Norio Kono

Examination of islets in the pancreas biopsy specimens from newly diagnosed Type 1 (insulin-dependent) diabetic patients

SummaryWe attempted to examine the immunopathological change of the pancreatic islets of newly diagnosed Type 1 (insulin-dependent) diabetic patients and thereby to obtain useful information for the therapy of the patients. For this purpose, pancreas biopsy under laparoscopy was performed 2–4 months after the onset of Type 1 diabetes in seven newly diagnosed patients. All biopsies were performed safely without any complications. Immunohistochemical examination of the biopsy specimens revealed a marked decrease of insulin-containing cells, preservation of glucagon-containing cells, and various degrees of expression of MHC class I and class II antigens in islet cells and in endothelial cells within and around the islets. Signs of active autoimmune phenomena, e. g. lymphocytic infiltration or immunoglobulin deposition in islets, were not detected in any of these patients by light microscopical evaluation. We conclude that pancreas biopsy under laparoscopy has shown various immunological changes in the islets of newly diagnosed Type 1 diabetic patients. Pancreas biopsy, however, may not be suitable under the present protocol for the selection of patients for immunotherapy because of problems including sampling errors.

Cloning of human muscle phosphofructokinase cDNA

Three overlapping cDNA clones for human muscle phosphofructokinase (HMPFK) covering the complete coding sequence were isolated. The sequence included a poly(A) tail, a 399 bp 3′‐untranslated region, a 2337 bp coding region for 779 amino acid residues and a part of the 5′‐untranslated region. Homologies between HMPFK and rabbit muscle phosphofructokinase (RMPFK) were 96% of the amino acids and 89% of the nucleotides in the coding region. Like RMPFK, HMPFK also possessed the internal homology between C‐ and N‐halves in its primary structure. Cloning of HMPFK cDNA will help to identify the molecular defect in patients with glycogenosis type VII (HMPFK deficiency).

Structure of the entire human muscle phosphofructokinase-encoding gene: a two-promoter system

We have recently shown that three types (A,B, and C) of mRNA species are transcribed from a single gene encoding human muscle phosphofructokinase (hPFK-M) through alternative splicing [Nakajima et al., Biochem. Biophys. Res. Commun. 166 (1990) 637-641]. To determine its complete structure and elucidate the mechanism of alternative RNA splicing, we isolated the hPFK-M gene, which spans about 30 kb, and contains 24 exons. Transcription start points were observed for both exon 1 and exon 2 by S1 nuclease protection assay and primer extension. Motifs of an Sp1-binding site were observed in the upstream region of exon 1 (promoter 1). A TATA-box-like sequence and a CAAT-box-like sequence were identified in the upstream region of exon 2 (promoter 2). Reporter assay revealed that the promoter 1 region was functional both in HeLa cells and myoblastic clonal cells, and that the promoter 2 region was active only in myoblastic cells. Motifs of M-CAT known as a muscle-specific enhancer, were observed in the promoter 2 region. These results indicated that the hPFK-M gene contains at least two promoter regions, facilitating the expression of the heterogeneous gene transcripts in a cell-type-specific manner.

Tissue specificity in expression and alternative RNA splicing of human phosphofructokinase-M and -L genes

Mode of the expression of phosphofructokinase (PFK) -M and -L genes was examined in various human tissues including muscle, placenta, liver, kidney, pancreas, stomach and reticulocytes. The gross level of mRNA expression of PFK-M and -L genes was estimated by Northern analysis. Polymerase chain reaction was used to detect mRNA expressed at low levels in these tissues. Tissue-specific expression of alternatively spliced PFK-M gene transcripts was also determined by polymerase chain reaction. The results indicated that alternative splicing of PFK-M gene transcripts was controlled in a tissue-specific manner.

The Exercise Test

A “semi-ischemic” forearm exercise test is useful for screening patients with muscle enzyme defects in the metabolic pathway from glycogen (glucose) to lactate. McArdle [1] first described the “ischemic” forearm exercise test; he observed no rise in plasma lactate concentration in the original patient. The test was later modified [2] and has since been used to screen patients with McArdle’s disease (glycogenosis type V). This test, in which the exercising arm is maintained at conditions of complete ischemia, is also available as a diagnostic test for other types of glycogenosis, including types VII [3] and III [4]. However, patients with type V glycogen storage disease (GSD) developed myoglobinemia, massive myoglobinuria, and marked serum creatine kinase elevation subsequent to routinely ischemic forearm exercise tests. Substantial evidence has been accumulated that the ischemic forearm exercise test is potentially hazardous to type V patients, as it might induce massive myoglobinuria sufficient to result in acute myoglobinuric renal failure [5, 6]. Therefore, the test had to be modified in the following fashion [7, 8]: after 30 min rest, blood is drawn from the antecubital vein of the nonexercising arm. A small size sphygmomanometer cuff applied around the wrist of the exercising arm is inflated to 200 mmHg. A second standard cuff around the upper arm is then inflated to mean arterial pressure, and the patient squeezes a hand manometer as powerfully as possible 120 times during a period of 2 min. Immediately after the exercise, the second cuff is rapidly inflated to 200 mmHg. Blood is drawn with a butterfly needle from the antecubital vein of the exercising arm 2 min after the end of the exercise, and the cuff around the upper arm is released. Then blood is obtained every 1 or 2 min four to five times (Table 1). This semi-ischemic forearm exercise test has been used for many patients with types V, VII, and III GSD, including cases of our own, and no severe signs or symptoms appeared during or after the test.

Low glucose-1, 6-bisphosphate and high fructose-2, 6-bisphosphate concentrations in muscles of patients with glycogenosis types VII and V

The level of glucose-1, 6-bisphosphate, a potent allosteric activator of phosphofructokinase, was markedly decreased in muscles of patients with glycogenosis type VII (muscle phosphofructokinase deficiency) and type V (muscle phosphorylase deficiency). Glucose-1-phosphate kinase activity in muscle was virtually absent in a patient with glycogenosis type VII, whereas it was normal in a patient with type V glycogenosis. Glucose-1-phosphate level was increased in type VII glycogenosis, whereas it was decreased in type V glycogenosis. Another activator of phosphofructokinase, fructose-2, 6-bisphosphate was increased in muscles of patients with both types of glycogenosis although it was much higher in type VII than in type V. This finding may be partly related to the difference of fructose-6-phosphate concentrations. The results suggest that phosphofructokinase would contribute to the major glucose-1-phosphate kinase activity in normal human muscle and would also form a kind of self-activating system.

Molecular Aspect of Myogenic Hyperuricemia: Cloning of Human Muscle Phosphofructokinase cDNA

In the last decade, molecular biology has made a breakthrough in our understanding of gene regulations, protein structures and mutant gene constitutions. Inborn errors of metabolism would be representatives of the fields of interest in which molecular biological techniques are expected to be the most powerful tools for analysis.

Autoantibodies to the insulin receptor impair clearance of plasma endogenous insulin

Plasma insulin clearance was studied in a patient with autoantibodies to the insulin receptor, manifesting persistent hyperinsulinemia associated with alternating hyper- and hypoglycemia. In the postabsorptive period, the plasma glucose level gradually decreased. To prevent the development of hypoglycemia, glucose was infused and the glycemic level was clamped at 50 mg/dl without insulin infusion. The plasma C-peptide level was below the detectable range during the clamp, indicating no appreciable secretion of insulin. The plasma insulin level declined exponentially with a markedly prolonged disappearance rate (half-time: 3.0 h) during the study. These results indicate that hyperinsulinemia in the postabsorptive period in this patient is attributable to the impairment of plasma insulin clearance through receptor-mediated mechanisms, and also confirm that the receptor plays the principal role in plasma insulin removal.

Exercise Induced Alteration of Erythrocyte Glycolysis Associated with Myogenic Hyperuricemia

Recently we reported ‘myogenic hyperuricemia’ in muscle glycogenosis types III, V and VII (Kono et al., 1986; Kono et al., 1987; Mineo et al., 1985; Mineo et al., 1987). The mechanism of myogenic hyperuricemia is: when energy production does not fill its requirement for continuing exercise, purine nucleotide degradation is accelerated (Fig. 1). The degradation of purine nucletide occurs even with mild exercise in these diseases. Its degradative metabolites such as inosine, hypoxanthine, and ammonia are released from working muscles into blood stream. Inosine and hypoxanthine are taken up by liver and metabolized to uric acid, causing hyperuricemia. In this study, we report exercise-induced alteration of erythrocyte glycolysis in muscle glycogenoses(Fig. 1), which is another metabolic consequence caused by accelerated purine nucleotide degradation in muscle.

Myogenic Hyperuricemia: A Comparative Study between Type V and Type VII Glycogenosis

Glycolysis subsequent to glycogen breakdown is one of the major energy(ATP)-generating systems necessary for muscle exercise. The metabolic process of glycolysis depends on the functional integrity of many enzymes. Glycogenosis types V and VII are genetic errors resulting in deficiencies of muscle phosphorylase and muscle phosphofructokinase, respectively. Patients with these diseases have common muscle symptoms such as easy fatigability, stiffness and pain during exercise. Hyper-uricemia or gout has been documented in some patients with glycogenosis types V and VII. We recently showed that excess purine degradation in exercising muscles due to impaired glycolysis or glycogen breakdown causes hyperuricemia (myogenic hyperuricemia) in these patients (Kono et al., 1986; Mineo et al., 1987). Interestingly, the incidence of hyperuricemia seems to be far greater in type VII than in type V. At least 9 of 26 patients with glycogenosis type VII have been reported to be hyperuricemic (Agamanolis et al., 1980; Hays et al., 1981; Zanella et al., 1982; Vora et al., 1983; Mineo et al., 1985; Fogelfeld et al., 1985; Kono et al., 1986). However, only a few among more than 100 patients with type V have been reported to be hyperuricemic (Hardiman et al., 1987; Kono et al., 1987). In order to elucidate the metabolic basis for the different incidence of hyperuricemia, we compared purine degradation in exercising muscles between type V and type VII glycogenosis.