Rosa Campione

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)

Abstract. Human liver slices (surgery biopsies) were preincubated with glucagon or epinephrine for 10 min at 37°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 μM glucagon (n= 7) or 10 μM epinephrine (n= 7) resulted in a reduction of PFK activity of 25% (P < 0.02) and 29% (p < 0.05), respectively. Addition of 2 μM glibenclamide in the preincubation mixture reduced the inhibitory effect of glucagon by 99% (P < 0.05) and that of epinephrine by 70% (p < 0.01). Likewise, 10 μM glucagon (n= 6) or 10 μM epinephrine (n= 4) reduced PK activity by 40% (P < 0.01) and 46% (P < 0.01), respectively. Addition of 2 μM glibenclamide significantly reduced the inhibitory effect of glucagon by 77% (P < 0.05) and that of epinephrine by 33% (P < 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.

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.