Metabolism of dioctanoylglycerol by isolated cardiac myocytes

Diacylglycerol (DG) metabolism by intact cardiac myocytes isolated from adult rat hearts and by broken cell preparations from myocytes was investigated in experiments with [3H]dioctanoylglycerol (diC8), a cell-permeable diacylglycerol analog. In a low-speed supernatant fraction, the Km and Vmax for DG kinase activity (formation of phosphatidic acid) was 22 microM and 110 nmol/h/mg, respectively, whereas the Km and Vmax for DG lipase activity (formation of monoacylglycerol) was 80 microM and 1000 nmol/h/mg. At a substrate concentration of 80 microM diC8, lipase activity was 7-fold greater than kinase activity. The majority of DG kinase activity was recovered in the soluble subcellular fraction; DG lipase activity was localized in a microsomal fraction. When [3H]diC8 was incubated with intact cardiac myocytes, 10-fold more radioactivity was incorporated into the products of the lipase pathway (monoacylglycerol and free glycerol) as compared to incorporation into the total phospholipid fraction which contained phosphatidic acid. This predominance of metabolism by hydrolysis through the lipase pathway was observed consistently when the incubation time, content of cardiac myocytes and concentration of exogenous diC8 was varied. Therefore, results from both in vitro determinations of enzyme activities in broken cell preparations and flux studies with intact cells have indicated that the lipase pathway is the principal enzymatic mechanism for the metabolism of diC8 in cardiac myocytes.     

Protein kinase inhibitor blocks the activation of a myocardial triacylglycerol lipase

Purified inhibitor of the cyclic AMP-dependent protein kinase (PKI) has been used as a probe to determine if hormone and cyclic AMP-induced activation of the cardiac alkaline triacylglycerol (TG) lipase is mediated through the cAMP-dependent protein kinase. Addition of CAM (cyclic AMP, Mg-ATP, and 3-isobutyl, 1-methylxanthine) to any of the four fractions (homogenate, 10,000 g supernatant, 105,000 g supernatant, or heparin-Sepharose eluate) from heparin perfused heart activated the TG lipase 60% to 110%. Preincubation of these fractions with 33 ng of PKI had no effect on control enzyme activity. Addition of PKI (33 ng) to extracts following CAM activation had little effect on homogenate TG lipase activity, but reduced activities in 10,000 g and 105,000 g supernatant fractions to their respective control levels, and inhibited TG hydrolase activity of activated heparin-Sepharose eluate to 50% below the control activity. If extracts were preincubated with PKI prior to CAM addition, TG lipase activity was reduced to approximately 50% below control levels in all fractions. PKI addition (33 ng) to 105,000 g supernatant obtained from hearts stimulated 60% by epinephrine perfusion reduced activity to 50% below the control level. PKI inhibition of TG lipase activity of 105,000 g supernatant could be reversed by adding 0.5 microgram of catalytic subunit of protein kinase (PKC) to the extract. The inhibition below control levels caused by CAM and PKI indicate that the PKI-PKC complex by itself or in combination with other extract molecules, has an inhibitory effect on the TG lipase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of insulin on the utilization of 14C-glycerol and 14C-glucose in hepatectomized nephrectomized rats

Insulin (i.v.) administration to functionally hepatectomized-nephrectomized rats did not alter circulating levels of glycerol and only slightly affected plasma radioactivity when animals received (U-14C)-glycerol, whereas after (U-14C)-glucose administration insulin enhanced hypoglycemia and greatly accelerated the rate of radioactivity loss from plasma. At 15 min after i.v. injection of (U-14C)-glycerol, radioactivity in total lipids was reduced in heart and lungs by insulin administration and enhanced in carcass and brown adipose tissue. These effects involved the 14C-glyceride glycerol fraction in the case of heart and 14C-fatty acids in carcass and adipose tissue. When (U-14C)-glucose was administered, insulin enhanced the appearance of 14C-water-soluble material in heart and carcass and 14C-total lipids in heart, carcass, and both brown and white adipose tissue. The effect in heart corresponded mainly to the 14C-glyceride glycerol fraction whereas it corresponded to the 14C-fatty acids in the other tissues. Therefore, insulin effects on glycerol metabolism substantially differ from those on glucose. Opposite effects on heart and lung glycerol utilization as compared to those in carcass and brown adipose tissue may account for the difficulties in observing changes in plasma glycerol levels after insulin treatment.     

Lipoprotein lipase is produced, regulated, and functional in rat brain.

Lipoprotein lipase (LP lipase, triacylglycero-protein acylhydrolase EC 3.1.1.34) activity was found in four dissimilar brain regions (hypothalamus, cortex, cerebellum, and midbrain) of adult male rats. Progressive accumulation of LP lipase activity in cultured fetal rat hypothalamic cells was also observed, indicating de novo synthesis of the lipase. The brain LP lipase activity was serum-dependent and was inhibited by 1 M NaCl and by protamine sulfate. Kinetic analysis revealed an apparent Km of 0.79 mM very similar to that of rat adipose tissue LP lipase. That the lipase was functioning in the cultured brain cells was indicated by uptake and incorporation of radioactivity from tri[( 1-14C]oleoyl)glycerol into cellular triacylglycerols, and into more polar lipids, such as phosphatidylcholine. Furthermore, brain LP lipase activity in adult rats was decreased in all four regions examined, most significantly in the hypothalamus, after 72 hr of food deprivation. Thus, authentic LP lipase is present in adult rat brain and can be synthesized by isolated brain cells in vitro. LP lipase also mediates the uptake of triacylglycerol fatty acids and their subsequent incorporation into cellular lipids of cultured brain cells. Decreased brain LP lipase activity after fasting suggests that this enzyme may be regulated by metabolic or nutritional factors. Because the largest changes in LP lipase activity in response to food deprivation occurred in the hypothalamus, the enzyme may have a role in hypothalamic control of food intake or in body-weight regulation.     

Regulation of rat heart triacylglycerol ester hydrolases by free fatty acids,fatty acyl CoA and fatty acyl carnitine

The regulation of triacylglycerol ester hydrolase (TG lipase) activity was studied in a pH 5.2 precipitate fraction from rat hearts. Neutral TG lipase activity (assayed at pH 7.5) was measured with both ethanolic and glycerol-dispersed triolein substrate preparations; acid lipase activity was determined at pH 5 with a glycerol-dispersed triolein substrate preparation containing lecithin. Neutral TG lipase activity was inhibited by free fatty acids (oleic acid) and by fatty acyl CoA compounds (oleoyl CoA, palmitoyl CoA); concentrations required for half-maximal inhibition were 100 μm and 20 to 25 μm, respectively. Palmitoyl carnitine resulted in an increase in neutral TG lipase activity. The inhibition of neutral TG lipase activity by oleoyl CoA could be prevented by increasing the content of albumin in the assay, and was reduced when the triolein substrate concentration was increased. Fatty acyl CoA compounds also inhibited acid TG lipase activity, but palmitoyl carnitine was the most effective inhibitor (concentration for half-maximal inhibition of 30 μm). The inhibition of neutral TG lipase activity by fatty acyl CoA is consistent with the observation that metabolic interventions that increase tissue levels of fatty acyl CoA result in an inhibition of rates of cardiac lipolysis.     

Prevention of diet-induced obesity by dietary isomerized hop extract containing isohumulones, in rodents

OBJECTIVE: Isomerized hop extract (IHE), which consists mainly of isohumulones and is required in the beer brewing process, was investigated for its effects on diet-induced obesity in two strains of mice. DESIGN: C57BL/6N and KK-A(y) mice were fed a standard or high-fat diet containing IHE and their body and tissue weights were measured at various time points. Oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT) were carried out in high-fat diet-fed C57BL/6N mice. The effects of IHE on intestinal lipid absorption were examined in Wistar rats using a plasma triacylglycerol assay after oral administration of a lipid emulsion. Fecal lipid levels were also measured in these animals after they were fed a high-fat diet containing IHE for 15 days. The effects of IHE on pancreatic lipase activity and the expression of genes involved in hepatic lipid metabolism were also examined using an in vitro assay and quantitative RT-PCR, respectively. RESULTS: Supplementation of high-fat-containing chow with IHE reduced body weight gain and improved glucose tolerance in our experimental mice. A reduction in body weight gain was also observed in C57BL/6N mice fed a standard diet containing IHE. Wistar rats fed a high-fat diet containing IHE showed reduced plasma triacylglycerol levels and an increase in their fecal lipid excretion. Similarly, their pancreatic lipase activity was inhibited and their elevation in plasma triacylglycerol levels seen after the oral administration of lipid emulsion was significantly suppressed. IHE-fed mice showed an increased expression in their lipid oxidation genes and a decreased expression in genes involved in triacylglycerol biosynthesis. CONCLUSION: The inhibition of intestinal dietary fat absorption may be the mechanism by which IHE induces its weight-lowering effects in high-fat diet-fed mice. The modulatory effect of IHE on lipid metabolism may also, at least partly, be responsible for its beneficial effects on body weight gain. These results suggest that IHE may be helpful in humans in preventing diet-induced obesity and perhaps even metabolic syndrome, the latter of which is known to be associated with obesity.     

Effects of nutritional state, insulin, and glucagon on lipid mobilization in rainbow trout, Oncorhynchus mykiss.

The effects of nutritional state, insulin, and glucagon on lipid mobilization were determined in rainbow trout, Oncorhynchus mykiss. In nutritional state experiments, fish were either fed continuously (except 24 to 36 hr prior to experimentation) with commercial trout chow or fasted for 4 weeks. Lipase activity in liver tissue isolated from fasted fish and cultured for 5 hr was greater than that in tissue isolated from fed fish and cultured. The presence of glucose (5.55 mM) in the incubation medium accentuates lipolytic activity in both liver and adipose tissue. Hormone response was assessed both in vivo and in vitro. Salmon insulin was injected into anesthetized fish (fed continuously except 24 hr prior to injections) in 10 microliters of saline/g body weight; final hormone dose was 100 ng/g body weight. Tissue and plasma were sampled 1 and 3 hr after injection. Insulin resulted in depressed plasma FA concentration and reduced hepatic triacylglycerol lipase activity. In vitro effects of hormones were evaluated by incubating liver and adipose tissue pieces in Hanks-MEM. Glucagon (bovine/porcine) directly stimulated lipid breakdown in both liver and adipose tissue. These actions were manifested by enhanced FA and glycerol released into the culture medium and by elevated triacylglycerol lipase activity. Insulin (bovine) generally appeared antilipolytic as this agent inhibited glucagon-stimulated lipase activity and glucagon-stimulated FA release. Furthermore, insulin (in the presence of glucose) reduced net lipolysis, as indicated by glycerol release, compared to control cultures. These results indicate that nutritional state and glucose are important modulators of lipid mobilization and that glucagon and insulin act directly on lipid storage sites to coordinate lipolysis in rainbow trout.     

Effects of epinephrine and norepinephrine on lipid mobilization from coho salmon liver incubated in vitro

The direct effects of epinephrine and norepinephrine (NE) on lipid mobilization were studied in coho salmon liver slices incubated in vitro. Fatty acid (FA) release from liver slices was measured continuously by pH-stat titration. The pH-stat assay system was validated by simultaneous colorimetric measurement of FA and glycerol release. Liver slices incubated in a glucose-free, low buffering capacity medium and stimulated with NE (10(-6) M) exhibited a one proton (titrimetric) to one FA (colorimetric) release profile. Under similar conditions, NE administration also stimulated glycerol release, in the expected three FA (colorimetric) to one glycerol (colorimetric) ratio. NE stimulated FA release from liver slices in a dose-dependent manner; epinephrine did not have a lipolytic effect. The beta-agonist isoproterenol stimulated FA release, whereas alpha-agonists had no effect. Furthermore, the beta-antagonist propranolol inhibited both NE- and beta-agonist-stimulated FA release. Liver triacylglycerol lipase activity was also stimulated by NE. Propranolol inhibited NE-stimulated lipase activity. These results establish the presence of hormone-sensitive lipase in salmon liver and suggest that NE-stimulated lipid mobilization in salmon liver is mediated through beta-adrenergic pathways.     

Characterization of triglyceride lipase activities in rat heart.

Triglyceride lipase activity was determined in particulate and soluble fractions from rat heart homogenates. Lipoprotein lipase activity was identified in the 100 000 × g supernatant fraction by the following in vitro criteria: stimulation of lipase activity by serum; maximal activity at alkaline pH (pH 8.2 to 8.4); and inhibition of lipase activity with NaCl. Triglyceride lipase activity was also determined under conditions in which the contribution of lipoprotein lipase to the assay was minimal. Particulate fractions (17 000 × g and 100 000 × g pellets) exhibited both an acid (pH 4.5 to 5) and a neutral (pH 7.5 to 8) pH optimum; the soluble (100 000 × g supernatant) fraction was characterized as having a single pH optimum of 7.5. The acid pH optimum for triglyceride lipase in the particulate fractions suggests that this activity originated in cardiac lysosomes, however the distribution of lipase activity determined at pH 5 did not correlate well with the lysosomal marker enzyme, N-acetyl-β-glucosaminidase. Chloroquine inhibited the acid triglyceride lipase (half maximal inhibition at 3 to 4 mm). In contrast, low concentrations (2 to 5 mm) of chloroquine stimulated lipase activity determined at pH 7.5; higher concentrations then inhibited enzyme activity. Similar results were obtained with NaCl; acid lipase activity was inhibited by all concentrations of NaCl whereas neutral lipase activity was first stimulated (25 to 150 mm NaCl) and then inhibited (200 to 500 mm NaCl). The addition of MgCl₂ (2 to 25 mm) resulted in an inhibition of lipase activity determined at pH 5 and a stimulation of activity determined at pH 7.5. Preincubation of the various rat heart fractions with ATP, cyclic AMP and protein kinase resulted in no change in lipase activity determined at either pH 5 or pH 7.5.     

Intracellular activation of protein kinase C and regulation of the surface transferrin receptor by diacylglycerol is a spontaneously reversible process that is associated with rapid formation of phosphatidic acid.

The effect of the synthetic diacylglycerol, sn-1,2-dioctanoylglycerol (diC8), on the expression of the surface transferrin receptor reveals that exogenous diC8 can act as an intracellular activator of protein kinase C and stimulate both down-regulation and increased receptor phosphorylation in a manner similar to that induced by the active tumor promotor, 4 beta-phorbol 12,13-dibutyrate. Unlike the spontaneously irreversible effect noted when 4 beta-phorbol 12, 13-dibutyrate is added, this same effect mediated by diC8 is brief, lasting only minutes, and is spontaneously reversible. The rate of reversibility is dependent on the concentration of diC8 added, and it is associated with rapid formation of a newly detected intracellular phospholipid that corresponds to sn-1,2-dioctanoyl phosphatidic acid. These data, in conjunction with findings that demonstrate that exogenous diacylglycerols (including diC8) when added to cells do not stimulate cellular phospholipase A2 or C, argue that protein kinase C is activated only briefly in this system since exogenous diC8 is subject to rapid intracellular metabolism to phosphatidic acid.     

Regulation of lipolysis in normoxic and hypoxic rat myocytes.

The mechanism involved in ischemia-induced myocardial lipolysis is still a matter of controversy. To elucidate the regulation of lipolysis at the cellular level, we incubated isolated rat myocytes in normoxic or hypoxic medium containing 11.1 mM glucose. Rates of lipolysis (glycerol output) were significantly (P less than 0.05, n = 12) higher in hypoxic than in normoxic myocytes (34.9 +/- 3.9 vs. 17.7 +/- 3.4 nmol/10(6) cells.30 min). However, there was no change in the content of cellular triacylglycerol (TG) in normoxic myocytes whereas it fell slightly (8 +/- 2 nmol/10(6) cells.30 min, P less than 0.05, n = 12) in hypoxic myocytes. On a molar basis glycerol output was significantly higher than the corresponding fall in TG (P less than 0.05, n = 12, both normoxic and hypoxic myocytes). This difference (glycerol output--TG reduction) amounted to 17.1 +/- 3.4 nmol/10(6) cells.30 min in normoxic myocytes and 27.6 +/- 5.1 nmol/10(6) cells.30 min in hypoxic myocytes (P less than 0.05, n = 12, normoxic vs. hypoxic). The hypoxia-induced rise in glycerol output was paralleled by an increased intracellular level of glycerol-3-phosphate. Both these responses were, however, dose-dependently inhibited by addition of pyruvate to the incubation medium, giving rise to a close correlation between cellular glycerol-3-phosphate and glycerol output (r = 0.75, P less than 0.05). This indicates mass action of glycerol-3-phosphate on fatty acid-TG cycling under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytosolic rat brain synapsin I is a diacylglycerol kinase.

The phosphorylation of diacylglycerol (DG), a reaction catalyzed by DG kinase, may be critical in the termination of effector-induced signals mediated by protein kinase C. Synapsin I is a principal target of intracellular protein kinases and is thought to be involved in the release of neurotransmitter from axon terminals. We present several lines of evidence which indicate that rat brain synapsin, in addition to this role, may function as a DG kinase. Purified rat brain DG kinase was digested with trypsin, which produced three major fragments whose sequence was identical to three regions in synapsin I. Using a rabbit anti-synapsin polyclonal antiserum, the elution profile of synapsin immunoreactivity coincided exactly with that of DG kinase activity in column fractions from the final step in the DG kinase purification procedure. As is the case with synapsin, the purified enzyme was a strongly basic protein with an isoelectric point greater than 10.0. Finally, incubating the DG kinase with highly purified bacterial collagenase, an enzyme that partially degrades the proline- and glycine-rich synapsin, resulted in the simultaneous loss of DG kinase activity and synapsin immunoreactivity. We conclude that cytosolic rat brain synapsin is capable of functioning as a DG kinase.     

Delayed accumulation of diacylglycerol in platelets as a mechanism for regulation of onset of aggregation and secretion

An important mechanism of platelet regulation is the formation of the second messenger diacylglycerol (DAG) and the activation of protein kinase C (PKC). Our previous studies suggested that the DAG/PKC pathway plays an important role in the induction of secretion and secondary aggregation rather than the earlier events of shape change and primary aggregation. We therefore examined the hypothesis that the delayed effects of PKC on platelets may result from delayed accumulation of DAG. The kinetics of DAG formation in human platelets were determined. When platelets were stimulated with gamma-thrombin, the largest phase of DAG accumulation was delayed for 0.6 to 0.8 minutes and DAG mass levels remained elevated for at least 2 minutes. In platelets stimulated with collagen, DAG accumulation was delayed for 1.0 to 1.2 minutes and DAG mass levels remained elevated for at least 3 minutes after stimulation. Sustained DAG production was also associated with sustained activation of PKC as monitored by phosphorylation of the 40-Kd substrate of PKC. The physiologic significance of delayed DAG accumulation was investigated using the cell-permeable DAG analog, dioctanoylglycerol (diC8). In synergy with subthreshold gamma-thrombin or collagen, exogenous diC8 reconstituted platelet activation. The optimal timing of addition of diC8 was 0.5 minutes after stimulation with gamma-thrombin or collagen. These kinetics were similar to those of endogenous DAG accumulation. These studies underscore the importance of a delayed accumulative phase of DAG generation as a mechanism controlling the onset of platelet secretion and irreversible aggregation.     

Domain exchange: characterization of a chimeric lipase of hepatic lipase and lipoprotein lipase.

Hepatic lipase and lipoprotein lipase hydrolyze fatty acids from triacylglycerols and are critical in the metabolism of circulating lipoproteins. The two lipases are similar in size and amino acid sequence but are distinguished by functional differences in substrate preference and cofactor requirement. Presumably, these distinctions result from structural differences in functional domains. To begin localization of these domains, a chimeric lipase was constructed composed of the N-terminal 329 residues of rat hepatic lipase linked to the C-terminal 136 residues of human lipoprotein lipase. The chimera hydrolyzed both monodisperse short-chain (esterase) and emulsified long-chain (lipase) triacylglycerol substrates with catalytic and kinetic properties closely resembling those of native hepatic lipase. However, monoclonal antibodies to lipoprotein lipase inhibited the lipase activity, but not the esterase function, of the chimera. Therefore, the chimeric molecule is a functional lipase and contains elements and characteristics from both parental enzymes. It is proposed that the N-terminal domain, containing the active center from hepatic lipase, governs the catalytic character of the chimera, and the C-terminal domain is essential for hydrolysis of long-chain substrates.     

Effect of chloroquine on rates of lipolysis in the isolated perfused rat heart and in rat epididymal fat pads

Triglyceride lipase (hydrolase) activity was determined in particulate (17 000 g and 100 000 g pellets) and soluble (100 000 g supernatant) subcellular fractions from rat epididymal fat pad homogenates. Particulate fractions exhibited both an acid (pH 4.5 to 5) and an alkaline (pH 8) pH optimum; in contrast, the soluble fraction was characterized as having a single pH optimum of 7.5. The addition of chloroquine to the triglyceride lipase assay resulted in an inhibition of acid lipase activity (half maximal inhibition at 20 to 25 mm chloroquine) whereas lipase activity measured at pH 7.5 was first stimulated by low concentrations of chloroquine (2 to 10 mm) and then inhibited with higher concentrations. The addition of chloroquine (10 μm or 50 μm) to Krebs-Henseleit bicarbonate buffer resulted in negative inotropic and chronotropic effects in isolated perfused rat hearts. Perfusion of rat hearts or preincubation of epididymal fat pads with Krebs-Henseleit bicarbonate buffer containing 10 μm or 50 μm chloroquine for 30 min did not decrease basal or epinephrine-stimulated rates of lipolysis as determined by measurements of glycerol release. Similarly, pretreatment of rats with chloroquine 2 h prior to killing had no effect on basal or epinephrine-stimulated rates of lipolysis in perfused hearts or in epididymal fat pads. Since chloroquine is a lysosomotropic agent, it is concluded that the lysosomal acid triglyceride lipase is unlikely to be involved in determining basal or hormone-stimulated rates of lipolysis.     

Comparison of lipoprotein lipase activity in heart myocytes and perfused hearts

This study was initiated to compare lipoprotein lipase activity in isolated heart myocytes and the heparin residual compartment of perfused hearts from adult rats. Heart lipoprotein lipase activity was divided into two fractions by 1 min of heparin perfusion. Heparin-residual and myocyte lipoprotein lipase activity were lower in hearts obtained from fasted compared to fed rats. In each case, the myocyte enzyme activity was 55 to 60% of heparin-residual levels. The difference between myocyte and heparin-residual activity may be a consequence of the time and treatment required to isolate cells in that long-term in vitro exposure of heart tissue to heparin also reduces residual activity. In vivo treatment with endotoxin decreased both heparin-residual and myocyte lipoprotein lipase activities; whereas, colchicine administration increased both activities compared to saline injected rats. In this latter experiment heart heparin-residual and myocyte lipoprotein lipase activities were positively correlated (r = 0.90). The results indicate that in the mature heart intracellular lipoprotein lipase activity is primarily associated with myocytes.     

Clearance of chylomicron triacylglycerol and cholesteryl ester from the plasma of streptozotocin-induced diabetic and hypercholesterolemic hypothyroid rats.

Labeled chylomicrons in thoracic duct lymph were collected after test meals containing 14C cholesterol and 2-3H glyceryl trioleate and were given by intravenous injection to groups of control rats, rats made diabetic by treatment with streptozotocin, and rats made hypothyroid and hypercholesterolemic by a diet containing cholesterol, peanut oil, cholic acid, and thiouracil. In the diabetic rats clearances from the plasma of chylomicron triacylglycerol and cholesteryl ester were impaired. A large variability in triacylglycerol clearance in diabetic rats was ascribed to variability in plasma triacylglycerol concentrations. Adipose tissue lipoprotein lipase activity was not impaired in the female diabetic rats used in this study. In the hypothyroid hypercholesterolemic rats chylomicron cholesteryl ester clearance from the plasma was impaired but chylomicron triacylglycerol was cleared efficiently, and adipose tissue lipoprotein lipase activity was similar to or greater than activity in controls. Ten minutes after intravenous injection most plasma radioactivity was recovered in lipoproteins of density less than 1.006 g/ml in all groups of rats, but relatively more was recovered at this density in both treatment groups. We suggest that chylomicron remnants accumulate in the plasma and contribute to the development of hyperlipemia in both treatment groups, but that the remnants formed in the diabetic rat are less depleted of triacylglycerol than the remnants formed in the hypothyroid hypercholesterolemic rat. It is suggested that factors other than measured lipoprotein lipase activities of adipose tissues may be important in determining the initial extent of hydrolysis of chylomicron triacyglycerol. We propose that the hypercholesterolemic hypothyroid rat is a useful model for the experimental production of the remnants of triacylglycerol-rich primary lipoproteins.     

Biological and functional characteristics of a novel low-molecular weight antagonist of glucose-dependent insulinotropic polypeptide receptor, SKL-14959, in vitro and in vivo

Aim: We recently discovered a glucose‐dependent insulinotropic polypeptide (GIP) receptor antagonist, SKL‐14959. GIP plays a role in the glucose and lipid metabolism, and is associated with obesity and insulin resistance. Therefore, we aimed to ascertain the inhibitory potency and glucose and lipid metabolism of SKL‐14959. Methods: SKL‐14959 was evaluated for its binding affinity to each GIP, glucagon‐like peptide‐1 (GLP‐1) and glucagon receptors by each labelled and non‐labelled ligand; GIP‐stimulated cyclic AMP (cAMP) production in CHO cells expressing human GIP receptor in vitro. Oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT) were performed to examine the insulinotropic effect on endogenous and exogenous GIP. Oil tolerance tests were also conducted to examine the lipid metabolism and the postheparin plasma lipase activity, lipoprotein lipase (LPL) and hepatic lipase (HL). Result: SKL‐14959 selectively bound to GIP receptor and inhibited GIP‐stimulated cAMP production with the Ki value of 55 nM and an IC₅₀ value of 2.9 µM, respectively. SKL‐14959·Na significantly increased blood glucose levels, inhibited insulin secretion in OGTT and inhibited the plasma glucose lowering of exogenous GIP in IPGTT. Furthermore, SKL‐14959 increased plasma triacylglycerol (TG) levels as well as suppressed the postheparin plasma lipase activity in an oil load test. Conclusion: These data indicate that SKL‐14959 is distinguished in the physiological phenotype of GIP following direct binding to the receptor.     

Myocardial metabolic regulation through peroxisome proliferator-activated receptor alpha after myocardial infarction

Although peroxisome proliferator-activated receptor alpha (PPARα) is closely associated with myocardial fatty acid metabolism, the pathophysiological role of PPARα in myocardial infarction (MI) is not yet known. The aim of the present study was to clarify the relationship between cardiac energy metabolism and PPARα expression in the remodelling of myocardium after MI. We assayed the expression of PPARα and several metabolic genes in cultured cardiac cells (myocytes and nonmyocytes) and in MI hearts. PPARα was strongly expressed in cardiac myocytes but not in nonmyocytes (mainly fibroblasts). In MI rats, PPARα and PPARα-regulated genes (lipoprotein lipase, heart-type fatty acid binding protein, long-chain acyl-CoA dehydrogenase and uncoupling protein-3) were decreased concomitantly, whereas uncoupling protein-2 was not decreased in severely ischemic regions. Immunohistochemical staining for PPARα was less decreased in borderline myocardium than in sham-operated hearts. Furthermore, in electron microscopic study, there were no lipid droplet accumulations in surviving myocardium after MI. Our results suggest that the reduced expression of PPARα is closely related to that of fatty acid metabolism genes in infarcted myocardium, and PPARα may play an important role in cardiac energy metabolism during remodelling after MI.