Keisuke Kosugi

Metformin or gliclazide, rather than glibenclamide, attenuate progression of carotid intima-media thickness in subjects with type 2 diabetes

Aim/hypothesisMetformin is a well-known oral hypoglycaemic agent and has been commonly used, in combination with sulphonylurea, to treat type 2 diabetes. However, the advantageous effect of metformin plus sulphonylurea on diabetic macroangiopathy has yet to be clarified. To evaluate whether sulphonylurea or sulphonylurea plus metformin prevent diabetic macroangiopathy, we examined the progression of carotid artery intima-media thickness (IMT) as a surrogate end point.MethodsSubjects with type 2 diabetes were divided into three groups, receiving the following treatments: (i) glibenclamide (n=59); (ii) gliclazide (n=30); and (iii) glibenclamide + metformin (n=29). Maximum IMT and average IMT (the greatest value among 6 average values of each 3 points including greatest thickness) were measured at the beginning and end of the observation period.ResultsFor the follow-up period of 3 years, the annual change in average IMT of the glibenclamide plus metformin group (0.003±0.048xa0mm) was smaller than that of the glibenclamide group (0.064±0.045xa0mm) and gliclazide group (0.032±0.036xa0mm) (p<0.0001 and p=0.043 respectively). In the gliclazide group, average IMT increased during the follow-up period, but annual change in average IMT was significantly smaller than that of the glibenclamide group (p=0.005). Glibenclamide + metformin or gliclazide also attenuated the progression of maximum IMT, compared with that of glibenclamide (0.041±0.105, 0.044±0.106, 0.114±0.131xa0mm/year respectively, p=0.029 and p=0.035 respectively). Multivariable regression analysis implied that administration of metformin or gliclazide significantly and independently (p<0.05) reduces the progression of average IMT, compared with glibenclamide monotherapy.Conclusions/interpretationThese data indicate that metformin or gliclazide, rather than glibenclamide, have a potent anti-atherogenic effect in type 2 diabetes.

Ketone bodies as markers for type 1 (insulin-dependent) diabetes and their value in the monitoring of diabetic control.

SummarySerum levels of acetoacetate, 3-hydroxybutyrate and the 3-hydroxybutyrate/acetoacetate ratio were determined in Type 1 (insulin-dependent) and Type 2 (non-insulin-dependent) diabetic patients by a new sensitive method. Efforts were made to differentiate Type 1 and Type 2 diabetes by serum levels of ketone bodies and to determine whether their measurement is a useful way of monitoring diabetic control. In Type 2 diabetes, serum levels of total ketone bodies did not exceed 2.0 mmol/l even if the patients were untreated or poorly controlled. In Type 1 diabetic subjects, treated with once or twice daily injections of insulin, morning serum levels of acetoacetate, 3-hydroxybutyrate and total ketone bodies were significantly elevated by four-, ten- and sevenfold, respectively. In Type 2 diabetic subjects treated with diet or sulphonylureas, serum levels of 3-hydroxybutyrate were highest before breakfast, next highest before dinner and decreased after each meal. The changes were roughly inversely proportional to serum insulin levels. In addition, insulin treatment normalized fasting serum levels of ketone bodies better than diet or sulphonylurea treatment. Acetoacetate was also significantly increased in both types of diabetes to a lesser extent, but no apparent diurnal rhythm was observed. Determination of serum levels of ketone bodies is useful for the diagnosis of Type 1 diabetes (those with total ketone bodies > 2 mmol/l) and for detecting insufficient insulin therapy.

Mechanism of adrenergic stimulation of hepatic ketogenesis

The effects of alpha- and beta-adrenergic stimulation on ketogenesis were examined in freshly isolated rat hepatocytes in order to determine which alpha- or beta-adrenergic stimulation is involved in the enhancement of ketogenesis. In the presence of 0.3 mmol/L (U-14C)-palmitate, epinephrine, norepinephrine, and phenylephrine at 500 ng/mL increased ketogenesis by 25% (16.0 +/- 0.17 v 12.8 +/- 0.13 nmol/mg protein per hour), 20% (15.3 +/- 0.28) and 20% (15.4 +/- 0.36), respectively. However, isoproterenol even at 1 microgram/mL did not stimulate ketogenesis. Phentolamine (5 micrograms/mL) almost completely abolished the effect of epinephrine on ketogenesis (13.7 +/- 0.30 v 16.0 +/- 0.17) but propranolol did not inhibit the stimulation by epinephrine (15.6 +/- 0.38 v 16.0 +/- 0.17). Trifluoperazine (10 mumol/L), presumably an inhibitor of calcium-dependent protein kinase, abolished the effect of epinephrine (13.6 +/- 0.22 v 16.0 +/- 0.17). These results indicate that catecholamines increase ketogenesis predominantly through the alpha-adrenergic system independent of cyclic AMP, and calcium-dependent protein kinase is thought to be involved in the activation of ketogenesis. On the other hand, glucagon stimulated ketogenesis with an increase of cyclic AMP, which was not inhibited by alpha- and beta-adrenergic antagonists. Alpha-adrenergic stimulation increased hepatic glycogenolysis much more at much lower concentrations when compared with ketogenesis. Stimulation of ketogenesis by catecholamines seemed to be less sensitive and responsive compared with hepatic glycogenolysis.

Association of soluble CD40 ligand with carotid atherosclerosis in Japanese type 1 diabetic patients

Aims/hypothesisIt has recently been shown that the soluble form of CD40 ligand (sCD40L) interacts with CD40 on vascular cells, leading to a variety of proinflammatory responses, and that serum sCD40L levels can be a predictive marker of cardiovascular events. The aim of this study was to estimate sCD40L levels in type 1 diabetic patients to examine a possible association with carotid atherosclerosis.Subjects and methodsHuman sCD40L levels in serum and intima–media thickness (IMT) of carotid artery were examined in 80 Japanese type 1 diabetic patients (27 men and 53 women, age 22.8±3.4xa0years (mean±SD), duration of diabetes 13.2±6.1xa0years) and 20 healthy age-matched non-diabetic individuals.ResultsSerum sCD40L levels were significantly (p=0.0185) higher in subjects with type 1 diabetes (2.10±1.33xa0ng/ml) compared with non-diabetic subjects (1.35±0.88xa0ng/ml). The greatest IMT (Max-IMT) and averaged IMT (Mean-IMT) were also significantly greater in patients with type 1 diabetes than in control subjects (0.73±0.14 vs 0.64±0.07xa0mm, p=0.0041, 0.63±0.09 vs 0.57±0.06xa0mm, p=0.0066, respectively). Levels of sCD40L were statistically significantly associated with Max-IMT (r=0.383, p<0.001) and Mean-IMT (r=0.275, p=0.0058). Furthermore, stepwise multivariate regression analyses demonstrated that sCD40L is a determinant of both Max- and Mean-IMT, independently of conventional risk factors.Conclusions/interpretationIt is suggested that increased levels of serum sCD40L are associated with accelerated atherosclerotic change observed in young patients with type 1 diabetes.

Hyperlipidemia and atherosclerosis in experimental insulinopenic diabetic monkeys.

Chronic insulinopenic diabetes was induced by i.v. streptozotocin in the non-human primate Macaca fuscata. Five diabetic monkeys were kept for 8-19 months and nine for 24-48 months without any insulin treatment. Hyperglycemia (241 +/- 22 mg/dl, M +/- SE less than or equal to 1 year) progressed to 376 +/- 34 mg/dl (greater than 2 years) and ketosis to 3.5 mM (greater than 2 years) during the course of diabetes; this was roughly inversely proportional to hypoinsulinemia (3.4 microU/ml, 2 years). Serum cholesterol increased from 184 +/- 11 (less than or equal to 1 year) to 328 +/- 66 mg/dl (greater than 2 years) with the major increase in LDL-cholesterol (2.7-fold over control, greater than 2 years). HDL-cholesterol did not change at all throughout the experimental period. TG increased from 144 +/- 25 (less than or equal to 1 year) to 676 +/- 116 (greater than 2 years) with a major increase in the VLDL fraction (15-fold over control, greater than 2 years). Serum levels of apo B increased to 141 +/- 16 (less than or equal to 2 years) and 223 +/- 8 mg/dl (greater than 2 years) in contrast to control, 73 +/- 2. Morphologically, lipid deposition in the intima and fatty streaks have been observed in the abdominal aorta of all the diabetic monkeys with duration of more than 2 years. In six of the diabetic monkeys atheromatous changes such as intimal and medial thickening with smooth muscle cell proliferation were observed with foam cell formation. Similar atherosclerotic lesions were observed in renal and coronary arteries in at least six of these monkeys. In diabetic monkeys with duration of less than 2 years, mild atherosclerotic lesions were observed in two out of five. The results indicate that long standing insulinopenia leads to metabolic derangements characterized by hyperglycemia, ketonemia and hyperlipidemia. Elevation of LDL-cholesterol and VLDL TG with an increase of apo B is a characteristic of lipoprotein disorder. Morphologically, early to moderately advanced lesions of atherosclerosis were observed in aorta, renal and coronary arteries as a result of metabolic derangement due to insulin deficiency.

A suppressive role of c-kinase for the stimulation of hepatic ketogenesis by glucagon and epinephrine

Long‐chain fatty acid oxidation Ketogenesis c‐Kinase Glucagon Epinephrine Carnitine palmitoyltransferase

Chronically streptozocin-diabetic monkey does not closely mimic human diabetic neuropathy.

In order to evaluate the value of diabetic Japanese monkeys (Macaca fuscatus) as an animal model for studying the pathogenesis of diabetic neuropathy, morphological examinations were performed on myelinated nerve fibers and endoneurial microvessels at three levels of the lower limb nerve in eight streptozocin (STZ)-diabetic monkeys with the duration of diabetes up to 36 months and in four roughly age-matched control monkeys using a computer-assisted image analyzer. Nerve fiber loss was not found, although a tendency for nerve fiber atrophy was found in diabetic monkeys. Endoneurial microvessels did not show either endothelial or pericyte proliferation or basement membrane thickening. The results suggest that chronically STZ-diabetic Japanese monkeys with the duration of diabetes up to 36 months might be useful for studying diabetic axonopathy, but do not closely mimic the nerve pathology found in human diabetic neuropathy.

Effect of insulin on the synthesis and release of lipid peroxide by cultured hepatocytes isolated from normal and diabetic rats

Lipid peroxide content in hepatocytes isolated from ketotic diabetic rats was higher than normal, and the release of peroxide into the media was also elevated for the initial 18 h. Insulin suppressed both peroxide release and synthesis by cultured hepatocytes isolated from normal and from diabetic rats.

In vitro stimulation of glucose utilization by insulin in primary cultures of rat hepatocytes

The effect of glucose concentration and insulin on glucose incorporation was studied in primary cultures of rat hepatocytes. The rate of glucose incorporation into hepatocytes was proportional to the medium glucose concentration from 100 to 800 mg/dl. At 800 mg/dl glucose the rate reached a plateau. Of the glucose taken up by hepatocytes, 16 and 18% was incorporated into glycogen and lipid, respectively, and 58% into the nucleotide fraction after incubation for 4 h. In the medium, lactate was the major product found. Insulin stimulates glucose incorporation by 20-112% into all the above pathways at glucose concentrations between 100 and 800 mg/dl. The insulin effect was noted as early as 2-4 h (early effect) and up to 24 h (delayed effect). This effect of insulin was observed to be dose dependent from 5 to 200 ng/ml insulin. While the delayed insulin effect was abolished by cycloheximide, the early effect of insulin was not affected. With respect to the key enzyme activities of glucose utilization, activation of glycogen synthase (increase of I-activity/total activity) and pyruvate kinase (activation at 0.2 mM phosphoenolpyruvate) was noted 4 h after insulin addition, and these effects were not abolished by cycloheximide. These two enzymes increased in total activity after 24 h. Both glucokinase and glucose-6-phosphate dehydrogenase activities increased by 30-35% and 65-93% at 4 and 24 h, respectively. The results indicate that hepatocytes directly utilize glucose in a dose-dependent manner with respect to glucose and insulin. A major early and delayed effect of insulin appeared due to the activation and induction of the key hepatic enzymes of glucose utilization, respectively.