Effects of Salvia officinalis L. and Chamaemelum nobile (L.) extracts on inflammatory responses in two models of human cells: Primary subcutaneous adipocytes and neuroblastoma cell line (SK-N-SH).

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia officinalis L. (sage), and Chamaemelum nobile (L.) (chamomile) have been used traditionally to treat various inflammatory conditions. AIMS: Our study aims to investigate the anti-inflammatory properties of both plant extracts in IL-1ß-stimulated neuroblastoma cells (SK-N-SH) and human subcutaneous mature adipocytes, as well as their potential protective effects against mature adipocytes conditioned media (ACM)-induced neuro-inflammation. MATERIALS AND METHODS: Human subcutaneous mature adipocytes and neuroblastoma cells were treated with 5 µg/ml (low dose: LD) and 50 µg/ml (high dose: HD) of each extract, with or without 0.5 ng/ml of human recombinant IL-1ß. To understand the cross talk between fat tissue and neuronal cells, SK-N-SH cell line was incubated with ACM 10%, in presence or absence of both extracts LD and HD. Following 4, and 24 h incubation, the released MCP-1, IL-6, IL-8, TNF-α, ICAM-1, VCAM-1 and SAA levels were measured using MSD Cytokines and Chemokines assay kits, and the cells were used for gene expression. RNA was quantified using Qubit™ RNA HS Assay. RNA aliquots were shipped to Eurofins Genomics (Aarhus, Denmark) for expression analysis on the human Clariom™ GO Screen Assay (952,361; ThermoFisher). RESULTS: Chamomile showed stronger effects compared to sage in both cell lines, at 4 and 24 h. Adipocytes acute treatment with sage decreased MCP-1, IL-6, IL-8 (p < 0.001), and TNF-α (p < 0.05) basal levels. This was mirrored at MCP-1 transcriptional level. Chronic treatment with both extracts resulted in a significant reduction in ICAM-1, VCAM-1 and SAA (p < 0.001) levels, in IL-1ß-stimulated adipocytes. However, in SK-N-SH cells, sage increased the basal levels of many cytokines and chemokines on both protein and transcriptional levels. This was also observed in IL-1ß-stimulated cells. In chamomile treated SK-N-SH cells, acute and chronic treatments decreased MCP-1 (p < 0.001), IL-6 (p < 0.01), TNF-α (p < 0.01), and IL-8 (p < 0.001) basal levels. In IL1-ß-stimulated SK-N-SH cells, chamomile HD induced a significant reduction in TNF-α after both acute and chronic treatments respectively, by 52% and 81%. At transcriptional level, this effect was only reflected at 4 h. ICAM-1, VCAM-1 and SAA levels were reduced in most of the studied conditions. In IL-1ß treated adipocytes, chamomile showed stronger reduction in MCP-1, ICAM-1 and VCAM-1 expression, however no significant reduction in TNF-α and IL-8 was observed, despite the decrease in basal levels. In SK-N-SH cells, ACM increased MCP-1, IL-6, IL-8, TNF-α, VCAM-1 and SAA levels. Sage HD acute treatment resulted in a reduction of ACM effect on IL-6, IL-8 and VCAM-1, with greater effect of chamomile on MCP-1 (p < 0.05); IL-6 (p < 0.001); TNF-α (p < 0.001); VCAM-1 (p < 0.001); and SAA (p < 0.001). This protective effect was also observed after chronic treatment. However, both extracts potentiated significantly the ACM-pro-inflammatory effect on IL-8 (p < 0.001). CONCLUSIONS: Sage decreased the pro-inflammatory markers mostly in human adipocytes, whereas chamomile showed a strong reduction in both cell populations. Both extracts reduced the ACM-induced inflammation effect and might be used as a preventive treatment for late-life cognitive impairment related to low-grade chronic inflammation associated with obesity. Further studies are needed to investigate their combination on other chronic inflammation-related diseases such as type 2 diabetes or rheumatoid arthritis.

Sixty Years of Drug Discovery for Type 2 Diabetes: Where Are We Now?

Today, excluding insulin, there are eight classes of anti-diabetic medicines that have been added to the pharmacy since the introduction of metformin in the mid-1950s; the sulfonylureas, biguanides, thiazolidinediones, α-glucosidase inhibitors, meglitinides, incretins, and sodium glucose transport 2 inhibitors. Does the fact that metformin is still first-line treatment suggest that our drug discovery efforts over the past 60 years have not been good enough? Or does it suggest that diabetes is such a complex disorder that no single treatment, other than gastric bypass surgery, can affect true normalization of not only blood sugar but also the underlying pathologies? Our understanding of the disease has most definitely improved which may bring hope for the future in terms of science, but for it to be beneficial, this science has to be translated into better drug treatments for the disease. In this review, I have examined the eight classes of anti-diabetes drugs from a drug discovery perspective.

Liver-directed overexpression of mitochondrial glycerol-3-phosphate acyltransferase results in hepatic steatosis, increased triacylglycerol secretion and reduced fatty acid oxidation.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first committed step in triacylglycerol (TAG) and phospholipid biosynthesis. GPAT activity has been identified in both ER and mitochondrial subcellular fractions. The ER activity dominates in most tissues except in liver, where the mitochondrial isoform (mtGPAT) can constitute up to 50% of the total activity. To study the in vivo effects of hepatic mtGPAT overexpression, mice were transduced with adenoviruses expressing either murine mtGPAT or a catalytically inactive variant of the enzyme. Overexpressing mtGPAT resulted in massive 12- and 7-fold accumulation of liver TAG and diacylglycerol, respectively but had no effect on phospholipid or cholesterol ester content. Histological analysis showed extensive lipid accumulation in hepatocytes. Furthermore, mtGPAT transduction markedly increased adipocyte differentiation-related protein and stearoyl-CoA desaturase-1 (SCD-1) in the liver. In line with increased SCD-1 expression, 18:1 and 16:1 in the hepatic TAG fraction increased. In addition, mtGPAT overexpression decreased ex vivo fatty acid oxidation, increased liver TAG secretion rate 2-fold, and increased plasma TAG and cholesterol levels. These results support the hypothesis that increased hepatic mtGPAT activity associated with obesity and insulin resistance contributes to increased TAG biosynthesis and inhibition of fatty acid oxidation, responses that would promote hepatic steatosis and dyslipidemia.

The effect of high-fat feeding on intramuscular lipid and lipid peroxidation levels in UCP3-ablated mice.

Uncoupling protein-3 (UCP3) has been suggested to protect against lipid-induced oxidative damage. Therefore, we studied intramuscular lipid peroxide levels and high-fat diet induced alterations in muscle lipid metabolism of UCP3-ablated mice. UCP3-/- mice showed approximately 3-fold higher levels of intramuscular lipid peroxides upon standard chow feeding, compared to wild-type littermates. Remarkably, this difference was no longer apparent on the high-fat diet. However, upon high-fat feeding, intramuscular triacylglycerol levels were approximately 50% lower in UCP3-/- mice, in comparison to UCP3+/+ animals. Succinate dehydrogenase activity, and total protein content of the muscle fatty acid transporter FAT/CD36 were however similar between UCP3-/- and UCP3+/+ mice.

Inverse agonism or neutral antagonism at G-protein coupled receptors: a medicinal chemistry challenge worth pursuing?

The identification of constitutive, or intrinsic, activity of G-protein coupled receptors has had major impact on receptor theory, the identification of agents that inhibit this ligand-independent receptor activity has led, in turn, to the concept of inverse agonism. It has subsequently emerged that the majority, around 85%, of all known G-protein coupled receptor antagonists are, in fact, inverse agonists. Agents that affect only ligand-dependent receptor activation, i.e. have no effect on constitutive receptor signalling, are termed neutral antagonists and turn out to be relatively rare in pharmacology. Is this relevant for medicinal chemistry? That question is difficult to answer with certainty because there has been little or no effort to understand the structure activity relationships of neutral antagonist vs. inverse agonist molecules. In this review, we suggest that these pharmacological differences may well be translated to differential effects in the whole animal and in medicine. We argue that having either option to inhibit a particular receptor may reveal differences in efficacy and tolerability thus increasing the potential value of a G-protein coupled receptor inhibitor programme. However, since inverse agonists appear to constitute a default inhibitor mode, a systematic survey of the structure activity relationships around what makes a neutral antagonist will be an essential first step towards this goal.

Glucose and fatty acid metabolism in McA-RH7777 hepatoma cells vs. rat primary hepatocytes: responsiveness to nutrient availability.

The overabundance of dietary fats and simple carbohydrates contributes significantly to obesity and metabolic disorders associated with obesity. The liver balances glucose and lipid distribution, and disruption of this balance plays a key role in these metabolic syndromes. We investigated (1) how hepatocytes balance glucose and fatty acid metabolism when one or both nutrients are supplied in abundance and (2) whether rat hepatoma cells (McA-RH7777) reflect nutrient partitioning in a similar manner as compared with primary hepatocytes. Increasing media palmitate concentration increased fatty acid uptake, triglyceride synthesis and beta-oxidation. However, hepatoma cells had a 2-fold higher fatty acid uptake and a 2-fold lower fatty acid oxidation as compared with primary hepatocytes. McA-RH7777 cells did not synthesize significant amounts of glycogen and preferentially metabolized the glucose into lipids or into oxidation. In primary hepatocytes, the glucose was mostly spared from oxidation and instead partitioned into both de novo glycogen and lipid synthesis. Overall, lipid production was rapidly induced in response to either glucose or fatty acid excess and this may be one of the earliest indicators of metabolic syndrome development associated with nutrient excess.

Treating obesity: pharmacology of energy expenditure.

The pharmacological treatment options for obesity are currently very limited but the prevalence of the disease is increasing rapidly. Obesity has many serious sequelae, the most common of which is type-2-diabetes. The benefits of weight loss on health are established but the major impediment to weight loss treatments is maintenance of weight lost over the long term. The reduced- or post-obese individual undergoes physiological changes that are geared towards energy storage and weight regain. One of the physiological changes is a reduced capacity to oxidise fatty acids pushing them through pathways of triacylglycerol synthesis. In this review, some of the past drug treatments aimed at increasing energy expenditure, such as dinitrophenol and ephedrine. are discussed. Current, or nearly current therapies such as sibutramine and rimonabant are also discussed in the context of increased energy expenditure. The main part of the review focuses on future prospects with discussion around a selection of targets with potential in energy expenditure that lie in pathways with AMP-kinase at their centre and ending at the mitochondrion.

Enhanced diabetogenic effect of streptozotocin in mice overexpressing UCP-3 in skeletal muscle.

Diabetic patients exhibit varying degrees of increased muscle UCP-3 expression in skeletal muscle and, in rodents, the pancreatoxin streptozotocin (STZ) upregulates UCP-3 mRNA in skeletal and cardiac muscles. We have investigated the development of STZ-induced diabetes in transgenic mice overexpressing UCP-3 in skeletal muscle in order to provide further insight on the functional role of muscle UCP-3. UCP-3 transgenic mice treated with STZ (UCP3-STZ) showed a significant increase in blood glucose concentration 3 days after the last dose of STZ with a progressive induction of diabetes, attaining blood glucose concentrations of 24.7 +/- 1.5 mmol/L on day 17. Wild-type mice treated with STZ (WT-STZ) only started to show an increase in blood glucose concentration 6 days after the last dose of STZ and peaked on day 17 at a lower concentration than in the UCP-STZ mice. The pancreatic insulin content of UCP-3 control mice (UCP3-CON) was decreased relative to wild-type control mice (WT-CON), and STZ reduced the total pancreatic insulin content by 72% in WT-STZ mice and by 88% in UCP3-STZ mice. In an insulin tolerance test, blood glucose concentrations declined more in the UCP-3 transgenic mice than in the wild-type mice. Mice overexpressing UCP-3 in skeletal muscle have a lower pancreatic insulin content, but tend to be more insulin-sensitive. These twin actions result in an increased susceptibility to STZ-induced diabetes in UCP-3 transgenic mice.

Development of glucose intolerance in male transgenic mice overexpressing human glycogen synthase kinase-3beta on a muscle-specific promoter.

Glycogen synthase kinase-3 (GSK-3) protein levels and activity are elevated in skeletal muscle in type 2 diabetes, and inversely correlated with both glycogen synthase activity and insulin-stimulated glucose disposal. To explore this relationship, we have produced transgenic mice that overexpress human GSK-3beta in skeletal muscle. GSK-3beta transgenic mice were heavier, by up to 20% (P < .001), than their age-matched controls due to an increase in fat mass. The male GSK-3beta transgenic mice had significantly raised plasma insulin levels and by 24 weeks of age became glucose-intolerant as determined by a 50% increase in the area under their oral glucose tolerance curve (P < .001). They were also hyperlipidemic with significantly raised serum cholesterol (+90%), nonesterified fatty acids (NEFAs) (+55%), and triglycerides (+170%). At 29 weeks of age, GSK-3beta protein levels were 5-fold higher, and glycogen synthase activation (-27%), glycogen levels (-58%) and insulin receptor substrate-1 (IRS-1) protein levels (-67%) were significantly reduced in skeletal muscle. Hepatic glycogen levels were significantly increased 4-fold. Female GSK-3beta transgenic mice did not develop glucose intolerance despite 7-fold overexpression of GSK-3beta protein and a 20% reduction in glycogen synthase activation in skeletal muscle. However, plasma NEFAs and muscle IRS-1 protein levels were unchanged in females. We conclude that overexpression of human GSK-3beta in skeletal muscle of male mice resulted in impaired glucose tolerance despite raised insulin levels, consistent with the possibility that elevated levels of GSK-3 in type 2 diabetes are partly responsible for insulin resistance.

Orexins and feeding: special occasions or everyday occurrence?

Neurons expressing prepro-orexin, the precursor of orexin-A and -B, are found in the lateral hypothalamic area, a region classically implicated in driving feeding. Orexin-A induces feeding transiently when injected centrally, and food intake can be decreased when orexin action is disrupted by immunoneutralization of orexin-A, or by pharmacological blockade of orexin receptors, or by transgenic knockout of orexin. Here, we argue that orexin neurons may act to stimulate feeding in the short term, and that important regulatory signals may be a fall in plasma glucose (stimulatory), countered by satiety signals generated by eating, such as gastric distention (inhibitory).

Anorectic, thermogenic and anti-obesity activity of a selective orexin-1 receptor antagonist in ob/ob mice.

A single dose of the orexin-1 (OX1) receptor antagonist 1-(2-methylbenzoxazol-6-yl)-3-[1,5] naphthyridin-4-yl urea hydrochloride (SB-334867-A) reduces orexin-A-induced feeding and natural feeding in Sprague Dawley rats. In this study, the anti-obesity effects of SB-334867-A were determined in genetically obese (ob/ob) mice dosed with SB-334867-A (30 mg/kg, i.p.) once daily for 7 days, and then twice daily for a further 7 days. SB-334867-A reduced cumulative food intake and body weight gain over 14 days. Total fat mass gain, determined by Dual Emission X-ray Absorptiometry, was reduced, while gain in fat-free mass was unchanged. Fasting (5 h) blood glucose was also reduced at the end of the study, with a trend to reduced plasma insulin. Interscapular brown adipose tissue (BAT) weight was reduced, the tissue was noticeably darker in colour and quantitative PCR (TaqMan) analysis of this tissue showed a trend to an increase in uncoupling protein-1 mRNA expression, suggesting that SB-334867-A might stimulate thermogenesis. This was confirmed in a separate study in which a single dose of SB-334867-A (30 mg/kg, i.p.) increased metabolic rate over 4 h in ob/ob mice. OX1 receptor mRNA was detected in BAT, and its expression was increased by 58% by treatment with SB-334867-A. This is the first demonstration that OX1 receptor antagonists have potential as both anti-obesity and anti-diabetic agents.

Fat oxidation in obesity: druggable or risky enterprise?

Two potential approaches for the treatment of obesity are presented, in which modulation of a target increases fatty acid oxidation. These two methods, which are here referred to as 'pull' and 'push' routes, involve creating a demand for fuel by uncoupling oxidation from ATP production ('pulling'), or shuttling fatty acyl CoA into the mitochondria by an afferent mechanism ('pushing'). Proof-of-principle studies in humans are required to assess the use of these approaches in weight loss maintenance.

Effects of a specific MCHR1 antagonist (GW803430) on energy budget and glucose metabolism in diet-induced obese mice.

OBJECTIVE: The melanin-concentrating hormone (MCH) is a centrally acting peptide implicated in the regulation of energy homeostasis and body weight, although its role in glucose homeostasis is uncertain. Our objective was to determine effects of MCHR1 antagonism on energy budgets and glucose homeostasis in mice. METHODS: Effects of chronic oral administration of a specific MCHR1 antagonist (GW803430) on energy budgets and glucose homeostasis in diet-induced obese (DIO) C57BL/6J mice were examined. RESULTS: Oral administration of GW803430 for 30 days reduced food intake, body weight, and body fat. Circulating leptin and triglycerides were reduced but insulin and nonesterified fatty acids were unaffected. Despite weight loss there was no improvement in glucose homeostasis (insulin levels and intraperitoneal glucose tolerance tests). On day 4-6, mice receiving MCHR1 antagonist exhibited decreased metabolisable energy intake and increased daily energy expenditure. However these effects had disappeared by day 22-24. Physical activity during the dark phase was increased by MCHR1 antagonist treatment throughout the 30-day treatment. CONCLUSIONS: GW803430 produced a persistent anti-obesity effect due to both a decrease in energy intake and an increase in energy expenditure via physical activity but did not improve glucose homeostasis.

NFκB as a potent regulator of inflammation in human adipose tissue, influenced by depot, adiposity, T2DM status, and TNFα.

OBJECTIVE: Central obesity and sub-clinical inflammation increase metabolic risk, this study examined the intracellular inflammatory pathways in adipose tissue (AT) that contribute to this risk. DESIGN AND METHODS: This study therefore addressed the influence of NFκB and JNK activation in human abdominal subcutaneous (AbdSc) and omental (Om) AT, the effect of adiposity, T2DM status and the role of TNFα in vitro, using molecular biology techniques. RESULTS: Our data showed NFκB activity is increased in Om AT versus AbdSc AT (P<0.01), which was reversed with respect to depot specific activation of JNK (P<0.01). However, T2DM status appeared to preferentially activate NFκB (P<0.001) over JNK. Furthermore, in vitro studies showed recombinant human (rh) TNFα treated AbdSc adipocytes increased NFκB activity over time (2-48 h, P<0.05) whilst JNK activity reduced (2 h, 4 h, P<0.05); inhibitor studies supported a preferential role for NFκB as a modulator of TNFα secretion. CONCLUSIONS: These studies suggest distinct changes in NFκB and JNK activation, dependent upon AT depot, adiposity and T2DM status, with in vitro use of rh TNFα leading to activation of NFκB. Consequently NFκB appears to play a central role in inflammatory mediated metabolic disease over JNK, highlighting NFκB as a potential key target for therapeutic intervention.

Central control of thermogenesis.

In mammals and birds, conservation of body heat at around 37 °C is vital to life. Thermogenesis is the production of this heat which can be obligatory, as in basal metabolic rate, or it can be facultative such as the response to cold. A complex regulatory system has evolved which senses environmental or core temperature and integrates this information in hypothalamic regions such as the preoptic area and dorsomedial hypothalamus. These areas then send the appropriate signals to generate and conserve heat (or dissipate it). In this review, the importance of the sympathetic nervous system is discussed in relation to its role in basal metabolic rate and adaptive thermogenesis with a particular emphasis to human obesity. The efferent sympathetic pathway does not uniformly act on all tissues; different tissues can receive different levels of sympathetic drive at the same time. This is an important concept in the discussion of the pharmacotherapy of obesity. Despite decades of work the medicine chest contains only one pill for the long term treatment of obesity, orlistat, a lipase inhibitor that prevents the absorption of lipid from the gut and is itself not systemically absorbed. The central controlling system for thermogenesis has many potential intervention points. Several drugs, previously marketed, awaiting approval or in the earlier stages of development may have a thermogenic effect via activation of the sympathetic nervous system at some point in the thermoregulatory circuit and are discussed in this review. If the balance is weighted to the "wrong" side there is the burden of increased cardiovascular risk while a shift to the "right" side, if possible, will afford a thermogenic benefit that is conducive to weight loss maintenance. This article is part of a Special Issue entitled 'Central Control Food Intake'

Factors predicting nongenetic variability in body weight gain induced by a high-fat diet in inbred C57BL/6J mice.

Inbred C57BL/6J mice displayed large individual variations in weight gain when fed a high-fat diet (HFD). The objective of this study was to examine whether this predominantly nongenetic variability could be predicted by relevant baseline features and to explore whether variations in these significant features were influenced during pregnancy and/or lactation. Fat mass (FM), fat-free mass (FFM), food intake (FI), resting metabolic rate (RMR), physical activity (PA), and body temperature (T(b)) were all evaluated at baseline in 60 mice (aged 10-12 weeks) before HFD feeding. Regression analyses showed that baseline FM was a strong positive predictor of weight gain between 4 and 16 weeks of HFD. Baseline PA was negatively associated with weight gain at week 8, 12, and 16, and baseline FFM had a positive effect at week 12 and 16. In a second experiment, 40 female mice were mated and litter sizes (LS) were manipulated on day 3 of lactation. Weaning weight and postweaning growth rate (GR) had positive impacts on FM and FFM at age 9 weeks (FM, P = 0.001; FFM, P < 0.001: n = 97). Lactation LS had a negative effect on weaning weight and a positive effect on postweaning GR. In conclusion, our results show that obesity induced by HFD was associated with a higher baseline FM, a higher baseline FFM and a lower baseline PA level before the exposure of HFD. Two of these traits (FM and FFM) were influenced by lactation LS via weaning weight and postweaning GR.

Physiological and behavioral responses to intermittent starvation in C57BL/6J mice.

The dual intervention point model states that body mass is controlled by upper and lower intervention points, above and below which animals (and humans) intervene physiologically to bring their body mass back into the acceptable range. It has been further suggested that the lower intervention point may be defined by the risk of starvation, while the upper intervention point may be defined by the risk of predation. The objective of the present study was to test whether the risk of starvation determines the lower intervention point and to examine the physiological and behavioral mechanisms that underpin the regulation of body mass, when the risk of starvation is increased. Sixty-four mice were exposed to random days of complete fasting or 50% food restriction and their body mass and fat mass responses were measured. Food intake, physical activity and body temperature were measured throughout the experiment. In addition, plasma leptin and insulin, triglyceride and non-esterified fatty acids, along with hypothalamic neuropeptides gene expression in the arcuate nucleus were assessed after 13 and 42 days of treatment. We found that C57BL/6J mice increased body mass and fatness in response to a short-term (13 days) intermittent fasting, which was restored to baseline as the treatment was prolonged. In contrast, intermittently 50% food restricted mice showed no significant changes in body mass or fatness. Over the first 13 days of treatment the data were consistent with the dual intervention point model as the mice showed both increased body mass and adiposity over this period. Over the more protracted period of 42 days the effect waned and was therefore inconsistent with the model. The body mass and fat mass gains in intermittently fasted mice were mainly accounted for by increased food intake. Elevated NPY gene expression after 13 days (three 24 h fasting events) may have driven the increase in food intake. However, no changes were observed in such neuropeptides as POMC, CART, AgRP, Ob-Rb and SOCS 3 or circulating levels of leptin, insulin, NEFA and TG. Hypothermia during fasting days may have also contributed to the increase in body mass. Over 42 days of treatment (nine 24 h fasting events) cumulative food intake was not affected by intermittent starvation. However physical activity, mainly activity during the light phase was lowered suggesting an adaptation to unpredictable starvation. Overall, mice exhibited different behavioral and physiological responses to intermittent starvation depending on the duration of treatment.

Effects of chronic oral rimonabant administration on energy budgets of diet-induced obese C57BL/6 mice.

The endocannabinoids have been recognized as an important system involved in the regulation of energy balance. Rimonabant (SR141716), a selective inverse agonist of cannabinoid receptor 1 (CB1), has been shown to cause weight loss. However, its suppressive impact on food intake is transient, indicating a likely additional effect on energy expenditure. To examine the effects of rimonabant on components of energy balance, we administered rimonabant or its vehicle to diet-induced obese (DIO) C57BL/6 mice once daily for 30 days, by oral gavage. Rimonabant induced a persistent weight reduction and a significant decrease in body fatness across all depots. In addition to transiently reduced food intake, rimonabant-treated mice exhibited decreased apparent energy absorption efficiency (AEAE), reduced metabolizable energy intake (MEI), and increased daily energy expenditure (DEE) on days 4-6 of treatment. However, these effects on the energy budget had disappeared by days 22-24 of treatment. No chronic group differences in resting metabolic rate (RMR) or respiratory quotient (RQ) (P > 0.05) were detected. Rimonabant treatment significantly increased daily physical activity (PA) levels both acutely and chronically. The increase in PA was attributed to elevated activity during the light phase but not during the dark phase. Taken together, these data suggested that rimonabant caused a negative energy balance by acting on both energy intake and expenditure. In the short term, the effect included both reduced intake and elevated PA but the chronic effect was only on increased PA expenditure.

Targeting thermogenesis and related pathways in anti-obesity drug discovery.

The health consequences of the obesity epidemic are a huge burden on patients and society. Yet it remains an unmet therapeutic need. Lifestyle or behaviour modification, although desirable, seems to benefit only a few and bariatric surgery is not an option for all and not without risks. Nevertheless, bariatric surgery is currently the gold standard in terms of weight loss therapy and any weight loss agent will be in combination with management of lifestyle modification. Sadly, there is a poor history for the pharmacological treatment of obesity and repeated safety concerns have attracted intense regulatory scrutiny. Indeed, recent market withdrawals leave us with just one agent approved for the long term treatment of obesity and that is only mildly efficacious in terms of weight loss, although it is beneficial in terms of metabolic health. There are two broad pharmacological approaches that can be applied in obesity drug discovery: reduce intake (or absorption) or increase expenditure (thermogenesis) of calories. In this review we will look at the latter approach. We will cover regulatory requirements and the rationale for this approach. We believe that post-obese subjects display abnormal metabolic responses to weight loss that almost inevitably leads to weight regain. We will then explore a number of approaches that potentially increase thermogenesis in humans. The challenge we have is in accumulating enough human data to validate this approach using drugs.

Binding properties of antagonists to cannabinoid receptors in intact cells.

The implication of the cannabinoid receptor 1 (CB(1) receptor) in several pathophysiological states has sparked the development of selective antagonists. Here we compare binding of the antagonists [(3) H]-AZ12491187, [(3) H]-taranabant and [(3) H]-rimonabant to intact human embryonic kidney cells stably expressing recombinant human CB(1) receptors (CB1r cells). Unlabelled ligands decreased the total binding of the three radioligands with closely the same order of potency: i.e. AZ12288553∼AZ12491187∼taranabant>rimonabant. Nondisplaceable (i.e. nonspecific) binding to the CB1r cells was the same as total binding to the wells containing untransfected cells and it was more pronounced for [(3) H]-AZ12491187 and [(3) H]-rimonabant than for [(3) H]-taranabant. [(3) H]-Rimonabant and (to a lesser extent) [(3) H]-AZ12491187 were also prone to bind nonspecifically to the walls of the wells. Compared to the other radioligands, [(3) H]-rimonabant displayed lower potency for the CB(1) receptors in saturation binding studies and faster association and dissociation in kinetic experiments. When dissociated, the three radioligands also showed prominent rebinding to the cells in medium only. This could be relieved by the presence of excess of unlabelled ligand and of bovine serum albumin (BSA) but a combination thereof was most efficient. The long 'residence time' of AZ12491187 at the CB(1) receptor (because of slow dissociation and prominent rebinding) and its pronounced incorporation into the membranes of the cells could contribute to long-lasting in vivo CB(1) receptor blockade.