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Relationship between various surrogate indices of insulin resistance and mitochondrial DNA content in the peripheral blood of 18 healthy volunteers
Mitochondrion 1 (2001) 71±77
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Relationship between various surrogate indices of insulin resistance and mitochondrial DNA content in the peripheral blood of 18 healthy volunteers Soo Lim a,b,c, Kyong Soo Park a,c, Min Seon Kim a,b, Bo Youn Cho a,b, Hong Kyu Lee a,b,c,d,* a Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea Center for Hormone Research, Clinical Research Institute, Seoul National University Hospital, Seoul, South Korea c The Institute of Endocrinology, Nutrition and Metabolism, Seoul National University Medical Research Center, Seoul, South Korea d Department of Biomedical Sciences, National Institute of Health, Seoul, South Korea b
Received 19 October 2000; received in revised form 6 February 2001; accepted 7 February 2001
Abstract Mutations or deletions of mitochondrial DNA (mtDNA) are associated with diabetes mellitus. In this study, we investigated the relationships between the mtDNA content in peripheral blood and surrogate indices of insulin resistance in 18 healthy young women (mean age 20.8 ^ 1.5 years). The mtDNA content was signi®cantly correlated with the area under the curve of insulin during an oral glucose tolerance test (r 20:622), the homeostasis model assessment for insulin resistance (r 20:616), the ratio of fasting glucose to insulin concentration (r 0:586) and the fasting insulin level (r 20:552). Further study is warranted to elucidate the mechanism by which the mtDNA content is associated with insulin resistance. q 2001 Elsevier Science B.V. and Mitochondria Research Society. All rights reserved. Keywords: Insulin resistance; Mitochondrial DNA; Homeostasis model assessment; Oral glucose tolerance test; Fasting insulin
1. Introduction The mitochondrion is an intracytoplasmic powerhouse organelle and has its own genome. Originally, mitochondria were independent cells which had aerobic respiration, but later became symbiotic with anaerobic cells, retaining their own DNA (Gray et al., 1999). Mitochondrial DNA (mtDNA) encodes 13 genes, which are essential for oxidative phosphorylation, and also codes ribosomal RNA and transfer RNA * Corresponding author. Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong Chongno-gu, Seoul 110-744, South Korea. Tel.: 182-2-760-2266; fax: 182-2-765-7966. E-mail address: [email protected] (H.K. Lee).
which are involved in the expression of these genes. There are about ten mitochondrial genomes per mitochondrion and each cell has hundreds to thousands of mitochondria. The number of mitochondria per cell varies according to the energy requirement of the tissue. It is well known that mutation in the gene for tRNALeu at position 3243 of mtDNA is associated with maternally inherited diabetes mellitus with deafness (Reardon et al., 1992; Alcolado et al., 1994; Kadowaki et al., 1994; van den Ouweland et al., 1994). There is also considerable evidence that point mutations in other sites of mtDNA and deletions of mtDNA are related to diabetes mellitus (Ballinger et al., 1992; Liang et al., 1997; Vialettes et al., 1997). Since mito-
1567-7249/01/$20.00 q 2001 Elsevier Science B.V. and Mitochondria Research Society. All rights reserved. PII: S 1567-724 9(01)00003-4
72
S. Lim et al. / Mitochondrion 1 (2001) 71±77
chondria are organelles producing adenosine triphosphate (ATP) and insulin secretion from pancreatic beta cells is dependent on ATP generation, abnormality in mtDNA may result in an insulin secretory defect. It appears that diabetes mellitus is associated with quantitative changes as well as qualitative changes in mtDNA. It was reported that the mtDNA copy number was decreased to 50% in muscle of diabetic patients (Antonetti et al., 1995). We have shown that the mtDNA content was decreased in peripheral blood leukocytes of diabetic patients (Lee et al., 1998). We have also found in our prospective populationbased study that subjects who became diabetics during a 2 year follow-up period had a signi®cantly lower mtDNA content in the prediabetic state (Lee et al., 1998). Interestingly, we have observed that there were negative correlations between the mtDNA content and clinical parameters of insulin resistance syndrome such as the waist-to-hip ratio and blood pressure. Insulin resistance has been known as a major pathogenic mechanism for type 2 diabetes mellitus. Moreover, insulin resistance is a common metabolic abnormality of common chronic diseases of a modern society such as hypertension, obesity, and hyperlipidemia, which increase cardiovascular diseases. As Reaven (1997) has pointed out, insulin resistance is not only a determinant of type 2 diabetes, but also of a cluster of metabolic syndrome including hypertension, dyslipidemia, obesity, and hyperuricemia. Since insulin resistance precedes the onset of metabolic syndrome as well as diabetes, an early detection of insulin resistance may be important to reduce the risk of this metabolic disease. Although the mtDNA content is clearly associated with diabetes mellitus, it is unclear whether the mtDNA content is related to insulin resistance itself. This study was conducted to investigate the relationship of the mtDNA content in peripheral blood with various surrogate indices of insulin resistance in healthy subjects.
range 18±24 years) were randomly recruited from students who had attended Ewha Woman's University. They were admitted to the Seoul National University Hospital Clinical Research Institute after 3 days on a diet that contained at least 150 g of carbohydrates. All subjects were in good health, as determined by medical history, physical examination, and screening routine laboratory analyses. Their cardiopulmonary function was evaluated by a pulmonary function test and exercise electrocardiogram before the study. None of the subjects displayed evidence of metabolic disorder, cardiovascular disease, or a history of smoking or regular medication. There was no family history of diabetes mellitus among their ®rst-degree relatives. Their socioeconomic levels were moderate to high. All subjects participated in the study voluntarily and informed consent was obtained. The study protocol was approved by Seoul National University Hospital Ethics Committee. 2.2. Measurement of anthropometric and biochemical parameters
2. Subjects and methods
Height, body weight, and waist and hip circumference were measured by the usual formula. Body mass index (BMI) was calculated as weight divided by squared height (kg/m 2). Recording of blood pressure took place between 08:00 and 09:00 h, when the subjects had been in a relaxed state for at least 1 h. After 14 h overnight fasting, venous blood samples were drawn from the antecubital vein in the morning (08:00±09:00 h). Plasma was separated immediately by centrifugation (2000 rev./min for 20 min at 48C). Biochemical measurements were conducted immediately after sampling. The fasting plasma concentration of glucose, total cholesterol, triglyceride, and high density lipoprotein (HDL)-cholesterol were measured using enzymatic methods with a Hitachi 747 chemistry analyzer (Hitachi, Tokyo, Japan). The level of low density lipoprotein (LDL)-cholesterol was calculated by the following formula: total cholesterol 2 HDL-cholesterol 2 triglyceride/2.2. The fasting plasma insulin concentration was determined using radioimmunoassay (LINCO kit, St. Louis, MO).
2.1. Subjects
2.3. Quanti®cation of mtDNA
Eighteen healthy women (mean age 20.8 years,
Blood samples for measurement of the mtDNA
S. Lim et al. / Mitochondrion 1 (2001) 71±77 Table 1 Sequences of primers Primer
Sequence
HmtF2 HmtR2 JF1
CAG GAC ATC CCG GGA GGC CTA GGT GGC AGA GCC C TAC CCA TGG CCA GCC ATG GGT A GGG CTC TGC CAT
JR1
Position ATG GTG CA TGA GGT TGA ACC TCC TA CTT AAC AA
2999±3018 3613±3593 3238±3247 3308±3327 3317±3308 3247±3228
content were centrifuged at 2000 rev./min for 20 min at 48C. The buffy-coat layer was separated and stored at 2708C until extraction of DNA. Total DNA was extracted using a QIAmp tissue kit (QIAGEN, Chatworth, CA) and concentrations of DNA were measured with a spectrophotometer (Beckman, Fullerton, CA). The mtDNA content was measured using competitive polymerase chain reaction (PCR) as described previously (Lim et al., 2001). In brief, the internal standard was designed to use the same primer set as the target gene but to yield a different sized PCR product (555 versus 615 bp). It was prepared by two independent PCR ampli®cations using specially designed primers shown in Table 1, producing 259 and 316 bp, respectively. Secondary PCR ampli®cation using the above products and primers hmtF2 and hmtR2 produced a 555 bp fragment containing sequences from mtDNA positions 2999±3247 and 3308±3613, with deletion of the intervening 60 bp (from positions 3248±3307). The known amounts of the serially diluted internal standard were added to 5 ng of total cellular DNA and subjected to PCR using a set of primers. The ®nal volume of the PCR reaction was 20 ml, containing 0.4 mmol/l of each primer, 200
Fig. 1. Electrophoresis by PCR product. Known amounts of the serially diluted internal standard DNA were added to total cellular DNA extracted from peripheral blood leukocytes and ampli®ed with primers hmtF1 and hmtR1. As indicated, two products were generated, the upper one derived from mtDNA (615 bp) and the lower one derived from internal standard DNA (555 bp). Lanes 1±5 are coampli®cations of varying amounts of internal standard DNA.
73
mmol/l of each dNTP, 1 unit of Taq polymerase, 20 mmol/l Tris±Cl, 1.5 mmol/l MgCl2, 50 mmol/l KCl, 0.05% Tween-20, and 0.0001% gelatin. The reaction took place under the following conditions: one cycle of 5 min at 948C, and 30 cycles of 30 s at 948C, 40 s at 578C and 40 s at 728C and a ®nal extension of 7 min at 728C. The PCR product was analyzed on agarose gel by electrophoresis and gels were stained with ethidium bromide and photographed under UV light (Fig. 1). The intensities of the target DNA band (615 bp) and competitor band (555 bp) were quanti®ed using NIH Image (image software available from the National Institutes of Health, Bethesda, MD). The ratio of each target mtDNA product/internal standard product was plotted against log (internal standard) to yield the equivalence point between internal standard and target mtDNA (Fig. 2). The r values of the standard curves were between 0.95 and 1.00. The interassay variance of mtDNA measurement was 12.2%. 2.4. Homeostasis model assessment of insulin resistance score The homeostasis model assessment of insulin resistance (HOMA-IR) score was calculated by the following formula, as described by Matthew et al. (1985): fasting plasma insulin (mIU/ml) £ fasting plasma glucose (mmol/l)/22.5.
Fig. 2. Quanti®cation of mtDNA in the peripheral blood leukocytes by competitive PCR. The ratio of each target/internal standard product was plotted against log (internal standard) to yield the equivalence point between internal standard and target DNA.
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S. Lim et al. / Mitochondrion 1 (2001) 71±77
2.5. Area under the curve of insulin during the oral glucose tolerance test (OGTT) Fasting plasma glucose and insulin concentrations were measured before and 30, 60, 90, and 120 min after the ingestion of a 75 g oral glucose challenge. The total integrated insulin response was quanti®ed by calculating the area under the curve of insulin (AUCinsulin) by use of the trapezoidal method. The identical methods used for determining plasma glucose and insulin concentrations were applied over the whole period of the study. 2.6. Statistics All data are presented as the mean ^ S.D. Correlations between variables were analyzed using Pearson's correlation. Statistical signi®cance was de®ned as P , 0:05. 3. Results The anthropometric and biochemical characteristics of the subjects are shown in Table 2. The mean value (^S.D.) of the mtDNA content in peripheral blood was 28.1 ^ 9.9 amol/5 ng genomic DNA. The mtDNA content showed a negative correlation with the waist-to-hip ratio (r 20:529, P 0:024) and a positive correlation with lean body mass (r 0:476, P 0:046). However, there is no signi®cant correlation between the mtDNA content and age, BMI, blood Table 2 Anthropometric and biochemical parameters of all subjects (n 18) Mean ^ S.D. Age (years) BMI (kg/m 2) Waist-to-hip ratio Lean body mass (kg) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Fasting plasma glucose (mmol/l) Fasting plasma insulin (mIU/ml) Total cholesterol (mmol/l) Triglyceride (mmol/l) LDL-cholesterol (mmol/l) HDL-cholesterol (mmol/l)
20.8 ^ 1.5 22.4 ^ 1.3 0.77 ^ 0.02 50.8 ^ 3.4 110.1 ^ 9.7 69.6 ^ 9.2 4.7 ^ 0.5 6.6 ^ 2.2 4.6 ^ 0.6 1.9 ^ 0.9 2.3 ^ 0.7 1.9 ^ 0.4
pressure, fasting plasma glucose concentration and lipid pro®les. The values of various surrogate indices of insulin resistance for the subjects are shown in Table 3. The mtDNA content was negatively correlated with AUCinsulin, HOMA-IR score and fasting plasma insulin concentration (Fig. 3A±C). There was a positive correlation between the mtDNA content and the ratio of the fasting plasma glucose/fasting plasma insulin concentration (FG/FI) (Fig. 3D). 4. Discussion Mitochondrion, a site of intracellular respiration, has its own genome, which encodes 13 proteins involving respiratory chain reactions and 24 structural RNAs (two ribosomal RNAs and 22 transfer RNAs). Thus, mutations of mtDNA may result in a defect in oxidative phosphorylation and in turn, cellular dysfunction. It is well known that point mutation of mtDNA at 3243 (A to G) is associated with a neuromuscular disorder, MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) (Suzuki et al., 1994; DiMauro et al., 1996). Interestingly, Kadowaki et al. (1994) have shown that this mutation was also commonly associated with maternal inherited diabetes. These patients were more likely to have a mother with diabetes, were younger at the time of diagnosis, had a lower frequency of obesity in the past, had a higher frequency of treatment with insulin, and had a higher frequency of hearing loss. Thus, it has been suggested that pancreatic beta cell dysfunction may be related to diabetes although the mechanism is unclear. However, there are several reports suggesting that insulin resistance is associated with mtDNA abnormalities. van den Ouweland et al. (1994) have shown Table 3 Values of surrogate indices of insulin resistance in 18 healthy volunteers Mean ^ S.D. Fasting plasma insulin concentration (mIU/ml) AUCinsulin (mIU/ml per 120 min) a HOMA-IR score FG/FI (mol/IU) a
AUCinsulin during a 75 g OGTT.
6.6 ^ 2.2 53.7 ^ 22.4 1.4 ^ 0.4 0.7 ^ 0.2
S. Lim et al. / Mitochondrion 1 (2001) 71±77
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Fig. 3. The correlation between the mtDNA content and the various surrogate indices of insulin resistance. (A) vs. AUCinsulin during an oral glucose challenge. (B) vs. HOMA-IR. (C) vs. FG/FI. (D) vs. fasting plasma insulin concentration. aamol/5 ng genomic DNA. bHOMA-IR fasting plasma insulin (mIU/ml) £ fasting plasma glucose (mmol/l)/22.5.
that the insulin secretory function is normal in diabetic patients having point mutation of mtDNA at 3243 (A to G), suggesting the involvement of peripheral insulin resistance. Moreover, Poulton et al. (1998) have demonstrated that the 16 189 variant in the Dloop of mtDNA, which is the transcriptional regulatory region, is associated with insulin resistance. While there are considerable data regarding the qualitative changes of mtDNA in diabetic patients, there is little attention given to quantitative changes of mtDNA in diabetes mellitus. We have demonstrated that the mtDNA content is decreased in type 2 diabetic patients. It was also observed that a decrease in the mtDNA content in peripheral blood precedes type 2
diabetes mellitus in our prospective population-based study (Lee et al., 1998). Although a decreased mtDNA content was clearly associated with diabetes, the underlining mechanism is unclear. The major pathogenic mechanism of type 2 diabetes mellitus is insulin resistance and an insulin secretory defect. A considerable body of evidence exists regarding the mtDNA content and abnormal beta cell dysfunction. In the pancreatic islet of the adult GK rat, which is a genetic diabetic model of defective insulin secretion, the mtDNA content is signi®cantly decreased (Serradas et al., 1995). In this study, we investigated the relationship between insulin resistance and the mtDNA content. We
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S. Lim et al. / Mitochondrion 1 (2001) 71±77
measured several surrogate indices of insulin resistance: the AUCinsulin response to a 75 g oral glucose challenge, fasting insulin, FG/FI and HOMA-IR score, since considerable data have demonstrated good correlations between these surrogate indices and insulin resistance measured with the euglycemic hyperinsulinemic clamp, which is a gold standard for insulin resistance (Matthew et al., 1985; Hollenbeck and Reaven, 1987; Laakso, 1992; Yeni-Komshian et al., 2000). In this study, we found signi®cant correlations between the mtDNA content in the peripheral blood and various surrogate indices of insulin resistance such as AUCinsulin, HOMA-IR score, FG/FI and the fasting insulin level in healthy young women. The mtDNA content also negatively correlated with the waist-to-hip ratio, which is a marker of central obesity. This is consistent with our previous study that showed a negative correlation of the mtDNA content with clinical phenotypes related to insulin resistance (waist-to-hip ratio and diastolic blood pressure) (Lee et al., 1998). Therefore, a decrease in the mtDNA content may be associated with both clinical and metabolic parameters of insulin resistance. It is unclear how the change in the mtDNA content is associated with insulin resistance and an insulin secretory defect. There is considerable evidence that oxidative stress seen in aging is associated with mtDNA damage (Corral-Debrinski et al., 1992; Fukagawa et al., 1999). mtDNA is susceptible to oxidative damage because mitochondria are a major organ generating reactive oxygen species and have a poor antioxidant defense mechanism (Richter, 1992). The mtDNA content was negatively correlated with age in our previously study (Lim et al., 2001). However, in this study, we included healthy young women with a mean age of 20.8 years (range 18±24 years). Thus, the effect of aging-associated oxidative stress may not totally explain the correlation between mtDNA and insulin resistance parameters. Insulin resistance and mtDNA could also be affected by ethnicity, culture and diet. Since this study included the homogenous population who were young healthy Korean women of middle class with a controlled diet before study, insulin resistance and the mtDNA content may not be signi®cantly affected by ethnicity, diet and culture. It is also possible that the content of mtDNA could be determined by genetic factors or physical ®tness. We have recently found that the mtDNA content is
lower in the offspring of type 2 diabetic patients with normal glucose tolerance, compared with control subjects without a family history of diabetes (Song et al., 2001). This may support a role of genetic factors in the determination of the mtDNA content although the importance of genetic factors in determining the mtDNA content needs to be investigated in the future. Physical ®tness may also affect the mtDNA content. In this study, we found a positive correlation between the mtDNA content and lean body mass. Moreover, we have previously demonstrated that the mtDNA content correlates well with aerobic exercise capacity (VO2max) and both are increased by aerobic exercise training (Lim et al., 2000). It is well known that exercise improves insulin resistance and both physical ®tness and the mtDNA content are decreased in diabetic patients (Antonetti et al., 1995; Bottini et al., 1995; Serradas et al., 1995). Thus, the mtDNA content is closely related to insulin resistance as well as physical ®tness, although causal relationships remain unclear. In summary, we have shown that the mtDNA content in the peripheral blood is signi®cantly correlated with various surrogate indices of insulin resistance such as AUCinsulin during OGTT, HOMA-IR score, FG/FI, and fasting insulin concentration. Thus, it could be concluded that the mtDNA content status has a close relationship with insulin resistance. This study is signi®cant in that it is the ®rst study focusing on the quantity of mtDNA in connection with insulin resistance. Further study is warranted to elucidate the mechanism by which the mtDNA content and insulin resistance are associated. Acknowledgements The authors thank Bong Sun Kang, Jeong Mi Kim, and Soo Jin Jeong for their technical assistance. The work was supported by a grant from the Korean Ministry of Health and Welfare. References Alcolado, J.C., Majid, A., Brockington, M., et al., 1994. Mitochondrial gene defects in patients with NIDDM. Diabetologia 37, 372±376. Antonetti, D.A., Reynet, C., Kahn, C.R., 1995. Increased expression
S. Lim et al. / Mitochondrion 1 (2001) 71±77 of mitochondrial-encoded genes in skeletal muscle of humans with diabetes mellitus. J. Clin. Invest. 95, 1383±1388. Ballinger, S.W., Shoffner, J.M., Hedaya, E.V., et al., 1992. Maternally transmitted diabetes and deafness associated with a 10.4 kb mitochondrial DNA deletion. Nat. Genet. 1, 11±15. Bottini, P., Tantucci, C., Scionti, L., et al., 1995. Cardiovascular response to exercise in diabetic patients: in¯uence of autonomic neuropathy of different severity. Diabetologia 38, 244±250. Corral-Debrinski, M., Shoffner, J.M., Lott, M.T., Wallace, D.C., 1992. Association of mitochondrial DNA damage with aging and coronary atherosclerotic heart disease. Mutat. Res. 275, 169±180. DiMauro, S., Hirano, M., Bonilla, E., De Vivo, D.C., 1996. The mitochondrial disorders. In: Berg, B.O. (Ed.), Principles of Child Neurology. McGraw-Hill, New York, pp. 1201±1232. Fukagawa, N.K., Li, M., Ling, P., Russell, J.C., Sobel, B.E., Absher, P.M., 1999. Aging and high concentrations of glucose potentiate injury to mitochondrial DNA. Free Radic. Biol. Med. 27, 1437± 1443. Gray, M.W., Burger, B., Lang, B.F., 1999. Mitochondrial evolution. Science 283, 1476±1481. Hollenbeck, C.B., Reaven, G.M., 1987. Variations in insulin-stimulated glucose uptake in healthy individuals with normal glucose tolerance. J. Clin. Endocrinol. Metab. 64, 1169±1173. Kadowaki, T., Kadowaki, H., Mori, Y., et al., 1994. A subtype of diabetes mellitus associated with a mutation of mitochondrial DNA. N. Engl. J. Med. 330, 962±968. Laakso, M., 1992. How good a marker is insulin level for insulin resistance? Am. J. Epidemiol. 137, 959±965. Lee, H.K., Song, J.H., Shin, C.S., et al., 1998. Decreased mitochondrial DNA content in peripheral blood precedes the development of non-insulin-dependent diabetes mellitus. Diabetes Res. Clin. Pract. 42, 161±167. Liang, P., Hughes, V., Fukagawa, N.K., 1997. Increased prevalence of mitochondrial DNA deletions in skeletal muscle of older individuals with impaired glucose tolerance. Diabetes 46, 920±923. Lim, S., Kim, S.K., Park, K.S., et al., 2000. Effect of exercise on the mitochondrial DNA content of peripheral blood in healthy women. Eur. J. Appl. Physiol. 82, 407±412. Lim, S., Kim, M.S., Park, K.S., et al., 2001. Correlation of plasma homocysteine and mitochondrial DNA content in peripheral blood in healthy women. Atherosclerosis, in press. Matthew, D.R., Hosker, J.P., Rudenski, A.S., Naylor, B.A., Treacher, D.F., Turner, R.C., 1985. Homeostasis model assessment:
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insulin resistance and b-cell function form fasting plasma glucose and insulin concentrations in man. Diabetologia 28, 412±419. Poulton, J., Brown, M.S., Cooper, A., Marchington, D.R., Phillips, D.I., 1998. A common mitochondrial DNA variant is associated with insulin resistance in adult life. Diabetologia 41, 54± 58. Reardon, W., Ross, R.J., Sweeney, M.G., et al., 1992. Diabetes mellitus associated with a pathogenic point mutation in mitochondrial DNA. Lancet 340, 1376±1379. Reaven, G., 1997. Banting lecture 1988. Role of insulin resistance in human disease. Nutrition 13 (1), 65. Richter, C., 1992. Reactive oxygen and DNA damage in mitochondria. Mutat. Res. 275, 249±255. Serradas, P., Giroix, M., Saulnier, C., et al., 1995. Mitochondrial deoxyribonucleic acid content is speci®cally decreased in adult, but not fetal, pancreatic islets of the Goto-Kakizaki rat, a genetic model of noninsulin-dependent diabetes. Endocrinology 136, 5623±5631. Song, J., Oh, J.Y., Sung, Y., Pak, Y.K., Park, K.S., Lee, H.K., 2001. Peripheral blood mitochondrial DNA content is related to the insulin sensitivity in offspring of type 2 diabetic patients. Diabetes Care, in press. Suzuki, S., Hinokio, Y., Hirai, S., et al., 1994. Pancreatic beta-cell secretory defect associated with mitochondrial point mutation of the tRNA(LEU(UUR)) gene: a study in seven families with mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes (MELAS). Diabetologia 37, 818±825. van den Ouweland, J.M., Lemkes, H.H., Trembath, R.C., et al., 1994. Maternally inherited diabetes and deafness is a distinct subtype of diabetes and associates with a single point mutation in the mitochondrial tRNA (Leu(UUR)) gene. Diabetes 43, 746± 751. Vialettes, B.H., Paquis-Flucklinger, V., Pelissier, J.F., et al., 1997. Phenotypic expression of diabetes secondary to a T14709C mutation of mitochondrial DNA. Comparison with MIDD syndrome (A3243G mutation): a case report. Diabetes Care 20, 1731±1737. Yeni-Komshian, H., Carantoni, M., Abbasi, F., Reaven, G.M., 2000. Relationship between several surrogate estimates of insulin resistance and quanti®cation of insulin-mediated glucose disposal in 490 healthy nondiabetic volunteers. Diabetes Care 23, 171±175.
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Relationship between various surrogate indices of insulin resistance and mitochondrial DNA content in the peripheral blood of 18 healthy volunteers Soo Lim a,b,c, Kyong Soo Park a,c, Min Seon Kim a,b, Bo Youn Cho a,b, Hong Kyu Lee a,b,c,d,* a Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea Center for Hormone Research, Clinical Research Institute, Seoul National University Hospital, Seoul, South Korea c The Institute of Endocrinology, Nutrition and Metabolism, Seoul National University Medical Research Center, Seoul, South Korea d Department of Biomedical Sciences, National Institute of Health, Seoul, South Korea b
Received 19 October 2000; received in revised form 6 February 2001; accepted 7 February 2001
Abstract Mutations or deletions of mitochondrial DNA (mtDNA) are associated with diabetes mellitus. In this study, we investigated the relationships between the mtDNA content in peripheral blood and surrogate indices of insulin resistance in 18 healthy young women (mean age 20.8 ^ 1.5 years). The mtDNA content was signi®cantly correlated with the area under the curve of insulin during an oral glucose tolerance test (r 20:622), the homeostasis model assessment for insulin resistance (r 20:616), the ratio of fasting glucose to insulin concentration (r 0:586) and the fasting insulin level (r 20:552). Further study is warranted to elucidate the mechanism by which the mtDNA content is associated with insulin resistance. q 2001 Elsevier Science B.V. and Mitochondria Research Society. All rights reserved. Keywords: Insulin resistance; Mitochondrial DNA; Homeostasis model assessment; Oral glucose tolerance test; Fasting insulin
1. Introduction The mitochondrion is an intracytoplasmic powerhouse organelle and has its own genome. Originally, mitochondria were independent cells which had aerobic respiration, but later became symbiotic with anaerobic cells, retaining their own DNA (Gray et al., 1999). Mitochondrial DNA (mtDNA) encodes 13 genes, which are essential for oxidative phosphorylation, and also codes ribosomal RNA and transfer RNA * Corresponding author. Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong Chongno-gu, Seoul 110-744, South Korea. Tel.: 182-2-760-2266; fax: 182-2-765-7966. E-mail address: [email protected] (H.K. Lee).
which are involved in the expression of these genes. There are about ten mitochondrial genomes per mitochondrion and each cell has hundreds to thousands of mitochondria. The number of mitochondria per cell varies according to the energy requirement of the tissue. It is well known that mutation in the gene for tRNALeu at position 3243 of mtDNA is associated with maternally inherited diabetes mellitus with deafness (Reardon et al., 1992; Alcolado et al., 1994; Kadowaki et al., 1994; van den Ouweland et al., 1994). There is also considerable evidence that point mutations in other sites of mtDNA and deletions of mtDNA are related to diabetes mellitus (Ballinger et al., 1992; Liang et al., 1997; Vialettes et al., 1997). Since mito-
1567-7249/01/$20.00 q 2001 Elsevier Science B.V. and Mitochondria Research Society. All rights reserved. PII: S 1567-724 9(01)00003-4
72
S. Lim et al. / Mitochondrion 1 (2001) 71±77
chondria are organelles producing adenosine triphosphate (ATP) and insulin secretion from pancreatic beta cells is dependent on ATP generation, abnormality in mtDNA may result in an insulin secretory defect. It appears that diabetes mellitus is associated with quantitative changes as well as qualitative changes in mtDNA. It was reported that the mtDNA copy number was decreased to 50% in muscle of diabetic patients (Antonetti et al., 1995). We have shown that the mtDNA content was decreased in peripheral blood leukocytes of diabetic patients (Lee et al., 1998). We have also found in our prospective populationbased study that subjects who became diabetics during a 2 year follow-up period had a signi®cantly lower mtDNA content in the prediabetic state (Lee et al., 1998). Interestingly, we have observed that there were negative correlations between the mtDNA content and clinical parameters of insulin resistance syndrome such as the waist-to-hip ratio and blood pressure. Insulin resistance has been known as a major pathogenic mechanism for type 2 diabetes mellitus. Moreover, insulin resistance is a common metabolic abnormality of common chronic diseases of a modern society such as hypertension, obesity, and hyperlipidemia, which increase cardiovascular diseases. As Reaven (1997) has pointed out, insulin resistance is not only a determinant of type 2 diabetes, but also of a cluster of metabolic syndrome including hypertension, dyslipidemia, obesity, and hyperuricemia. Since insulin resistance precedes the onset of metabolic syndrome as well as diabetes, an early detection of insulin resistance may be important to reduce the risk of this metabolic disease. Although the mtDNA content is clearly associated with diabetes mellitus, it is unclear whether the mtDNA content is related to insulin resistance itself. This study was conducted to investigate the relationship of the mtDNA content in peripheral blood with various surrogate indices of insulin resistance in healthy subjects.
range 18±24 years) were randomly recruited from students who had attended Ewha Woman's University. They were admitted to the Seoul National University Hospital Clinical Research Institute after 3 days on a diet that contained at least 150 g of carbohydrates. All subjects were in good health, as determined by medical history, physical examination, and screening routine laboratory analyses. Their cardiopulmonary function was evaluated by a pulmonary function test and exercise electrocardiogram before the study. None of the subjects displayed evidence of metabolic disorder, cardiovascular disease, or a history of smoking or regular medication. There was no family history of diabetes mellitus among their ®rst-degree relatives. Their socioeconomic levels were moderate to high. All subjects participated in the study voluntarily and informed consent was obtained. The study protocol was approved by Seoul National University Hospital Ethics Committee. 2.2. Measurement of anthropometric and biochemical parameters
2. Subjects and methods
Height, body weight, and waist and hip circumference were measured by the usual formula. Body mass index (BMI) was calculated as weight divided by squared height (kg/m 2). Recording of blood pressure took place between 08:00 and 09:00 h, when the subjects had been in a relaxed state for at least 1 h. After 14 h overnight fasting, venous blood samples were drawn from the antecubital vein in the morning (08:00±09:00 h). Plasma was separated immediately by centrifugation (2000 rev./min for 20 min at 48C). Biochemical measurements were conducted immediately after sampling. The fasting plasma concentration of glucose, total cholesterol, triglyceride, and high density lipoprotein (HDL)-cholesterol were measured using enzymatic methods with a Hitachi 747 chemistry analyzer (Hitachi, Tokyo, Japan). The level of low density lipoprotein (LDL)-cholesterol was calculated by the following formula: total cholesterol 2 HDL-cholesterol 2 triglyceride/2.2. The fasting plasma insulin concentration was determined using radioimmunoassay (LINCO kit, St. Louis, MO).
2.1. Subjects
2.3. Quanti®cation of mtDNA
Eighteen healthy women (mean age 20.8 years,
Blood samples for measurement of the mtDNA
S. Lim et al. / Mitochondrion 1 (2001) 71±77 Table 1 Sequences of primers Primer
Sequence
HmtF2 HmtR2 JF1
CAG GAC ATC CCG GGA GGC CTA GGT GGC AGA GCC C TAC CCA TGG CCA GCC ATG GGT A GGG CTC TGC CAT
JR1
Position ATG GTG CA TGA GGT TGA ACC TCC TA CTT AAC AA
2999±3018 3613±3593 3238±3247 3308±3327 3317±3308 3247±3228
content were centrifuged at 2000 rev./min for 20 min at 48C. The buffy-coat layer was separated and stored at 2708C until extraction of DNA. Total DNA was extracted using a QIAmp tissue kit (QIAGEN, Chatworth, CA) and concentrations of DNA were measured with a spectrophotometer (Beckman, Fullerton, CA). The mtDNA content was measured using competitive polymerase chain reaction (PCR) as described previously (Lim et al., 2001). In brief, the internal standard was designed to use the same primer set as the target gene but to yield a different sized PCR product (555 versus 615 bp). It was prepared by two independent PCR ampli®cations using specially designed primers shown in Table 1, producing 259 and 316 bp, respectively. Secondary PCR ampli®cation using the above products and primers hmtF2 and hmtR2 produced a 555 bp fragment containing sequences from mtDNA positions 2999±3247 and 3308±3613, with deletion of the intervening 60 bp (from positions 3248±3307). The known amounts of the serially diluted internal standard were added to 5 ng of total cellular DNA and subjected to PCR using a set of primers. The ®nal volume of the PCR reaction was 20 ml, containing 0.4 mmol/l of each primer, 200
Fig. 1. Electrophoresis by PCR product. Known amounts of the serially diluted internal standard DNA were added to total cellular DNA extracted from peripheral blood leukocytes and ampli®ed with primers hmtF1 and hmtR1. As indicated, two products were generated, the upper one derived from mtDNA (615 bp) and the lower one derived from internal standard DNA (555 bp). Lanes 1±5 are coampli®cations of varying amounts of internal standard DNA.
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mmol/l of each dNTP, 1 unit of Taq polymerase, 20 mmol/l Tris±Cl, 1.5 mmol/l MgCl2, 50 mmol/l KCl, 0.05% Tween-20, and 0.0001% gelatin. The reaction took place under the following conditions: one cycle of 5 min at 948C, and 30 cycles of 30 s at 948C, 40 s at 578C and 40 s at 728C and a ®nal extension of 7 min at 728C. The PCR product was analyzed on agarose gel by electrophoresis and gels were stained with ethidium bromide and photographed under UV light (Fig. 1). The intensities of the target DNA band (615 bp) and competitor band (555 bp) were quanti®ed using NIH Image (image software available from the National Institutes of Health, Bethesda, MD). The ratio of each target mtDNA product/internal standard product was plotted against log (internal standard) to yield the equivalence point between internal standard and target mtDNA (Fig. 2). The r values of the standard curves were between 0.95 and 1.00. The interassay variance of mtDNA measurement was 12.2%. 2.4. Homeostasis model assessment of insulin resistance score The homeostasis model assessment of insulin resistance (HOMA-IR) score was calculated by the following formula, as described by Matthew et al. (1985): fasting plasma insulin (mIU/ml) £ fasting plasma glucose (mmol/l)/22.5.
Fig. 2. Quanti®cation of mtDNA in the peripheral blood leukocytes by competitive PCR. The ratio of each target/internal standard product was plotted against log (internal standard) to yield the equivalence point between internal standard and target DNA.
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S. Lim et al. / Mitochondrion 1 (2001) 71±77
2.5. Area under the curve of insulin during the oral glucose tolerance test (OGTT) Fasting plasma glucose and insulin concentrations were measured before and 30, 60, 90, and 120 min after the ingestion of a 75 g oral glucose challenge. The total integrated insulin response was quanti®ed by calculating the area under the curve of insulin (AUCinsulin) by use of the trapezoidal method. The identical methods used for determining plasma glucose and insulin concentrations were applied over the whole period of the study. 2.6. Statistics All data are presented as the mean ^ S.D. Correlations between variables were analyzed using Pearson's correlation. Statistical signi®cance was de®ned as P , 0:05. 3. Results The anthropometric and biochemical characteristics of the subjects are shown in Table 2. The mean value (^S.D.) of the mtDNA content in peripheral blood was 28.1 ^ 9.9 amol/5 ng genomic DNA. The mtDNA content showed a negative correlation with the waist-to-hip ratio (r 20:529, P 0:024) and a positive correlation with lean body mass (r 0:476, P 0:046). However, there is no signi®cant correlation between the mtDNA content and age, BMI, blood Table 2 Anthropometric and biochemical parameters of all subjects (n 18) Mean ^ S.D. Age (years) BMI (kg/m 2) Waist-to-hip ratio Lean body mass (kg) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Fasting plasma glucose (mmol/l) Fasting plasma insulin (mIU/ml) Total cholesterol (mmol/l) Triglyceride (mmol/l) LDL-cholesterol (mmol/l) HDL-cholesterol (mmol/l)
20.8 ^ 1.5 22.4 ^ 1.3 0.77 ^ 0.02 50.8 ^ 3.4 110.1 ^ 9.7 69.6 ^ 9.2 4.7 ^ 0.5 6.6 ^ 2.2 4.6 ^ 0.6 1.9 ^ 0.9 2.3 ^ 0.7 1.9 ^ 0.4
pressure, fasting plasma glucose concentration and lipid pro®les. The values of various surrogate indices of insulin resistance for the subjects are shown in Table 3. The mtDNA content was negatively correlated with AUCinsulin, HOMA-IR score and fasting plasma insulin concentration (Fig. 3A±C). There was a positive correlation between the mtDNA content and the ratio of the fasting plasma glucose/fasting plasma insulin concentration (FG/FI) (Fig. 3D). 4. Discussion Mitochondrion, a site of intracellular respiration, has its own genome, which encodes 13 proteins involving respiratory chain reactions and 24 structural RNAs (two ribosomal RNAs and 22 transfer RNAs). Thus, mutations of mtDNA may result in a defect in oxidative phosphorylation and in turn, cellular dysfunction. It is well known that point mutation of mtDNA at 3243 (A to G) is associated with a neuromuscular disorder, MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) (Suzuki et al., 1994; DiMauro et al., 1996). Interestingly, Kadowaki et al. (1994) have shown that this mutation was also commonly associated with maternal inherited diabetes. These patients were more likely to have a mother with diabetes, were younger at the time of diagnosis, had a lower frequency of obesity in the past, had a higher frequency of treatment with insulin, and had a higher frequency of hearing loss. Thus, it has been suggested that pancreatic beta cell dysfunction may be related to diabetes although the mechanism is unclear. However, there are several reports suggesting that insulin resistance is associated with mtDNA abnormalities. van den Ouweland et al. (1994) have shown Table 3 Values of surrogate indices of insulin resistance in 18 healthy volunteers Mean ^ S.D. Fasting plasma insulin concentration (mIU/ml) AUCinsulin (mIU/ml per 120 min) a HOMA-IR score FG/FI (mol/IU) a
AUCinsulin during a 75 g OGTT.
6.6 ^ 2.2 53.7 ^ 22.4 1.4 ^ 0.4 0.7 ^ 0.2
S. Lim et al. / Mitochondrion 1 (2001) 71±77
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Fig. 3. The correlation between the mtDNA content and the various surrogate indices of insulin resistance. (A) vs. AUCinsulin during an oral glucose challenge. (B) vs. HOMA-IR. (C) vs. FG/FI. (D) vs. fasting plasma insulin concentration. aamol/5 ng genomic DNA. bHOMA-IR fasting plasma insulin (mIU/ml) £ fasting plasma glucose (mmol/l)/22.5.
that the insulin secretory function is normal in diabetic patients having point mutation of mtDNA at 3243 (A to G), suggesting the involvement of peripheral insulin resistance. Moreover, Poulton et al. (1998) have demonstrated that the 16 189 variant in the Dloop of mtDNA, which is the transcriptional regulatory region, is associated with insulin resistance. While there are considerable data regarding the qualitative changes of mtDNA in diabetic patients, there is little attention given to quantitative changes of mtDNA in diabetes mellitus. We have demonstrated that the mtDNA content is decreased in type 2 diabetic patients. It was also observed that a decrease in the mtDNA content in peripheral blood precedes type 2
diabetes mellitus in our prospective population-based study (Lee et al., 1998). Although a decreased mtDNA content was clearly associated with diabetes, the underlining mechanism is unclear. The major pathogenic mechanism of type 2 diabetes mellitus is insulin resistance and an insulin secretory defect. A considerable body of evidence exists regarding the mtDNA content and abnormal beta cell dysfunction. In the pancreatic islet of the adult GK rat, which is a genetic diabetic model of defective insulin secretion, the mtDNA content is signi®cantly decreased (Serradas et al., 1995). In this study, we investigated the relationship between insulin resistance and the mtDNA content. We
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S. Lim et al. / Mitochondrion 1 (2001) 71±77
measured several surrogate indices of insulin resistance: the AUCinsulin response to a 75 g oral glucose challenge, fasting insulin, FG/FI and HOMA-IR score, since considerable data have demonstrated good correlations between these surrogate indices and insulin resistance measured with the euglycemic hyperinsulinemic clamp, which is a gold standard for insulin resistance (Matthew et al., 1985; Hollenbeck and Reaven, 1987; Laakso, 1992; Yeni-Komshian et al., 2000). In this study, we found signi®cant correlations between the mtDNA content in the peripheral blood and various surrogate indices of insulin resistance such as AUCinsulin, HOMA-IR score, FG/FI and the fasting insulin level in healthy young women. The mtDNA content also negatively correlated with the waist-to-hip ratio, which is a marker of central obesity. This is consistent with our previous study that showed a negative correlation of the mtDNA content with clinical phenotypes related to insulin resistance (waist-to-hip ratio and diastolic blood pressure) (Lee et al., 1998). Therefore, a decrease in the mtDNA content may be associated with both clinical and metabolic parameters of insulin resistance. It is unclear how the change in the mtDNA content is associated with insulin resistance and an insulin secretory defect. There is considerable evidence that oxidative stress seen in aging is associated with mtDNA damage (Corral-Debrinski et al., 1992; Fukagawa et al., 1999). mtDNA is susceptible to oxidative damage because mitochondria are a major organ generating reactive oxygen species and have a poor antioxidant defense mechanism (Richter, 1992). The mtDNA content was negatively correlated with age in our previously study (Lim et al., 2001). However, in this study, we included healthy young women with a mean age of 20.8 years (range 18±24 years). Thus, the effect of aging-associated oxidative stress may not totally explain the correlation between mtDNA and insulin resistance parameters. Insulin resistance and mtDNA could also be affected by ethnicity, culture and diet. Since this study included the homogenous population who were young healthy Korean women of middle class with a controlled diet before study, insulin resistance and the mtDNA content may not be signi®cantly affected by ethnicity, diet and culture. It is also possible that the content of mtDNA could be determined by genetic factors or physical ®tness. We have recently found that the mtDNA content is
lower in the offspring of type 2 diabetic patients with normal glucose tolerance, compared with control subjects without a family history of diabetes (Song et al., 2001). This may support a role of genetic factors in the determination of the mtDNA content although the importance of genetic factors in determining the mtDNA content needs to be investigated in the future. Physical ®tness may also affect the mtDNA content. In this study, we found a positive correlation between the mtDNA content and lean body mass. Moreover, we have previously demonstrated that the mtDNA content correlates well with aerobic exercise capacity (VO2max) and both are increased by aerobic exercise training (Lim et al., 2000). It is well known that exercise improves insulin resistance and both physical ®tness and the mtDNA content are decreased in diabetic patients (Antonetti et al., 1995; Bottini et al., 1995; Serradas et al., 1995). Thus, the mtDNA content is closely related to insulin resistance as well as physical ®tness, although causal relationships remain unclear. In summary, we have shown that the mtDNA content in the peripheral blood is signi®cantly correlated with various surrogate indices of insulin resistance such as AUCinsulin during OGTT, HOMA-IR score, FG/FI, and fasting insulin concentration. Thus, it could be concluded that the mtDNA content status has a close relationship with insulin resistance. This study is signi®cant in that it is the ®rst study focusing on the quantity of mtDNA in connection with insulin resistance. Further study is warranted to elucidate the mechanism by which the mtDNA content and insulin resistance are associated. Acknowledgements The authors thank Bong Sun Kang, Jeong Mi Kim, and Soo Jin Jeong for their technical assistance. The work was supported by a grant from the Korean Ministry of Health and Welfare. References Alcolado, J.C., Majid, A., Brockington, M., et al., 1994. Mitochondrial gene defects in patients with NIDDM. Diabetologia 37, 372±376. Antonetti, D.A., Reynet, C., Kahn, C.R., 1995. Increased expression
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