val-Leu) and 2) to determine the effects of this antibody on the $\beta$-adrenergic receptor ligand interaction. The peptide sequence contains an amino acid residue such as Asp-113 which was identified as one of important component for receptor-ligand interaction in site-directed mutagenesis studies. Production of antibody was performed by immunization of rabbits through popliteal lymph node with the peptide coupled with Keyhole Limpet Hemocyanin (KLH). The titer of antibody against this peptide was 1 : 1000. The anti-peptide antibody was able to detect a 67 kDa protein band in western blot corresponding to the molecular weight of the $\beta$-adrenergic receptor in partially purified receptor fraction derived from guinea pig lung. The antisera inhibited the specific binding of [$^3$H]dihydroalprenolol to $\beta$-adrenergic receptor in a concentration-dependent manner. The results from this study suggest that the peptide sequence selected in the present study is important for the receptor ligand interaction.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.2
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• pp.161-167
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• 1997

This study compares the subtypes of central beta adrenergic receptors (ARs) of brains of untreated rats with those of imipramine-treated rats. Beta adrenergic receptors were measured by quantitative autoradiography of the binding of $^3H$-dihydroalprenolol ($^3H$-DHA) in coronal sections of rat brain. Repeated treatment of rats with imipramine significantly reduced the binding of $^3H$-DHA to beta-1 AR in many brain areas, especially throughout the cerebral cortex, hippocampus, thalamus, and amygdala. Significant reductions of the binding of $^3H$-DHA to beta-2 AR were not found in any area of the brain. These data suggests that a selective down-regulation of beta-1 AR may be involved in the adaptive changes occurring after prolonged imipramine treatment.

• Biomolecules & Therapeutics
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• v.3 no.4
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• pp.266-272
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• 1995

The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently through the use of specific antibodies. To generate and characterize a moloclonal antibody against $\beta$-adrenergic receptor, a synthetic $\beta$2-adrenergic receptor peptide (Phe-Gly-Asn-Phe-Trp-Cys-Phe-Trp-Thr-Ser-lle-Asp-Val-Leu) which may comprise part of $\beta$-adrenergic receptor ligand binding pocket was coupled to Keyhole Limpet Hemocyanin (KLH) and used as an immunogen. Male BALB/C mice were immunized with this antigen and the immunized spleen was fused with myeloma SP2/0-Ag14 cells to produce monoclonal antibodies. Two clones were obtained but one of monoclonal antibodies, mAb5G09, was used throughout in this study because the other clone, mAb5All showed weak immunoreactivity against KLH as well. The mouse monoclonal antibody mAb5G09 produced in this study showed immunoreactivity to peptide-KLH conjugates and also to human A43l cells and guinea pig lung $\beta$2-adrenergic receptor as revealed by ELISA and western blot. In the course of determination of the effects of mAb5G09 on $\beta$-receptor ligand binding, it was observed that mAb5G09 specifically bound $\beta$-adrenergic radioligand [$^3$H]dihydroalprenolol (DHA) with a dissociation constant (Kd) of 60 nM. The [$^3$H]DHA binding activity of mAb5G09 had characteristics of immunoglobulins and the binding activity was not observed in the control anti-KLH monoclonal antibody. The monoclonal antibody, mAb5G09 produced in this study may provide useful models for the study of the structure of receptor binding sites.

• Preventive Nutrition and Food Science
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• v.9 no.1
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• pp.71-78
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• 2004

Adrenergic receptors play a major role in thermogenesis and lipolysis in brown and visceral adipose tissues, and have been implicated in the pathogenesis of obesity and metabolic disorders. The purpose of this study was to estimate the effects of $\beta$2-adrenergic receptor ($\beta$2AR) and $\beta$3-adrenergic receptor ($\beta$3AR) genotypes on hyperglycemia and obesity in the Korean population. A representative sample consisting of 530 Korean men and women were measured for height, weight, BMI, WHR, obesity index and body composition. The genotypes of $\beta$2AR polymorphism in codon 27 and $\beta$3AR polymorphism in codon 64 were analyzed by the PCR RFLP method. Serum concentrations of fasting glucose, total cholesterol, HDL cholesterol and triglyceride were determined. The frequencies of $\beta$2AR and $\beta$3AR genotype were: both wild type, 62.5% ; only $\beta$2AR variant type, 12.8% ; only $\beta$3AR variant type, 18.8% ; and both variant type, 5.8% ; the frequency of E and R alleles were 0.098 and 0.137, respectively. Among the physiological parameters, fasting glucose level was significantly higher in subjects with both variant type compared with the three other types (p <0.05), Subjects with both variant type had 12%, 12% and 9.3% increases in serum glucose levels compared with wild type, only $\beta$2AR variant type, and only $\beta$3AR variant type, respectively. When logistic regression analysis was conducted to estimate the risk for hyperglycemia, the subjects were selected for fasting blood glucose concentrations of more than 6.105 m㏖/L (110 mg/dL), and the odds ratios were 1.215 (p=0.636) for only $\beta$2AR variant type,1.659 (p=0.089) for only $\beta$3AR variant type, and 3.078 (p=0.011) for both variant type. These results suggest that the interaction of $\beta$2AR and $\beta$3AR variant genotypes has a strong association with increased glucose levels, and might be a significant risk factor for hyperglycemia among Korean subjects.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.86-86
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• 1995

The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently by the use of specific anti-receptor antibodies. A 14-mer peptide (from Phe102 to Leu115 of ${\beta}$2-adrenergic receptor) was synthesized and this peptide was coupled to carrier protein Keyhole Limpet Hemocyanin(KLH) by glutaraldehyde method. A 0.5mg of KLH-coupled peptide was emulsified with equal volume of complete Freund's adjuvant and injected via popliteal lymph node to each of the three Newzealnd White rabbits. Booster injections were repeated at 4 weeks interval for three times with incomplete Freund's adjuvants. One week after the final injection, serum was prepared from ear artery. Nonspecific immunoglobulins were removed by passing the serum through KLH-Sepharose 6B affinity matrix and further by incubation with bovine lung aceton powder. The titer of the antibody for synthetic peptide which was determined by enzyme linked immunosorbent assay(ELISA) was about l/l,000. The antibody produced in this study revealed 67kDa protein band in the western blot of partially purified guinea pig lung ${\beta}$-adrenergic receptor preparation. The antibody inhibited ${\beta}$-adrenergic antaginist [3H] Dihydroalprenolol binding to soluble ${\beta}$-adrenergic receptor by 25% while control sera did not show any inhibitory effects, The result of this study suggests that the peptide sequence selected in this study may play some important roles in adrenergic receptor-ligand interaction.