J.F. Kuo

Insulin-like activity of a microbial protease on isolated fat cells

Abstract A protease produced by Streptomyces griseus has been found to display insulin-like effects on the metabolism of isolated fat cells from rat epididymal tissue. These effects include enhanced conversion of glucose to carbon dioxide and lipid, and repression of the lipolysis stimulated by corticotropin and norepinephrine. Greater than 95% of the proteolytic activity could be abolished without significantly altering the insulin-like activity of the protease. The significance of these observations is discussed.

Stimulation of glucose utilization and inhibition of lipolysis by polyene antibiotics in isolated adipose cells

Abstract Polyene antibiotics, such as filipin, pimaricin, and nystatin, like insulin, were found to stimulate the uptake and the metabolism of extracellular glucose by isolated adipose cells. Such stimulation, however, was abolished when the polyenes were present at high concentrations. The filipin-stimulated lipogenesis from glucose was inhibited by phlorizin, 3- O -methylglucose, 2-deoxyglucose and puromycin. Polyenes neither enhanced nor inhibited the insulin- or the protease-stimulated glucose utilization, but inhibited the deoxyfrenolicin-stimulated process to variable degrees. No significant addition to the stimulatory effects of glucose utilization was observed in the copresence of phospholipase C and polyenes. Phospholipase C, at concentrations high enough to abolish its own stimulation of glucose utilization, also abolished the stimulatory effects of filipin or nystatin, but not of pimaricin. Like insulin, polyenes stimulated still further the lipogenesis from glucose in the presence of palmitic acid. Polyenes blocked lipolysis mediated by either norepinephrine, corticotropin, caffeine or theophylline. It is suggested that the interactions of polyenes with the phospholipid-cholesterol constituent of the lipoprotein membrane imparted to adipose cells activities characteristic of those of the insulin-treated cells.

Inhibition by phlorizin of insulin- and protease-stimulated glucose utilization in isolated adipose cells

1. n1. Utilization of extracellular glucose, determined by its conversion to CO2 and total lipid, has been employed to study the effect of phlorizin on isolated adipose cells incubated with and without insulin, α-chymotrypsin, trypsin and pronase (Streptomyces griseus protease, Type VI). n n2. n2. In dilute glucose medium, the effect of insulin on glucose utilization is to lower the apparent Km, and also to increase the vmax. Phlorizin competitively inhibited glucose utilization without affecting intracellular metabolism, including the reactions involved in lipolysis and oxidation of 14C-labeled cellular materials. n n3. n3. Phlorizin at 0.92 mM inhibited glucose utilization by isolated adipose cells to the same extent in the presence and absence of insulin, α-chymotrypsin or trypsin. Moreover, the degree of inhibition was essentially identical for both parameters measured (CO2 production and lipid synthesis), and remained constant over a wide range of concentrations of these agents. It appears that phlorizin blocks only the process of glucose entry, and does not affect the mechanisms whereby glucose utilization is increased by insulin or proteolytic enzymes.

Effects of deoxyfrenolicin on isolated adipose cells—II: Lipolysis, adenosine 3', 5'-monophosphate levels, and comparison with the effects of vitamin K5

Abstract Deoxyfrenolicin, an antifungal naphthoquinone, inhibited lipolysis induced by norepinephrine, corticotropin, caffeine or theophylline in isolated adipose cells. It was unexpectedly found that deoxyfrenolicin augmented still higher the intracellular adenosine 3, 5-monophosphate (cyclic AMP) levels that were maximally elevated by theophylline plus norepinephrine or corticotropin. Deoxyfrenolicin was without effect on the basal levels of cyclic AMP. Vitamin K 5 , not vitamin K 1 , was found to mimic not only the effects of deoxyfrenolicin on inhibiting lipolysis and augmenting cyclic AMP levels elevated by hormones but also its effects on stimulating glucose oxidation by adipose cells. Vitamin K 1 could not counteract the effects of deoxyfrenolicin or vitamin K 5 . No addition was observed for the effects of deoxyfrenolicin and vitamin K 5 . It seemed that the mode of action of deoxyfrenolicin on adipose cells is similar to that of vitamin K 5 , of which inhibition of electron transport or oxidative phosphorylation may be one of the sites of action.

Effects of avenaciolide on lipolysis and on glucose, amino acid and palmitic acid metabolism in isolated adipose cells

Abstract Avenaciolide, an antifungal lactone, inhibited the lipolysis in adipose cells mediated by lipolytic hormones (adrenocorticotropin and norepinephrine) and phosphodiesterase inhibitors (caffeine and theophylline). Although without effect on the basal oxidation of glucose, avenaciolide abolished the stimulation of glucose oxidation caused by insulin or proteolytic enzymes. It also eliminated the stimulatory effects of these substances on lipogenesis, but inhibited this process to some extent per se as well. Oxidation and esterification of palmitic acid were inhibited, but little effect on amino acid metabolism was noted.

Effects of arsenite of lipolysis and metabolism of glucose, palmitic acid and amino acids by isolated adipose cells

Abstract 1. 1. Arsenite at an optimum concentration of 0.05 mM was found to stimulate about 2-fold the basal glucose utilization (oxidation and lipogenesis) by isolated fat cells. Stimulation of lipogenesis by arsenite at or below the optimum concentration resulted from enhanced synthesis of both fatty acid and glyceride-glycerol, the former being greater than the latter. Reduction in lipogenesis by arsenite at a concentration slightly greater than optimum resulted primarily from inhibition of fatty acid synthesis; glycerol synthesis was not affected. 2. 2. Arsenite at an optimum concentration of 0.1 mM stimulated esterification of palmitic acid, while no significant enhancement in protein synthesis or amino acid oxidation was noted. Metabolism of glucose, palmitic acid and amino acid is greatly inhibited by arsenite at concentrations exceeding 0.1 mM. 3. 3. Lipolysis mediated by lipolytic hormones (adrenocorticotropin and norepinephrine) and phosphodiesterase inhibitors (caffeine and theophylline), employed either singly or in combination, was inhibited by arsenite.

Effects of Bacillus subtilis protease, type VIII (subtilopeptidase A), On mannose and galactose utilization by isolated adipose cells

Abstract 1. 1. Bacillus subtilis protease, Type VIII (subtilopeptidase A, EC 2.4.4.16), like insulin, stimulated the uptake of mannose and the incorporation of the isotope from [1- 14 C]mannose into CO 2 , fatty acid, glyceride-glycerol, protein, and glycogen in isolated adipose cells. [1- 14 C]Galactose was also utlized, however, its utilization was not stimulated by either insulin or B. subtilis protease. Insulin, but not B. subtilis protease, lowered the apparent K m of the mannose oxidation by adipose cells, whereas insulin and the protease were without effect on the apparent K m of galactose oxidation. 2. 2. Mannose reduced the oxidation, but stimulated the esterification, of [1- 14 C]-palmitic acid by adipose cells incubated with or without insulin and B. subtilis protease. The insulin- or the protease-stimulated oxidation of [1- 14 C]mannose was inhibited, but the lipogenesis from it was stimulated in the presence of palmitic acid. 3. 3. Avenaciolide was without effect on the basal lipogenesis from either sugar, but the process stimulated by insulin or B. subtilis protease was inhibited. Puromyin and 2-deoxyglucose inhibited the insulin- or the protease-stimulated oxidation of [1- 14 C]-mannose to a greater extent than the basal process. It is suggested that there may exist functionally distinct pathways for the basal and the insulin- and/or the protease-stimulated pathways for mannose entry into adipose cells. Moreover, the pathways for mannose entry may be similar to those for fructose, but distinguishable from those for glucose.

Effects of Bacillus subtilis protease, type VIII (subtilopeptidase A) on isolated adipose cells Fructose utilization and its inhibition by avenaciolide, piericidin A and puromycin

Abstract 1. 1. It was found that Bacillus subtilis protease, Type VIII (subtilopeptidase A, EC 3.4.4.16), like insulin, stimulated fructose utilization (oxidation and lipogenesis) by isolated adipose cells. The optimum concentration was comparable to the required for maximal stimulation of glucose utilization, about 5 μg/ml, and higher enzyme concentrations resulted in a reduced stimulation. Insulin and B. subtilis protease induced a higher υ max for the utilization of glucose and fructose. Insulin, but not B. subtilis protease, lowered the apparent K m values of either glucose or fructose utilization to about one half of the basal values. 2. 2. The basal and the elevated oxidation of fructose was not affected by palmitic acid, although its conversion to fatty acid was greatly reduced, and its conversion to glyceride-glycerol was stimulated. The protease, like insulin, reduced the oxidation of palmitic acid and enhanced its esterification in the presence of fructose. 3. 3. From the evidence obtained through the use of avenaciolide, piericidin A and puromycin, it is suggested that the basal and the insulin- or the protease-stimulated glucose and fructose uptake pathways may be different in isolated adipose cells, and that glucose and fructose uptake may again not be effected through identical mechanisms. Moreover, the kinetic data also suggested that B. subtilis protease stimulated glucose and fructose utilization by a mechanism quite distinct from that of insulin.

Effects of deoxyfrenolicin on isolated adipose cells. I. Glucose and fructose utilization.

Abstract Deoxyfrenolicin, at an optimal concentration of about 15 to 20 μg ml , stimulated to a greater extent than did insulin the oxidation of glucose and fructose, presumably via the pentose cycle, by isolated adipose cells. Stimulation of glucose uptake and lipogenesis from glucose and fructose were lower than by insulin, however. At a higher concentration (60 μg ml ) of deoxyfrenolicin, the stimulatory effects were reduced or abolished. Deoxyfrenolicin, at a suboptimal concentration (10.5 μg ml ), stimulated still further the maximally stimulated lipogenesis from glucose and fructose in response to insulin or several “insulin-like” proteases. It is suggested that deoxyfrenolicin stimulated sugar utilization in isolated adipose cells, at least in part, through a mechanism different from the actions of insulin or proteases.

Effects of piericidin A on the metabolism of isolated adipose cells

Abstract The effects of piericidin A, a structural analog of coenzyme Q, on the metabolism of isolated adipose cells were studied. Piericidin A inhibited both the basal and the insulin-stimulated glucose and fructose utilization (oxidation and lipogenesis). Piericidin A completely abolished the stimulatory effects of proteases ( Bacillus subtilis protease, Streptomyces griseus protease and α-chymotrypsin) on glucose utilization. The utilization of palmitic acid was inhibited by piericidin A to a greater extent than amino acids. Piericidin A accelerated glycogenolysis, but exerted no significant effects on the level of lipid content or the oxidation of the cellular components. The lipolysis induced by lipolytic hormones (corticotropin and norepinephrine) or phosphodiesterase inhibitors (caffeine and theophylline) or by both was also blocked by piericidin A. The effects of peiricidin A on the isolated adipose cells were interpreted as probably being due to plasma membrane effects and possible effects on the adenyl cyclase system.