Interventions against obesity through increased lipolysis in adipose tissue

Obesity is a disease of excess adiposity affecting> 17% of men and >20% of women in Britain. Clinically, it is defined by a Body Mass Index (BMI, kg/m2) of 2:30. Obesity is a confounding factor that promotes insulin resistance, hyperinsulinaemia and type 2 diabetes. Type 2 diabetes accounts for >90% of all cases of diabetes, with a prevalence of 2-6% of adults in most western societies, a majority of which are overweight or obese. Weight loss in obese patients reduces the risk of developing diabetes by >50%. This thesis has investigated the first part of a two-stage therapeutic intervention against obesity in which adipose tissue lipolysis will be combined with increased energy expenditure: the approach is also designed to consider agents that will benefit glycaemic control in coexistent obesity and diabetes by improving insulin sensitivity. Rodent and human in vitro models of adipocyte biology and skeletal muscle have been developed, characterised and evaluated. They include isolated epididymal and parametrial adipocytes of lean and obese diabetic ob/ob mice, cultured 3T3-Ll preadipocytes, isolated human omental and subcutaneous adipocytes and rat L6 cultured muscle cells. Compounds investigated for anti-obesity and anti-diabetic properties include M2 (sibutramine metabolite), 3-guanidinopropionic acid and mazindol. In vivo studies were undertaken to investigate these compounds further in lean and ob/ob mice. In vivo studies indicated that M2 and 3-guanidinopropionic acid reduced body weight gain in ob/ob mice. The three compounds increased lipolysis in adipocytes isolated from lean and ob/ob mice and human adipose depots. The direct action of these compounds was mediated via a pathway involving the f3 adrenoceptors and components of the lipolytic signalling pathway, including protein kinase A and p38 MAP kinase. In addition, M2 and mazindol were capable of increasing glucose uptake into insulin sensitive tissues. M2 and mazindol can act directly on adipose tissue and skeletal muscle to increase glucose uptake via a pathway involving new protein synthesis and activation of the glucose transporters. The M2-stimulated pathway is activated by the conversion of phosphatidylinositol bisphosphate to phosphatidylinositol trisphosphate by phosphatidylinositol 3-kinase. Thus, M2, mazindol and 3-GPA showed pharmacodynamic properties which suggested they might be potential therapeutic treatments for obesity and diabetes.

Inhibitors of lipolysis in tumor-induced cachexia

The MAC16 tumour produces a factor which exhibits lipid-mobilizing activity in vitro in addition to causing extensive depletion of host lipid stores. The mechanism of the anti-lipolytic effect of two anti-cachectic agents, eicosapentaenoic acid, an ω-3 polyunsaturated fatty acid (PUFA), and N-(3-phenoxycinnamyl)acetohydroxamic acid (BW A4C), a 5-lipoxygenase inhibitor, has been investigated. These two agents reduce tumour growth and reverse the weight loss which accompanies transplantation of the MAC16 murine colon adenocarcinoma into NMRI mice. Mice transplanted with the MAC16 tumour exhibited weight loss which was directly proportional to the serum lipolytic activity measured in vitro up to a weight loss corresponding to 16% of the original body weight. After this time, an inverse relationship between weight loss and lipolytic activity was observed. Body composition analysis revealed a large decrease in body fat relative to other body compartments. The anti-tumour/anti-cachectic effect of EPA did not appear to be due to its ability to inhibit the production of prostaglandin E2. The MAC16 lipolytic factor increased adenylate cyclase activity in adipocyte plasma membranes in a concentration-dependent manner. EPA inhibited the production of cAMP attributed to this lipid-mobilizing factor. EPA produced alterations in Gi , the guanine nucleotide binding protein which mediates hormonal inhibition of adenylate cyclase, in addition to altering cAMP production in adipocyte plasma membranes in response to hormonal stimulation. The alterations in adenylate cyclase activity were complex and not specific to EPA. EPA stimulated adenylate cyclase activity when in a relatively high fatty acid : membrane ratio and inhibited activity when this ratio was lowered. The inhibitory effect of EPA on adenylate cyclase activity may be the underlying mechanism which explains its anti-lipolytic and anti-cachectic effect. The inability of the related ω-3 PUFA, docosahexaenoic acid (DHA), to inhibit cachexia may be due to a difference in the metabolic fates of these two fatty acids. BW A4C inhibited lipolysis in isolated adipocytes which suggests that this compound may possess the potential for an anti-cachectic effect which is independent of its inhibitory effect on tumour growth.

The primary amine metabolite of sibutramine stimulates lipolysis in adipocytes isolated from lean and obese mice and in isolated human adipocytes

Sibutramine is a satiety-inducing serotonin-noradrenaline reuptake inhibitor that acts predominantly via its primary and secondary metabolites. This study investigates the possibility that sibutramine and/or its metabolites could act directly on white adipose tissue to increase lipolysis. Adipocytes were isolated by a collagenase digestion procedure from homozygous lean (+/+) and obese-diabetic ob/ob mice, and from lean nondiabetic human subjects. The lipolytic activity of adipocyte preparations was measured by the determination of glycerol release over a 2-hour incubation period. The primary amine metabolite of sibutramine M2, caused a concentration-dependent stimulation of glycerol release by murine lean and obese adipocytes (maximum increase by 157 ± 22 and 245 ± 1696, respectively, p < 0.05). Neither sibutramine nor its secondary amine metabolite M1 had any effect on lipolytic activity. Preliminary studies indicated that M2-induced lipolysis was mediated via a beta-adrenergic action. The non-selective beta-adrenoceptor antagonist propranolol (10-6M) strongly inhibited M2-stimulated lipolysis in lean and obese murine adipocytes. M2 similarly increased lipolysis by isolated human omental and subcutaneous adipocytes (maximum increase by 194 ± 33 and 136 ± 4%, respectively, p < 0.05) with EC50 values of 12 nM and 3 nM, respectively. These results indicate that the sibutramine metabolite M2 can act directly on murine and human adipose tissue to increase lipolysis via a pathway involving beta-adrenoceptors. © Georg Thieme Verlag KG Stuttgart.

Role of β3-adrenergic receptors in the action of a tumour lipid mobilizing factor

Induction of lipolysis in murine white adipocytes, and stimulation of adenylate cyclase in adipocyte plasma membranes, by a tumour-produced lipid mobilizing factor, was attenuated by low concentrations (10-7-10-5M) of the specific β3-adrenoceptor antagonist SR59230A. Lipid mobilizing factor (250 nM) produced comparable increases in intracellular cyclic AMP in CHOKI cells transfected with the human β3-adrenoceptor to that obtained with isoprenaline (1 nM). In both cases cyclic AMP production was attenuated by SR59230A confirming that the effect is mediated through a β3-adrenoceptor. A non-linear regression analysis of binding of lipid mobilizing factor to the β3-adrenoceptor showed a high affinity binding site with a Kd value 78±45 nM and a Bmax value (282±1 fmole mg protein-1) comparable with that of other β3-adrenoceptor agonists. These results suggest that lipid mobilizing factor induces lipolysis through binding to a β3-adrenoceptor. © 2002 The Cancer Research Campaign.

Modulation of adipocyte G-protein expression in cancer cachexia by a lipid-mobilizing factor (LMF)

Adipocytes isolated from cachectic mice bearing the MAC 16 tumour showed over a 3-fold increase in lipolytic response to both low concentrations of isoprenaline and a tumour-derived lipid mobilizing factor (LMF). This was reflected by an enhanced stimulation of adenylate cyclase in plasma membrane fractions of adipocytes in the presence of both factors. There was no up-regulation of adenylate cyclase in response to forskolin, suggesting that the effect arose from a change in receptor number or G-protein expression. Immunoblotting of adipocyte membranes from mice bearing the MAC16 tumour showed an increased expression of Gαs up to 10% weight loss and a reciprocal decrease in Gα. There was also an increased expression of Gαs and a decrease in Gα in adipose tissue from a patient with cancer-associated weight loss compared with a non-cachectic cancer patient. The changes in G-protein expression were also seen in adipose tissue of normal mice administered pure LMF as well as in 3T3L1 adipocytes in vitro. The changes in G-protein expression induced by LMF were attenuated by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). This suggests that this tumour-derived lipolytic factor acts to sensitize adipose tissue to lipolytic stimuli, and that this effect is attenuated by EPA, which is known to preserve adipose tissue in cancer cachexia. © 2001 Cancer Research Campaign.

The role of eicoapentaenoic acid in cancer cahexia

Cachexia is a wasting syndrome often associated with malignancy, characterised by alterations in host metabolism and significant catabolism of host adipose tissue and skeletal muscle. The MAC16 murine adenocarcinoma is profoundly cachexigenic, inducing host weight-loss at relatively small tumour burden without the induction of anorexia. A 4DkDa factor capable of inducing lipolysis in vitro via an activation of adenylate cyclase (AC) has been isolated from the MAC16 tumour, and the urine of cachectic cancer patients, using a series of ion exchange and gel exclusion chromatography procedures. This lipid-mobilising factor (LMF) has been demonstrated to stimulate lipolysis in adipocytes dose-dependently via a signal transduction pathway involving, possibly, β3-adrenoceptors. Oral administration of the n-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) attenuated the progression of cachexia, but not the production of LMF, in MAC16 tumour-bearing mice, and was significantly incorporated into plasma phospholipids, skeletal muscle and adipose tissue. EPA supplemented cancer patients also demonstrated significantly increased plasma EPA concentrations. Decreased plasma membrane AC activity in response to LMF was observed in adipocytes isolated from mice receiving EPA. Incubation in vitro of adipocytes, or plasma membranes, with PUFAs significantly altered membrane fatty acid composition and attenuated the induction of both lipolysis, and AC activity, by LMF. The inhibitory actions of EPA, but not docosahexaenoic acid, are probably the consequence of an interaction with guanine nucleotide binding proteins (G-proteins). Progression of the cachectic state induced an up-regulation of adipocyte membrane expression of stimulatory G-proteins, allied with a concomitant down-regulation of inhibitory G-proteins, thus facilitating the catabolic actions of LMF, implying some tumour-mediated effect. A reversal of such alterations was observed upon oral administration of EPA, suggesting that the primary mechanism of action of this fatty acid is an inhibition of the end organ effects of LMF.

Second messenger pathways in the action of cachectic factors

Cachexia in cancer is characterised by progressive depletion of both adipose tissue stores and skeletal muscle mass. Two catabolic factors produced by cachexia-inducing tumours have the potential for inducing these changes in body composition: (i) proteolysis-inducing factor (PIF) which acts on skeletal muscle to induce both protein degradation and inhibit protein synthesis, (ii) lipid-mobilising factor (LMF), which has been shown to directly induce lipolysis in isolated epididymal murine white adipocytes. Administration of lipid-mobilising factor (LMF) to mice produced a specific reduction in carcass lipid with a tendency to increase non-fat carcass mass. Treatment of murine myoblasts, myotubes and tumour cells with tumour-produced LMF, caused concentration dependent stimulation of protein synthesis, within a 24hr period. It produced an increase in intracellular cyclic AMP levels, which was linearly related to the increase in protein synthesis. The observed effect was attenuated by pretreating cells with the adenylate cyclase inhibitor, MDL12330A and was additive with stimulation produced by forskolin. Both propranolol and a specific 3 adrenergic antagonist SR59230A, significantly reduced the stimulation of protein synthesis induced by LMF. LMF also affected protein degradation in vitro, as demonstrated by a reduction in proteasome activity, a key component of the ubiquitin-dependent proteolytic pathway. These effects were opposite to those produced by PIF which caused both a decrease in the rate of protein synthesis and an elevation on protein breakdown when incubated in vitro.Incubation of LMF with a fat cell line produced alterations in the levels of guanine-nucleotide binding proteins (G proteins). This was also evident in adipocyte plasma membranes isolated from mice bearing the tumour model of cachexia, MAC16 adenocarcinoma and from patients with cancer cachexia. Progression through the cachectic state induced an upregulation of stimulatory G proteins paralleled with a downregulation of inhibitory G proteins. These changes would contribute to the increased lipid mobilisation seen in cancer cachexia.

Sensing and control of adipocyte function

Obesity has become a global epidemic. Approximately 15% of the world population is either overweight or obese. This figure rises to 75% in many westernised countries including the United Kingdom. Health costs in the UK to treat obesity and associated disease are conservatively estimated at 6% of the National Health Service (NHS) budget equating to 3.33 billion Euros. Excess adiposity, especially in visceral depots, increases the risk of type 2 diabetes, cardiovascular disease, gall stones, hypertension and cancer. Type 2 diabetes mellitus accounts for >90% of all cases of diabetes of which the majority can be attributed to increased adiposity, and approximately 70% of cardiovascular disease has been attributed to obesity in the US. Weight loss reduces risk of these complications and in some cases can eliminate the condition. However, weight loss by conventional non-medicated methods is often unsuccessful or promptly followed by weight regain. This thesis has investigated adipocytes development and adipokine signalling with a view to enhance the understanding of tissue functionality and to identify possible targets or pathways for therapeutic intervention. Adipocyte isolation from human tissue samples was undertaken for these investigative studies, and the methodology was optimised. The resulting isolates of pre-adipocytes and mature adipocytes were characterised and evaluated. Major findings from these studies indicate that mature adipocytes undergo cell division post terminal differentiation. Gene studies indicated that subcutaneous adipose tissue exuded greater concentrations and fluctuations of adipokine levels than visceral adipose tissue, indicating an important adiposensing role of subcutaneous adipose tissue. It was subsequently postulated that the subcutaneous depot may provide the major focus for control of overall energy balance and by extension weight control. One potential therapeutic target, 11ß-hydrosteroid dehydrogenase (11ß-HSD1) was investigated, and prospective inhibitors of its action were considered (BVT1, BVT2 and AZ121). Selective reduction of adiposity of the visceral depot was desired due to its correlation with the detrimental effects of obesity. However, studies indicated that although the visceral depot tissue was not unaffected, the subcutaneous depot was more susceptible to therapeutic inhibition by these compounds. This was determined to be a potentially valuable therapeutic intervention in light of previous postulations regarding long-term energy control via the subcutaneous tissue depot.

Induction of lipolysis in vitro and loss of body fat in vivo by zinc-alpha(2)-glycoprotein

Loss of adipose tissue in cancer cachexia has been associated with tumour production of a lipid-mobilizing factor (LMF) which has been shown to be homologous with the plasma protein zinc-a2-glycoprotein (ZAG). The aim of this study was to compare the ability of human ZAG with LMF to stimulate lipolysis in vitro and induce loss of body fat in vivo, and to determine the mechanisms involved. ZAG was purified from human plasma using a combination of Q Sepharose and Superdex 75 chromatography, and was shown to stimulate glycerol release from isolated murine epididymal adipocytes in a dose-dependent manner. The effect was enhanced by the cyclic AMP phosphodiesterase inhibitor Ro20-1724, and attenuated by freeze/thawing and the specific ß3-adrenoreceptor antagonist SR59230A. In vivo ZAG caused highly significant, time-dependent, decreases in body weight without a reduction in food and water intake. Body composition analysis showed that loss of body weight could be attributed entirely to the loss of body fat. Loss of adipose tissue may have been due to the lipolytic effect of ZAG coupled with an increase in energy expenditure, since there was a dose-dependent increase in expression of uncoupling protein-1 (UCP-1) in brown adipose tissue. These results suggest that ZAG may be effective in the treatment of obesity.

Zinc-α2-glycoprotein, a lipid mobilizing factor, is expressed in adipocytes and is up-regulated in mice with cancer cachexia

Zinc-α2-glycoprotein (ZAG), a 43-kDa protein, is overexpressed in certain human malignant tumors and acts as a lipid-mobilizing factor to stimulate lipolysis in adipocytes leading to cachexia in mice implanted with ZAG-producing tumors. Because white adipose tissue (WAT) is an endocrine organ secreting a wide range of protein factors, including those involved in lipid metabolism, we have investigated whether ZAG is produced locally by adipocytes. ZAG mRNA was detected by RT-PCR in the mouse WAT depots examined (epididymal, perirenal, s.c., and mammary gland) and in interscapular brown fat. In WAT, ZAG gene expression was evident in mature adipocytes and in stromal-vascular cells. Using a ZAG Ab, ZAG protein was located in WAT by Western blotting and immunohistochemistry. Mice bearing the MAC16-tumor displayed substantial losses of body weight and fat mass, which was accompanied by major increases in ZAG mRNA and protein levels in WAT and brown fat. ZAG mRNA was detected in 3T3-L1 cells, before and after the induction of differentiation, with the level increasing progressively after differentiation with a peak at days 8-10. Both dexamethasone and a β 3 agonist, BRL 37344, increased ZAG mRNA levels in 3T3-L1 adipocytes. ZAG gene expression and protein were also detected in human adipose tissue (visceral and s.c.). It is suggested that ZAG is a new adipose tissue protein factor, which may be involved in the modulation of lipolysis in adipocytes. Overexpression in WAT of tumor-bearing mice suggests a local role for adipocyte-derived ZAG in the substantial reduction of adiposity of cancer cachexia.

The role of glucocorticoids in the induction of zinc-α2-glycoprotein expression in adipose tissue in cancer cachexia

Loss of adipose tissue in cancer cachexia in mice bearing the MAC16 tumour arises from an increased lipid mobilisation through increased expression of zinc-α2-glycoprotein (ZAG) in white (WAT) and brown (BAT) adipose tissue. Glucocorticoids have been suggested to increase ZAG expression, and this study examines their role in cachexia and the mechanisms involved. In mice bearing the MAC16 tumour, serum cortisol concentrations increased in parallel with weight loss, and the glucocorticoid receptor antagonist RU38486 (25 mg kg-1) attenuated both the loss of body weight and ZAG expression in WAT. Dexamethasone (66 μg kg-1) administration to normal mice produced a six-fold increase in ZAG expression in both WAT and BAT, which was also attenuated by RU38486. In vitro studies using 3T3-L1 adipocytes showed dexamethasone (1.68 μM) to stimulate lipolysis and increase ZAG expression, and both were attenuated by RU38486 (10 μM), anti-ZAG antibody (1 μ gml-1), and the β3-adrenoreceptor (β3-AR) antagonist SR59230A (10 μM). Zinc-α2-glycoprotein also increased its own expression and this was attenuated by SR59230A, suggesting that it was mediated through the β3-AR. This suggests that glucocorticoids stimulate lipolysis through an increase in ZAG expression, and that they are responsible for the increase in ZAG expression seen in adipose tissue of cachectic mice. © 2005 Cancer Research UK.

Antidiabetic properties of zinc-alpha2-glycoprotein in ob/ob mice

Zinc-alpha(2)-glycoprotein (ZAG) is an adipokine associated with fat loss in cancer cachexia. The purpose of this study was to evaluate the ability of recombinant human ZAG to attenuate type 2 diabetes in the ob/ob mouse model. ZAG (50 microg daily, iv) induced a progressive loss of body weight (3.5 g in 5 d), without an effect on food or water intake but with a 0.4 C rise in body temperature, suggesting an increased energy expenditure. Despite an increased plasma glycerol, indicative of increased lipolysis, levels of glucose, triglycerides, and nonesterified fatty acids were decreased by 17, 25, and 62%, respectively, due to an increased use of both glucose and lipids by muscle and brown adipose tissue. The weight of the latter increased 2-fold, and there was increased expression of uncoupling proteins-1 and -3. Plasma insulin levels were reduced by 36%, whereas pancreatic insulin was increased 4-fold, and there was a 53% decrease in the total area under the glucose curve in the glucose tolerance test and reduced insulin requirement. There was an increase in skeletal muscle mass due to an increase in protein synthesis and a decrease in protein degradation. These results suggest that ZAG may potentially be effective in the treatment of type 2 diabetes.

Mechanisms of Cancer Cachexia

Up to 50% of cancer patients suffer from a progressive atrophy of adipose tissue and skeletal muscle, called cachexia, resulting in weight loss, a reduced quality of life, and a shortened survival time. Anorexia often accompanies cachexia, but appears not to be responsible for the tissue loss, particularly lean body mass. An increased resting energy expenditure is seen, possibly arising from an increased thermogenesis in skeletal muscle due to an increased expression of uncoupling protein, and increased operation of the Cori cycle. Loss of adipose tissue is due to an increased lipolysis by tumor or host products. Loss of skeletal muscle in cachexia results from a depression in protein synthesis combined with an increase in protein degradation. The increase in protein degradation may include both increased activity of the ubiquitin-proteasome pathway and lysosomes. The decrease in protein synthesis is due to a reduced level of the initiation factor 4F, decreased elongation, and decreased binding of methionyl-tRNA to the 40S ribosomal subunit through increased phosphorylation of eIF2 on the a-subunit by activation of the dsRNA-dependent protein kinase, which also increases expression of the ubiquitin-proteasome pathway through activation of NF?B. Tumor factors such as proteolysis-inducing factor and host factors such as tumor necrosis factor-a, angiotensin II, and glucocorticoids can all induce muscle atrophy. Knowledge of the mechanisms of tissue destruction in cachexia should improve methods of treatment. Copyright © 2009 the American Physiological Society

Weight loss in tumour-bearing mice is not associated with changes in resistin gene expression in white adipose tissue

Resistin, a product of white adipose tissue, is postulated to induce insulin resistance in obesity and regulate adipocyte differentiation. The aim of this study was to examine resistin gene expression in adipose tissue from mice bearing the MAC16 adenocarcinoma, which induces cancer cachexia with marked wasting of adipose tissue and skeletal muscle mass. MAC16-bearing mice lost weight progressively over the period following tumour transplantation, while the weight of control mice remained stable. Leptin mRNA in gonadal fat was 50% lower in MAC16 mice than in controls (p<0.05). Plasma insulin concentrations were also significantly lower in the MAC16 group (p<0.05). However, resistin mRNA level in gonadal fat in MAC16 mice was similar to controls (94% of controls). Thus, despite severe weight loss and significant falls in leptin expression and insulin concentration, resistin gene expression appears unchanged in white adipose tissue of mice with MAC16 tumour. Maintenance of resistin production may help inhibit the formation of new adipocytes in cancer cachexia.

The challenge of managing coexistent type 2 diabetes and obesity

The presence of obesity with type 2 diabetes increases morbidity and mortality from each condition. Excess adiposity accentuates insulin resistance and complicates the treatment of type 2 diabetes. Glucagon-like peptide 1 receptor agonists promote weight loss, whereas metformin, dipeptidyl peptidase 4 inhibitors, and a glucosidase inhibitors are typically weight neutral. The anabolic effects of increased insulin secretion and action restrict the benefits of treatment in obese patients. New treatments should ideally reduce hyperglycaemia and excess adiposity. Potential new treatments include analogues of intestinal and adipocyte hormones, inhibitors of renal glucose reabsorption and cellular glucocorticoid activation, and activators of cellular energy production.