Effect of a protein and energy dense n-3 fatty acid enriched oral supplement on loss of weight and lean tissue in cancer cachexia:A randomised double blind trial

Aim: N-3 fatty acids, especially eicosapentaenoic acid (EPA), may possess anticachectic properties. This trial compared a protein and energy dense supplement enriched with n-3 fatty acids and antioxidants (experimental: E) with an isocaloric isonitrogenous control supplement (C) for their effects on weight, lean body mass (LBM), dietary intake, and quality of life in cachectic patients with advanced pancreatic cancer. Methods: A total of 200 patients (95 E; 105 C) were randomised to consume two cans/day of the E or C supplement (480 ml, 620 kcal, 32 g protein ± 2.2 g EPA) for eight weeks in a multicentre, randomised, double blind trial. Results: At enrolment, patients' mean rate of weight loss was 3.3 kg/month. Intake of the supplements (E or C) was below the recommended dose (2 cans/day) and averaged 1.4 cans/day. Over eight weeks, patients in both groups stopped losing weight (Δweight E: -0.25 kg/month versus C: -0.37 kg/month; p=0.74) and LBM (ΔLBM E: +0.27 kg/month versus C: +0.12 kg/month; p=0.88) to an equal degree (change from baseline E and C, p<0.001). In view of evident non-compliance in both E and C groups, correlation analyses were undertaken to examine for potential dose-response relationships. E patients demonstrated significant correlations between their supplement intake and weight gain (r=0.50, p<0.001) and increase in LBM (r=0.33, p=0.036). Such correlations were not statistically significant in C patients. The relationship of supplement intake with change in LBM was significantly different between E and C patients (p=0.043). Increased plasma EPA levels in the E group were associated with weight and LBM gain (r=0.50, p<0.001; r=0.51, p=0.001). Weight gain was associated with improved quality of life (p<0.01) only in the E group. Conclusion: Intention to treat group comparisons indicated that at the mean dose taken, enrichment with n-3 fatty acids did not provide a therapeutic advantage and that both supplements were equally effective in arresting weight loss. Post hoc dose-response analysis suggests that if taken in sufficient quantity, only the n-3 fatty acid enriched energy and protein dense supplement results in net gain of weight, lean tissue, and improved quality of life. Further trials are required to examine the potential role of n-3 enriched supplements in the treatment of cancer cachexia.

An FTO variant is associated with Type 2 diabetes in South Asian populations after accounting for body mass index and waist circumference

Aims - A common variant, rs9939609, in the FTO (fat mass and obesity) gene is associated with adiposity in Europeans, explaining its relationship with diabetes. However, data are inconsistent in South Asians. Our aim was to investigate the association of the FTO rs9939609 variant with obesity, obesity-related traits and Type 2 diabetes in South Asian individuals, and to use meta-analyses to attempt to clarify to what extent BMI influences the association of FTO variants with diabetes in South Asians. Methods - We analysed rs9939609 in two studies of Pakistani individuals: 1666 adults aged = 40 years from the Karachi population-based Control of Blood Pressure and Risk Attenuation (COBRA) study and 2745 individuals of Punjabi ancestry who were part of a Type 2 diabetes case–control study (UK Asian Diabetes Study/Diabetes Genetics in Pakistan; UKADS/DGP). The main outcomes were BMI, waist circumference and diabetes. Regression analyses were performed to determine associations between FTO alleles and outcomes. Summary estimates were combined in a meta-analysis of 8091 South Asian individuals (3919 patients with Type 2 diabetes and 4172 control subjects), including those from two previous studies. Results - In the 4411 Pakistani individuals from this study, the age-, sex- and diabetes-adjusted association of FTO variant rs9939609 with BMI was 0.45 (95% CI 0.24–0.67) kg/m2 per A-allele (P = 3.0 × 10-5) and with waist circumference was 0.88 (95% CI 0.36–1.41) cm per A-allele (P = 0.001). The A-allele (30% frequency) was also significantly associated with Type 2 diabetes [per A-allele odds ratio (95% CI) 1.18 (1.07–1.30); P = 0.0009]. A meta-analysis of four South Asian studies with 8091 subjects showed that the FTO A-allele predisposes to Type 2 diabetes [1.22 (95% CI 1.14–1.31); P = 1.07 × 10-8] even after adjusting for BMI [1.18 (95% CI 1.10–1.27); P = 1.02 × 10-5] or waist circumference [1.18 (95% CI 1.10–1.27); P = 3.97 × 10-5]. Conclusions - The strong association between FTO genotype and BMI and waist circumference in South Asians is similar to that observed in Europeans. In contrast, the strong association of FTO genotype with diabetes is only partly accounted for by BMI.

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

Double-blind, placebo-controlled, randomized study of eicosapentaenoic acid diester in patients with cancer cachexia

Purpose: Eicosapentaenoic acid (EPA) has been proposed to have specific anticachectic effects. This trial compared EPA diethyl ester with placebo in cachectic cancer patients for effects on weight and lean body mass. Patients and Methods: Five hundred eighteen weight-losing patients with advanced gastrointestinal or lung cancer were studied in a multicenter, double-blind, placebo controlled trial. Patients were randomly assigned to receive a novel preparation of pure EPA at a dose of 2 g or 4 g daily or placebo (2g EPA, n = 175; 4 g EPA, n = 172; placebo, n = 171). Patients were assessed at 4 weeks and 8 weeks. Results: The groups were well balanced at baseline. Mean weight loss at baseline was 18% (n = 518). Over the 8-week treatment period, both intention-to-treat analysis and per protocol analysis revealed no statistically significant improvements in survival, weight, or other nutritional variables. There was, however, a trend in favor of EPA with analysis of the primary end point, weight, at 8 weeks showing a borderline, nonsignificant treatment effect (P = .066). Relative to placebo, mean weight increased by 1.2 kg with 2 g EPA (95% CI, 0 kg to 2.3 kg) and by 0.3 kg with 4g EPA (-0.9 kg to 1.5 kg). Conclusion: The results indicate no statistically significant benefit from single agent EPA in the treatment of cancer cachexia. Future studies should concentrate on other agents or combination regimens. © 2006 by American Society of Clinical Oncology.

Attenuation of proteasome-induced proteolysis in skeletal muscle by beta-hydroxy-beta-methylbutyrate in cancer-induced muscle loss

Loss of skeletal muscle is an important determinant of survival in patients with cancer-induced weight loss. The effect of the leucine metabolite beta-hydroxy-beta-methylbutyrate (HMB) on the reduction of body weight loss and protein degradation in the MAC16 model of cancer-induced weight loss has been compared with that of eicosapentaenoic acid (EPA), a recognized inhibitor of protein degradation. HMB was found to attenuate the development of weight loss at a dose greater than 0.125 g/kg accompanied by a small reduction in tumor growth rate. When EPA was used at a suboptimal dose level (0.6 g/kg) the combination with HMB seemed to enhance the anticachectic effect. Both treatments caused an increase in the wet weight of soleus muscle and a reduction in protein degradation, although there did not seem to be a synergistic effect of the combination. Proteasome activity, determined by the "chymotrypsin-like" enzyme activity, was attenuated by both HMB and EPA. Protein expression of the 20S alpha or beta subunits was reduced by at least 50%, as were the ATPase subunits MSS1 and p42 of the 19S proteasome regulatory subunit. This was accompanied by a reduction in the expression of E2(14k) ubiquitin-conjugating enzyme. The combination of EPA and HMB was at least as effective or more effective than either treatment alone. Attenuation of proteasome expression was reflected as a reduction in protein degradation in gastrocnemius muscle of cachectic mice treated with HMB. In addition, HMB produced a significant stimulation of protein synthesis in skeletal muscle. These results suggest that HMB preserves lean body mass and attenuates protein degradation through down-regulation of the increased expression of key regulatory components of the ubiquitin-proteasome proteolytic pathway, together with stimulation of protein synthesis.

Biochemical mechanisms of cellular catabolism

Purpose of review: To provide an in-depth analysis of current developments concerning biochemical mechanisms of cellular catabolism. There have been a number of important developments in this area over the past 12 months, particularly with respect to protein catabolism. Recent findings: Protein degradation in a range of catabolic conditions is mediated primarily through the ubiquitin-proteasome proteolytic pathway. Glucocorticoids have been suggested to activate this system in sepsis, while in cancer cachexia a tumour-produced sulphated glycoprotein, proteolysis-inducing factor, induces protein catabolism in skeletal muscle by increasing expression of proteasome subunits and the ubiquitin carrier protein, E214k. Apoptosis may also be important in the loss of muscle protein during the early stage of cachexia. Induction of proteasome expression by glucocorticoids appears to be a direct result of the downregulation of the activity of nuclear factor ?B, while proteolysis-inducing factor acts through 15-hydroxyeicosatetraenoic acid as an intracellular transducer. Summary: Formation of 15-hydroxyeicosatetraenoic acid is inhibited by eicosapentaenoic acid, which has been shown to attenuate the development of weight loss in patients with pancreatic cancer. When eicosapentaenoic acid is combined with an energy dense nutritional supplement, there is an increase in body weight of cachectic cancer patients through an increase in lean body mass. Eicosapentaenoic acid also prevents protein catabolism and activation of the ubiquitin-proteasome proteolytic pathway during acute starvation in mice, suggesting a similar pathway is involved. Thus eicosapentaenoic acid may be effective in the treatment of protein catabolism in conditions other than cancer.

Cleavage of caspases-1, -3, -6, -8 and -9 substrates by proteases in skeletal muscles from mice undergoing cancer cachexia

A prominent feature of several type of cancer is cachexia. This syndrome causes a marked loss of lean body mass and muscle wasting, and appears to be mediated by cytokines and tumour products. There are several proteases and proteolytic pathways that could be responsible for the protein breakdown. In the present study, we investigated whether caspases are involved in the proteolytic process of skeletal muscle catabolism observed in a murine model of cancer cachexia (MAC16), in comparison with a related tumour (MAC13), which does not induce cachexia. Using specific peptide substrates, there was an increase of 54% in the proteolytic activity of caspase-1, 84% of caspase-8, 98% of caspase-3 151% to caspase-6 and 177% of caspase-9, in the gastrocnemius muscle of animals bearing the MAC16 tumour (up to 25% weight loss), in relation to muscle from animals bearing the MAC13 tumour (1-5% weight loss). The dual pattern of 89 kDa and 25 kDa fragmentation of poly (ADP-ribose) polymerase (PARP) occurred in the muscle samples from animals bearing the MAC16 tumour and with a high amount of caspase-like activity. Cytochrome c was present in the cytosolic fractions of gastrocnemius muscles from both groups of animals, suggesting that cytochrome c release from mitochondria may be involved in caspase activation. There was no evidence for DNA fragmentation into a nucleosomal ladder typical of apoptosis in the muscles of either group of mice. This data supports a role for caspases in the catabolic events in muscle involved in the cancer cachexia syndrome. © 2001 Cancer Research Campaign.

Loss of skeletal muscle in cancer: biochemical mechanisms

Patients with cancer often undergo a specific loss of skeletal muscle mass, while the visceral protein reserves are preserved. This condition known as cachexia reduces the quality of life and eventually results in death through erosion of the respiratory muscles. Nutritional supplementation or appetite stimulants are unable to restore the loss of lean body mass, since protein catabolism is increased mainly as a result of the activation of the ATP-ubiquitin-dependent proteolytic pathway. Several mediators have been proposed. An enhanced protein degradation is seen in skeletal muscle of mice administered tumour necrosis factor (TNF), which appears to be mediated by oxidative stress. There is some evidence that this may be a direct effect and is associated with an increase in total cellular-ubiquitin-conjugated muscle proteins. Another cytokine, interleukin-6 (IL-6), may play a role in muscle wasting in certain animal tumours, possibly through both lysosomal (cathepsin) and non-lysosomal (proteasome) pathways. A tumour product, proteolysis-inducing factor (PIF) is produced by cachexia-inducing murine and human tumours and initiates muscle protein degradation directly through activation of the proteasome pathway. The action of PIF is blocked by eicosapentaenoic acid (EPA), which has been shown to attenuate the development of cachexia in pancreatic cancer patients. When combined with nutritional supplementation EPA leads to accumulation of lean body mass and prolongs survival. Further knowledge on the biochemical mechanisms of muscle protein catabolism will aid the development of effective therapy for cachexia.

Studies on the anti-obesity activity of zinc-α2-glycoprotein in the rat

OBJECTIVE: To investigate the anti-obesity effect of the adipokine zinc-a(2)-glycoprotein (ZAG) in rats and the mechanism of this effect. SUBJECTS: Mature male Wistar rats (540 ± 83 g) were administered human recombinant ZAG (50 µg per 100 g body weight given intravenously daily) for 10 days, while control animals received an equal volume of phosphate-buffered saline (PBS). RESULTS: Animals treated with ZAG showed a progressive decrease in body weight, without a decrease in food and water intake, but with a 0.4 °C rise in body temperature. Body composition analysis showed loss of adipose tissue, but an increase in lean body mass. The loss of fat was due to an increase in lipolysis as shown by a 50% elevation of plasma glycerol, accompanied by increased utilization of non-esterified fatty acids, as evidenced by the 55% decrease in plasma levels. Plasma levels of glucose and triglycerides were also reduced by 36-37% and there was increased expression of the glucose transporter 4 in both skeletal muscle and adipose tissue. Expression of the lipolytic enzymes adipose triglyceride lipase and hormone-sensitive lipase in the white adipose tissue (WAT) were increased twofold after ZAG administration. There was almost a twofold increased expression of uncoupling proteins 1 and 3 in brown adipose tissue and WAT, which would contribute to increased substrate utilization. Administration of ZAG increased ZAG expression twofold in the gastrocnemius muscle, BAT and WAT, which was probably necessary for its biological effect. CONCLUSION: These results show that ZAG produces increased lipid mobilization and utilization in the rat.

Attenuation of muscle atrophy by an N-terminal peptide of the receptor for proteolysis-inducing factor (PIF)

Background: Atrophy of skeletal muscle in cancer cachexia has been attributed to a tumour-produced highly glycosylated peptide called proteolysis-inducing factor (PIF). The action of PIF is mediated through a high-affinity membrane receptor in muscle. This study investigates the ability of peptides derived from the 20 N-terminal amino acids of the receptor to neutralise PIF action both in vitro and in vivo. Methods: Proteolysis-inducing factor was purified from the MAC16 tumour using an initial pronase digestion, followed by binding on DEAE cellulose, and the pronase was inactivated by heating to 80°C, before purification of the PIF using affinity chromatography. In vitro studies were carried out using C2C12 murine myotubes, while in vivo studies employed mice bearing the cachexia-inducing MAC16 tumour. Results: The process resulted in almost a 23?000-fold purification of PIF, but with a recovery of only 0.004%. Both the D- and L-forms of the 20mer peptide attenuated PIF-induced protein degradation in vitro through the ubiquitin-proteosome proteolytic pathway and increased expression of myosin. In vivo studies showed that neither the D- nor the L-peptides significantly attenuated weight loss, although the D-peptide did show a tendency to increase lean body mass. Conclusion: These results suggest that the peptides may be too hydrophilic to be used as therapeutic agents, but confirm the importance of the receptor in the action of the PIF on muscle protein degradation.

Editorial : which weight loss diet?

Full editorial: A recent study evaluating the long-term (2 yr) weight reducing efficacy of different types of diets – high or low in carbohydrates (CHOs), protein or fat - confirmed that it is calorie deficit not dietary composition that determines the loss and maintenance of body weight.1 Is there any advantage in following a specific weight loss diet? Short-term use of nutritionally complete commercially available (very) low calorie diets has benefited people with diabetes when  supported by education programmes.2 Initial weight loss has been encouraging with some fad diets eg the Atkins and the South Beach diets, but these diets are difficult to maintain and there are safety issues regarding their short- and long-term use – especially in people with diabetes.3 The types of macronutrients consumed can have a considerable impact on glycaemic control and energy metabolism. Although a low CHO diet additionally enhances initial weight loss by reducing cellular water content, if fat is not proportionally reduced the diet may not benefit the lipid profile for vascular disease risk. High fat and high protein diets – which are simultaneously low in CHOs – increase vulnerability to hypoglycaemia in people taking insulin secretagogues or on insulin therapy, and may promote excess fat metabolism and ketogenesis, particularly in people vulnerable to lack of insulin. Very low protein diets are not recommended as lean body mass tends to be reduced in diabetes. Altering the macronutrient balance has implications for the micronutrient mix: deficiencies are higher if more foods are excluded and conversely specific micronutrient excess can occur with some fad diets. The altered nutrient mix affects intestinal fauna and flora, and gut motility and glycaemic control are influenced by the quantity and type of fibre consumed. Support programmes help individuals achieve long term weight loss and there is mounting evidence that community schemes which educate and promote lifestyle changes may stem the rising tide of obesity and consequent type 2 diabetes.4 Consuming smaller portions of a balanced diet (and adjusting antidiabetic medications accordingly) will create an energy deficit to promote healthy weight loss. Increased movement/exercise will enhance this energy deficit. Knowledge (eg 1g fat has 2.25 times more energy than 1g CHO) allows sensible food choices and compensation for inclusion of small volumes of  ‘naughty but nice’ foods. Ultimately weight control requires self control. References 1. Sacks FM, Bray GA, Carey VJ et al. Comparison of weight-loss diets with different compositions of fat, protein, and carbohydrates. N Engl J Med 2009;360:859–73. 2. Bennett P. Obesity, diabetes and VLCD. Br J Diabetes Vasc Dis 2004;4:328–30. 3. Baldwin EJ. Fad diets in diabetes. Br J Diabetes Vasc DIs 2004;4:333–7. 4. Romon M, Lommoz A, Tafflet M et al. Downward trends in the prevalence of childhood overweight in the setting of 12-year school- and community-based programmes. Public Health Nutr 2008; Dec 28, 1–8 [Epub ahead of print].

Cancer cachexia

Causative factors: Nutritional supplementation or pharmacological manipulation of appetite are unable to control the muscle atrophy seen in cancer cachexia. This suggests that tumour and/or host factors might be responsible for the depression in protein synthesis and the increase in protein degradation. An increased expression of the ubiquitin-proteasome proteolytic pathway is responsible for the increased degradation of myofibrillar proteins in skeletal muscle, and this may be due to tumour factors, such as proteolysis-inducing factor (PIF), or host factors such as tumour necrosis factor-α (TNF-α). In humans loss of adipose tissue is due to an increase in lipolysis rather than a decrease in synthesis, and this may be due to tumour factors such as lipid-mobilising factor (LMF) or TNF-α, both of which can increase cyclic AMP in adipocytes, leading to activation of hormone-sensitive lipase (HSL). Levels of mRNA for HSL are elevated twofold in adipose tissue of cancer patients, while there are no changes in lipoprotein lipase (LPL), involved in extraction of fatty acids from plasma lipoproteins for storage. Treatment for cachexia: This has concentrated on increasing food intake, although that alone is unable to reverse the metabolic changes. Agents interfering with TNF-α have not been very successful to date, although more research is required in that area. The only agent tested clinically that is able to interfere with the action of PIF is eicosapentaenoic acid (EPA). EPA attenuates protein degradation in skeletal muscle by preventing the increased expression of the ubiquitin-proteasome pathway, but has no effect on protein synthesis. When used alone EPA prevents further wasting in cachectic patients, and, when it is combined with an energy- and protein-dense nutritional supplement, weight gain is seen, which is totally lean body mass. These results suggest that mechanistic studies into the causes of cancer cachexia will allow appropriate therapeutic intervention.

The 'cancer cachectic factor'

The object of this study was to summarize information on catabolic factors produced by tumours which lead to tissue catabolism in cancer cachexia and to use this information for the development of effective therapy. The study population was made up of patients with cancer cachexia and weight loss greater than 1 kg month-1. They had a varied range of carcinomas, particularly pancreatic, but also of the breast, ovary, lung, colon and rectum. Cachectic factors were isolated by standard biochemical methods, and the mechanism of tissue catabolism was evaluated in vitro and in vivo. We isolated a 24-kDa sulphated glycoprotein produced by cachexia-inducing murine and human tumours, which induces catabolism of myofibrillar proteins in skeletal muscle and for this reason has been named proteolysis-inducing factor (PIF). PIF was shown to be present in a diverse range of carcinomas in patients whose rate of weight loss exceeded 1.0 kg month-1. Administration of PIF to normal mice produced a rapid decrease in body weight, which arose primarily from a loss of skeletal muscle, accompanied by increased mRNA levels for ubiquitin, the ubiquitin-carrier protein (E214k), and proteasome subunits. This suggests that PIF induces protein catabolism through an increased expression of the key components of the ATP-ubiquitin-dependent proteolytic pathway. The action of PIF was attenuated both in vitro and in vivo by eicosapentaenoic acid (EPA). Oral EPA has been found to stabilize the body weight of patients with advanced pancreatic cancer and, when combined with an energy- and protein-rich nutritional supplement, to produce weight gain arising solely from an increase in lean body mass. Nutritional supplementation alone is unable to reverse the process of muscle wasting in cancer patients, since this arises from activation of the ubiquitin proteasome pathway by PIF, which is independent of nutrient intake. EPA is able to down-regulate the increased expression of this pathway and prevents muscle wasting in cancer patients.

In vitro assessment of the combined effect of eicosapentaenoic acid, green tea extract and curcumin C3 on protein loss in C2C12 myotubes

EPA has been clinically shown to reduce muscle wasting during cancer cachexia. This study investigates whether curcumin or green tea extract (GTE) enhances the ability of low doses of eicosapentaenoic acid (EPA) to reduce loss of muscle protein in an in vitro model. A low dose of EPA with minimal anti-cachectic activity was chosen to evaluate any potential synergistic effect with curcumin or GTE. Depression of protein synthesis and increase in degradation was determined in C2C12 myotubes in response to tumour necrosis factor-α (TNF-α) and proteolysis-inducing factor (PIF). EPA (50 μM) or curcumin (10 μg ml−1) alone had little effect on protein degradation caused by PIF but the combination produced complete inhibition, as did the combination with GTE (10 μg ml−1). In response to TNF-α (25 ng ml−1)-induced protein degradation, EPA had a small, but not significant effect on protein degradation; however, when curcumin and GTE were combined with EPA, the effect was enhanced. EPA completely attenuated the depression of protein synthesis caused by TNF-α, but not that caused by PIF. The combination of EPA with curcumin produced a significant increase in protein synthesis to both agents. GTE alone or in combination with EPA had no effect on the depression of protein synthesis by TNF-α, but did significantly increase protein synthesis in PIF-treated cells. Both TNF-α and PIF significantly reduced myotube diameter from 17 to 13 μm for TNF-α (23.5%) and 15 μm (11.8%) for PIF However the triple combination of EPA, curcumin and GTE returned diameters to values not significantly different from the control. These results suggest that either curcumin or GTE or the combination could enhance the anti-catabolic effect of EPA on lean body mass.

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.