Effects of chronic treatment with noradrenaline or a specific beta3-adrenergic agonist, CL 316 243, on energy expenditure and epididymal adipocyte lipolytic activity in rat

1. The effects of 7 days exposure to a specific beta3-adrenergic agonist, CL 316 243 (1 mg/kg x 24 hr), or to the physiological hormone, noradrenaline (5 mg/kg x 24 hr), were tested on energy expenditure and on in vitro lipolysis in male Sprague-Dawley rats. 2. At the second day of treatment, the total energy expenditure and the resting metabolic rate were increased by 20 and 30%, respectively, in the CL-treated group. Under the same conditions, a dose five times higher of NA increased the resting metabolic rate by 11% without any significant change in the total daily energy expenditure. 3. The CL-treated group showed a lower weight gain, correlated with a significant reduction in retroperitoneal adipose tissue weight. Both treatments resulted in a marked desensitization (increased EC50 values) of the NA stimulated lipolysis of epididymal adipocytes. The effects of both treatments on maximal lipolysis were opposite. Indeed, chronic NA-treatment decreased the responsiveness of lipolysis while chronic treatment with CL increased the maximal stimulation of lipolysis to NA. Furthermore, dose-response curve for CL on lipolysis showed a marked functional desensitization of beta3-adrenergic response. 4. Our results demonstrate the high selectivity of beta3-adrenergic agonists to stimulate whole body energy expenditure and lipid mobilization in rodents. The present results point out for the first time an adrenergic desensitization of the lipolytic response after chronic administration of a beta3-agonist.     

Oleoyl-estrone increases adrenal corticosteroid synthesis gene expression in overweight male rats

Oleoyl-estrone (OE) induces a marked loss of body fat in rats by maintaining energy expenditure, body protein and blood glucose despite decreasing food intake. OE increases glucocorticoids, but they arrest OE lipid-mobilization. We studied here whether OE induces a direct effect on adrenal glands function as part of this feedback regulation. Dietary overweight male rats were given oral 10 nmol/g OE gavages for ten days. A group (PF) of pair-fed to OE rats, and controls received vehicle-only gavages. OE rats lost slightly more body than PF, but had larger adrenal glands. Tissue corticosterone levels, and gene expressions for glucocorticoid-synthesizing enzymes were increased in OE versus controls and PF; thus, we assumed that adrenal growth affected essentially its cortex since OE also lowered the expression of the medullar catecholamine synthesis enzyme genes. Serum corticosterone was higher in PF than in OE and controls, but liver expression of corticosteroid-disposing steroid 5α-reductase was 3× larger in OE than PF and controls. Circulating glucocorticoids changed little under OE, in spite of higher adrenal gland and liver content, hinting at modulation of glucocorticoid turnover as instrumental in their purported increased activity. In conclusion, we have observed that OE considerable enhanced the expression of the genes controlling the synthesis of glucocorticoids from cholesterol in the rat and increasing the adrenal glands’ corticosterone, size and cellularity, but also the liver disposal of corticosteroids, suggesting that OE increases corticosterone synthesis and degradation (i.e. serum turnover), a process not driven by limited energy availability but directly related to the administration of OE.     

Effect of oral oleoyl-estrone on the energy balance of diabetic rats.

We studied the effects of a 10-day oral 10 micromol/kg oleoyl-estrone (OE) treatment on streptozotocin-diabetic Wistar, Goto-Kakizaki and control Wistar rats. Streptozotocin rats lost more than half the energy ingested as urine glucose. Oleoyl-estrone induced the loss of body weight (mainly body fat) in all groups. Energy expenditure was similar in the three groups of rats studied. Water turnover was deranged in streptozotocin rats, which spent 14% of energy available heating the water drunk. Body lipids were highest in Goto-Kakizaki; lipid levels in streptozotocin rats were very low. Oleoyl-estrone decreased body lipid content in Wistar and Goto-Kakizaki; oleoyl-estrone decreased triacylglycerols (44% in Wistar and Goto-Kakizaki and 22% in streptozotocin rats) and phospholipids but did not affect body cholesterol. Oleoyl-estrone decreased insulin and leptin, did not affect blood glucose but decreased plasma glucose in all groups. There were no changes in plasma triacylglycerols or fatty acids, but HDL, LDL and cholesterol decreased in all groups. The same effects of OE on insulin, plasma (but not blood) glucose and leptin were observed in both models, but the presence of insulin seems to be needed for OE to normalise glycaemia and to facilitate the uptake and utilisation of glucose by tissues. This different handling of glucose and triacylglycerol energy accounts for the disparate effects of OE on energy balance. The main conclusion of this study is that OE function as a lipid-mobilising hormone is dependent on the mass of reserves available, which in turn is closely related to insulin status. Lack of insulin thus results in limited OE effects, and insulin resistance does not prevent or limit the effects of OE on energy homeostasis or the mobilisation of fat.     

Citrate diminishes hypothalamic acetyl-CoA carboxylase phosphorylation and modulates satiety signals and hepatic mechanisms involved in glucose homeostasis in rats

The hypothalamic AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway is known to play an important role in the control of food intake and energy expenditure. Here, we hypothesize that citrate, an intermediate metabolite, activates hypothalamic ACC and is involved in the control of energy mobilization. Initially, we showed that ICV citrate injection decreased food intake and diminished weight gain significantly when compared to control and pair-fed group results. In addition, we showed that intracerebroventricular (ICV) injection of citrate diminished (80% of control) the phosphorylation of ACC, an important AMPK substrate. Furthermore, citrate treatment inhibited (75% of control) hypothalamic AMPK phosphorylation during fasting. In addition to its central effect, ICV citrate injection led to low blood glucose levels during glucose tolerance test (GTT) and high glucose uptake during hyperglycemic-euglycemic clamp. Accordingly, liver glycogen content was higher in animals given citrate (ICV) than in the control group (23.3+/-2.5 vs. 2.7+/-0.5 microg mL(-1) mg(-1), respectively). Interestingly, liver AMPK phosphorylation was reduced (80%) by the citrate treatment. The pharmacological blockade of beta3-adrenergic receptor (SR 59230A) blocked the effect of ICV citrate and citrate plus insulin on liver AMPK phosphorylation. Consistently with these results, rats treated with citrate (ICV) presented improved insulin signal transduction in liver, skeletal muscle, and epididymal fat pad. Similar results were obtained by hypothalamic administration of ARA-A, a competitive inhibitor of AMPK. Our results suggest that the citrate produced by mitochondria may modulate ACC phosphorylation in the hypothalamus, controlling food intake and coordinating a multiorgan network that controls glucose homeostasis and energy uptake through the adrenergic system.     

Discovery of potent and orally bioavailable heterocycle-based beta3-adrenergic receptor agonists, potential therapeutics for the treatment of obesity

A novel series of heterocycle-based analogs were prepared and evaluated for their in vitro and in vivo biological activity as human beta(3)-adrenergic receptor (AR) agonists. Several analogs demonstrated potent agonist activity at the beta(3)-AR, functional selectivity against beta(1)- and beta(2)-ARs, and favorable pharmacokinetic profiles in vivo. Compound 17 increased oxygen consumption in rats, a measure of energy expenditure, with an ED(20%) of 2mg/kg.     

Treatment of pregnant rats with oleoyl-estrone slows down pup fat deposition after weaning

Background  

In rats, oral oleoyl-estrone (OE) decreases food intake and body lipid content. The aim of this study was to determine whether OE treatment affects the energy metabolism of pregnant rats and eventually, of their pups; i.e. changes in normal growth patterns and the onset of obesity after weaning.     

Oleoyl-oestrone inhibits lipogenic, but maintains thermogenic, gene expression of brown adipose tissue in overweight rats

In the present study we intended to determine how BAT (brown adipose tissue) maintained thermogenesis under treatment with OE (oleoyl-oestrone), a powerful slimming hormone that sheds off body lipid but maintains the metabolic rate. Overweight male rats were subjected to daily gavages of 10 nmol/g of OE or vehicle (control) for 10 days. A PF (pair-fed) vehicle-receiving group was used to discount the effects attributable to energy availability limitation. Interscapular BAT mass, lipid, DNA, mRNA and the RT-PCR (real-time PCR) expression of lipid and energy metabolism genes for enzymes and regulatory proteins were measured. BAT mass and lipid were decreased in OE and PF, with the latter showing a marked reduction in tissue mRNA. Maintenance of perilipin gene expression in PF and OE rats despite the loss of lipid suggests the preservation of the vacuolar interactive surface, a critical factor for thermogenic responsiveness. OE and, to a lesser extent, PF maintained the expression of genes controlling lipolysis and fatty acid oxidation, but markedly decreased the expression of those genes involved in lipogenic and acyl-glycerol synthesis. OE did not affect UCP1 (uncoupling protein 1) (decreased in PF), beta(3) adrenergic receptors or hormone-sensitive lipase gene mRNAs, which may translate in maintaining a full thermogenic system potential. OE rats were able to maintain a less energetically stressed BAT (probably through glucose utilization) than PF rats. These changes were not paralleled in PF rats, in which lower thermogenesis and glucose preservation resulted in a heavier toll on internal fat stores. Thus the mechanism of action of OE is more complex and tissue-specific than previously assumed.     

Beta 3-adrenergic receptor is involved in feeding regulation in chicks

We examined whether the brain beta 3-adrenergic receptor (B3-AR) is involved in the feeding regulation of chicks. Intracerebroventricular (ICV) injection of BRL37344, a B3-AR agonist, reduced food intake of chicks under ad libitum, but not fasting, feeding conditions. The ICV injection of BRL37344 did not affect chick posture or locomotion activity suggesting that BRL37344 inhibited feeding without induction of sleep-like behavior as caused by norepinephrine. Furthermore, the rectal temperature increased following the ICV injection of BRL37344. Intraperitoneal administration of BRL37344 did not reduce food intake under ad libitum feeding condition. The present study demonstrated that the brain B3-AR is involved in the inhibition of feeding in chicks. We also suggested that activation of the brain affects the energy metabolism in chicks.     

Oleoyl-estrone affects lipid metabolism in adrenalectomized rats treated with corticosterone through modulation of SREBP1c expression

Oleoyl-estrone (OE) elicits a decrease in body fat, which is blocked by glucocorticoids. In order to analyze this counterregulatory effect, we studied the effects of oral OE on adrenalectomized female rats simultaneously receiving corticosterone (subcutaneous pellets). Circulating corticosteroids, liver glycogen, lipids and the expressions in whole liver, soleus muscle, interscapular brown adipose tissue (BAT), and the inguinal and periovaric white adipose tissue (WAT) of genes controlling lipid metabolism were analyzed. Corticosterone reversed OE lipid mobilization, storing fat in liver and subcutaneous WAT. This was not simply the predominance of corticosteroid enhancement of lipogenesis against OE inhibition, but a synergy to enhance lipogenesis. Periovaric WAT showed a different effect, with corticosterone inhibiting OE arrest of lipogenic gene expressions. The data presented suggests that interaction of OE and glucocorticoids (and the metabolic response) depends on the organ or WAT site; there was a direct relationship on the direction and extent of change of SREBP1c expression with those of important energy and lipid handling genes. Our results confirm that corticosterone blocks – and even reverses – OE effects on body lipids in a dose-dependent way, a process mediated, at least in part, by modulation of SREBP1c expression.     

Oleoyl-estrone is a precursor of an estrone-derived ponderostat signal

Oleoyl-estrone (OE) is a powerful anti-obesity compound that decreases food intake, decreases insulin resistance and circulating cholesterol. OE stimulates a severe loss of body fat by decreasing adipose tissue lipid synthesis and maintaining lipolysis. Therefore, the body economy loses lipid energy because energy expenditure is maintained. This study analyses the discrepancy between OE effects and the distribution of labelled OE in plasma. Estrone radioimmunoassay of organic solvent plasma extracts of rats treated with OE showed the massive presence of acyl-estrone, but saponification did not release estrone, but containing similar unknown compound. Analysis of label distribution in plasma after oral gavages of (3)H-OE showed the presence of a more hydrophilic compound than OE or any estrogen as well as (3)H(2)O, formed from (3)H-OE in the acidic stomach medium. OE was not attached to a specific transporter in plasma. Through serum HPLC analysis we found W, a labelled derivative more hydrophilic than OE or estrone. The results were confirmed using (14)C-OE. HPLC-MS/MS studies showed that plasma OE levels were one order of magnitude lower than those of W. When liver cell cytosols from rats laden with (3)H-OE were incubated with nuclei from untreated rats, the OE-derived label (i.e., Ws) was found attached to nuclear DNA. Neither estradiol nor estrone interfered with its binding. W is a fairly hydrophilic compound of low molecular weight containing the estrone nucleus, but it is not an ester because saponification or esterases do not yield estrone as OE does. It is concluded that OE acts through its conversion to W, its active form; which binds to a nuclear receptor different from that of estrogen. The estimated W serum levels are proportional to the pharmacological OE effects in vivo. We postulate W as a new type of hormone that exerts the full range of in vivo effects thus far attributed to OE. The full identification of W is anticipated to open the way for the development of new OE-like anti-obesity drugs.     

Electroacupuncture suppresses expression of gastric ghrelin and hypothalamic NPY in chronic food restricted rats

Electroacupuncture (EA) has been reported to reduce body weight in overweight subjects in clinical practice, as well as in rats and mice with diet-induced obesity. In the present study, this effect of EA was tested in lean rats subjected to long-term food restriction (FR, food was offered only 1 h/day). Two hertz EA administered once every other day produced a further reduction in body weight in FR rats. Exploration of the mechanism involved revealed significant downregulation of the orexigenic peptides: ghrelin in the stomach, and neuropeptide Y (NPY) but not Agouti-related peptide (AgRP) in the hypothalamus, which was in line with the reduction in food intake in rats receiving EA stimulation as compared with those receiving restraint only. Uncoupling protein 3 (UCP3), involved in accelerating energy expenditure, was not significantly altered. These results suggest that the EA-induced body weight reduction was due mainly to a decrease in food intake rather than an increase in energy expenditure. A reduction in the orexigenic peptides ghrelin and NPY may be involved in the underlying mechanism.     

Chemical sympathectomy alters regulation of body weight during prolonged ICV leptin infusion

To assess the importance of the sympathetic nervous system in regulating body weight during prolonged leptin infusion, we evaluated food intake, body weight, and physical activity in conscious, unrestrained rats. Initial studies illustrated that prolonged intracerebroventricular (ICV) infusion of leptin enhanced substrate oxidation so that adipose tissue lipid stores were completely ablated, and muscle triglyceride and liver glycogen stores were depleted. After neonatal chemical sympathectomy, changes in weight and food intake were compared in groups of sympathectomized (SYM) and control (CON) adult animals during ICV infusion of leptin. CON animals lost 60 +/- 9 g over 10 days vs. 25 +/- 3 g in the SYM animals when food intake was matched between the two groups. Greater weight loss despite similar energy intake points to an important role of the sympathetic nervous system in stimulating energy expenditure during ICV leptin infusion by increasing the resting metabolic rate, since no differences in physical activity were observed between CON and SYM groups. In conclusion, activation of the SNS by leptin increases energy expenditure by augmenting the resting metabolic rate.     

Effect of adipocyte beta3-adrenergic receptor activation on the type 2 diabetic MKR mice

The antiobesity and antidiabetic effects of the beta3-adrenergic agonists were investigated on nonobese type 2 diabetic MKR mice after injection with a beta3-adrenergic agonist, CL-316243. An intact response to acute CL-316243 treatment was observed in MKR mice. Chronic intraperitoneal CL-316243 treatment of MKR mice reduced blood glucose and serum insulin levels. Hyperinsulinemic euglycemic clamps exhibited improvement of the whole body insulin sensitivity and glucose homeostasis concurrently with enhanced insulin action in liver and adipose tissue. Treating MKR mice with CL-316243 significantly lowered serum and hepatic lipid levels, in part due to increased whole body triglyceride clearance and fatty acid oxidation in adipocytes. A significant reduction in total body fat content and epididymal fat weight was observed along with enhanced metabolic rate in both wild-type and MKR mice after treatment. These data demonstrate that beta3-adrenergic activation improves the diabetic state of nonobese diabetic MKR mice by potentiation of free fatty acid oxidation by adipose tissue, suggesting a potential therapeutic role for beta3-adrenergic agonists in nonobese diabetic subjects.     

Maternal treatment with oleoyl-estrone induces resistance to lipid accrual in their descendants

Objective: To determine whether treatment of rat dams with oleoyl‐estrone (OE) has an effect on the offspring's long‐term response to diet restriction during lactation. Methods and Procedures: Control, OE‐treated, and diet‐restricted dams were treated up to day 15 of lactation. Changes in food intake and body weight were recorded for dams and their pups. After weaning, pups received a 4‐week standard diet followed by a 4‐week period of high‐fat diet. Lipid, protein, and energy content of pups plus energy intake and efficiency. Serum metabolites (glucose, urea, and cholesterol) and serum hormones (adiponectin, leptin, insulin, and sexual hormones). Results: Neither pups from dams in the OE‐treated nor in the diet‐restricted group showed significant changes in weight, though these two groups ingested 79% of food ingested by controls. At weaning, the pups from OE‐treated rats were smaller than those of the control or diet‐restricted groups. These pups maintained the differences in size and lipid content during the 4‐week standard‐diet period, whereas pups from diet‐restricted dams showed a sharp decrease in their lipid content. During the 4 weeks of high‐fat diet, the male offspring from OE‐treated dams increased the difference in lipid content in relation to the pups from control dams whereas in females the differences decreased. Female offspring from diet‐restricted dams showed the most marked changes in metabolite and hormone levels in relation to controls. Discussion: Treatment of lactating dams with OE programs the metabolic response of their offspring to resist the challenge of a high‐fat diet that would lead to obesity in adulthood.     

Prolonged treatment with the β3-adrenergic agonist CL 316243 induces adipose tissue remodeling in rat but not in guinea pig: 1) fat store depletion and desensitization of β-adrenergic responses

Beta3-adrenergic agonists have been considered as potent antiobesity and antidiabetic agents mainly on the basis of their beneficial actions discovered twenty years ago in obese and diabetic rodents. The aim of this work was to verify whether prolonged treatment with a beta3-adrenergic agonist known to stimulate lipid mobilisation, could promote desensitization of beta-adrenergic responses. Wistar rats and guinea pigs were treated during one week with CL 316243 (CL, 1 mg/kg/d) by implanted osmotic minipumps. In control animals, beta3-adrenergic agonists were lipolytic in rat but not in guinea pig adipocytes. CL-treatment did not alter body weight gain in both species, but reduced fat stores in rats. Lipolysis stimulation by forskolin was unmodified but responses to beta1-, beta2- and beta3-agonists were reduced in visceral or subcutaneous white adipose tissues of CL-treated rats. Similarly, the beta3-adrenergic-dependent impairment of insulin action on glucose transport and lipogenesis in rat adipocytes was diminished after CL-treatment. In rat adipocytes, [125I]ICYP binding and beta3-adrenoceptor mRNA levels were reduced after sustained CL administration. These findings show that CL 316243 exerts (beta3-adrenergic lipolytic and antilipogenic effects in rat adipocytes. These actions, which are likely involved in the fat depletion observed in rat, also lead to the desensitization of all beta-adrenergic responses. Therefore this desensitization, together with the lack of slimming action in guinea pig, seriously attenuates the usefulness of beta3-agonists as antiobesity agents, and may explain why such agonists have not been conducted to a widespread clinical use.     

Acute and chronic effects of FR-149175, a beta 3-adrenergic receptor agonist, on energy expenditure in Zucker fatty rats

Clinical therapies for both obesity and obese non-insulin-dependent diabetes mellitus require maintenance of reduced body weight after the initial successful reduction resulting from calorie control, exercise, or medication. Although beta(3)-adrenergic receptor (beta(3)-AR) agonists have been shown to stimulate whole body energy expenditure and lipid mobilization, whether stimulatory effects on oxygen consumption and lipolysis are influenced by chronic exposure to agonists has not been fully characterized. We therefore examined the acute and chronic effects of FR-149175, a selective beta(3)-AR agonist, on whole body oxygen consumption in genetically obese Zucker fatty rats. Chronic treatment with FR-149175 caused a decrease in both body weight gain and white fat pad weight at doses that induced lipolysis in acute treatment (1 and 3.2 mg/kg p.o.). Single administration of FR-149175 (0.1, 1, and 3.2 mg/kg p.o.) dose dependently increased whole body oxygen consumption. Repetitive administration did not cause attenuation of the thermogenic response at lower doses (0.1 and 1 mg/kg 2 times daily), whereas the highest dose (3.2 mg/kg 2 times daily) induced a progressive increase in oxygen consumption. PCR analyses of retroperitoneal white adipose tissue indicated little or no change in beta(3)-AR mRNA levels. Uncoupling protein 1 gene expression increased at 1 mg/kg, and drastic upregulation was detected at 3.2 mg/kg. FR-149175 also increased HSL mRNA levels in a dose-related manner, whereas there was no effect on genes involved in beta-oxidation. These results support that the thermogenic effect of beta(3)-AR agonists is not attenuated by chronic exposure to agonists.     

Prolonged treatment with the β3-adrenergic agonist CL 316243 induces adipose tissue remodeling in rat but not in guinea pig: 2) modulation of glucose uptake and monoamine oxidase activity

Beta3-adrenergic agonists are well-recognited to promote lipid mobilisation and adipose tissue remodeling in rodents, leading to multilocular fat cells enriched in mitochondria. However, effects of beta3-adrenergic agonists on glucose transport are still controversial. In this work, we studied in white adipose tissue (WAT) the influence of sustained beta3-adrenergic stimulation on the glucose transport and on the mitochondrial monoamine oxidase (MAO) activity. As one-week administration of CL 316243 (CL, 1 mg/kg/d) induces beta-adrenergic desensitization in rat but not in guinea pig adipocytes, attention was paid to compare these models. When expressing glucose uptake as nmoles of 2-deoxyglucose/100 mg cell lipids, maximally stimulated uptake was increased in adipocytes of WAT from treated rats but not from treated guinea pigs. However, basal hexose uptake was also increased in CL-treated rats and, as a consequence, the dose-dependent curves for insulin stimulation were similar in control and CL-treated rats when expressed as fold increase over basal. Insulin-induced lipogenesis was unchanged in rat or guinea pig adipocytes after CL-treatment. The glucose carriers GLUT4 and corresponding mRNA were increased in subcutaneous WAT or in brown adipose tissue (BAT) but not in visceral WAT or muscles of CL-treated rats. There was an increase of MAO activity in WAT and BAT, but not in liver, of CL-treated rats while no change was detected in guinea pigs. These findings show that only rat adipocytes, which are beta3-adrenergic-responsive, respond to chronic beta3-AR agonist by an increase of GLUT4 content and MAO activity, despite a desensitization of all beta-adrenoceptor subtypes.     

Central angiotensin II has catabolic action at white and brown adipose tissue

Considerable evidence implicates the renin-angiotensin system (RAS) in the regulation of energy balance. To evaluate the role of the RAS in the central nervous system regulation of energy balance, we used osmotic minipumps to chronically administer angiotensin II (Ang II; icv; 0.7 ng/min for 24 days) to adult male Long-Evans rats, resulting in reduced food intake, body weight gain, and adiposity. The decrease in body weight and adiposity occurred relative to both ad libitum- and pair-fed controls, implying that reduced food intake in and of itself does not underlie all of these effects. Consistent with this, rats administered Ang II had increased whole body heat production and oxygen consumption. Additionally, chronic icv Ang II increased uncoupling protein-1 and β(3)-adrenergic receptor expression in brown adipose tissue and β3-adrenergic receptor expression in white adipose tissue, which is suggestive of enhanced sympathetic activation and thermogenesis. Chronic icv Ang II also increased hypothalamic agouti-related peptide and decreased hypothalamic proopiomelanocortin expression, consistent with a state of energy deficit. Moreover, chronic icv Ang II increased the anorectic corticotrophin- and thyroid-releasing hormones within the hypothalamus. These results suggest that Ang II acts in the brain to promote negative energy balance and that contributing mechanisms include an alteration in the hypothalamic circuits regulating energy balance, a decrease in food intake, an increase in energy expenditure, and an increase in sympathetic activation of brown and white adipose tissue.