Mitochondrial DNA Mutations Are Associated with Both Decreased Insulin Secretion and Advanced Microvascular Complications in Japanese Diabetic Subjects

doi:10.1016/S1056-8727(99)00060-4

Journal of Diabetes and its Complications

Volume 13, Issues 5–6, September–December 1999, Pages 277-283

https://doi.org/10.1016/S1056-8727(99)00060-4 Get rights and content

Abstract

To assess the roles of various mitochondrial (Mt) DNA mutations in diabetic and nondiabetic subjects, we screened Mt DNAs at the 3243 base pair (bp) and its adjacent portion in unrelated Japanese diabetic and nondiabetic subjects. Furthermore, to clarify the clinical features of diabetic subjects harboring a Mt DNA mutation, we evaluated the ability of insulin secretion and microvascular complications in diabetic subjects. Five hundred thirty-seven diabetic patients and 612 unrelated nondiabetic subjects were recruited into this study. In Mt DNA analyses, Mt DNA was isolated from peripheral leukocytes of the subjects, and then an Mt DNA fragment surrounding the tRNA^Leu(UUR) site was amplified by the polymerase chain reaction (PCR) using two sets of primers. These fragments were further digested with three kinds of restriction endonucleases and were subjected to agarose gel electrophoresis. When a mutation was present, Mt DNA fragments were directly sequenced with an autosequencer. Baseline characteristics in all subjects were examined, and microvascular complications and insulin secretory capacity in diabetic subjects were newly evaluated. Eight kinds of Mt DNA mutations, which were point mutations, were found in 74 subjects. Each affected subject had only one mutation in the Mt DNA examined. Among them, the mutations at np 3316, 3394, 3593, and 3391 were accompanied by amino acid replacement. Thirty-eight diabetic patients were affected (7.1%), including two subjects with a point mutation at np 3243, and 26 nondiabetic subjects were affected (4.2%). Thus, there was a higher prevalence in diabetic subjects than in nondiabetic subjects. There was no significant difference in the prevalence of maternally inherited diabetes between these two groups. The mean level of urinary C-peptide excretion was lower in diabetic subjects with an Mt DNA mutation (DM+) than in those without it (DM−). Although the prevalence of hypertension in DM+ was higher than that in DM−, diabetic retinopathy and nephropathy in DM+ were problematic, in comparison with those in DM−, when statistical corrections were performed for the effect of hypertension. Furthermore, a strategy based on logistic regression analysis revealed that advanced retinopathy and decreased urinary C-peptide excretion in all diabetic subjects studied were strongly related to the presence of Mt DNA mutation. Our results suggest that Mt DNA mutations in Japanese diabetic subjects are related to the development of diabetes, and also that these mutations are associated with not only a decrease in insulin secretion but also advanced diabetic microvascular complications.

Introduction

Many kinds of mutations and deletions in mitochondrial (Mt) DNA have been reported to be associated with the development of diabetes mellitus, in concert with other genetic factors and/or environmental factors.¹⁻⁴ Among them, tRNA^Lue(UUR) and its adjacent region within the Mt genome are linked to high susceptibility to diabetes mellitus.² A point mutation at 3243 base pair (bp) in the Mt tRNA^Lue(UUR) gene is commonly referred to as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), and it has been recognized as a causative factor in a new clinical entity: maternally inherited diabetes and deafness (MIDD).5, 6 Mt dysfunction through the involvement of Mt oxidative phosphorylation may lead to impairment of glucose-induced insulin secretion from pancreatic β cells in subjects harboring these mutations.2, 3, 7 The clinical phenotypes of diabetes mellitus with these mutations are generally heterogeneous.5, 8 However, there is little information about the degree of microvascular complications in diabetic subjects with an Mt DNA mutation.

To assess the roles of various Mt DNA mutations in diabetic and nondiabetic subjects, we screened for Mt DNAs at the 3243 bp and its adjacent portion in Japanese unrelated 537 diabetic and 612 nondiabetic subjects. Furthermore, to clarify the clinical features of diabetic subjects harboring an Mt DNA mutation, we evaluated the ability of insulin secretion and microvascular complications in diabetic subjects.

Section snippets

Subjects

The Japanese subjects studied were 537 diabetic subjects (299 men, 238 women; including 24 type I diabetes, 12 men and 12 women) and 612 nondiabetic subjects (197 men, 415 women) who were inpatients or outpatients of Kanazawa Medical University Hospital. Nondiabetic subjects were patients who were diagnosed as having mild hypertension, hyperlipidemia, or thyroid disease, and were under treatment. Informed consent was obtained from each participant. Type II diabetes was diagnosed according to

Clinical Characteristics of Diabetic and Nondiabetic Subjects Studied

Clinical characteristics of the diabetic and nondiabetic subjects studied are shown in Table 1. There were significant differences in age, gender, and HbA_1c between diabetic and nondiabetic subjects. The median (range) duration of diabetes in the diabetic subjects was 10 (0–42) years. BMI was similar between the two groups.

Mt DNA Analysis in Diabetic and Nondiabetic Subjects Studied

As shown in Table 2, eight kinds of Mt DNA point mutations were identified, all of which were single mutations. Each affected subject had only one mutation in the Mt DNA

Discussion

In this study, we demonstrated that the prevalence of Mt DNA mutation was higher in unrelated Japanese diabetic subjects than in nondiabetic subjects, showing that mutated Mt DNA may be linked to the development of diabetes. Furthermore, we showed that the prevalence of maternally inherited diabetes in diabetic subjects, even with Mt DNA mutation, was low, and that microvascular complications in diabetic subjects with an Mt DNA mutation were more pronounced than those in diabetic subjects

Acknowledgements

This work was in part supported by a Grant for Project Research from Kanazawa Medical University (P98-2).

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It was reported that human populations can be divided into several mtDNA haplogroups on the basis of specific nucleotide polymorphisms, and some studies have pointed out the possible impact of haplogroups on mitochondrial physiology.32,33 Thus, mtDNA haplogroups might be the possible reason for the difference between Western and East Asian populations.3 Previous studies also demonstrated that macular pattern dystrophy might protect against the development of diabetic retinopathy through a reduction in retinal metabolism and a decrease in oxygen consumption in the retina.10,16
This study presents nine patients with mitochondrial tRNA Leu (UUR) m.3243A > G mutation and compares the clinical characteristics and diabetes complications with type 1 diabetes (T1DM) or early onset type 2 diabetes (T2DM).
The study covers 9 patients with MIDD, 33 patients with T1DM and 86 patients (age of onset ≤ 35 years) with early onset T2DM, matched for sex, age at onset of diabetes, duration of diabetes. All patients with MIDD were confirmed as carrying the m.3243A > G mitochondrial DNA mutation. Serum HbA1c, beta-cell function, retinal and renal complications of diabetes, bone metabolic markers, lumbar spine and femoral neck BMD bone mineral density were compared to characterize the clinical features of all patients.
Nine patients were from five unrelated families, and the mean (SD) onset age of those patients was 31.2 ± 7.2 year. Two patients required insulin at presentation, and six patients progressed to insulin requirement after a mean of 7.2 years. β-Cell function in the MIDD group was intermediate between T1DM and early-onset T2DM. In MIDD, four patients were diagnosed as diabetic retinopathy (4/9) and five patients (5/9) had macroalbuminuria. The number of patients with diabetic retinopathy and macroalbuminuria in the MIDD group was comparable to T1DM or early-onset T2DM. The rate of osteoporosis (BMD T-score < − 2.5 SD) in the patient with MIDD was higher than the T1DM or early-onset T2DM group.
Our study indicates that of the nine subjects with MIDD, three patients (1-II-1, 1-II-3, 1-II-4) who came from the same family had a history of acute pancreatitis. Compared with T1DM or early-onset T2DM matched for sex, age, duration of diabetes, MIDD patients had the highest rate of osteoporosis.
Usefulness of postmortem biochemistry in identification of ketosis: Diagnosis of ketoacidosis at the onset of autoimmune type 1 diabetes in an autopsy case with cold exposure and malnutrition
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It is thought that she was placed under low temperature exposure as for this result [25]. The mitochondrial (mt) DNA 3243 mutation [26] was not detected in the whole blood. No blood alcohol was detected in several body fluids.
A severely malnourished, Japanese female in her twenties was found dead in her apartment. On autopsy, most of the findings from the internal examination were suggestive of hypothermia. Postmortem biochemistry, however, showed severely increased levels of glycated hemoglobin (HbA1c) and blood and urine glucose levels. Levels of acetone, 3-hydroxybutyric acid, and acetoacetate in various body fluids were also highly increased, indicating ketosis. The serum insulin and c-peptide levels were severely low, and subsequent testing was positive for anti-GAD antibodies. Immunohistochemical examination of the pancreatic islet cells revealed few insulin-positive cells but many glucagon-positive cells on staining. Furthermore, slight invasion of CD8-positive lymphocytes in the pancreatic islets of Langerhans was observed. Results of immunostaining of the pancreatic and bronchial epithelial tissues were partly positive for the Influenza A virus. We concluded that severe ketoacidosis associated with rapid-onset hyperglycemia due to autoimmune type 1 diabetes (AT1D) had occurred shortly before death. However, the ketosis was accompanied by hypothermia and malnutrition as well as diabetic ketoacidosis (DKA). Therefore, we retrospectively collected biochemical data on cases of hypothermia and malnutrition and compared them with the present case. Serum glucose, acetone, 3-hydroxybutyric acid, and acetoacetic acid can be used for screening and diagnosis to distinguish DKA from ketosis due to hypothermia and malnutrition. Therefore, in the present case, we diagnosed that the natural cause of death was due to AT1D. In conclusion, screening investigations for relevant biochemical markers can provide essential information for the diagnosis of metabolic disturbances, which fail to demonstrate characteristic autopsy findings.
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Mitochondria provide cells with most of the energy in the form of ATP. Mutations in mitochondrial DNA (mtDNA) are associated with type 2 diabetes mellitus (T2DM) because ATP plays a critical role in the production and the release of insulin. To systematically determine mutant loci and to investigate their association with T2DM in Chinese Han population, 17 commonly reported mutant loci were screened in 236 cases of T2DM and 240 normal controls by PCR-RFLP, allele-specific PCR (AS-PCR) and DNA sequencing methods. Biological softwares were used to analyze the secondary structure of DNA, RNA and the corresponding proteins for missense mutations. Sixteen mutant loci were detected in total, of which five were novel, GenBank accession nos. were DQ092356, DQ473644 and DQ473645; they were mainly in16S rRNA, ND1 and ND4 gene. There was significant difference between the two groups for ND1 and ND4 genes mutation frequencies (ND1: P = 0.001, OR = 3.944, 95% CI 1.671–9.306; ND4: P = 0.010, OR = 5.818, 95% CI 1.275–26.537). No significant association was observed between the two groups for 5178A/C polymorphisms (P = 0.418). Our study suggested that T3394C and A12026G might be associated with T2DM in Chinese Han population, and T2DM with mtDNA variant should be considered mitochondrial diabetes.
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Original articlesMitochondrial DNA Mutations Are Associated with Both Decreased Insulin Secretion and Advanced Microvascular Complications in Japanese Diabetic Subjects

Abstract

Introduction

Section snippets

Subjects

Clinical Characteristics of Diabetic and Nondiabetic Subjects Studied

Mt DNA Analysis in Diabetic and Nondiabetic Subjects Studied

Discussion

Acknowledgements

Biochim Biophys Acta

Int J Biochem Cell Biol

Diabetes Res Clin Pract

Biochem Biophys Res Commun

Biochem Biophys Res Commun

Biochem Biophys Res Commun

Mitochondrial DNA polymorphism in Japanese. II. Analysis with restriction enzymes of four or five base pair recognition

Hum Genet

Mitochondria and diabetes. Genetic, biochemical, and clinical implications of the cellular energy circuit

Diabetes

Mitochondrial DNA and disease

Ann Med

Maternally inherited diabetes and deafnessA new diabetes subtype

Diabetologia

Maternally inherited diabetes and deafness (MIDD)A distinct subtype of diabetes associated with a mitochondrial tRNALue(UUR) gene point mutation

Muscle Nerve Suppl

Stimulation by D-glucose of mitochondrial oxidative events in ilet cells

Biochem J

A subtype of diabetes mellitus associated with a mutation of mitochondrial DNA

N Engl J Med

Report of the Expert Committee on the Diagnosis and Classification of Diabetes mellitus

Diabetes Care

Original articles
Mitochondrial DNA Mutations Are Associated with Both Decreased Insulin Secretion and Advanced Microvascular Complications in Japanese Diabetic Subjects

Maternally inherited diabetes and deafness (MIDD)A distinct subtype of diabetes associated with a mitochondrial tRNA^Lue(UUR) gene point mutation