To study the correlations between insulin degradation and the effect of insulin on glucose metabolism, insulin clearance, glucose uptake and output, and glucose oxidation were observed in perfused rat liver, kidney, heart and skeletal muscle (hindquarter) at various concentrations of insulin. Each organ was cyclically perfused for 60 min at a flow rate set proportionally to the physiological blood supply (liver, kidney, heart and skeletal muscle were set at 1. 8, 12. 6, 2.5 and 0.08 ml/min/g, respectively) with Krebs-Ringer bicarbonate solution (pH 7.4) containing 10 mM glucose and 2 % bovine serum albumin.
All of these tissues extracted insulin from the perfusates in a manner following first-order kinetics at insulin concentrations below 2000 μU/ml. Insulin clearance rates of liver, kidney, heart and skeletal muscle were 157, 1063, 142 and 6.3 p//min/g, respectively.
Insulin effect on glucose uptake and oxidation in the heart and skeletal muscle increased dosedependently within a range of 0-2000μU/ml insulin. Insulin effects on perfusate glucose in the liver reached the maximum at 100μU/ml insulin, while insulin had no effect on glucose metabolism in the kidney.
These results indicated that the mode of insulin degradation is similar in each tissue, but there is no common correlation between insulin degradation and the effect of insulin on glucose metabolism in each tissue, and that the priority in insulin degradation may be given to the liver and kidney in part through large blood supplies to those tissues.

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Intensive insulin therapy is known to maintain good overall control of blood glucose levels in diabetic patients. Little is known about the relationship between the in vivo effect of insulin and different tims of administration. We therefore investigated the differences in metabolic improvement from the diabetic state when NPH insulin (8 unit/day) was administered at different times: just before the light cycle (am 6: 00)(L-NPH) or just before the dark cycle (pm 6: 00)(D-NPH) in streptozotocin (60 mg/kg)-induced diabetic rats (DM).
The following results were obtained:
1) Body weight: Body weight of DM groups increased after NPH insulin administration for 7 days. The increase was immediate and marked in the L-NPH group.
2) Food intake: Hyperphagia in diabetic state was significantly decreased only in the L-NPH group (p<0.05).
3) Blood glucose: Following insulin administration the D-NPH group decreased to the level of 23% at 12 hrs and 63% at 24 hrs as compared with the DM-saline group. In contrast. the L-NPH group decreased to the level of 12% at 12 hrs and 77% at 24 hrs.
4) Weight of fat tissue: Weight of retroperitoneal and brown adipose tissue increased after insulin administration but recovered to the control level only in the L-NPH group.
5) Total lipid content (TLC) in liver: Much more TLC in DM was decreased to the control level by insulin administration, and TLC in the L-NPH group was significantly less than that in D-NPH group (p<0.05). The present studies suggest that there are differences in metabolic improvement from the diabetic state with insulin administation time. Further investigation is needed in clinical patients.

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The myo-inositol (MI) was measured in various nerve tissues obtained from streptozotocininduced hyperglycemic, insulin-induced hypoglycemic and lithium-treated rats. Also studied was the effect of atropine on the MI content in hypoglycemic and of thyrotropin-releasing hormone (TRH) in hyperglycemic and in lithium-treated rats.
In the hyperglycemic group, the MI content was significantly reduced in the spinal cord and sciatic nerve, but not in the cerebral cortex and cerebellar cortex, compared with the control group. In the hyperglycemia plus TRH-treated group, the MI content was not different from the hyperglycemic group in each of the nerve tissues. Compared with the control group, the MI in the hypoglycemic group was significantly reduced in the cerebral cortex, thalamus-caudate nucleus, brain stem and spinal cord, but not in the cerebellar cortex, cranial nerves and sciatic nerve. Atropine therapy prevented the reduction of MI in hypoglycemic rats. Lithium treatment significantly reduced the MI in the cerebral cortex and spinal cord, but not in the sciatic nerve, compared with the control group. In the lithium-and TRH-treated group, the MI was also reduced in the cerebellar cortex, but not in the sciatic nerve.
These results show that the reduction of MI in the cerebral cortex, thalamus-caudate nucleus, brainstem and spinal cord may be induced by both the decrease of MI synthesis from glucose and the activation of MI metabolism by cholinergic neurons. In the cerebellar cortex, however, the reduction of MI may be induced by exogenously superimposed TRH activating MI metabolism. The reduction of MI in the sciatic nerve was not thought to be induced by the decrease of MI synthesis. These observations of the difference of MI metabolism in various nerve tissues support a role of MI in the pathogenesis of hyperglycemic or hypoglycemic nerve damage.

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To establish the clinical significance of plasma free amino acids (PFAA) for protein metabolism in 31 patients with non-insulin-dependent diabetes mellitus (NIDDM), plasma concentrations of major free neutral amino acids, including valine (Val), isoleucine (Ileu), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe) and methionine (Met), were examined by a new type of rapid, simplified high performance liquid chromatography.
Significant increases in branched-chain amino acids (BCAA) and Phe were observed, whereas Tyr and Met showed no alterations in patients with NIDDM compared with healthy controls. In addition, significant correlations were found between the ratio of Val +Ileu+Leu (BCAA) to Tyr+ Phe (AAA)(Fischer's ratio) and fasting plasma glucose (FPG) and hemoglobin A₁c (HbA₁c). We also found that Fischer's ratios were significantly elevated in patients with poor diabetic control compared with patients with good control and healthy controls. However, after corrective therapy. Fischer's ratio was restored toward normal in parallel to improvements in FPG and HbAic levels. Furthermore, patiens with diabetic triopathy, especially diabetic nephropathy, showed a significant increase in Fischer's ratio compared with diabetic patients without complications and healthy controls.
These data suggest that Fischer's ratio may well be used as a measure of insulin-dependent alterations of protein metabolism and represents a good indicator for assessing the state of diabetic control, the course and the prognosis of patients with NIDDM, in addition to FPG and HbA₁c.

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It has been emphsized that glucagon, as one of the counter-regulatory hormones, plays a major role in worsening diabetic ketoacidosis and an important role in producing ketone bodies in the liver. The present study was undertaken to determine whether the glucagon action on ketogenesis in the liver is mediated through the cyclic AMP system, using isolated perfused rat liver preparations.
Fed male Wistar rats weighing 200-250 g were used. Experimental detailed procedures were as reported previously. The initial circulating solution was the Krebs-Ringer bicarbonate buffer, containing 2.5% bovine albumin, 25% bovine erythrocytes, 10 mmol/l glucose and 5 mmol/l lactate. Oleate, octanoate, glucagon and dibutyryl cyclic AMP were added to the circulating solution after 30 min of pre-perfusion. Then metabolites such as acetoacetate and β-hydroxybutyrate in the perfusate were determined serially during 90 min of the liver perfusion.
Glucagon significantly promoted the production of ketone body from oleate, but not from octanoate. When glucagon was replaced by dibutyryl cyclic AMP, ketogenesis increased beyond the increase with either oleate or octanoate alone. Emeriamine, an inhibitor of carnitine acyltransferase I, significantly suppressed the ketone body production from oleate alone. Emeriamine completely removed the net increase of ketogenesis from oleate in the presence of either glucagon or dibutyryl cyclic AMP. However, it did not suppress the ketogenesis from octanoate alone. In addition, the additive effect of dibutyryl cyclic AMP on ketogenesis from octanoate remained.
This indicates that the main site of glucagon action on ketogenesis exists in the system mediated through the carnitine acyltransferase I which is located on the outer surface of the internal membrane of the mitochondria. Secondly, this action of glucagon is not always mediated through the cyclic AMP system. Finally, the dibutyryl cyclic AMP stimulation of ketogenesis of the liver in the presence of free fatty acid may be in the metabolic process beyond the carnitine acyltransferase I.

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Nonenzymatic glycation of tissue protein has received attention in relation to the development of diabetic complications.
In the present study, we investigated the formation of lens glycation in diabetic patients using furosine which is formed by acid hydrolysis of fructose-lysine, glucose bound to lysine residues protein, as an indicator. Cataractous lenses were collected from 22 non-diabetic senile subjects and diabetic patients. The isolated cataractous lens was divided into three parts, capsule, cortex and nucleus. Furosine was determined by high-performance liquid chromatography and expressed the ratio of furosine peak area to tyrosine peak area.
The glycation level of the cataractous lens protein in diabetic patients was significantly higher than that in non-diabetic patients. In both groups, the furosine content was decreased in the following order: capsule, cortex and nucleus. A significant positive correlation was found between the HbA1 level and the furosine value in the lens nucleus.
It is considered that diabetes may become a precipitating factor in the aging process through glycation of the lens protein. Furthermore, it suggests that glycation of tissue is an important subject in the studies of the development of diabetic complications and the aging mechanism.

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To investigate the daily difference in diurnal profiles of blood glucose and to set a standard for glycemic control during treatment with sulfonylurea (glibenclamide) in noninsulin-dependent diabetic patients, 12 subjects including six newly treated and six previously treated with sulfonylurea underwent the following examinations. Diurnal profiles of blood glucose were examined when they were poorly controlled with or without glibenclamide. And then, monitoring of three consecutive diurnal profiles was attempted when they were well controlled, a needle-type glucose sensor inserted into the subcutaneous tissue of the forearm using.
It was revealed that the daily difference in glycemic excursions judged by an index of MODD (absolute means of daily difference) was so small as to represent very reproducible diurnal glycemic patterns. Moreover, it was also revealed that postprandial blood glucose exceeded 200 mg/dl when fasting blood glucose stayed between 120 and 140 mg/dl, and postprandial blood glucose remained below 200 mg/dl when fasting blood glucose was below 120 mg/dl. From these results, it is suggested that fasting blood glucose below 120 mg/dl is necessary to attain sufficiently strict glycemic control with sufonylurea to keep postprandial glycemic levels below 200 mg/dl.

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The present study was undertaken to elucidate the role of plasma apoprotein in vascular complications of pancreatic diabetes. In eight pancreatic diabetics, plasma apoprotein Al, AIL B, CII, CIII, and E were determined by the single radial immunodiffusion method. Eachvalue in the pancreatic diabetics was compared statistically with the value for 23 age-matched primary diabetics and 13 healthy controls.
Apoprotein B was 80 ± 10 mg/dl (mean ± SD) in the pancreatic diabetics. This value was significantly lower than that in the primary diabetics (107 ± 28 mg/dl)(p<0.05). Apoprotein atherogenic index estimated as the ratio of apoprotein B to AI or All in the pancreatic diabetics (0.68± 0.22, 2.63±0.87) was lower than that in the primary diabetics (0.98±0.44, 3.82 ±1.43). These results indicate that the changes in plasma approprotein are likely to ensure a low incidence of vascular complications in pancretic diabetes.
Aporprotein B was significantly correlated with LDL cholesterol in healthy controls (r=0.911, p<0.001), in primary diabetics (r=0.829, p<0.001), and in pancreatic diabetics (r=0.901, p<0.01). There were no significant differences for each level of apoprotein CII, CIII, or E in the three study groups.

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We have developed a simple but quantitative method for measuring anti-insulin antibody with beads coated with the antibody, and have applied this newly devised method to the samples obtained from 50 NIDDM and IDDM under insulin therapy. The mean duration of insulin therapy was 5.8 years and the average insulin dosage was 46.4 U/day. Nine of these patients have changed their insulin therapy from porcine or bovine to human insulin. Our bead method showed good reproducibility, a better correlation with total insulin concentration, and a binding capacity with a higher affinity site indicated by Scatchard analysis than the conventional PEG method. This is considered to be more quantitative than the PEG method. Seven of the diabetics studied showed negative insulin antibody, while others had insulin antibody titers distributed getween 14.1 to 10335.6μU /ml by our method. The antibody titers of diabetics decreased upon changing to human insulin theapy. So it is thought to be consistent with the clinical course.

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A series of patients who had suffered from diabetic gangrene was analyzed. Out of 4, 131 diabetics who were admitted to our ward during between 1970 and 1985 (16 years), 47 cases suffered from diabetic gangrene (1.14%). There was a trend that those with gangrene had been diabetic for a long period and were poorly controlled. They frequently had accompanying diabetic complications such as neuropathy, retinopathy and nephropathy. Eighty-five percent improved with conservative therapy but others needed surgical treatment including amputation. Our study indicates that good control of diabetes mellitus is essential for the prevention of diabetic gangrene.

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The aim of this study is to evaluate the influence of prescribed exercises during daily training, and to assess the longterm training effect. Temporal changes in insulin sensitivity following acute exercise were examined by the euglycemic insulin cramp technique.
Eleven healthy male volunteers, 19 to 20 years of age, were involved in the study. Six of these subjects were assigned to an M group with exercise on a treadmill at a moderate load (V0_2max 40-50%) and the remaining 5, to an S group with severe exercise (VO_2max 60-70%) on a bicycle ergometer. All of these subjects were examined by the euglycemic insulin cramp test before the exercise, then immediately after and 24 and 72 hours after the exercise, which invariably lasted 120 minutes.
The mean metabolic rates of glucose for the M group at the 4 consecutive time points were5.53±0.59, 5.71±0.47, 6.61±0.60 and 5.59±0.64 mg/kg/min, respectively. A slight upward tendency appeared only at 24 hours after exercise. The corresponding values for the S group were 6.55±0.61, 6.02±0.36, 8.21±0.94 and 8.32±0.88 mg/kg/min, respectively. The values at 24 and 72 hours post exercise were significantly increased (p<0.05). These results may be interpreted as indicating that while insulin sensitivity improves for at least 3 days following exercise at an increased load of VO_2max 60-70%, exercise at such load levels as are employed in ordinary exercise treatment (V0_2max 40-50%) would not be sufficient to induce a significant favorable change in insulin sensitivity. In view of the tendency for the glucose metabolic rate to increase 24 hours after excercise, it seems advisable to discontinue training for 2 days prior to the euglycemic insulin cramp test in assessing the effect of long-term training programs.

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Although the absolute retention time is a fundamental value in HPLC, there is usually some small variation in every chromatogram, even though the operating conditions are the same. For this reason, the most widespread method used today is the internal standard methd, in which the retention time of a solute is measured relative to that of a reference solute (internal standard). An internal standard added in the sample is also useful as a visible reference during the HPLC run. Such situat.ons often exist in clinical studies such as those of abnormal insulinemia or hyperproinsu linemia, in which the sample concentrations are usually too low to be detected by spectrophotometry.
We prepared three peptide fragments of cytochrome-C by trypsin or protease digestion for use as internal standards for HPLC of human insulin, proinsulin and C-peptide. Under our experimental conditions, coefficients of variability (C.V.) of the absolte retention time were 1.46% for insulin, 1.36% for proinsulin and 0.91% for C-peptide, while the C.V. values of the relaive retention time were much lower: 0.84%, 0.56% and 0.65%, respectively. These results suggest the usefulness of the internal stadard method for HPLC of these hormones.

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