Response of insulin, glucagon, lactate, and nonesterified fatty acids to glucose in visceral obesity with and without NIDDM: relationship to hypertension.

Insulin, glucagon, glucose, nonesterified fatty acids (NEFA), and lactate response to oral glucose tolerance test (OGTT, 75 g glucose) and their correlation with mean blood pressure (BP), were studied in 10 normal subjects (N), 25 subjects with abdominal obesity (O), and 9 subjects with abdominal obesity and IGT or non-insulin-dependent diabetes (OD). O and OD patients, as compared to N subjects, showed increased fasting NEFA, lactate, insulin, and glucagon. NEFA area and insulin total and incremental areas were increased in O and OD (P < 0.001 in all instances). Glucagon total areas were increased only in OD (P < 0.01). Lactate total areas were increased in O (P < 0.001) and in OD (P < 0.01), while lactate incremental area was diminished in O and, even more, in OD subjects (P < 0.001 in both instances) and was inversely correlated with the basal level (P < 0.001). In all subjects as a whole, increase in NEFA area was weakly correlated with total and incremental insulinemic areas (P < 0.05) and more strongly correlated with glucagon and lactate areas (P < 0.01). Conversely, the incremental areas of lactate were negatively correlated with total insulin (P < 0.05), NEFA (P < 0.05), and glucagon (P < 0.001) areas. BP was increased in O (103.62 +/- 2.37) and, even more, in OD (109.41 +/- 5.22) compared to that seen in N (92.55 +/- 0.94 mm Hg), with P < 0.01, and was correlated with fasting insulin (P < 0.01) and glucose (P < 0.05) and, even more, with total (P < 0.001) and incremental (P < 0.01) insulin areas and NEFA areas (P < 0.001). Conversely, BP also was negatively correlated with incremental lactate area (P < 0.01) (similarly to insulin and NEFA area). Our data would suggest that in O and OD patients, insulin resistance is associated with elevated NEFA, insulin and glucagon as well as with high BP. since NEFA are inhibitors of Na,K-ATPase, they could contribute to elevate BP through the repression of this enzyme (which we have shown previously to be reduced in adipose tissue of obese subjects and correlated negatively with BP.

Rabbit lens and retina phosphorylate glucose through a glucokinase-like enzyme: study in normal and spontaneously hyperglycemic animals.

After having previously shown that some noninsulin-sensitive tissues (capillaries and optic nerve) phosphorylate glucose in a concentration-dependent manner through a glucokinase-like enzyme, here, we report data on glucose phosphorylation in rabbit lens and retina at various glucose concentrations (1, 5, 10, 25, 50, and 100 mmol/L). In the 3000 g supernatant of lens and retina homogenates from two separate groups of female albino rabbits ten animals in each group; 1.8-2.0 kg body weight; mean +/- SEM morning glycemia: 8.19 +/- 0.28 and 8.12 +/- 0.24 mmol/L, respectively) was assayed glucose phosphorylating activity (NADP reduction measured as change in optical density at 366 nm at pH 7.5). The enzyme activity did not reach the maximum at low glucose concentration (1 mmol/L), as it occurs in several tissues, but increased progressively in both tissues with the increase in glucose concentration. Values (mean +/- SEM) for lens were 0.197 +/- 0.031 nmol/min/mg protein at 1 mmol/L and 0.327 +/- 0.051 (the highest value) at 50 mmol/L glucose (+65.99%, p < 0.01; r = 0.31, p < 0.05). Values for retina were 36.02 +/- 2.12 at 1 mmol/L glucose and 42.48 +/- 2.79 (the highest value) at 25 mmol/L glucose (+17.93%, p < 0.001; r = 0.32, p < 0.05). These kinetic characteristics, somewhat reminiscent of those shown by hepatic glucokinase, are still more pronounced when we calculated the "glucokinase component," obtained by subtracting the activity at 1 mmol/L glucose (hexokinase component) from that at the highest glucose concentration (total glucose phosphorylating activity). In five rabbits of similar age and weight, with spontaneous hyperglycemia (mean +/- SEM morning glycemia: 11.71 +/- 0.60) glucose phosphorylation in the retina was lower than normal, value at pH 7.5 and 1 mmol/L glucose being 24.52 +/- 2.20 versus 36.02 +/- 2.12 of normal animals (-31.93%, p < 0.01). This, if occurs also in other tissues, could contribute to the hyperglycemia by reducing glucose utilization. In these animals, however, the glucose phosphorylating activity retained the responsivity to increasing glucose concentrations, with value at 100 mmol/L of 28.65 +/- 2.10, corresponding to + 16.84% over the value at 1 mmol/L (p < 0.01). Therefore, the actual glucose phosphorylation in the retina of these animals would depend both upon the enzyme level (which is reduced) and glucose concentration (which is increased). Due to the in vivo inhibition of the hexokinase component by glucose 6-phosphate, the glucokinase component in retina and lens may be predominant in vivo, making the stimulating effect of hyperglycemia much more important than it would appear from our in vitro data. This might play a role in the chronic diabetic complications.

Rabbit optic nerve phosphorylates glucose through a glucokinase-like enzyme: studies in normal and spontaneously hyperglycemic animals.

We investigated glucose phosphorylation at various concentrations of glucose (1, 5, 10, 25, 50, 100 mmol/liter) in rabbit optic nerve. In the 3000 g supernatant of whole rabbit optic nerve homogenates from female albino rabbits (n = 10, 1.8-2.0 kg body weight, mean +/- SEM morning glycemia: 8.25 +/- 0.29 mmol/liter), the glucose phosphorylating activity (NADP reduction measured as change in optical density at 366 nm at pH 7.5) increased progressively with the increase in glucose concentration (r = 0.89; P < 0.05) and approached the maximum at a very high glucose level (100 mmol/liter), with values (mean +/- SEM) of 8.75 +/- 0.97 nanomol/min/mg protein and 11.57 +/- 1.15 at 1 and 100 mmol/liter glucose, respectively (+32.23%; P < 0.01). At a more alkaline pH (8.2; n = 5, mean +/- SEM morning glycemia: 8.83 +/- 0.07 mmol/liter) glucose phosphorylation was higher than at pH 7.5 and retained the glucose concentration dependence (r = 0.95, P < 0.01). These kinetic characteristics are reminiscent of those of the low-affinity enzyme glucokinase, which is typically present in the liver. By subtracting the activity at 1 mmol/liter glucose from that at higher glucose concentrations, we calculated the "glucokinase component," forms the "total" glucose phosphorylating activity. In five rabbits (of similar age and weight) with spontaneous hyperglycemia (mean +/- SEM: 11.71 +/- 0.60 mmol/liter), the optic nerve glucose phosphorylating activity was lower (value at 1 mmol/liter glucose: 5.42 +/- 1.31, -38.06%, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

[Protein S deficiency and thrombophilia: presentation of a clinical case and review of the literature]. / Deficit di proteina S e trombofilia: presentazione di un caso clinico e revisione della letteratura.

We report the case of a 22-year-old obese woman with severe protein S deficiency, probably genetic in nature, associated with recurrent venous thrombosis. Protein S deficiency is a rather rare disease: it may be an inherited, either homozygous (purpura fulminans at neonatal age), heterozygous, or acquired disorder. The thrombophilic state may be manifested as deep vein thrombosis or thrombophlebitis of the superficial veins with a high risk of pulmonary embolism in the young, and it is often exacerbated by pregnancy. In our case, the presenting event, bilateral deep venous (iliac-femoral) thrombosis complicated by disseminated intravascular coagulation, had occurred when the patient was 13 years old. We started long-term therapy with oral coagulants, i.e. warfarin even if the latter may cause skin necrosis ("warfarin dermatitis") in some patients with protein S deficiency. The clinician must consider protein S deficiency in cases of recurrent thrombosis, particularly in young patients: the importance of early implementation of long-term preventive therapy should not be underestimated.

A glucokinase-like enzyme carries out glucose phosphorylation in capillaries of normal and spontaneously hyperglycemic rabbits.

We have studied glucose phosphorylation at increasing glucose concentrations (1, 5, 10, 25, 50, and 100 mmol/liter) in capillaries of the choroidocapillary lamina from the eye of normal female albino rabbits (n = 10; body wt 1800-2000 g; mean +/- SEM morning glycemia: 147.77 +/- 4.02 mg/dl) and from the eye of spontaneously hyperglycemic rabbits (n = 5, body wt 1800-2000 g, mean +/- SEM morning glycemia; 211.00 +/- 10.76 mg/dl). In the 3000g supernatant of capillary homogenates, the glucose phosphorylating activity (NADP reduction measured as optical density change at 366 nm at pH 7.5) increased progressively with the rise of glucose concentration (r = 0.36; P < 0.05), approaching the peak at high glucose level (25 mmol/liter), with values ranging from 5.32 +/- 0.46 (SEM) nmol/min/mg protein to 7.14 +/- 0.74 (+34.21%, P < 0.01). When measured at a more alkaline pH (8.2) the glucose phosphorylation was higher than at pH 7.5 and retained the responsiveness to increasing glucose concentrations. These kinetic characteristics differ from those seen in most tissues and are somewhat reminiscent of those shown by hepatic glucokinase. Indeed, by subtracting the activity at 1 mmol/liter glucose from that at higher glucose concentrations, we calculated the "glucokinase component" which together with the "hexokinase component" form the total glucose phosphorylating activity. Glucose phosphorylation in capillaries from spontaneously hyperglycemic rabbits was lower than normal (values: 3.66 +/- 0.31 vs 5.32 +/- 0.46 of the normal rabbits; -31.20%; P < 0.05). This could contribute to the hyperglycemia by reducing glucose utilization. However, in these animals the enzyme activity retained the responsivity to increasing glucose concentrations (r = 0.41, P < 0.05). Therefore, the actual capillary glucose phosphorylation in these animals would depend upon both the enzyme level (which is reduced) and the glucose concentration (which is increased). Due to the in vivo inhibition of the hexokinase component, the glucokinase component may be predominant in vivo, making the stimulating effects of hyperglycemia much more pronounced than it would appear from our data in vitro. This may lead to glucose overutilization. These kinetic characteristics of glucose phosphorylation in capillaries might be relevant to the mechanisms leading to diabetic microangiopathy.

[Use of an automatic blood glucose measurement system for the assessment of insulin resistance during the oral glucose tolerance test]. / Utilizzazione di un sistema di controllo automatico della glicemia per la valutazione della insulino-resistenza durante OGTT.

An automatic glycemic control system (Beta-like, Esaote) was used to calculate the insulin area (IA) required to keep glycemia within the normal range during OGTT (using NDDG criteria). IA was calculated by adding total endogenous insulin to insulin infused by the Betalike system (Actrapid HM, Novo). During the test, glycemia was obliged to follow a mean normal curve using an insulin infusion according to a special algorithm which automatically adapted to individual parameter variations during the different stages of OGTT. Fourteen blood samples were collected to assay metabolites (glucose, NEFA, lactate and alanine) and hormones (insulin, C peptide, glucagon). Data on insulinemia and glycemia were used to calculate the respective areas under the total and incremental curve (IA expressed in UL-1 min-1 and GA expressed in mM.L-1.min-1); an insulin resistance index was then calculated (total and incremental) using the following formula: IA/(normal GA/patient GA). This test allows us: a) to evaluate the insulin secretory response to a standard glycemic stimulus represented by a glycemic curve within the normal range; b) to calculate the quantity of insulin necessary to maintain the glycemic curve within the normal range; c) to evaluate the body's total insulin resistance according to an index calculated on the basis of the insulin area required; d) to compare the calculated insulin resistance index with NEFA and glucagon data obtained during the test; e) to identify the exact evolution of these events over time during OGTT.

Capillaries phosphorylate glucose in a concentration-dependent manner through a glucokinase-like enzyme: a study in the eel.

Glucose phosphorylation was studied in a pure capillary preparation obtained from the rete mirabile of the eel swimbladder. In the 3000g supernatant of capillary homogenates, the glucose phosphorylating activity did not reach the maximum at low glucose concentration (1 mmole/liter), as it occurs in most tissues, but increased with the increase in glucose concentration and approached the maximum at very high (300 mmole/liter) glucose levels, with values (mean +/- SEM, n = 10) of 5.85 +/- 0.94 nmole.min-1.mg-1 protein and 19.97 +/- 1.89 at 1 and 300 mmole/liter glucose, respectively. The apparent Km value for glucose was about 50 mmole/liter, i.e., at supraphysiological glucose concentration, like the enzyme glucokinase, typically present in the liver but absent from most other tissues. This new enzyme did not phosphorylate fructose (similar to glucokinase from liver, which is rather specific for glucose) but was not inhibited by N-acetyl-glucosamine (in contrast to hepatic glucokinase). Thus, capillaries phosphorylate glucose in a concentration-dependent manner, which suggests that they are equipped with a glucokinase-like enzyme. This may explain the reported increase in glucose uptake during capillary exposure to high glucose concentrations and would suggest that the hyperglycemia of the diabetic state may be associated with increased glucose utilization, which may play a role in the development of microangiopathy.

Extra-pancreatic action of glibenclamide in man: reduction in vitro of the inhibitory effect of glucagon and epinephrine on the hepatic key glycolytic enzymes phosphofructokinase (PFK) and pyruvate kinase (PK).

Human liver slices (surgery biopsies) were preincubated with glucagon or epinephrine for 10 min at 37 degrees C in Krebs-Henseleit solution at pH 7.4, in the absence or presence of glibenclamide, and then homogenized and assayed for phosphofructokinase (PFK) and pyruvate kinase (PK) activity at subsaturating, near physiological, substrate concentrations (suitable for detecting regulatory effects). Preincubation with 10 microM glucagon (n = 7) or 10 microM epinephrine (n = 7) resulted in a reduction of PFK activity of 25% (P less than 0.02) and 29% (P less than 0.05), respectively. Addition of 2 microM glibenclamide in the preincubation mixture reduced the inhibitory effect of glucagon by 99% (P less than 0.05) and that of epinephrine by 70% (P less than 0.01). Likewise, 10 microM glucagon (n = 6) or 10 microM epinephrine (n = 4) reduced PK activity by 40% (P less than 0.01) and 46% (P less than 0.01), respectively. Addition of 2 microM glibenclamide significantly reduced the inhibitory effect of glucagon by 77% (P less than 0.05) and that of epinephrine by 33% (P less than 0.05). In the absence of the hormones, glibenclamide was without effect. Thus, glibenclamide opposes the inhibitory effect of glucagon and epinephrine on two key hepatic glycolytic enzymes. Since the inhibition of key glycolytic enzymes favours gluconeogenesis, the observed action of glibenclamide, if it occurs also in vivo, might reduce the glucagon- and epinephrine-stimulated gluconeogenesis, and could be regarded as an insulin-like action.

Metabolic effects of high glucose concentrations: inhibition of hepatic pyruvate kinase.

We tested the in vitro effects of various glucose concentrations on the activity of hepatic pyruvate kinase, assayed at subsaturating, near physiological concentration (0.20 mmol/l) of the substrate phosphoenolpyruvate, to detect the "active" form of the enzyme. A 10-min incubation of mouse liver slices (n = 18) with increasing glucose concentrations (5, 10 and 20 mmol/l) resulted in a significant (p less than 0.01), progressive pyruvate kinase inhibition of 15, 28 and 41%, respectively. Similar data were obtained by incubating mouse liver homogenates (n = 7) with glucose, although with this material (which was supplemented with the pyruvate kinase activator fructose-1,6-diphosphate) the inhibition at the highest glucose concentration used was lower (24%, p less than 0.02). Addition of 10 nmol/l insulin during slice incubation (n = 8) prevented by 98% and 69% the inhibition exerted by 10 and 20 mmol/l glucose, respectively. Insulin alone was without effect on the enzyme activity. Glucose might inhibit pyruvate kinase by competing with the activator fructose-1,6-diphosphate. Insulin might overcome the glucose effect by activating pyruvate kinase through the known mechanism of enzyme dephosphorylation. Thus, in decompensated diabetes the high level of blood glucose may contribute, together with the counterregulatory hormones, to inhibit hepatic pyruvate kinase and therefore to stimulate gluconeogenesis.

Increased hexokinase/glucose-6-phosphatase ratio in the diabetic kidney as index of glucose overutilization.

In mice with streptozotocin-induced diabetes of 3 days' duration, the hexokinase/glucose-6-phosphatase (HK/G6Pase) ratio in the kidney was enhanced by 52% (mean +/- SEM: 0.40 +/- 0.04 vs. 0.26 +/- 0.03; p less than 0.02) compared to control mice as a result of a 25% increase of HK (16.68 +/- 0.93 vs. 13.31 +/- 1.04 nmol/min/mg protein; p = 0.05) and a 17% decrease of G6Pase (42.51 +/- 2.75 vs. 51.25 +/- 1.89; p less than 0.05). In contrast, as expected, the corresponding ratio (HK + glucokinase/G6Pase) was strikingly reduced in the liver. In 9-day diabetic mice, the kidney enzyme changes were much smaller; however, in a chronic disease such as diabetes, even minimal deviations from the normal may lead to significant metabolic changes with time. The enhanced HK/G6Pase ratio in the diabetic kidney suggests an increase in glucose utilization. This may contribute to the increased synthesis of glycogen, glycoproteins (including basement membrane) and RNA (via provision of ribose-phosphate) occurring in the diabetic kidney and supports the view that the kidney (as opposed to other tissues) shows an 'anabolic response' to diabetes.

Metabolic effects of short-term fasting in obese hyperglycaemic humans and mice.

The aim of this study was to investigate the metabolic effects of short-term fasting in obese diabetic patients and to correlate the observed changes with the activity of hepatic key enzymes in an animal model of obesity-associated diabetes (ob/ob mice, C57BL/6J strain). In obese diabetic patients (ODP), a 72-h fast (causing slight change in body weight) decreased fasting glycemia by 3.82 +/- 0.79 mmoles/l and significantly improved glucose tolerance (OGTT) while reducing basal and stimulated insulinemia, whereas in obese non-diabetic patients (ONDP) only a small decrease in fasting glycemia (1.24 +/- 0.51 mmoles/l) occurred. This suggests that in ODP hyperphagia is a factor contributing to maintain hyperglycaemia and glucose intolerance (in the face of hyperinsulinaemia, indicating insulin resistance). In fed obese hyperglycaemic mice (OHM), which are a good model of the human obesity-associated diabetes, hepatic fructose-1,6-diphosphatase (F16Pase) and glucose-6-phosphatase (G6Pase), involved in glucose production, showed increased activity (+52 and +200 per cent, respectively) compared to control mice (CM), and the ratios of F16Pase and G6Pase to the opposing enzymes phosphofructokinase (PFK1) and glucokinase (GK), i.e. the F16Pase/PFK1 and G6Pase/GK ratios, were increased by 38 and 101 per cent, respectively, suggesting increase in gluconeogenesis and perhaps in glycogenolysis. In the 48-h fasted OHM, F16Pase activity was decreased (-30 per cent) compared to the fed animals, while the activity of G6Pase showed a smaller and statistically not significant change (-22 per cent). In contrast, in the CM a 48-h fasting was associated with a trend toward increased F16Pase (+22 per cent) and G6Pase (+173 per cent). However, since PFK1 and GK decreased to a similar extent in OHM and CM, the F16Pase/PFK1 and G6Pase/GK ratios, basally elevated in the OHM, did not change with fasting, whereas in the CM they showed a striking elevation (+71 and +274 per cent, respectively). The basally elevated F16Pase/PFK1 and G6Pase/GK ratios (functionally linked to glucose production) in the OHM may contribute to maintain hyperglycaemia; in these mice, the lack of further increase in the glucose production-related F16Pase/PFK1 and G6Pase/GK ratios (which occurs in CM) with fasting might allow that the interruption of the afflux of dietary carbohydrates ameliorates the glycaemic level. Similar mechanisms might occur also in the ODP.

Anabolic response of some tissues to diabetes.

In contrast to liver, adipose tissue, and muscle, in which the diabetic state is associated with a "catabolic response," some tissues, typically the kidney and perhaps the intestinal mucosa and some vascular cell types, show an "anabolic response" to diabetes, with enhanced activity of the anabolic pathways and diminished activity of the catabolic ones. The kidney of alloxan or streptozotocin diabetic rats is hypertrophied, and shows enrichment in intracellular glycogen and abundant accumulation of glycoprotein material at the basement membrane level. Accordingly, protein synthesis and the enzymes of glucose utilization as well as those engaged in UDP sugar formation or in the hydroxylation and glycosylation processes (required for glycoprotein synthesis) show increased activity in the diabetic kidney, while the catabolic, lysosomal enzymes (cathepsin D and several glycosidases) are depressed. We observed a reduction of -24% in the activity of cathepsin D and -23% in that of galactosidase in the kidney of streptozotocin diabetic mice, as opposed to increases of +135 and +32%, respectively, found in liver. It is not known which factor(s) may be responsible for such an anabolic response of some tissues to diabetes, but persistent hyperglycemia and/or some hormonal abnormalities may be involved. The above data refer to changes in tissue enzyme content caused by induction-repression mechanisms, but rapid (activation-inhibition) effects may also occur. We observed that preincubation of slices of mouse kidney cortex for 10 min with 20.8 mmole/liter glucose resulted in a 80% activation of phosphofructokinase, as assayed in the tissue homogenate at physiological (50 mumole/liter) concentration of the substrate fructose-6-P, suggesting that hyperglycemia may be responsible for some of the metabolic changes occurring in the diabetic kidney.

Cathepsin D and other hydrolases in the kidney of streptozotocin-diabetic mice. Possible relevance to microangiopathy.

The lysosomal enzymes cathepsin D (E.C. 3.4.23.5), alpha-glucosidase (E.C. 3.2.1.20) and beta-galactosidase (E.C. 3.2.1.23), potentially involved in the breakdown of the peptide component and the disaccharide units of basement membrane glycoproteins, were studied in the kidney cortex and liver of streptozotocin-diabetic mice. In the liver of diabetic mice, as compared to controls, an increase was found for the total activity (measured in frozen-thawed homogenates) of cathepsin D (+135%, P less than 0.01) and beta-galactosidase (+32%, P less than 0.05). In the kidney a decrease was observed for both the free activity (measured in 12,000 g supernatant) and the total activity of these two enzymes (cathepsin D: -62% and -24%; beta-galactosidase: -29% and -23%; P less than 0.05 in all instances). Alpha-glucosidase did not show significant changes in either tissues. Total protein content of the two organs did not change significantly with diabetes and therefore cannot account for the enzyme alterations observed. These data indicate that the response of kidney to diabetes is opposite to that of liver (decrease versus increase in catabolic enzymes), and suggest decreased degradation of basement membrane in some tissues in diabetes, which may contribute to the thickening of basement membrane and therefore to the development of microangiopathy.

Insulin resistance in the obese hyperglycemic (ob/ob) mouse. Failure of hyperinsulinemia to activate hepatic pyruvate kinase (PK).

In obese hyperglycemic (ob/ob) mice, as compared to controls, hepatic pyruvate kinase (PK) activity was enhanced by 35.63% (214.75 +/- 13.60 nmol/min/mg protein v 158.33 +/- 10.47, P less than 0.01) when measured at saturating (6.6 mmol/L) concentration of the substrate phosphoenolpyruvate (total activity), but the activity recorded at subsaturating (1.3 mmol/L) substrate concentration (active fraction) was unchanged (86.37 +/- 6.42 v 85.66 +/- 13.59) or even decreased if expressed as percent of the total activity (40.21 +/- 2.56% v 54.10 +/- 5.07, P less than 0.05). Since insulin induces the synthesis of hepatic PK and favors the conversion of the inactive (phosphorylated) to the active (dephosphorylated) form, these findings suggest that in ob/ob mice the striking hyperinsulinemia, although it is able to increase the hepatic content of PK, fails to activate this enzyme. This may favor gluconeogenesis in these animals. The hepatic concentration of PK effectors (fructose-1,6-P2 and phosphoenolpyruvate) was unchanged in ob/ob mice, and the in vitro effect of the activator fructose-1,6-P2 (15 mumol/L), which would favor the activation (dephosphorylation) of PK, was preserved. It is suggested that hepatic PK in ob/ob mice is resistant to activation by insulin.