Inhibition of adipocyte lipolysis by papaverine: papaverine can inhibit the redistribution of hormone-sensitive lipase

Papaverine, despite being a potent phosphodiesterase inhibitor, actually blocks adipocyte lipolysis. The present study was designed to clarify the mechanism of the inhibitory effect of papaverine on lipolysis. Lipolysis, stimulated by either 10 microM isoproterenol or 5 mM dibutyryl cAMP, was significantly inhibited by papaverine (100 microM and above). Papaverine, however, did not affect the isoproterenol-induced increase in the protein kinase A (A-kinase) activity ratio. In cell-free extract from non-stimulated adipocytes, cAMP-stimulated A-kinase activities were almost completely blocked by H-89, a potent inhibitor of A-kinase, but not by papaverine. Thus, the inhibitory effect of papaverine on lipolysis could be responsible for a deficit in step(s) distal to A-kinase activity. Hormone-sensitive lipase activities in the infranatant fraction of centrifuged homogenates of cells, which were maximally stimulated with isoproterenol were significantly reduced. This result indicates that hormone-sensitive lipase redistributes from cytosol to its substrate in lipolytically stimulated cells. Papaverine completely blocked the isoproterenol-induced decrease in lipase activity in the infranatant fraction. These results suggest that papaverine blocks lipolysis through its inhibitory effect on the redistribution of hormone-sensitive lipase.     

Modulation by phenobarbital of the differentiation of 3T3 preadipocytes

The effect of phenobarbital upon the differentiation of two preadipocyte cell lines, 3T3 F442A and 3T3 L-1, was examined by measuring the synthesis and secretion of lipoprotein lipase. Extracellular enzyme was measured by treating intact cells with heparin, and the intracellular enzyme was subsequently assayed in cell homogenates. When confluent cultures of 3T3 F442A cells were treated with insulin, the cells underwent differentiation as indicated by increased activity of lipoprotein lipase within 6 days, followed in turn by increased levels of protein and triglyceride. Addition of phenobarbital with insulin enhanced total lipoprotein lipase, protein, and triglyceride content. The activity of lipoprotein lipase accumulated in the heparin-releasable fraction during differentiation was increased 2- to 3-fold and the intracellular enzyme was enhanced 15- to 20-fold by the addition of phenobarbital. The ability of phenobarbital to modulate differentiation was dependent upon the time of addition. When added early in the postconfluent period, there was a greater increase in lipoprotein lipase activity than when the drug was added at later times. Phenobarbital also stimulated lipoprotein lipase in differentiating 3T3 L-1 cells in the presence of insulin, although lipoprotein lipase activity was moderately enhanced by phenobarbital alone in these cells. These results suggest that phenobarbital may affect the conversion of adipoblasts into preadipocytes and thereby increase the proportion of cells susceptible to the differentiating stimulus.     

Relationship between cyclic AMP production and lipolysis induced by forskolin in rat fat cells.

Forskolin (7 beta-acetoxy-8, 13-epoxy-1 alpha,6 beta,9 alpha-trihydroxy-labd-14-ene-11-one) induced both cyclic AMP production and lipolysis in intact fat cells, but stimulated lipolysis without increasing cyclic AMP at a concentration of 10(-5) M. Homogenization of fat cells elicited lipolysis without elevation of cyclic AMP. Forskolin did not stimulate lipolysis in the homogenate. Forskolin stimulated both cyclic AMP production and lipolysis in a cell-free system consisting of endogenous lipid droplets and a lipoprotein lipase-free lipase fraction prepared from fat cells. However, at a concentration of 10(-6) M, it induced lipolysis without increase in the cyclic AMP content in this cell-free system. In the cell-free system, homogenization of the lipid droplets resulted in marked increase in lipolysis to almost the same level as that with 10(-4) M forskolin without concomitant increase in cyclic AMP. Addition of forskolin to a cell-free system consisting of homogenized lipid droplets and lipase did not stimulate lipolysis further. Phosphodiesterase activities were found to be almost the same both in the presence and absence of forskolin in these reaction mixtures. Although 10(-3) M forskolin produced maximal concentrations of cyclic AMP: 6.7 x 10(-7) M in fat cells and 2.7 x 10(-7) M in the cell-free system, 10(-4) M cyclic AMP did not stimulate lipolysis in the cell-free system. In a cell-free system consisting of lipid droplets and the lipase, pyrophosphate inhibited forskolin-induced cyclic AMP production, but decreased forskolin-mediated lipolysis only slightly. Based on these results, mechanism of lipolytic action of forskolin was discussed.     

Carboxylesterase 3 (EC 3.1.1.1) is a major adipocyte lipase

Hydrolysis of triglycerides is central to energy homeostasis in white adipose tissue (WAT). Hormone-sensitive lipase (HSL) was previously felt to mediate all lipolysis in WAT. Surprisingly, HSL-deficient mice show active HSL-independent lipolysis, suggesting that other lipase(s) also mediate triglyceride hydrolysis. To clarify this, we used functional proteomics to detect non-HSL lipase(s) in mouse WAT. After cell fractionation of intraabdominal WAT, most non-HSL neutral lipase activity is localized in the 100,000 x g infranatant and fat cake fractions. By oleic acid-linked agarose chromatography of infranatant followed by elution in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid gradient, we identified two peaks of esterase activity using p-nitrophenyl butyrate as a substrate. One of the peaks contained most of the lipase activity. In the corresponding fractions, gel permeation chromatography and SDS-PAGE, followed by tandem mass spectrometric analysis of excised Coomassie Blue-stained peptides, revealed carboxylesterase 3 (triacylglycerol hydrolase (TGH); EC 3.1.1.1). TGH is also the principle lipase of WAT fat cake extracts. Partially purified WAT TGH had lipase activity as well as lesser but detectable neutral cholesteryl ester hydrolase activity. Western blotting of subcellular fractions of WAT and confocal microscopy of fibroblasts following in vitro adipocytic differentiation are consistent with a distribution of TGH to endoplasmic reticulum, cytosol, and the lipid droplet. TGH is responsible for a major part of non-HSL lipase activity in WAT in vitro and may mediate some or all HSL-independent lipolysis in adipocytes.     

Cyclic AMP activation of a triglyceride lipase in broken cell preparations of rat heart

The effect of CAM [cyclic AMP, Mg-ATP, and 3-isobutyl, 1-methylxanthine (MIX)] on triacylglycerol (TG) lipase activity in extracts from heparin-perfused rat heart was determined. TG lipase activity in homogenate, 10,000g supernatant, 105,000g supernatant, ammonium sulfate supernatant, and the eluate from heparin-Sepharose was increased between 62 and 151% when incubated with a combination of 0.3 mM cyclic AMP, 5 mM MgCl2, and 2 mM ATP. The addition of Mg-ATP + cyclic AMP caused a greater activation of TG lipase in the various fractions than did Mg-ATP + MIX or cyclic AMP + MIX. These results suggest that activation may be mediated by the classical cyclic AMP-protein kinase cascade. Control and CAM-stimulated activities were increased by heparin and inhibited by NaCl and protamine sulfate. In the absence of serum in the assay, the CAM system caused a relatively greater stimulation of lipolytic activity in each fraction compared to when serum was present in the assay. However, the absolute values were 6.1 to 16.3-fold greater with serum in the assay than without serum. In a similar manner, TG lipase activity was stimulated by CAM between 1.75 and 4.26-fold at pH 7.4, and only between 1.62 and 2.51-fold at pH 8.1. However, the absolute values at pH 8.1 were 6.77 to 31.83-fold greater than those seen at pH 7.4. These data demonstrate, for the first time, the cyclic AMP activation of a TG lipase above basal levels in cell-free fractions of rat heart. It is intriguing to speculate that the intracellular fraction of lipoprotein lipase may play a role in the hormonal regulation of cardiac TG lipolysis.     

Lack of feedback regulation of cyclic 3':5'-AMP accumulation by free fatty acids in chicken fat cells.

Fat cells isolated from the mesenteric adipose tissue of chickens (pullets) responded to glucagon with an increase in lipolysis and a sustained rise in cyclic adenosine 3':5'-monophosphate (cyclic AMP) over a 30-min incubation. The prolonged accumulation of cyclic AMP due to glucagon in chicken fat cells was primarily intracellular. In addition, there was little increase in cyclic AMP accumulation due to theophylline alone or potentiation of the increase due to glucagon. These data indicate that chicken fat cells, unlike rat fat cells, are relatively insensitive to theophylline. Neither lipolysis nor cyclic AMP accumulation by chicken fat cells was inhibited by free fatty acid to albumin ratios (3 to 7) which markedly reduced both events in rat fat cells. However, in the absence of albumin from the medium, lipolysis in chicken fat cells was reduced, but not to the same extent as in rat fat cells. Chicken fat cells did accumulate more intracellular free fatty acids in response to lipolytic agents than did rat fat cells. The uptake of oleate by rat and chicken fat cells was identical. Glucagon-induced accumulation of cyclic AMP by chicken fat cell ghosts was unaffected by added oleate. Under identical conditions glucagon-induced adenylate cyclase activity of rat fat cell ghosts was markedly inhibited by added oleate. Triglyceride lipase activity of the pH 5.2 precipitate from a 40,000 x g infranatant of homogenized fat cells from chickens was less sensitive than that from rat fat cells to the ratio of oleate to albumin. These results suggest that the maintenance of cyclic AMP levels in chicken fat cells incubated with lipolytic agents results from the relative insensitivity of chicken fat cells to free fatty acid inhibition of cyclic AMP accumulation.     

Nuclear protein-kinase activity in perfused rat liver stimulated with dibutyryl-adenosine cyclic 3':5'-monophosphate.

Cytoplasmic and nuclear protein kinase activities from perfused rat liver have been studied in response to dibutyryl-adenosine cyclic 3':5'-monophosphate added at a concentration that stimulates hepatic gluconeogenesis (100 muM). Total nuclear protein kinase, as assayed using a mixed histone fraction as phosphate acceptor, is increased by 5-fold within 8 min of the addition of cyclic nucleotide to the perfusate. In contrast the total cytoplasmic protein kinase activity is decreased to 50% of the control value. The protein substrate specificity of the protein kinase that is present in the nucleus in response to dibutyryl-adenosine cyclic 3':5'-monophosphate stimulation is similar to that of cytoplasmic, adenosine cyclic 3':5'-monophosphate-dependent, protein kinase but is distinct from that of the enzyme(s) present in control nuclei. The predominant species to protein kinase from stimulated nuclei has a sedimentation constant of 3.9 S. This value is identical to that of the catalytic subunit of cytoplasmic adenosine 3':5'-monophosphate-dependent protein kinase. These data suggest that some of the effects of adenosine 3':5'-monophosphate on nuclear events may be mediated through its interaction with the inactive protein kinase holoenzyme in the cytoplasm and the subsequent redistribution of the active catalytic subunits generated by this interaction.     

Triacylglycerol lipase activity in the rabbit renal medulla

Although the renal medulla is rich in triacylglycerols, the lipolysis of these intracellular triacylglycerols by a renomedullary triacylglycerol lipase has not been directly demonstrated. The present study demonstrates triacylglycerol lipase activity localized in the particulate subcellular fractions of rabbit renal medullae. Renomedullary triacylglycerol lipase activity, as determined by the hydrolysis of [14C]triolein to [14C]oleic acid, was observed to have a pH optimum of 5.8. Addition of cAMP/ATP/magnesium acetate resulted in an 80% activation of crude homogenate triacylglycerol lipase activity; addition of exogenous cAMP-dependent protein kinase resulted in a further activation of lipolysis. 3 mM CaCl2 had no effect on basal triacylglycerol lipase activity. 1 M NaCl did not inhibit lipolysis, suggesting that the lipase activity measured was not due to lipoprotein lipase. Endogenous renomedullary triacylglycerols were hydrolysed by a lipase in the 100,000 X g pellet of renomedullary homogenates, resulting in the release of free fatty acids including arachidonic and adrenic acids. Dispersed renomedullary cells were prepared to monitor hormone-sensitive triacylglycerol lipase activity in intact cells. Addition of 10 microM forskolin and 10 microM epinephrine resulted in 8-fold and 50-fold increases in triacylglycerol lipase activity, respectively, as defined by release of free glycerol from the cells. These studies demonstrate that a cAMP-dependent hormone-sensitive triacylglycerol lipase is present in the renal medulla, and is responsible for the hydrolysis of renomedullary triacylglycerols.     

Cyclic AMP metabolism and lipolysis in cultured human adipocyte precursors

Triacylglycerol breakdown (lipolysis) results from a series of reactions culminated by activation of "hormone-stimulated" triacylglycerol lipase, an enzyme unique to adipose tissue. We have studied various components of the lipolytic process in human omental adipocyte precursors differentiating in culture. The levels of cyclic AMP, the "second messenger" of lipolytic hormones, were about sixfold higher in fat cell precursors than those in abdominal skin fibroblasts. L-Isoproterenol resulted in significant elevation of cyclic AMP levels in both cell types. Preincubation of intact adipocyte precursors with insulin resulted in significant enhancement of "low Km" cyclic AMP phosphodiesterase activity; in contrast, this hormone had no effect on fibroblast phosphodiesterase activity, a distinctive biochemical difference despite the morphological similarities between the two cell types during the early stages of adipocyte precursor maturation. Incubation of adipocyte precursors with isoproterenol resulted in the release of fatty acids into the medium, findings indicative of "hormone-stimulated" lipase activity and, hence, the operation of the entire "lipolytic cascade"; isoproterenol-stimulated lipolysis was inhibited by insulin. Release of fatty acids from fibroblasts was not observed. Thus, "hormone-stimulated" lipolysis and insulin stimulation of cyclic AMP phosphodiesterase activity are expressed during early stages of human adipocyte precursor differentiation.     

Site-directed fluorogenic modification of bacteriorhodopsin by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole

Use of a digitonin-permeabilized rat adipocyte preparation overcomes inherent problems which occur when currently used broken cell systems are utilized for studying the regulation of hormone-sensitive lipase. The effect of digitonin on plasma membrane permeability was concentration-dependent being nearly maximum at 20 micrograms/ml as assessed by (a) leakage of 85% cellular lactate dehydrogenase after 30 min, (b) the efflux of 72% preloaded cellular (86Rb) rubidium within 10 min and (c) immediate inhibition of glucose oxidation. Hormone-modulated rates of lipolysis were preserved in this preparation. Following maximal activation of lipolysis in adipocytes with catecholamines, the rate of lipolysis in intact cells and digitonin-treated cells was elevated 26-fold and 20-fold respectively, while the rate in homogenates from these cells was elevated only 2.8-fold. Insulin suppressed catecholamine-dependent activation of lipolysis by at least 90% when subsequently measured in intact cells and digitonin-treated cells. Insulin suppression was only 56% when measured in homogenates. The hormone-sensitive lipase in permeabilized cells, as opposed to intact cells and homogenates, was activated by cyclic AMP to a degree that approached activation by catecholamines. In homogenates, cyclic AMP (1.0 mM) plus ATP (0.25 mM) activated the lipase only 36%, while neither alone had any effect. In digitonin-permeabilized cells, however, exogenous cyclic AMP alone activated lipolysis in a concentration-dependent manner with 1 microM, 30 microM and 1.0 mM cyclic AMP activating lipolysis by 41%, 250% and 1300% respectively. In contrast, lipolysis in intact cells was activated by 0%, 25% and 250% by 1 microM, 30 microM and 1.0 mM cyclic AMP. Also in digitonin-treated preparations, ATP alone activated lipolysis 40%, but ATP plus cyclic AMP activated lipolysis to only 74% of the level due to cyclic AMP alone. These studies indicate that the permeabilized adipocyte preparation is an excellent system for investigating the mechanism of regulation of the hormone-sensitive lipase by permitting manipulation of the intracellular environment while preserving the physiological response of the lipase.     

Stimulation of the hormone-sensitive triacylglycerol lipase from adipose tissue by phosphatidylethanolamine

The activity of a pigeon adipose tissue hormone-sensitive triacylglycerol lipase preparation was increased from 2- to 5-fold by the presence of phosphatidylethanolamine in assays with three different methods of preparing triolein substrates. Phosphatidylethanolamine from egg yolk produced the greatest stimulation of lipase activity; the stimulation was concentration-dependent but was not time-dependent. A comparable increase in triacylglycerol lipase activity due to phosphatidylethanolamine was also observed with enzyme preparations from chicken and rat adipose tissue. Phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, Triton X-100 and sodium dodecyl sulfate all inhibited enzyme activity. Phosphatidylethanolamine had no effect on acid lipase activity in the pigeon adipose tissue preparation. Preincubation of the pigeon adipose tissue lipase with ATP, cyclic AMP and protein kinase resulted in a 2.15-fold activation of hydrolase activity determined in the absence of phosphatidylethanolamine. In contrast, non-activated and protein kinase-activated forms of the lipase were characterized as having very nearly the same activity in assays with substrate preparations containing phosphatidylethanolamine. The phosphatidylethanolamine-dependent stimulation of lipase activity was characterized kinetically as being due to an increase in maximal velocity. The modulation of the adipose tissue hormone-sensitive lipase activity by phospholipids could be involved in the hormonal regulation of lipolysis.     

Hexose transport stimulation and membrane redistribution of glucose transporter isoforms in response to cholera toxin, dibutyryl cyclic AMP, and insulin in 3T3-L1 adipocytes

Exposure of 3T3-L1 adipocytes to 100 ng/ml of cholera toxin or 1 mM dibutyryl cyclic AMP caused a marked stimulation of deoxyglucose transport. A maximal increase of 10- to 15-fold was observed after 12-24 h of exposure, while 100 nM insulin elicited an increase of similar magnitude within 30 min. A short term exposure (4 h) of cells to cholera toxin or dibutyryl cyclic AMP resulted in a 3- to 4-fold increase in deoxyglucose transport which was associated with significant redistribution of both the HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) glucose transporters from low density microsomes to the plasma membrane fraction. Total cellular amounts of both transporter proteins remained constant. In contrast, cells exposed to cholera toxin or dibutyryl cyclic AMP for 12 h exhibited elevations in total cellular contents of GLUT1 (but not GLUT4) protein to about 1.5- and 2.5-fold above controls, respectively. Although such treatments of cells with cholera toxin (12 h) versus insulin (30 min) caused similar 10-fold enhancements of deoxyglucose transport, a striking discrepancy was observed with respect to the content of glucose transporter proteins in the plasma membrane fraction. While insulin elicited a 2.6-fold increase in the levels of GLUT4 protein in the plasma membrane fraction, cholera toxin increased the amount of this transporter by only 30%. Insulin or cholera toxin increased the levels of GLUT1 protein in the plasma membrane fraction equally (1.6-fold). Thus, a greater number of glucose transporters in the plasma membrane fraction is associated with transport stimulation by insulin compared to cholera toxin. We conclude that: 1) at early times (4 h) after the addition of cholera toxin or dibutyryl cyclic AMP to 3T3-L1 adipocytes, redistribution of glucose transporters to the plasma membrane appears to contribute to elevated deoxyglucose uptake rates, and 2) the stimulation of hexose uptake after prolonged treatment (12-18 h) of cells with cholera toxin may involve an additional increase in the intrinsic activity of one or both glucose transporter isoforms.     

The lipolytic stimulation of 3T3-L1 adipocytes promotes the translocation of hormone-sensitive lipase to the surfaces of lipid storage droplets

Hormone-sensitive lipase catalyzes the rate-limiting step in the release of fatty acids from triacylglycerol-rich lipid storage droplets of adipocytes, which contain the body's major energy reserves. Hormonal stimulation of cAMP formation and the activation of cAMP-dependent protein kinase leads to the phosphorylation of hormone-sensitive lipase and a large increase in lipolysis in adipocytes. By contrast, phosphorylation of hormone-sensitive lipase by the kinase in vitro results in a comparatively minor increase in catalytic activity. In this study, we investigate the basis for this discrepancy by using immunofluorescence microscopy to locate hormone-sensitive lipase in lipolytically stimulated and unstimulated 3T3-L1 adipocytes. In unstimulated cells, hormone-sensitive lipase is diffusely distributed throughout the cytosol. Upon stimulation of cells with the beta-adrenergic receptor agonist, isoproterenol, hormone-sensitive lipase translocates from the cytosol to the surfaces of intracellular lipid droplets concomitant with the onset of lipolysis, as measured by the release of glycerol to the culture medium. Both hormone-sensitive lipase translocation and lipolysis are reversed by the incubation of cells with the beta-adrenergic receptor antagonist, propranolol. The treatment of cells with cycloheximide fails to inhibit lipase translocation or lipolysis, indicating that the synthesis of nascent proteins is not required. Cytochalasin D and nocodazole used singly and in combination also failed to have a major effect, thus suggesting that the polymerization of microfilaments and microtubules and the formation of intermediate filament networks is unnecessary. Hormone-sensitive lipase translocation and lipolysis were inhibited by N-ethylmaleimide and a combination of deoxyglucose and sodium azide. We propose that the major consequence of the phosphorylation of hormone-sensitive lipase following the lipolytic stimulation of adipocytes is the translocation of the lipase from the cytosol to the surfaces of lipid storage droplets.     

Evidence for a dual mechanism of lipolysis activation by epinephrine in rat adipose tissue

Whole homogenates prepared from tissue previously exposed to epinephrine displayed a 3-fold increased rate of lipolysis of endogenous substrate. When the aqueous infranatant phase of such homogenates was collected by centrifugation and assayed against exogenous triolein emulsions, no hormone effect could be demonstrated. Treatment of such infranatants with cAMP-dependent protein kinase prepared from muscle increased their lipase activity against exogenous triolein by 80%. Employing [3H]triolein emulsions as exogenous substrate, rates of lipolysis of both endogenous and exogenous glycerides were measured simultaneously in whole tissue homogenates. Prior treatment of the tissue with epinephrine increased the rate of lipolysis of endogenous glycerides an average of 3-fold but had no effect on the hydrolysis of exogenous triolein. By contrast, treatment of whole homogenates with protein kinase accelerated lipolysis of exogenous triolein without altering the rate of hydrolysis of endogenous glycerides. The data suggest that a second pathway of lipolysis activation occurs in response to epinephrine in addition to that involving a cAMP-mediated increase in the state of phosphorylation of the hormone-sensitive lipase.     

Modulation of lipoprotein lipase activity in cultured rat mesenchymal heart cells and preadipocytes by dibutyryl cyclic AMP, cholera toxin and 3-isobutyl-1-methylxanthine

We have compared the effects of cellular cyclic AMP modulation on the regulation of lipoprotein lipase in cultures of rat epididymal pad preadipocytes and mesenchymal heart cells. Addition of dibutyryl cyclic AMP (dibutyryl cAMP) or 3-isobutyl-1-methylxanthine (IBMX) to preadipocytes grown in serum-containing culture medium resulted in a progressive decrease in lipoprotein lipase activity released into the culture medium so that at 6-8 h enzyme activity ranged between 20 and 30% of that recovered in the control dishes. Similar short-term (6-8 h) studies of the heart cell cultures showed a variable and much less pronounced depression of lipoprotein lipase activity. Thus, following dibutyryl cAMP and IBMX treatment, lipoprotein lipase activity ranged between 70 and 95% of control values. Incubation for 6 h with cholera toxin was followed by a 4-fold rise in the concentration of cellular cyclic AMP in both types of culture, but while in heart cell cultures enzyme activity was unchanged, lipoprotein lipase activity in preadipocytes decreased to 30% of control value. After 24 h incubation with all three effectors, an increase in lipoprotein lipase activity was seen. In the preadipocytes the increase ranged between 50 and 150% above control value, in the heart cell cultures it was 100-250%. 24-h incubation of heart cell cultures with dibutyryl cAMP resulted in a 6-fold increase of heparin-releasable lipoprotein lipase activity while residual activity was doubled. The rise in surface-bound lipoprotein lipase was evidenced also by an increase in the lipolysis of chylomicron triacylglycerol. In the presence of cycloheximide, the dibutyryl cAMP-induced heparin-releasable and residual lipoprotein lipase activity declined at the same rate as the basal activity. The reason for the difference in response of cultured preadipocytes and heart cells to the effectors during the first 8 h of incubation has not been elucidated, but could be related to a possible absence of hormone-sensitive lipase in the heart cells, and hence in a difference in intracellular metabolism of triacylglycerol. On the other hand, a common mechanism can be postulated for the long-term effect of cyclic AMP on the induction of lipoprotein lipase activity in both types of cultures. It probably involves mRNA and protein synthesis, which culminates in an increase in enzyme activity.     

Partial purification and properties of guanosine 3':5'-monophosphate-dependent protein kinase from pig lung.

Guanosine 3':5'-monophosphate(cyclic GMP)-dependent protein kinase which catalyzes the phosphorylation of histone was purified about 200-fold from the soluble fraction of pig lung by pH 5.5 precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. The apparent Ka values for guanosine 3':5'-monophosphate and adenosine 3':5'-monophosphate were determined to be about 17 and 360 nM, respectively. Mg2+ was essential for the activity exhibiting biphasic stimulation behavior and neither Mn2+ nor Ca2+ could substitute for Mg2+. However, these divalent ions markedly inhibited the protein kinase activity stimulated by cyclic GMP in the presence of Mg2+.     

Demonstration that cyclic adenosine 3',5'-monophosphate mediates the lipolytic action of parathyroid hormone.

Studies were carried out with rat epididymal fat pads first to compare the effects of the synthetic N-terminal 1-34 peptide of bovine parathyroid hormone and of the native hormone to determine whether this portion of the molecule is responsible for the lipolytic action of the hormone and second to determine whether this biologic action of parathyroid hormone is mediated by cyclic adenosine 3',5'-monophosphate. The N-terminal polypeptide was as effective as the native hormone in stimulating lipolysis in the concentration range between 10(-8) M and 10(-6) M. Parathyroid hormone stimulated lipolysis by isolated fat cells. The concentration of cyclic adenosine 3',5'-monophosphate in the fat pads was significantly increased by the hormone (10(-6)M). Lipolytic stimulation by parathyroid hormone (10(-6)M) was diminished by insulin (100 muU/ml) and prostaglandin E1 (1 mug/ml), both of which are known inhibitors of lipolysis. The findings indicate that the amino-terminal 1-34 peptide portion of parathyroid hormone is responsible for the lipolytic action and that this effect is mediated through cyclic adenosine 3',5'-monophosphate.     

Effect of epinephrine and other lipolytic agents on intracellular lipolysis and lipoprotein lipase activity in 3T3-L1 adipocytes

3T3-L1 adipocytes were used to test the hypothesis that hormone-sensitive lipolysis and lipoprotein lipase activity might be regulated in a reciprocal manner. Intracellular lipolysis was stimulated by catecholamine, dibutyryl cAMP, and ACTH, but not by glucagon. The effects of epinephrine on lipolysis were blocked by the beta-antagonist propanolol but not by the alpha-antagonist phentolamine. Hormone-stimulated lipolysis was not changed by acute (45 min) or chronic (2 days) treatment of the cells with insulin whereas the latter treatment augmented lipoprotein lipase activity about fivefold. Epinephrine did not affect the lipoprotein lipase activity of insulin-stimulated cells. Withdrawal of glucose from the medium decreased lipoprotein lipase activity and the effect of epinephrine on lipolysis. Effects of lipolytic agents on activity of lipoprotein lipase were variable and concentration-dependent. Lipoprotein lipase activity was decreased only by concentrations of epinephrine greater than those inducing maximal intracellular lipolysis, and the decrease in activity occurred about 30 min after the increase in glycerol release. There seems to be no relationship between the level of activity of lipoprotein lipase and the maximal rate of hormone-stimulated lipolysis in 3T3-L1 cells. Unlike in adipose tissue and adipocytes of rats, hormone-stimulated lipolysis and lipoprotein lipase activity in murine 3T3-L1 adipocytes appear to be regulated independently.     

Secretion of triglyceride lipase from rat hepatocytes in culture: modulation by insulin and phenobarbital

Hepatic triglyceride lipase (HTGL) was measured in primary rat hepatocytes maintained for 3 days under three different culture conditions: basal medium, basal medium plus insulin, and basal medium plus insulin and phenobarbital. The activity of HTGL secreted by these cells was measured by treating intact cells with heparin; intracellular enzyme was subsequently measured in cell homogenates. Insulin stimulated intracellular triglyceride lipase activity by 48% and extracellular lipase by 30%. Phenobarbital, an enzyme-inducing drug, caused a further 15% increase in extracellular hepatic triglyceride lipase; whereas, the intracellular activity was reduced. The presence of insulin greatly stimulated the rate of enzyme secretion, and this rate was not notably affected by the presence of phenobarbital. After 3 days in culture, the short term (2-8 h) synthesis and secretion of enzyme from cultures treated with insulin or insulin plus phenobarbital were equally inhibited by cycloheximide. Monensin also inhibited enzyme secretion in both cultures and caused a similar increase in intracellular lipase activities. Insulin did not significantly affect the proportion of intracellular enzyme (17.7% basal vs. 15.8% insulin). On the other hand phenobarbital produced a 20-30% reduction in the proportion of intracellular enzyme (12.5 vs. 17.7% basal or 15.8% insulin). These findings suggest a drug-induced redistribution of triglyceride lipase.     

Stimulation of lipolysis and hormone-sensitive lipase via the extracellular signal-regulated kinase pathway

Hormonally stimulated lipolysis occurs by activation of cyclic AMP-dependent protein kinase (PKA) which phosphorylates hormone-sensitive lipase (HSL) and increases adipocyte lipolysis. Evidence suggests that catecholamines not only can activate PKA, but also the mitogen-activated protein kinase pathway and extracellular signal-regulated kinase (ERK). We now demonstrate that two different inhibitors of MEK, the upstream activator of ERK, block catecholamine- and beta(3)-stimulated lipolysis by approximately 30%. Furthermore, treatment of adipocytes with dioctanoylglycerol, which activates ERK, increases lipolysis, although MEK inhibitors decrease dioctanoylglycerol-stimulated activation of lipolysis. Using a tamoxifen regulatable Raf system expressed in 3T3-L1 preadipocytes, exposure to tamoxifen causes a 14-fold activation of ERK within 15-30 min and results in approximately 2-fold increase in HSL activity. In addition, when differentiated 3T3-L1 cells expressing the regulatable Raf were exposed to tamoxifen, a 2-fold increase in lipolysis is observed. HSL is a substrate of activated ERK and site-directed mutagenesis of putative ERK consensus phosphorylation sites in HSL identified Ser(600) as the site phosphorylated by active ERK. When S600A HSL was expressed in 3T3-L1 cells expressing the regulatable Raf, tamoxifen treatment fails to increase its activity. Thus, activation of the ERK pathway appears to be able to regulate adipocyte lipolysis by phosphorylating HSL on Ser(600) and increasing the activity of HSL.