The role of somatostatins in the regulation of metabolism in fish

Somatostatins (SS) are a structurally and functionally diverse family of peptide hormones. Somatostatins possess a wide variety of biological functions, including numerous secretotropic, developmental, and metabolic effects. Studies on fish have revealed considerable insight into the role of SS on the regulation of intermediary metabolism. Somatostatins promote both lipid and carbohydrate breakdown in fish and lamprey. Such actions are mediated by secretotropic effects of SS. For example, SS inhibit insulin (INS); insulin deficiency favors lipolysis and glycogenolysis over lipogenesis and glycogenesis. Somatostatins also directly stimulate the breakdown of stored triacylglycerols (TG) and glycogen in storage tissues. In addition, SS interact with the growth and reproductive axes of fish, findings that suggest SS serve to modulate energy partitioning among various growth, development and reproductive processes.     

Suppression of adipose lipolysis by long-chain fatty acid analogs

Agonist-induced lipolysis of adipose fat is robustly inhibited by insulin or by feedback inhibition by the long-chain fatty acids (LCFA) produced during lipolysis. However, the mode of action of LCFA in suppressing adipose lipolysis is not clear. β,β'-Tetramethyl hexadecanedioic acid (Mββ/ EDICA16) is a synthetic LCFA that is neither esterified into lipids nor β-oxidized, and therefore, it was exploited for suppressing agonist-induced lipolysis in analogy to natural LCFA. Mββ is shown here to suppress isoproterenol-induced lipolysis in the rat in vivo as well as in 3T3-L1 adipocytes. Inhibition of isoproterenol-induced lipolysis is due to decrease in isoproterenol-induced cAMP with concomitant inhibition of the phosphorylation of hormone-sensitive lipase and perilipin by protein kinase A. Suppression of cellular cAMP levels is accounted for by inhibition of the adenylate cyclase due to suppression of Raf1 expression by Mββ-activated AMPK. Suppression of Raf1 is further complemented by induction of components of the unfolded-protein-response by Mββ. Our findings imply genuine inhibition of agonist-induced adipose lipolysis by LCFA, independent of their β-oxidation or reesterification. Mββ suppression of agonist-induced lipolysis and cellular cAMP levels independent of the insulin transduction pathway may indicate that synthetic LCFA could serve as insulin mimetics in the lipolysis context under conditions of insulin resistance.     

Characterization of lipolytic responses of isolated white adipocytes from hamsters.

1. Lipolysis by isolated white adipocytes from hamsters, as measured by glycerol production, was stimulated by corticotropin, isopropylnorepinephrine (INE), norepinephrine, or epinephrine (EPI), in a dose-dependent fashion. 2. Lipolysis was stimulated by five inhibitors of cyclic 3',5'-adenosine monophosphate phosphodiesterase: caffeine, theophylline, 1-methyl-3-isobutyl xanthine, 1-ethyl-4-(isopropylidenehydrazine)-1H-pyrazolo-(3,4,-b)-pyridine-5-carboxylic acid ethyl ester (SQ 20009), and 4-(3,4-dimethoxybenzyl)-2-imidazolidinone (Ro 7-2956). Caffeine-stimulated lipolysis consistently attained higher rates than did hormone-stimulated lipolysis. However, when cells were stimulated by both caffeine and a hormone, lipolytic rates were consistently lower than those attained under the influence of caffeine alone. 3. Isolated white adipocytes from hamsters were sensitive to both alpha- and beta-adrenergic antagonists. The beta-adrenergic antagonist propranolol could completely inhibit norepinephrine-stimulated glycerol production. The alpha-adrenergic antagonist phentolamine, on the other hand, had a biphasic effect on the cells. At 5-10(-7) M or 5-10(-6) M, phentolamine enhanced norepinephrine-stimulated lipolysis, while concentrations higher than 5-10(-5) M caused inhibition. 4. The effects of two different concentrations of six antilipolytic agents, prostaglandin E1, nicotinic acid, phenylisopropyladenosine, 5-methylpyrazole-3-carboxylic acid, adenosine and insulin, were measured. With the exception of insulin, all of these agents showed much more potent inhibition of caffeine-stimulated lipolysis than of hormone-stimulated lipolysis. Insulin, in contrast, showed only modest inhibition of hormone-stimulated lipolysis and virtually no inhibition of caffeine-stimulated lipolysis.     

Absence of alpha-adrenergic inhibition of lipolysis in swine adipose tissue

Adipose tissue slices from young and older pigs and genetically obese pigs were incubated to demonstrate alpha-adrenergic inhibition of lipolysis as found by other investigators in dog, guinea-pig, hamster, human and rabbit adipose tissue. Purported alpha-adrenergic agonists (amidephrine, clonidine, methoxamine, phenylephrine) did not inhibit basal or catecholamine-stimulated lipolysis. Purported alpha-adrenergic antagonists (dihydroergotamine, phenoxybenzamine, phentolamine, prazosin, yohimbine) did not enhance basal or stimulated lipolysis. Adipose tissue from pigs is different from that of most species but similar to that of rats with no alpha-adrenergic inhibition of lipolysis.     

Differential inhibition of lipolysis by 2-bromopalmitic acid and 4-bromocrotonic acid in 3T3-L1 adipocytes

Two inhibitors of fatty acid oxidation, 2-bromopalmitic acid (Br-C16) and 4-bromocrotonic acid (Br-C4) were examined for their effect on lipolysis in 3T3-L1 adipocytes. Both agents inhibited in a dose-dependent manner the rate of oxidation of exogenously added [1-¹⁴C]palmitate with similar time-courses, reaching a plateau at 3–9 h. While Br-C16 at 50 μM and 100 μM inhibited palmitate oxidation by approximately 40% and 60%, respectively, pretreatment with both concentrations inhibited lipolysis in washed cells in an almost identical manner. The magnitude of inhibition increased with time of pretreatment. On the other hand, like inhibition of fatty acid oxidation, inhibition of lipolysis by Br-C4 pretreatment was dose-dependent with maximal inhibition reached after 3 h pretreatment. The finding that isoproterenol- and dibutyryl cAMP-stimulated lipolysis were similarly suppressed by either Br-C4 or Br-C16 pretreatment, suggesting that a step distal to cAMP formation was involved. In addition, while the inhibitory effect of Br-C16 was not significantly influenced, the inhibition of lipolysis caused by Br-C4 was attenuated by pretreating cells with crotonic acid, octanoate, or palmitate. The longer chain-length of the fatty acids the cells were exposed, the stronger attenuation of the inhibition caused by Br-C4 was observed. Moreover, whereas pretreatment with Br-C16 was without effect, pretreatment with Br-C4 significantly decreased hormone-sensitive lipase (HSL) activity in cell extracts, albeit to an extent much smaller than its inhibitory effect on lipolysis. In conclusion, these results indicate that irreversible inhibition of lipolysis by Br-C16 or Br-C4 cannot be attributed to their effect on fatty acid oxidation. Some factor capable of modulating HSL activity seems to be involved.     

Inhibitory effect and mode of action of somatostatin on lipolysis in chicken adipocytes

The effect of somatostatin on lipolysis was investigated utilizing isolated chicken adipocytes. Somatostatin-14 and -28 inhibited basal lipolysis. This ability to suppress glycerol release (used as an index of lipolysis) was emphasized in presence of stimulated lipolysis. Concentration of 1 ng/ml somatostatin-14 (0.625 nM) and somatostatin-28 (0.312 nM) was found to inhibit completely the glycerol release induced by concentrations of glucagon up to 2 ng/ml (0.58 nM). The percentage of inhibition was dose-dependent. The antilipolytic effect of somatostatin-14 was also observed during ACTH and aminophylline-stimulated lipolysis. Among the mechanisms which could account for the inhibition, a possible competitive effect of somatostatin-14 with 125I-labelled glucagon binding to adipocyte membranes was excluded. The small inhibiting effect of somatostatin-14 on glycerol release prompted by dibutyryl cyclic AMP, together with the significant inhibiting effect on aminophylline-stimulated lipolysis argued for a reduction of cyclic AMP accumulation. The increase of cyclic AMP levels induced by glucagon was substantially reduced in presence of somatostatin-14. It was concluded that in chicken adipocytes somatostatin inhibited the rate of lipolysis and that reduction on cyclic AMP could be responsible, at least in part, for the antilipolytic effect.     

Mechanisms of inhibition of triacylglycerol hydrolysis by human gastric lipase

In the human stomach, gastric lipase hydrolyzes only 10 to 30% of ingested triacylglycerols because of an inhibition process induced by the long chain free fatty acids generated, which are mostly protonated at gastric pH. The aim of this work was to elucidate the mechanisms by which free fatty acids inhibit further hydrolysis. In vitro experiments examined gastric lipolysis of differently sized phospholipid-triolein emulsions by human gastric juice or purified human gastric lipase, under close to physiological conditions. The lipolysis process was further investigated by scanning electron microscopy, and gastric lipase and free fatty acid movement during lipolysis were followed by fluorescence microscopy. The results demonstrate that: 1) free fatty acids generated during lipolysis partition between the surface and core of lipid droplets with a molar phase distribution coefficient of 7.4 at pH 5.40; 2) the long chain free fatty acids have an inhibitory effect only when generated during lipolysis; 3) inhibition of gastric lipolysis can be delayed by the use of lipid emulsions composed of small-size lipid droplets; 4) the release of free fatty acids during lipolysis induces a marked increase in droplet surface area, leading to the formation of novel particles at the lipid droplet surface; and 5) the gastric lipase is trapped in these free fatty acid-rich particles during their formation. In conclusion, we propose a model in which the sequential physicochemical events occurring during gastric lipolysis lead to the inhibition of further triacylglycerol lipolysis.     

Inducible nitric oxide synthase modulates lipolysis in adipocytes

The role of inducible nitric oxide synthase (iNOS) in the modulation of adipocyte lipolysis was investigated. Treatment of white and brown adipose cell lines and mouse adipose explants with a mixture of tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide (LPS) doubled the lipolytic rate, and this was associated with marked induction of iNOS expression and nitric oxide (NO) production. iNOS inhibition by 1400W, aminoguanidine, or L-NIL pretreatment further increased the cytokine/LPS-mediated lipolysis by 30% (P < 0.05) in cultured adipocytes and in adipose explants. However, this potentiating effect of iNOS inhibition was abolished in adipose explants isolated from iNOS knockout mice. Pharmacological inhibitors of adenylyl cyclase or protein kinase A reduced cytokine/LPS-induced lipolysis and also blunted the potentiating effect of iNOS inhibition on the lipolytic rate. Furthermore, addition of the antioxidants l-cystine and l-glutathione to cytokine/LPS-stimulated adipocytes mimicked the lipolytic effect of iNOS inhibition. In conclusion, inhibition of iNOS activity in adipocytes potentiates cytokine/LPS-induced lipolysis. This effect was fully reversed by adenylyl cyclase and protein kinase A inhibitors but was mimicked by cellular antioxidants. These data suggest that iNOS-mediated NO production counteracts cytokine/LPS-mediated lipolysis in adipocytes and that this feedback mechanism involves an oxidative process upstream of cAMP production in the signaling pathway.     

Inhibition of growth hormone-induced lipolysis by 3',5'-guanosine monophosphate in chicken adipose tissue in vitro

The influence of cyclic 3',5'-guanosine monophosphate (cGMP) on the lipolytic and antilipolytic (inhibition of glucagon-stimulated lipolysis) responses to GH (1 microgram/ml) was examined in chicken adipose tissue in vitro. Both 8-bromo-cGMP (0.1 mM) and sodium nitroprusside (1 mM) (a guanyl cyclase stimulator) completely inhibited the lipolytic effect of GH. A cGMP-lowering agent, LY83583 (10 microM), reversed the inhibitory effect of sodium nitroprusside on GH-stimulated lipolysis. Furthermore, the suppressive effects of insulin (100 ng/ml), insulin-like growth factor I (IGF-I) (100 ng/ml), or insulin-like growth factor II (IGF-II/MSA) (100 ng/ml), but not somatostatin (1 ng/ml), on GH-stimulated lipolysis were prevented by LY83583 addition. Neither 8-bromo-cGMP, sodium nitroprusside, nor LY83583 altered GH-induced inhibition of glucagon (1 ng/ml)-stimulated lipolysis. It is proposed that cGMP may mediate inhibitory control of GH-stimulated lipolysis by insulin, IGF-I, and IGF-II in chicken adipose tissue.     

In vitro effects of leptin on human adipocyte metabolism

OBJECTIVE: Leptin receptors are expressed in adipocytes, suggesting potential autocrine/paracrine effects. Studies on the direct effects of leptin on adipose tissue metabolism in different species have yielded controversial data. To assess the in vitro effects of leptin on human adipocyte metabolism: lipolysis, the insulin-induced inhibition of lipolysis and lipogenesis were studied in adipocytes obtained from infants and adults. METHODS: Lipolysis was studied by incubating adipocytes with increasing concentrations of leptin or isoprenaline. Glycerol in the incubation medium was measured as an indicator of lipolysis. For the lipogenesis and insulin-induced inhibition of lipolysis experiments, the cells were preincubated with 0, 25, or 250 ng/ml of leptin for 2 h. RESULTS: Leptin did not stimulate lipolysis in human adipocytes, either in children or adults. Preincubation with leptin did not affect the insulin-induced inhibition of lipolysis, but decreased the insulin-induced lipogenesis (p < 0.05). CONCLUSIONS: This study shows that leptin has no direct lipolytic effect in human adipocytes. The lack of effect on the insulin-induced inhibition of lipolysis and the negative effect on lipogenesis indicates that the effect of leptin is not at the proximal insulin-signalling pathway but further downstream.     

Modulation of the leptin-induced white adipose tissue lipolysis by nitric oxide

The present study tested the hypothesis that nitric oxide (NO) is involved in the leptin-induced stimulation of lipolysis. The effect of intravenous (iv) administration of leptin (10, 100 and 1000 microg/kg body weight) or vehicle on serum NO concentrations and glycerol release from white adipocytes of Wistar rats was examined. One hour after injection, the three leptin doses tested increased serum NO concentrations 15.1%, 23.4% and 60.0%, respectively (P<.001 vs. baseline). The effect of leptin on NO concentrations was significantly dose dependent on linear trend testing (P=.0001). Simple linear regression analysis showed that the lipolytic rate measured was significantly correlated with serum NO concentrations (P=.0025; r=.52). In order to gain further insight into the potential underlying mechanisms, the effect of leptin on lipolysis was studied in the setting of nitric oxide synthase (NOS) inhibition or acute ganglionic blockade. The stimulatory effect of leptin on lipolysis was significantly decreased (P<.05) under NOS inhibition. On the contrary, the leptin-induced lipolysis was unaltered in pharmacologically induced ganglionic blockade. The lack of effect on isoproterenol-, forskolin- and dibutyryl-cyclic AMP-stimulated lipolysis suggests that leptin does not interfere with the signal transduction pathway at the beta-adrenergic receptor, the adenylate cyclase and the protein kinase A levels. These findings suggest that NO is a potential regulator of leptin-induced lipolysis.     

The biological activity of duck 'big' somatostatin on chicken adipose tissue

A peptide, eluted with cytochrome c, called 'big' somatostatin, is the only somatostatin-like immunoreactivity present in the peripheral plasma of the duck. The metabolic action of partially purified fractions of 'big' somatostatin was investigated on glucagon-stimulated lipolysis in chicken adipocytes. Significant inhibition of glycerol release (an index of lipolysis) induced by physiological concentrations of glucagon was observed with physiological concentrations of 'big' somatostatin; the percentage of inhibition was dose-dependent.     

Effect of PCO2 on epinephrine-induced lipolysis in isolated fat cells.

The effect of different pHs obtained by changing the PCO2 and the effect of PCO2 at constant pH on the lipolysis induced by epinephrine in isolated fat cells have been investigated. An inhibition of activated lipolysis was found in acidosis while in alkalosis no significant change was detected. When the experiments were performed at different PCO2s but at constant pH, the results showed an inhibition of lipolysis by high PCO2 whereas low PCO2 did not affect it. It is concluded that either acidosis or high PCO2 lead to an inhibition of the lipolysis induced by epinephrine in isolated fat cells. As regards alkalosis and low PCO2 it seems likely that the intracellular pH is not affected to the same extent as in alkalosis by high [HCO(-3)] or under the conditions of the present experiments the [H+] needed to alterate lipolysis was not reached.     

Mechanisms involved in the regulation of free fatty acid release from isolated human fat cells by acylation-stimulating protein and insulin.

The effects of acylation-stimulating protein (ASP) and insulin on free fatty acid (FFA) release from isolated human fat cells and the signal transduction pathways to induce these effects were studied. ASP and insulin inhibited basal and norepinephrine-induced FFA release by stimulating fractional FFA re-esterification (both to the same extent) and by inhibiting FFA produced during lipolysis (ASP to a lesser extent than insulin). Protein kinase C inhibition influenced none of the effects of ASP or insulin. Phosphatidylinositol 3-kinase inhibition counteracted the effects of insulin but not of ASP. Phosphodiesterase 3 (PDE3) activity was stimulated by ASP and insulin, whereas PDE4 activity was slightly increased by ASP only. Selective PDE3 inhibition reversed the effects of both ASP and insulin on fractional FFA re-esterification and lipolysis. Selective PDE4 inhibition slightly counteracted the ASP but not the effect of insulin on fractional FFA re-esterification and did not prevent the action of ASP or insulin on lipolysis. Thus, ASP and insulin play a major role in regulating FFA release from fat cells as follows: insulin by stimulating fractional FFA re-esterification and inhibiting lipolysis and ASP mainly by stimulating fractional FFA re-esterification. For both ASP and insulin these effects on FFA release are mediated by PDE3, and for ASP PDE4 might also be involved. The signaling pathway preceding PDE is not known for ASP but involves phosphatidylinositol 3-kinase for insulin.     

Natriuretic peptides: a new lipolytic pathway in human adipocytes.

Atrial natriuretic peptide (ANP) receptors have been described on rodent adipocytes and expression of their mRNA is found in human adipose tissue. However, no biological effects associated with the stimulation of these receptors have been reported in this tissue. A putative lipolytic effect of natriuretic peptides was investigated in human adipose tissue. On isolated fat cells, ANP and brain natriuretic peptide (BNP) stimulated lipolysis as much as isoproterenol, a nonselective beta-adrenergic receptor agonist, whereas C-type natriuretic peptide (CNP) had the lowest lipolytic effect. In situ microdialysis experiments confirmed the potent lipolytic effect of ANP in abdominal s.c. adipose tissue of healthy subjects. A high level of ANP binding sites was identified in human adipocytes. The potency order defined in lipolysis (ANP > BNP > CNP) and the ANP-induced cGMP production sustained the presence of type A natriuretic peptide receptor in human fat cells. Activation or inhibition of cGMP-inhibited phosphodiesterase (PDE-3B) (using insulin and OPC 3911, respectively) did not modify ANP-induced lipolysis whereas the isoproterenol effect was decreased or increased. Moreover, inhibition of adenylyl cyclase activity (using a mixture of alpha(2)-adrenergic and adenosine A1 agonists receptors) did not change ANP- but suppressed isoproterenol-induced lipolysis. The noninvolvement of the PDE-3B was finally confirmed by measuring its activity under ANP stimulation. Thus, we demonstrate that natriuretic peptides are a new pathway controlling human adipose tissue lipolysis operating via a cGMP-dependent pathway that does not involve PDE-3B inhibition and cAMP production.     

Regulation of cyclic AMP metabolism and lipolysis in isolated rat fat cells by insulin, N6-(phenylisopropyl) adenosine and 2',5'-dideoxyadenosine.

The increases in cyclic AMP accumulation and lipolysis by rat fat cells incubated in the presence of catecholamines were abolished by N6-(phenylisopropyl) adenosine. The same inhibition of cyclic AMP accumulation was seen in the presence of 2',5'-dideoxyadenosine but lipolysis was unaffected. In contrast, insulin inhibited lipolysis without affecting cyclic AMP accumulation by norepinephrine plus adenosine deaminase. These results suggest that there are either multiple pools of cyclic AMP or that ther exists some other mechanism which is involved in the regulation of lipolysis by hormones.     

The action of local anaesthetics on lipolysis and on adenosine 3′:5′-cyclic monophosphate content in isolated rat fat-cells

1. Local anaesthetics inhibited hormone-stimulated lipolysis in isolated rat fat-cells. The most potent anaesthetic was dibucaine, which inhibited adrenaline-stimulated lipolysis by 50% at a concentration of 0.16mm. 2. The amount of inhibition produced by a given concentration of anaesthetic was very similar with adrenaline, theophylline and dibutyryl cyclic AMP, at submaximal and maximal concentrations. 3. The inhibitory effect of dibucaine on lipolysis was apparent within 5 min and was constant over 1h. 4. Dibucaine inhibited basal, adrenaline-stimulated and insulin-stimulated glucose uptake at concentrations 6-10-fold higher than those inhibiting lipolysis. 5. The effects of dibucaine on lipolysis and glucose uptake were reversed after removal of anaesthetic and washing of cells. 6. Dibucaine further elevated the concentration of cyclic AMP in the presence of adrenaline or adrenaline plus theophylline. 7. Dibucaine had no effect on ATP content at concentrations causing 80% inhibition of lipolysis, but lowered ATP content at higher concentrations. 8. The relative potency of different local anaesthetics as inhibitors of hormone-stimulated lipolysis paralleled their potency as inhibitors of ion movements in other systems. 9. The possibility is discussed that Ca(2+) ions are involved in the regulation of lipolysis, and that local anaesthetics inhibit lipolysis by interfering with Ca(2+) translocation.     

Short-term fasting and lipolytic activity in rat adipocytes.

The aim of this experiment was to study the influence of 18-hour food deprivation on basal and stimulated lipolysis in adipocytes obtained from young male Wistar rats. Fat cells from fed and fasted rats were isolated from the epididymal adipose tissue by collagenase digestion. Adipocytes were incubated in Krebs-Ringer buffer (pH 7.4, 37 degrees C) without agents affecting lipolysis and with different lipolytic stimulators (epinephrine, forskolin, dibutyryl-cAMP, theophylline, DPCPX, amrinone) or inhibitors (PIA, H-89, insulin). After 60 min of incubation, glycerol and, in some cases, also fatty acids released from adipocytes to the incubation medium were determined. Basal lipolysis was substantially potentiated in cells of fasted rats in comparison to adipocytes isolated from fed animals. The inhibition of protein kinase A activity by H-89 partially suppressed lipolysis in both groups of adipocytes, but did not eliminate this difference. The agonist of adenosine A (1) receptor also did not suppress fasting-enhanced basal lipolysis. The epinephrine-induced triglyceride breakdown was also enhanced by fasting. Similarly, the direct activation of adenylyl cyclase by forskolin or protein kinase A by dibutyryl-cAMP resulted in a higher lipolytic response in cells derived from fasted animals. These results indicate that the fasting-induced rise in lipolysis results predominantly from changes in the lipolytic cascade downstream from protein kinase A. The antagonism of the adenosine A (1) receptor and the inhibition of cAMP phosphodiesterase also induced lipolysis, which was potentiated by food deprivation. Moreover, the rise in basal and epinephrine-stimulated lipolysis in adipocytes of fasted rats was shown to be associated with a diminished non-esterified fatty acids/glycerol molar ratio. This effect was presumably due to increased re-esterification of triglyceride-derived fatty acids in cells of fasted rats. Comparing fed and fasted rats for the antilipolytic effect of insulin in adipocytes revealed that short-term food deprivation resulted in a substantial deterioration of the ability of insulin to suppress epinephrine-induced lipolysis.     

Hormonal control of cardiac lipolysis by glyco(geno)lysis

The importance of the glucose/fatty acid cycle in the control of cardiac lipolysis is emphasized by the following observations. Addition of the glycogen debranching inhibitor deoxynojirimycin or an O2-vehicle, fluorocarbon F-43, to media perfusing paced, lipid-enriched, Langendorff hearts lower cardiac lactate and glycerol 3-phosphate levels together with inhibition of glucagon-stimulated glycerol (and lactate) release. The absence of fluorocarbon during perfusion of 5 Hz paced langendorff hearts probably results in limited tissue oxygenation, resulting in glycogenolysis and lipolysis. The results indicate hormonal control of cardiac lipolysis by glyco(geno)lysis.     

The biological activity of duck ‘big’ somatostatin on chicken adipose tissue

A peptide, eluted with cytochrome c, called ‘big’ somatostatin, is the only somatostatin-like immunoreactivity present in the peripheral plasma of the duck. The metabolic action of partially purified fractions of ‘big’ somatostatin was investigated on glucagon-stimulated lipolysis in chicken adipocytes. Significant inhibition of glycerol release (an index of lipolysis) induced by physiological concentrations of glucagon was observed with physiological concentrations of ‘big’ somatostatin; the percentage of inhibition was dose-dependent.