- Email: [email protected]
Reduced Skeletal Muscle Calpain-10 Transcript Level Is Due to a Cumulative Decrease in Major Isoforms
Molecular Genetics and Metabolism 73, 111–113 (2001) doi:10.1006/mgme.2001.3171, available online at http://www.idealibrary.com on
BRIEF COMMUNICATION Reduced Skeletal Muscle Calpain-10 Transcript Level Is Due to a Cumulative Decrease in Major Isoforms in response to physiological changes or pathological conditions (6). We have recently demonstrated a lower level of total calpain-10 mRNA in the skeletal muscle of nondiabetic Pima Indians homozygous for the G allele of SNP-43 and showed that this was associated with a decreased rate of insulin-mediated glucose turnover or insulin resistance (2). To determine whether or not the reduced level of calpain-10 mRNA in the G/G homozygotes was due to a change in levels of particular isoforms, we measured the abundance of the isoforms comprising total calpain-10 mRNA in skeletal muscle.
The DNA polymorphism SNP-43 in the calpain-10 gene is associated with insulin resistance and reduced skeletal muscle transcript in Pima Indians. Alternative splicing generates transcript isoforms calpain-10a to -10h. We determined the contribution of calpain-10 mRNA isoforms to the decreased total skeletal muscle calpain-10 mRNA levels observed in the G/G homozygotes. The expression levels of the major isoforms, calpain-10a and -10f, were positively correlated with the total calpain-10 mRNA levels, indicating a cumulative effect. © 2001 Academic Press
Key Words: type 2 diabetes; Pima Indians; Calpain-10; isoforms; skeletal muscle.
MATERIALS AND METHODS Subjects
Calpain-10 is a member of the calcium-activated neutral protease family and is expressed in many human tissues including skeletal muscle, liver, and pancreas (1). Variation in the gene encoding this enzyme (CAPN10) is associated with type 2 diabetes in Mexican Americans and northern Europeans (1). In addition, it is associated with insulin resistance in nondiabetic Pima Indians (2), a population that has a high prevalence of type 2 diabetes (3). In this population, insulin resistance has been determined as a familial predictor of the disease that is presumably genetically determined (4). The DNA polymorphism associated with insulin resistance is an A 3 G transition in intron 3 of the CAPN10 (g.4852G/A and designated SNP-43) and molecular biological studies suggested that it may function as a cis-acting element regulating transcription of this gene (1). Alternative splicing generates at least eight calpain-10 transcripts (calpain-10a to -10h) (1) and is one mechanism of regulating gene expression and the abundance of specific mRNA and protein isoforms (5). In general, the proportion of particular isoforms may change during development as well as
The study was approved by the Tribal Council of the Gila River Indian Community and by the Institutional Review Board of the National Institutes of Diabetes and Digestive and Kidney Diseases and written informed consent was obtained before participation. The 25 volunteers [18 males (2) and 7 females] were full heritage Pima or Tohono O’Odham Indians from the Gila River Indian Community. They were healthy by medical history, physical examination, and routine laboratory tests, and were not taking medications. All subjects were genotyped for the SNP-43 DNA polymorphism and were divided into two groups, the G/G and the G/A plus the A/A groups, as in previous reports (1,2). RNA Extraction and cDNA Synthesis Percutaneous muscle biopsies were obtained from the vastus lateralis after local anesthesia with 1% lidocaine as described previously (7). The biopsies were rinsed in saline and immediately frozen in liquid nitrogen and stored at ⫺70°C prior to RNA 111 1096-7192/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
112
BRIEF COMMUNICATION
extractions using a ToTALLY RNA kit (Ambion, Austin, TX). Integrity of RNA was confirmed by visualization on an agarose gel. Complementary DNA was synthesized using a Advantage RT-forPCR kit (CLONTECH, Palo Alto, CA). Efficiency of the cDNA synthesis was determined by PCR amplification of -actin or G3PDH transcripts. Real-Time PCR Real-time PCR probes and primers were designed using the Primer Express software (Applied Biosystems, Foster City, CA); their sequences are available upon request. The specific requirements for the realtime PCR probe/primer set for measuring total calpain-10 mRNA level only allowed the detection of isoforms calpain-10a to -10f and -10h (2). The probes were designed to span one or more introns and to hybridize to the appropriate alternative splice sites. Due to the transcript splicing configuration (1) and the specific requirements for probe/primer design, there were no suitable probe-primer sets to detect isoforms calpain-10a, -10b, and -10g individually. Therefore, the expression levels of calpain-10a and -10b isoforms were quantified by subtracting the expression levels of -10f from that of -10af and -10e from -10be, respectively. The primers were from Genosys (The Woodlands, TX); the probes were from Applied Biosystems and Annovis (Dulles, VA). Realtime PCR was performed using ABI PRISM 7700 (Applied Biosystems). For each primer/probe set, a standard curve was generated by a serial dilution of a cDNA sample synthesized from muscle RNA of a healthy subject. Each dilution of the standard curve was run in triplicate, and a mean value was calculated to represent the cDNA input. The samples were run in duplicate, and the mean value was normalized as a ratio to an endogenous reference, -actin RNA. Data Analysis The experimental data were calculated using the standard curve and comparative C T methods (Applied Biosystems) that produced comparable results. Data were expressed as means ⫾ SEM in relative units. Statistical significance was calculated using Student’s t test with a P value of ⬍ 0.05 being significant. RESULTS AND DISCUSSION We determined that calpain-10a to -10f and -10h isoforms were expressed in human skeletal muscle
FIG. 1. The relationship between RNA expression levels of the total calpain-10 and its major skeletal muscle isoforms. The expression levels of isoforms 10a (F) and 10f (Œ) are positively correlated with total calpain-10 expression (r ⫽ 0.823 and r ⫽ 0.848, respectively; both P ⬍ 0.01, n ⫽ 25).
(data not shown). Isoforms 10b, c, d, e, and h were very low in abundance and, therefore, did not substantially contribute to the measured total mRNA level of calpain-10. Calpain-10a and -10f mRNAs are the most abundant ones in human skeletal muscle and, thus, the major contributors to the level of total calpain-10 mRNA in this tissue. It is interesting to note that calpain-10f is produced by a different splice site of intron 3 where SNP-43 resides, resulting in a shorter exon 3 (1). Of the two major skeletal muscle calpain-10 isoforms, calpain-10a mRNA showed a higher expression level than -10f. The expression levels of calpain-10a and -10f correlated significantly with the total calpain-10 mRNA levels (Fig. 1). The G/G homozygotes (n ⫽ 10) had lower total calpain-10 mRNA (0.035 ⫾ 0.005 relative units) when compared to the G/A plus A/A individuals (n ⫽ 15; 0.061 ⫾ 0.009 relative units; P ⫽ 0.02). This difference was mainly due to the levels of calpain10a and -10f mRNA as the major isoforms. Taking the G/A plus A/A groups as a control with a mean expression level of 100%, the G/G group tended to have lower concentrations of calpain-10a (58 ⫾ 14%, P ⫽ 0.08) and calpain-10f isoforms (55 ⫾ 17%, P ⫽ 0.1). These findings further indicated that the decreased total calpain-10 mRNA in the G/G homozygotes resulted from an overall diminished transcription of the calpain-10 gene that could result from reduced transcription initiation rates and/or lower level of pre-mRNA stability, rather than altered mRNA splicing. In conclusion, the present study showed that the levels of the two major calpain-10 isoforms, -10a and
BRIEF COMMUNICATION
-10f, were correlated with the total calpain-10 mRNA levels. Decreased expression levels of these isoforms account for the lower level of the mean total calpain-10 mRNA levels in Pima Indians homozygous for the G allele of SNP-43 compared to those homozygous and heterozygous for the A allele. ACKNOWLEDGMENTS We thank the members of the Gila River Indian community for their helpful cooperation and participation. We acknowledge the excellent technical assistance of Chris Wiedrich, the assistance and patient care provided by Michael Milner, PA, Carol Massengil, RN, the nurses of the Clinical Research Ward, and Dr. Arline Salbe along with the metabolic kitchen staff.
REFERENCES 1.
2.
3.
4.
5.
Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner TH, Mashima H, Schwarz PEH, del Bosque-Plata L, Horikawa Y, Oda Y, Yoshiuchi I, Colilla S, Polonsky KS, Wei S, Concannon P, Iwasaki N, Schulze J, Baier LJ, Bogardus C, Groop L, Boerwinkle E, Hanis CL, Bell GI. Genetic variation in the calpain-10 gene (CAPN10) is associated with type 2 diabetes mellitus. Nature Genet 26: 163–175, 2000. Baier LJ, Permana PA, Yang X, Pratley RE, Hanson R, Shen G-Q, Mott DM, Knowler WC, Cox NJ, Horikawa Y, Oda N, Bell GI, Bogardus C. A calpain-10 gene polymorphism is associated with reduced muscle mRNA levels and insulin resistance. J Clin Invest 106:R69 –R73, 2000. Knowler WC, Bennett PH, Hamman RF, Miller M. Diabetes incidence and prevalence in Pima Indians: A 19-fold greater incidence than in Rochester, Minnesota. Am J Epidemiol 108:497–505, 1978. Sakul H, Pratley R, Cardon L, Ravussin E, Mott D, Bogardus C. Familiality of physical and metabolic characteristics that predict the development of non-insulin dependent diabetes mellitus in Pima Indians. Am J Hum Genet 60:651– 656, 1997. Inoue K, Ohno M, Shimura Y. Aspects of splice site selection
113
in constitutive and alternative pre-mRNA splicing. Gene Expression 4:177–182, 1995. 6.
Iida K, Hidaka K, Takeuchi M, Nakayama M, Tutani C, Mukai T, Morisaki T. Expression of MEF2 genes during human cardiac development. Tohoku J Exp Med 187:15–23, 1999.
7.
Kida Y, Puente ARE, Bogardus C, Mott DM. Insulin resistance is associated with reduced fasting and insulin-stimulated glycogen synthase phosphatase activity in human skeletal muscle. J Clin Invest 85:476 – 481, 1990.
Xiaolin Yang* Richard E. Pratley* ,1 Leslie J. Baier* Yukio Horikawa† Graeme I. Bell† Clifton Bogardus* Paska A. Permana* ,2 *Clinical Diabetes and Nutrition Section Phoenix Epidemiology and Clinical Research Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Phoenix, Arizona †The Howard Hughes Medical Institute and the Departments of Biochemistry and Molecular Biology, Medicine and Human Genetics The University of Chicago Chicago, Illinois Received December 20, 2000, and in revised form February 22, 2001; published online April 24, 2001
1 Current address: Department of Cardiovascular, Metabolic and Endocrine Clinical Research, Novartis Pharmaceutical Corp., East Hanover, NJ. 2 To whom correspondence should be addressed at Clinical Diabetes and Nutrition Section, Phoenix Epidemiology and Clinical Research Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 4212 N. 16th Street, Phoenix, AZ 85016. Fax: (602) 200-5335. E-mail: [email protected].
BRIEF COMMUNICATION Reduced Skeletal Muscle Calpain-10 Transcript Level Is Due to a Cumulative Decrease in Major Isoforms in response to physiological changes or pathological conditions (6). We have recently demonstrated a lower level of total calpain-10 mRNA in the skeletal muscle of nondiabetic Pima Indians homozygous for the G allele of SNP-43 and showed that this was associated with a decreased rate of insulin-mediated glucose turnover or insulin resistance (2). To determine whether or not the reduced level of calpain-10 mRNA in the G/G homozygotes was due to a change in levels of particular isoforms, we measured the abundance of the isoforms comprising total calpain-10 mRNA in skeletal muscle.
The DNA polymorphism SNP-43 in the calpain-10 gene is associated with insulin resistance and reduced skeletal muscle transcript in Pima Indians. Alternative splicing generates transcript isoforms calpain-10a to -10h. We determined the contribution of calpain-10 mRNA isoforms to the decreased total skeletal muscle calpain-10 mRNA levels observed in the G/G homozygotes. The expression levels of the major isoforms, calpain-10a and -10f, were positively correlated with the total calpain-10 mRNA levels, indicating a cumulative effect. © 2001 Academic Press
Key Words: type 2 diabetes; Pima Indians; Calpain-10; isoforms; skeletal muscle.
MATERIALS AND METHODS Subjects
Calpain-10 is a member of the calcium-activated neutral protease family and is expressed in many human tissues including skeletal muscle, liver, and pancreas (1). Variation in the gene encoding this enzyme (CAPN10) is associated with type 2 diabetes in Mexican Americans and northern Europeans (1). In addition, it is associated with insulin resistance in nondiabetic Pima Indians (2), a population that has a high prevalence of type 2 diabetes (3). In this population, insulin resistance has been determined as a familial predictor of the disease that is presumably genetically determined (4). The DNA polymorphism associated with insulin resistance is an A 3 G transition in intron 3 of the CAPN10 (g.4852G/A and designated SNP-43) and molecular biological studies suggested that it may function as a cis-acting element regulating transcription of this gene (1). Alternative splicing generates at least eight calpain-10 transcripts (calpain-10a to -10h) (1) and is one mechanism of regulating gene expression and the abundance of specific mRNA and protein isoforms (5). In general, the proportion of particular isoforms may change during development as well as
The study was approved by the Tribal Council of the Gila River Indian Community and by the Institutional Review Board of the National Institutes of Diabetes and Digestive and Kidney Diseases and written informed consent was obtained before participation. The 25 volunteers [18 males (2) and 7 females] were full heritage Pima or Tohono O’Odham Indians from the Gila River Indian Community. They were healthy by medical history, physical examination, and routine laboratory tests, and were not taking medications. All subjects were genotyped for the SNP-43 DNA polymorphism and were divided into two groups, the G/G and the G/A plus the A/A groups, as in previous reports (1,2). RNA Extraction and cDNA Synthesis Percutaneous muscle biopsies were obtained from the vastus lateralis after local anesthesia with 1% lidocaine as described previously (7). The biopsies were rinsed in saline and immediately frozen in liquid nitrogen and stored at ⫺70°C prior to RNA 111 1096-7192/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
112
BRIEF COMMUNICATION
extractions using a ToTALLY RNA kit (Ambion, Austin, TX). Integrity of RNA was confirmed by visualization on an agarose gel. Complementary DNA was synthesized using a Advantage RT-forPCR kit (CLONTECH, Palo Alto, CA). Efficiency of the cDNA synthesis was determined by PCR amplification of -actin or G3PDH transcripts. Real-Time PCR Real-time PCR probes and primers were designed using the Primer Express software (Applied Biosystems, Foster City, CA); their sequences are available upon request. The specific requirements for the realtime PCR probe/primer set for measuring total calpain-10 mRNA level only allowed the detection of isoforms calpain-10a to -10f and -10h (2). The probes were designed to span one or more introns and to hybridize to the appropriate alternative splice sites. Due to the transcript splicing configuration (1) and the specific requirements for probe/primer design, there were no suitable probe-primer sets to detect isoforms calpain-10a, -10b, and -10g individually. Therefore, the expression levels of calpain-10a and -10b isoforms were quantified by subtracting the expression levels of -10f from that of -10af and -10e from -10be, respectively. The primers were from Genosys (The Woodlands, TX); the probes were from Applied Biosystems and Annovis (Dulles, VA). Realtime PCR was performed using ABI PRISM 7700 (Applied Biosystems). For each primer/probe set, a standard curve was generated by a serial dilution of a cDNA sample synthesized from muscle RNA of a healthy subject. Each dilution of the standard curve was run in triplicate, and a mean value was calculated to represent the cDNA input. The samples were run in duplicate, and the mean value was normalized as a ratio to an endogenous reference, -actin RNA. Data Analysis The experimental data were calculated using the standard curve and comparative C T methods (Applied Biosystems) that produced comparable results. Data were expressed as means ⫾ SEM in relative units. Statistical significance was calculated using Student’s t test with a P value of ⬍ 0.05 being significant. RESULTS AND DISCUSSION We determined that calpain-10a to -10f and -10h isoforms were expressed in human skeletal muscle
FIG. 1. The relationship between RNA expression levels of the total calpain-10 and its major skeletal muscle isoforms. The expression levels of isoforms 10a (F) and 10f (Œ) are positively correlated with total calpain-10 expression (r ⫽ 0.823 and r ⫽ 0.848, respectively; both P ⬍ 0.01, n ⫽ 25).
(data not shown). Isoforms 10b, c, d, e, and h were very low in abundance and, therefore, did not substantially contribute to the measured total mRNA level of calpain-10. Calpain-10a and -10f mRNAs are the most abundant ones in human skeletal muscle and, thus, the major contributors to the level of total calpain-10 mRNA in this tissue. It is interesting to note that calpain-10f is produced by a different splice site of intron 3 where SNP-43 resides, resulting in a shorter exon 3 (1). Of the two major skeletal muscle calpain-10 isoforms, calpain-10a mRNA showed a higher expression level than -10f. The expression levels of calpain-10a and -10f correlated significantly with the total calpain-10 mRNA levels (Fig. 1). The G/G homozygotes (n ⫽ 10) had lower total calpain-10 mRNA (0.035 ⫾ 0.005 relative units) when compared to the G/A plus A/A individuals (n ⫽ 15; 0.061 ⫾ 0.009 relative units; P ⫽ 0.02). This difference was mainly due to the levels of calpain10a and -10f mRNA as the major isoforms. Taking the G/A plus A/A groups as a control with a mean expression level of 100%, the G/G group tended to have lower concentrations of calpain-10a (58 ⫾ 14%, P ⫽ 0.08) and calpain-10f isoforms (55 ⫾ 17%, P ⫽ 0.1). These findings further indicated that the decreased total calpain-10 mRNA in the G/G homozygotes resulted from an overall diminished transcription of the calpain-10 gene that could result from reduced transcription initiation rates and/or lower level of pre-mRNA stability, rather than altered mRNA splicing. In conclusion, the present study showed that the levels of the two major calpain-10 isoforms, -10a and
BRIEF COMMUNICATION
-10f, were correlated with the total calpain-10 mRNA levels. Decreased expression levels of these isoforms account for the lower level of the mean total calpain-10 mRNA levels in Pima Indians homozygous for the G allele of SNP-43 compared to those homozygous and heterozygous for the A allele. ACKNOWLEDGMENTS We thank the members of the Gila River Indian community for their helpful cooperation and participation. We acknowledge the excellent technical assistance of Chris Wiedrich, the assistance and patient care provided by Michael Milner, PA, Carol Massengil, RN, the nurses of the Clinical Research Ward, and Dr. Arline Salbe along with the metabolic kitchen staff.
REFERENCES 1.
2.
3.
4.
5.
Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner TH, Mashima H, Schwarz PEH, del Bosque-Plata L, Horikawa Y, Oda Y, Yoshiuchi I, Colilla S, Polonsky KS, Wei S, Concannon P, Iwasaki N, Schulze J, Baier LJ, Bogardus C, Groop L, Boerwinkle E, Hanis CL, Bell GI. Genetic variation in the calpain-10 gene (CAPN10) is associated with type 2 diabetes mellitus. Nature Genet 26: 163–175, 2000. Baier LJ, Permana PA, Yang X, Pratley RE, Hanson R, Shen G-Q, Mott DM, Knowler WC, Cox NJ, Horikawa Y, Oda N, Bell GI, Bogardus C. A calpain-10 gene polymorphism is associated with reduced muscle mRNA levels and insulin resistance. J Clin Invest 106:R69 –R73, 2000. Knowler WC, Bennett PH, Hamman RF, Miller M. Diabetes incidence and prevalence in Pima Indians: A 19-fold greater incidence than in Rochester, Minnesota. Am J Epidemiol 108:497–505, 1978. Sakul H, Pratley R, Cardon L, Ravussin E, Mott D, Bogardus C. Familiality of physical and metabolic characteristics that predict the development of non-insulin dependent diabetes mellitus in Pima Indians. Am J Hum Genet 60:651– 656, 1997. Inoue K, Ohno M, Shimura Y. Aspects of splice site selection
113
in constitutive and alternative pre-mRNA splicing. Gene Expression 4:177–182, 1995. 6.
Iida K, Hidaka K, Takeuchi M, Nakayama M, Tutani C, Mukai T, Morisaki T. Expression of MEF2 genes during human cardiac development. Tohoku J Exp Med 187:15–23, 1999.
7.
Kida Y, Puente ARE, Bogardus C, Mott DM. Insulin resistance is associated with reduced fasting and insulin-stimulated glycogen synthase phosphatase activity in human skeletal muscle. J Clin Invest 85:476 – 481, 1990.
Xiaolin Yang* Richard E. Pratley* ,1 Leslie J. Baier* Yukio Horikawa† Graeme I. Bell† Clifton Bogardus* Paska A. Permana* ,2 *Clinical Diabetes and Nutrition Section Phoenix Epidemiology and Clinical Research Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Phoenix, Arizona †The Howard Hughes Medical Institute and the Departments of Biochemistry and Molecular Biology, Medicine and Human Genetics The University of Chicago Chicago, Illinois Received December 20, 2000, and in revised form February 22, 2001; published online April 24, 2001
1 Current address: Department of Cardiovascular, Metabolic and Endocrine Clinical Research, Novartis Pharmaceutical Corp., East Hanover, NJ. 2 To whom correspondence should be addressed at Clinical Diabetes and Nutrition Section, Phoenix Epidemiology and Clinical Research Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 4212 N. 16th Street, Phoenix, AZ 85016. Fax: (602) 200-5335. E-mail: [email protected].