Upcoming M-1-T Cycle

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  1. Originally posted by jweave23


    (i.e like running one while on T3 for example,


    For answers to board issues, read the Suggestion and News forum at the bottom of the main page.


  2. That was giving me way too much trouble, so, eh, **** it:


    Obesity Reviews
    Volume 2 Issue 4 Page 255 - November 2001
    doi:10.1046/j.1467-789X.2001.00043.x


    Mitochondrial uncoupling as a target for drug development for the treatment of obesity
    J. A. Harper 1,2, K. Dickinson 3 and M. D. Brand 1
    Summary

    Mitochondrial proton cycling is responsible for a significant proportion of basal or standard metabolic rate, so further uncoupling of mitochondria may be a good way to increase energy expenditure and represents a good pharmacological target for the treatment of obesity. Uncoupling by 2,4-dinitrophenol has been used in this way in the past with notable success, and some of the effects of thyroid hormone treatment to induce weight loss may also be due to uncoupling. Diet can alter the pattern of phospholipid fatty acyl groups in the mitochondrial membrane, and this may be a route to uncoupling in vivo. Energy expenditure can be increased by stimulating the activity of uncoupling protein 1 (UCP1) in brown adipocytes either directly or through 3-adrenoceptor agonists. UCP2 in a number of tissues, UCP3 in skeletal muscle and the adenine nucleotide translocase have also been proposed as possible drug targets. Specific uncoupling of muscle or brown adipocyte mitochondria remains an attractive target for the development of antiobesity drugs.

    Pharmaceutical interest in uncoupling Go to: Choose Top of page Pharmaceutical interest i... << Standard metabolic rate What is uncoupling? Physiological significanc... Mechanism of proton leak:... Artificial uncoupling by ... Thyroid hormones Inducible uncoupling cata... beta3-Adrenergic receptor ago... Uncoupling proteins 2 and... Is the uncoupling of mito... Acknowledgements References

    Obesity is a disease resulting from a prolonged positive imbalance between energy intake and energy expenditure resulting in the storage of fat. The rapidly increasing world-wide incidence of obesity and its association with serious comorbid diseases means it is beginning to replace under-nutrition and infectious diseases as the most significant contributor to ill health in the developed world (1). Weight loss, induced by dieting, has been shown to be successful in reducing the health consequences of obesity but unfortunately >90% of individuals who lose weight through dietary control eventually return to their original weight (2). Pharmacological treatment may therefore be desirable for those patients with associated comorbid conditions who have been unable to control their obesity through diet and exercise.

    Any treatment for obesity has to reduce energy intake, increase energy expenditure or combine both effects. Current therapies for obesity predominantly lead to decreased energy intake either by acting at satiety centres in the brain (e.g. sibutramine) (3-5) or by reducing the efficiency of intestinal absorption (e.g. orlistat) (6,7). In addition to reducing energy intake, sibutramine increases standard metabolic rate (SMR) profoundly in rodents, but increased energy expenditure appears to be only a minor component of its activity in humans (8,9). Exercise is the most practical and potentially easiest way to increase energy output. Studies have shown a sevenfold-increased risk of the incidence of overweight in those with a physical activity ratio (total energy expenditure: resting metabolic rate) of <1.8 (10). The main benefit of exercise is to increase resting metabolic rate, and overall energy expenditure, by a greater amount than that resulting directly from the exercise (11). Pharmacological agents that increase metabolic rate by increasing uncoupling of mitochondrial oxidative phosphorylation are likely to mimic this beneficial effect of exercise on resting metabolic rate and could provide a useful adjunct to agents acting to reduce satiety; this review will consider how such uncoupling can be achieved.

    As discussed below, uncoupling of mitochondria represents a particularly attractive target, as there is already excellent proof of concept for this approach in humans using 2,4-dinitrophenol (DNP) (12) and in animals using 3-adrenoceptor agonists (13), or overexpression of UCP3 (uncoupling protein 3) (14). However, there are problems associated with the use of chemical uncouplers like DNP. Many of these problems may result from inappropriate activities in critical tissues, and more selective uncoupling would be desirable.

    Skeletal muscle represents a particularly attractive target for directed uncoupling due to the large muscle mass, which accounts for approximately 15-20% of SMR (15,16). The maximal aerobic capacity of a human is generally estimated to be up to 12 times SMR in untrained subjects (17). Most of this increase can be directly attributed to skeletal muscle respiratory activity and it is clear that muscle can greatly increase its metabolic activity. Doubling metabolic rate by modestly uncoupling skeletal muscle should produce few adverse side-effects as this increase would only be equivalent to mild exercise (actually equivalent to approximately the difference between lying down and standing up (17)). Indeed, support for this view has been obtained using proteins that may naturally uncouple mitochondria (uncoupling proteins 1 and 3). High expression of human UCP3 in mouse skeletal muscle led to decreased weight gain despite increased food intake (14), and expression of UCP1 in mouse skeletal muscle led to improvements in insulin sensitivity and resistance to obesity on a high fat diet (18).

    Standard metabolic rate Go to: Choose Top of page Pharmaceutical interest i... Standard metabolic rate << What is uncoupling? Physiological significanc... Mechanism of proton leak:... Artificial uncoupling by ... Thyroid hormones Inducible uncoupling cata... beta3-Adrenergic receptor ago... Uncoupling proteins 2 and... Is the uncoupling of mito... Acknowledgements References

    Basal metabolic rate (BMR) is the minimal calorific requirement for normal life in an organism in the absence of external stimulation, work and growth. It is measured under rigorous conditions at thermoneutrality, in a post-absorptive state. Many physiological processes continue in this minimal state, including ventilation and blood circulation. At the cellular level, the reactions that make up BMR include protein turnover, ion cycling across the plasma membrane, turnover of nucleic acids and lipids, and proton cycling across the mitochondrial inner membrane (uncoupling) (19,20). Resting metabolic rate (RMR) is measured as BMR but not in the post-absorptive state (17). These measurements are not suitable in animals (particularly ectotherms), therefore standard metabolic rate (SMR) is used. SMR is measured under defined conditions which are not necessarily those of BMR; in particular, temperature may vary. SMR varies considerably between species. It is primarily dependant on body mass to approximately the 0.75th power (21,22), so mass-specific SMR decreases as body mass increases (22,23). For example, the SMR per gram of a mouse (body mass 0.05 kg) is approximately 10-fold higher than the SMR per gram of a horse (body mass 500 kg). The field metabolic rate (FMR) is the metabolic rate of an animal in its natural environment. Factors such as digestion, hunting, reproduction, growth and temperature regulation increase the metabolic rate around twofold in humans and around fourfold in other mammals (20), irrespective of body size. In other words, around one quarter of field energy expenditure in mammals is due to SMR (21,24,25). The differences in mass-specific SMR between species indicate the existence of regulatory mechanisms that may be amenable to pharmacological manipulation. Some of these differences in SMR may be caused by differences in mitochondrial proton cycling.

    What is uncoupling? Go to: Choose Top of page Pharmaceutical interest i... Standard metabolic rate What is uncoupling? << Physiological significanc... Mechanism of proton leak:... Artificial uncoupling by ... Thyroid hormones Inducible uncoupling cata... beta3-Adrenergic receptor ago... Uncoupling proteins 2 and... Is the uncoupling of mito... Acknowledgements References

    Mitochondria are normally responsible for 90% of cellular oxygen consumption and the majority of adenosine triphosphate (ATP) production. The flow of electrons from reduced substrate to oxygen is coupled by a proton electrochemical gradient across the mitochondrial inner membrane to the synthesis of ATP from adenosine diphosphate (ADP) and phosphate (Fig. 1). This process of oxidative phosphorylation can be subdivided into two distinct parts: the generation of the proton electrochemical gradient by the respiratory chain and the synthesis of ATP by the Fo-F1 ATP synthase using the potential energy stored in the gradient. However, not all of the available energy is coupled to ATP synthesis. Instead, much is lost by uncoupled reactions when protons move from the cytosol back into the mitochondrial matrix via pathways which circumvent the ATP synthase and other uses of the electrochemical gradient. There are two sorts of uncoupling. Basal uncoupling is not acutely regulated and is present in all mitochondria, whereas inducible proton conductance is catalysed by proteins, tightly regulated, and found in discrete cell types (Fig. 1).

    Physiological significance of mitochondrial proton cycling Go to: Choose Top of page Pharmaceutical interest i... Standard metabolic rate What is uncoupling? Physiological significanc... << Mechanism of proton leak:... Artificial uncoupling by ... Thyroid hormones Inducible uncoupling cata... beta3-Adrenergic receptor ago... Uncoupling proteins 2 and... Is the uncoupling of mito... Acknowledgements References

    Proton cycling is a major contributor to SMR, responsible for around 26% of the oxygen consumed in resting rat hepatocytes (26) and about 52% in perfused, resting rat skeletal muscle (15). Multiplication of these values by the contribution of each tissue to SMR suggests that proton cycling accounts for 20-25% of rat SMR (15). This makes proton cycling the largest single contributor to SMR.

    Under field conditions, ATP turnover increases significantly and the ATP synthase competes with proton leak for the same proton electrochemical energy. It is important to discover whether proton cycling still represents a significant proportion of energy expenditure when ATP synthesis is increased. Rolfe and co-workers (16) showed that when respiration rate was doubled by stimulating ureagenesis and gluconeogenesis in hepatocytes and muscle contraction in perfused rat hindquarters, the rate of proton cycling stayed fairly constant. Of course, proton cycling dropped as a proportion of total respiration rate, but only to 22% in hepatocytes and 34% in muscle (16). Estimates from these experiments suggest that proton cycling in animals under more plausibly physiological conditions uses around 15-20% of total energy consumption and so remains a substantial proportion of SMR.

    SMR varies with body mass, and proton cycling is an important component of SMR. Does proton cycling change in parallel with SMR, or does it change less or more than SMR? Components of SMR such as the urinary excretion of endogenous nitrogen and sulphur (27) and the rates of respiration, circulation and renal activity (28,29) remain a constant proportion of SMR as body mass varies. Only relatively recently has the contribution of proton cycling to SMR been examined in animals of different body mass. Porter and Brand (30) found that proton conductance in liver mitochondria decreased with increasing body mass in mammals. However, the proportion of energy expended via proton cycling was approximately the same in hepatocytes from all mammals (31,32). Around 70% of the proton conductance difference was because of less mitochondrial inner membrane area in cells from larger animals (33). The remaining difference was some intrinsic property of the membrane. Because this intrinsic property can be altered according to body mass it might be modifiable by drugs. If proton leak could be stimulated in some way, then more energy would be dissipated during synthesis of ATP. This partial uncoupling would cause an increase in SMR and an alteration in the balance between energy input and output if energy intake did not rise to compensate. At comparable energy intakes, a 1% change in BMR in humans would lead to loss or gain of about 1 kg of adipose tissue per year, so over a long period it could cause or combat obesity.

    Mechanism of proton leak: passive diffusion Go to: Choose Top of page Pharmaceutical interest i... Standard metabolic rate What is uncoupling? Physiological significanc... Mechanism of proton leak:... << Artificial uncoupling by ... Thyroid hormones Inducible uncoupling cata... beta3-Adrenergic receptor ago... Uncoupling proteins 2 and... Is the uncoupling of mito... Acknowledgements References

    To consider how to increase mitochondrial uncoupling as a means to increase BMR and reduce obesity, it would be helpful to understand how protons re-enter the mitochondrial matrix without passing through the ATP synthase. Diffusion of protons through the phospholipid bilayer is one possible mechanism of basal proton conductance. To judge the physiological importance of this process, phospholipids from mitochondria with different proton conductance were extracted and reformed into liposomes and the proton conductance of the bilayer was examined (34). This experiment allowed two issues to be resolved; first, the percentage of proton conductance that could be explained by proton diffusion across the bilayer; and second, the effect of the phospholipid fatty acyl composition of the bilayer on the conductance. The results showed that passive diffusion in liposomes could account for only 2.5% to 25% of the proton conductance of mitochondria with the same phospholipid fatty acyl composition, implying that some other property of the membrane, such as the presence of proteins, was important in determining the conductance. There was no significant difference in proton conductance between liposomes with very different phospholipid fatty acyl compositions, despite a known relationship between the phospholipid fatty acyl composition of intact mitochondria and proton conductance (34). Perhaps acyl composition is important in mitochondria, but loss of phospholipid asymmetry or of some specific protein causes the effect to be lost in liposomes.

    Mitochondrial membranes containing a higher ratio of polyunsaturated fatty acyl groups (PUFA) to monounsaturated fatty acyl groups (MUFA) may allow a higher molecular activity of membrane proteins, so membrane acyl composition could affect metabolic rate. In particular, n-3 PUFAs have been strongly linked to SMR (35). As described above, mammals with a smaller body mass have higher mass-specific SMR and higher mitochondrial proton conductance than larger mammals. Surface area and fatty acyl composition of mitochondria both vary with body mass (33,35), so either factor could affect proton conductance. Around two-thirds of the difference in proton conductance of mitochondria from mammals of different body mass is due to the amount of mitochondrial membrane, and one-third is due to some difference (protein or phospholipid) in membrane composition (33). Dietary fatty acids can alter the fatty acyl composition of the mitochondrial inner membrane (36), and it is conceivable that diet, or pharmaceutical agents that alter the PUFA : MUFA ratio of fatty acyl groups in the membrane, could affect mitochondrial uncoupling and hence modify BMR and weight gain. For example, fish oil high in n-3 PUFA has been suggested to limit obesity (37,38). Oils that contain high levels of n-3 fatty acids and purport to be beneficial are already on sale, although evidence that they effect weight loss is lacking (39).

    Artificial uncoupling by dinitrophenol Go to: Choose Top of page Pharmaceutical interest i... Standard metabolic rate What is uncoupling? Physiological significanc... Mechanism of proton leak:... Artificial uncoupling by ... << Thyroid hormones Inducible uncoupling cata... beta3-Adrenergic receptor ago... Uncoupling proteins 2 and... Is the uncoupling of mito... Acknowledgements References

    Uncoupling of mitochondria as a treatment for obesity is not unprecedented; the artificial uncoupler 2,4-dinitrophenol (DNP) has been used for this purpose for many years (12,40). DNP is a lipid-soluble weak acid which acts as a protonophore because it can cross membranes protonated, lose its proton and return as the anion, then reprotonate and repeat the cycle. In this way, it increases the basal proton conductance of mitochondria and uncouples. The effect of DNP derivatives was first noted in 1885 when scientists saw thermogenic effects of Martius Yellow (a dinitro-alpha-napthol), a coal tar dye used in the 19th century to give the impression that food was rich in eggs (41).

    DNP was introduced as a drug in the 1930s and used with considerable success, though reports of side-effects (cataracts) and some deaths from overdose led to it being chased off the market by the US Food and Drug Administration (FDA) in 1938. This use of DNP to treat obesity was stimulated by observations of its toxicity in French munitions workers during World War I (the French commonly used a mixture of 40% dinitrophenol and 60% trinitrophenol for their munitions). In animals, it was shown that the drug promoted a direct stimulation of cellular respiration and a consequent rise in body temperature. It led to the almost immediate onset of rigor mortis when death was promoted by large doses (42,43).

    A series of controlled trials in obese patients were prompted by these promising mode of action studies. The most extensive were carried out during the 1930s at Stanford University, CA, USA (44-47), though others also performed careful studies (48). Interpretation of this work can be complicated, as the doses of DNP had to be optimized for each patient (due to the steep dose response and patient to patient variability) and were usually increased as the studies progressed. This means the DNP-sensitive patients on low doses of the drug would show enhanced efficacy compared with the population (and vice versa for patients on high does of DNP). However, there was a clear dependence of metabolic rate on DNP dose (Fig. 2). There was an average 11% increase in metabolic rate for each dosage increment of 0.1 g of DNP (44,48). Doses up to 0.5 g (about 5 mg kg 1) were generally well-tolerated apart from patients almost always reporting a feeling of warmth together with increased perspiration (45,46,48). Between about 5-10 mg kg 1, patients reported profuse sweating but, surprisingly, there was no evidence of increased body temperature or heart rate. Doses above 10 mg kg 1 led to increased heart rate, respiration rate and excessive body temperature rises (45). Single doses of 3-5 mg kg 1 produced an increase in resting metabolic rate of 20-30% within the first hour. This was maintained for 24 h after which a gradual fall became apparent. Daily dosing of 3-5 mg kg 1 produced a gradual increase of efficacy: an average 40% increase in metabolic rate was observed after a few weeks that was then maintained, with no sign of tolerance, for at least 10 weeks (45).

    Weight loss in these studies, where no attempt was made to control diet, was reported to be variable. Of 170 treated patients, a mean of 7.8 kg weight loss was recorded, averaging 0.64 kg week 1 (44) (Fig. 2). There were no clearly reported effects on food consumption. In contrast to the use of thyroid extract (also in common use at the time to treat obesity), DNP did not promote urinary nitrogen excretion, so the assumption was made that weight loss could be attributed to a specific loss of fat (47). One potential complication in the interpretation of weight loss data is the ability of the drug to promote oedema. This effect was reported to account for a common apparent failure to continue to lose weight in the face of a maintained increase in metabolic rate (48). For some patients, withdrawing DNP led to a rapid weight loss that was attributed to loss of excess body water after which DNP dosing could be resumed with its former effectiveness. Therapeutic doses of DNP had no effects on blood pressure or heart rate in normal patients. Interestingly, a subset of 30 hypertensive patients exhibited average falls of 9.4% in systolic and 12.6% in diastolic blood pressure (44). These improvements in hypertension were also noted at doses of DNP insufficient to cause weight loss (48). Some studies were also performed in diabetic patients with inconsistent results but there did appear to be improvements in glucose tolerance after long-term dosing (48). The reported potency of DNP to treat obesity (and associated comorbid conditions) in these early trials compares well with current treatments for obesity (3,4,6).

    This ability of dinitrophenol to produce good reductions in body weight, without the need for dietary restriction, led to its widespread use to treat obesity. In the absence of formal regulatory controls it is not surprising that it was soon prescribed by inexperienced physicians with no access to the metabolic rate measurements necessary to determine optimal doses. DNP was even included in a variety of 'antifat nostrums' that could be used by the public without medical consultation. By 1934 it was estimated that a total of 100 000 patients had been treated (40).

    Given the steep dose dependence of metabolic rate and the widespread use of DNP it is perhaps not surprising that a number of people were 'literally cooked to death' in the 1930s due to accidental or deliberate overdose (40). It was argued at the time that the reported deleterious effects of DNP were remarkably few (when given at the correct therapeutic doses), given the large number of patients who had taken it. The authors did, however, note a 7% incidence of severe skin rashes, necessitating discontinuation of treatment. There was no overt liver or kidney damage, but the same authors voiced some concerns about the incidence of agranulocytosis though they saw no cases in their own long-term studies (44). Of more concern, a number of cases of cataracts were reported in women in 1935 (49,50). In 1938 the FDA acquired more powers to prosecute manufacturers of misbranded therapies and announced that the use of a variety of patent medicines (including DNP) could lead to prosecution (12). These threats led to a withdrawal, in 1938, of the DNP-containing nostrums from the market as well as an end to the official clinical use of DNP. Interestingly, however, there are reports on the Internet that describe the use of DNP by US clinics (who avoided illegal interstate transportation of DNP for human use by synthesizing it within each state), and give detailed protocols for its use amongst the designer drug community of body-builders and those willing to risk self-administration of DNP to lower body mass.

    Given the age of the publications describing the mode of action in rodents, one of us (KD) recently re-evaluated the effects of DNP in rats and confirmed the relative insensitivity of this species and the particularly steep dose response to increase metabolic rate. In contrast to humans, DNP did not significantly increase metabolic rate in rats when dosed orally at 10 mg kg 1 (data not shown) but a good, comparatively long lasting, increase was seen at 30 mg kg 1 that was comparable in magnitude to a rodent 3-adrenoceptor agonist (Fig. 3) (51). Doses of 100 mg kg 1 produced an escalating hyperthermia that necessitated killing the animals to avoid distress.

    Thyroid hormones Go to: Choose Top of page Pharmaceutical interest i... Standard metabolic rate What is uncoupling? Physiological significanc... Mechanism of proton leak:... Artificial uncoupling by ... Thyroid hormones << Inducible uncoupling cata... beta3-Adrenergic receptor ago... Uncoupling proteins 2 and... Is the uncoupling of mito... Acknowledgements References

    Thyroid hormones have a long history in the treatment of obesity (52) and are positively correlated with BMR (53,54). At the cellular level, hepatocytes isolated from hyperthyroid rats have twice the respiration rate of euthyroid controls. About half of the increase is caused by increased mitochondrial proton cycling, which is partly due to greater proton conductance and partly to a greater driving force (proton electrochemical gradient) (55). At the subcellular level, mitochondria prepared from the liver of hyperthyroid animals have increased proton permeability compared with those from euthyroid animals (56,57).

    In the 1950s the respiratory stimulation in isolated mitochondria was attributed to direct uncoupling by thyroid hormones using the same mechanism as DNP (58). However, this effect only occurred in vitro with supra-physiological concentrations of hormones (59). There are now two main hypotheses for the stimulation of respiration and proton conductance in mitochondria and cells by thyroid hormones (60).

    In the first hypothesis, thyroid hormones act directly at the membrane level, rigidifying the bilayer and leading to a compensatory change in the fatty acyl composition of the phospholipids. Alternatively, thyroid hormones could alter membrane composition through transcriptional regulation of desaturases and other enzymes. In either case, the change in phospholipid fatty acyl composition alters the proton conductance and degree of uncoupling of the mitochondrial inner membrane. In support of this hypothesis, the polyunsaturation of mitochondrial phospholipid acyl chains increases in hyperthyroidism (36,60,61), and liver mitochondrial proton conductance correlates with polyunsaturation of mitochondrial phospholipid acyl chains (33,62). Hyperthyroid mitochondria also have less cholesterol and increased cardiolipin (63). Changes in the area of mitochondrial inner membrane are also implicated in the change in measured proton conductance (57).

    In the second hypothesis, thyroid hormones act through transcriptional regulation and affect proton conductance through expression of specific proteins (64). For example, the levels of UCP2 and UCP3 mRNA increase in response to thyroid treatment (65-69).

    Supra-physiological doses of thyroid hormones are no longer used in the treatment of obesity because of unwanted side-effects such as tachycardia, increased heart weight (70,71), thyroid atrophy (72) and a negative nitrogen balance, that is a loss of lean body mass (muscle) (73,74). They may cause loss of water and muscle rather than loss of fat and adipose tissue.

    Inducible uncoupling catalysed by specific proteins Go to: Choose Top of page Pharmaceutical interest i... Standard metabolic rate What is uncoupling? Physiological significanc... Mechanism of proton leak:... Artificial uncoupling by ... Thyroid hormones Inducible uncoupling cata... << beta3-Adrenergic receptor ago... Uncoupling proteins 2 and... Is the uncoupling of mito... Acknowledgements References


    Uncoupling protein 1

    The ability of hibernators, cold-adapted rodents and new-born mammals to produce heat and increase SMR by non-shivering thermogenesis results mostly or only from the activity of uncoupling protein 1 (UCP1) in mitochondria in brown adipose tissue (BAT) (75,76). The identification of UCP1 as the mediator of uncoupling followed from the observation of greater uncoupling of BAT mitochondria than of liver and heart mitochondria (77,78). Importantly, this increased uncoupling was abolished by albumin and by purine nucleotides, indicating that it could be regulated in vivo by free fatty acids and nucleotides (79). The ability of UCP1 to transport protons was demonstrated by mitochondrial swelling experiments (77,79). A 32-kDa protein was observed to be specifically and highly expressed in BAT mitochondria. Photo-affinity labelling identified a 32-kDa protein as the binding site of purine nucleotides and a putative uncoupling protein was proposed (80). Subsequently, the 32 kDa protein was purified from hamster and rat (81,82). The cDNA was sequenced and cloned and the protein's proton translocating activity was demonstrated in transgenic yeast mitochondria (83) and liposomes (84-87). After these experiments it was generally accepted that UCP1 was responsible for the regulated uncoupling of BAT.

    Further experiments were performed with transgenic mice (88). A mouse model in which all UCP1 had been removed by homologous recombination was created by Enerbäck and co-workers (89). These UCP1-deficient mice were more sensitive to cold and showed a greatly reduced effect of 3-adrenoreceptor agonists, indicating reduced thermogenic capacity. However, when they were fed normal or high-fat diets they were not obese. There was an increase in the adiposity of the BAT, as expected if it did not have to oxidize fatty acids as fast to maintain a normal proton electrochemical gradient. Interestingly, other studies in transgenic mice using a diphtheria toxin gene linked to a UCP1 promoter to genetically ablate BAT did show obesity linked to loss of BAT activity (90). As the obesity in this UCP1-DTA model seemed to result predominantly from hyperphagia it appears that the differences between the two models resides in other (non-thermogenic) activities of BAT. Other studies using targeted expression of UCP1 to mouse muscle (18) or white fat (91) demonstrated a resistance to the development of obesity and an improvement in comorbid conditions consistent with increased uncoupling activities in these tissues.


    3-Adrenergic receptor agonists Go to: Choose Top of page Pharmaceutical interest i... Standard metabolic rate What is uncoupling? Physiological significanc... Mechanism of proton leak:... Artificial uncoupling by ... Thyroid hormones Inducible uncoupling cata... beta3-Adrenergic receptor ago... << Uncoupling proteins 2 and... Is the uncoupling of mito... Acknowledgements References

    There has been considerable interest within the pharmaceutical industry in developing specific 3-adrenoceptor agonists that would selectively lead to the activation of uncoupling through UCP1 in brown fat. The ability to selectively uncouple should avoid many of the side-effects that might occur with DNP. The activity of the 3-adrenoceptor to activate lipolysis in both white and brown fat leading to a consequent activation of UCP1 mediated lipolysis in brown fat is now well understood (92-94). Numerous studies in rodents have shown that 3-adrenoceptor agonists produce profound improvements in insulin sensitivity in a number of diabetic animal models and lead to substantial weight loss due to selective fat loss (13,95). These rodent studies provide excellent evidence that activation of uncoupling by brown fat represents an important potential human therapeutic target. Unfortunately, clinical studies have yet to provide proof of concept in humans, though clinical trials are still in progress. It has proved particularly difficult to develop 3-adrenoceptor agonists that lack activity against 1 and 2-adrenoceptors (13,95). This difficulty, coupled with the very low levels of brown fat in adult humans has made the results of the various clinical trials difficult to interpret. However, if 3-adrenoceptor agonists prove capable of reactivating dormant brown fat, or if conversion of white to brown adipose tissue becomes possible, then a suitably selective agent may show relevant activity in humans.

    Uncoupling proteins 2 and 3 Go to: Choose Top of page Pharmaceutical interest i... Standard metabolic rate What is uncoupling? Physiological significanc... Mechanism of proton leak:... Artificial uncoupling by ... Thyroid hormones Inducible uncoupling cata... beta3-Adrenergic receptor ago... Uncoupling proteins 2 and... << Is the uncoupling of mito... Acknowledgements References

    UCP1 is restricted to brown adipocytes, and was originally thought to be the only transporter capable of uncoupling mitochondria (96-98). However, UCP1 probes occasionally gave weak signals in other tissues (93), suggesting the presence of homologues. UCP2 was discovered because of its relatively high sequence identity to UCP1 (59%). UCP2 mRNA has been found in many tissues, including cardiac muscle, BAT, white adipose tissue, skeletal muscle, kidney, non-parenchymal liver cells, lung, placenta, pancreatic cells and the immune system. However, mRNA expression does not necessarily imply protein expression, as UCP2 protein was not detected in heart, skeletal muscle, liver or BAT (99). UCP3 was also discovered by database searching. Human UCP3 is 59% identical to human UCP1 and 72% identical to human UCP2. UCP3 is restricted to BAT and skeletal muscle (100), both tissues that make a major contribution to thermogenesis, making it a more enticing target for drug development than UCP2.

    Evidence that UCP2 and UCP3 are responsible for basal proton conductance comes from experiments employing high expression of mammalian UCP2 and UCP3 in yeast (101-103) and transgenic overexpression of human UCP3 in mice (14). Such high expression lowers mitochondrial membrane potential and increases state 4 respiration, indicating uncoupling. It inhibits the growth of yeast and protects against obesity in hyperphagic mice. However, the normal concentrations of UCP2 (99) and UCP3 (104,105)protein expressed in mammalian tissues are very low. The amounts expressed in transgenic yeast (104) or UCP3-overexpressing mice (14) are strongly supra-physiological and there is good evidence that the uncoupling is an artefact not related to the physiological functions of UCP2 and UCP3 (105,106) (S. Cadenas et al. unpublished data; J. A. Harper et al. unpublished data). Proteoliposomes have also been used in studies of the function and regulation of UCP1 and its homologues (107-110). These studies found different nucleotide sensitivities and low turnover numbers, but they did identify ubiquinone as an essential cofactor for uncoupling in vitro by UCP1 and its homologues (111,112).

    The UCPs have been knocked out in mice, avoiding interpretational problems resulting from the requirement for overexpressed protein to be correctly inserted and folded in membrane. Two studies (113,114) found that UCP2 mRNA was increased in BAT of UCP1 knockout mice with no increased uncoupling. However, UCP2 protein was not measured and may not have changed. The proton conductance of skeletal muscle mitochondria from UCP3 knockout mice was decreased (115,116), indicating that UCP3 is responsible for at least some of the proton conductance in skeletal muscle. However, we have not been able to confirm these observations (S. Cadenas et al. unpublished data). Despite the reported effects in genetically modified mice, natural changes in UCP2 and UCP3 mRNA and UCP3 protein levels do not alter mitochondrial proton conductance. For example, UCP2 and UCP3 mRNA increase in muscle from starved rats (117) despite a known depression in thermogenesis (118). UCP3 mRNA increases fourfold and UCP3 protein doubles but there is no change in the mitochondrial proton conductance (119).

    Taken together, the evidence suggests that UCP1, UCP2 and UCP3 are not responsible for the basal proton conductance of mitochondria. Clearly, UCP1 causes inducible uncoupling in BAT mitochondria, and UCP2 and UCP3 may yet be found to catalyse a similar uncoupling that is induced by unidentified agonists. The UCPs remain important potential targets for specific pharmacological uncoupling as a treatment for obesity. Other members of the mitochondrial carrier family have also been implicated in the uncoupling of mitochondria. In particular, the adenine nucleotide transporter is responsible for uncoupling by free fatty acids (120,121) and AMP (122). These carriers may also be potential drug targets.

    Is the uncoupling of mitochondria a viable target for drug development? Go to: Choose Top of page Pharmaceutical interest i... Standard metabolic rate What is uncoupling? Physiological significanc... Mechanism of proton leak:... Artificial uncoupling by ... Thyroid hormones Inducible uncoupling cata... beta3-Adrenergic receptor ago... Uncoupling proteins 2 and... Is the uncoupling of mito... << Acknowledgements References

    Uncoupling mitochondria is an effective way to increase thermogenesis and basal metabolic rate and it can lead to a substantial reduction in body weight by loss of fat deposits. This has been shown in humans taking DNP orally and in transgenic mice overexpressing human UCP3 or UCP1 in muscle. Proof of concept has therefore been unambiguously established, showing that uncoupling can increase energy expenditure without compensatory mechanisms increasing food intake to the extent that they nullify the uncoupling.

    However, using pharmacological agents to uncouple all mitochondria throughout the body may be a high-risk treatment, because it might compromise energy homeostasis in tissues such as heart and brain. On the other hand, active tissues like these may be less susceptible to mild uncoupling than less active ones like resting muscle or resting BAT because proton conductance has much less control over respiration rate in active mitochondria (123). The small difference between the effective and the fatal doses of DNP, as well as side-effects resulting from its non-selective actions, mean that it is not itself a suitable antiobesity drug. Tissue selectivity and safety need to be improved.

    The UCP3-overexpressing mouse shows that selective uncoupling of muscle mitochondria is sufficient for a strong anti-obesity effect. Specific uncoupling of brown adipose tissue mitochondria through UCP1 or of muscle mitochondria through UCP3 or in some other way remains a viable and attractive target for the development of drugs for the treatment of obesity.


    Acknowledgements Go to: Choose Top of page Pharmaceutical interest i... Standard metabolic rate What is uncoupling? Physiological significanc... Mechanism of proton leak:... Artificial uncoupling by ... Thyroid hormones Inducible uncoupling cata... beta3-Adrenergic receptor ago... Uncoupling proteins 2 and... Is the uncoupling of mito... Acknowledgements << References

    JAH was supported by a BBSRC CASE award with Knoll Ltd.

    References Go to: Choose Top of page Pharmaceutical interest i... Standard metabolic rate What is uncoupling? Physiological significanc... Mechanism of proton leak:... Artificial uncoupling by ... Thyroid hormones Inducible uncoupling cata... beta3-Adrenergic receptor ago... Uncoupling proteins 2 and... Is the uncoupling of mito... Acknowledgements References <<

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    Obesity Reviews
    Volume 2 Issue 4 Page 255 - November 2001

    I have a feedling with this, it just might be able too. Lets just say I have a *ahem* hunch
    Then perhaps I'm comparing apples (DNP) to oranges (UA). I still maintain however, that uncouplers are best used off-cycle, and will deliver categorically better results when each is used separately with a different purpose in mind.

    I would certainly think an anabolic agent would do so, no? (i.e like running one while on T3 for example, although I realize T3 and uncouplers do not share the same mechanism of action).
    I can see where you're going with this, but thyroid is a whole different ball-game. I still hold to my initial recommendations.
    •   
       


  3. I think we already have that article somewhere. Maybe in The UA FAQ?


    Well your point is taken but Weave isn't exactly using this for gains, moreso than a cutting agent and from I've seen its a great one. It would be the same if he used Winny, Var or Tren but in this case I tihnk his curiosity is the main culprit for using M1T.
    For answers to board issues, read the Suggestion and News forum at the bottom of the main page.

  4. Okay, 1 week in @ 20mg.

    So far up 5lbs but slightly leaner.... very nice

    No major sides to report. Hair and libido is fine. Definitely feel "on" as expected, but no negative reactions at all. This definitely delivers results faster than other delivery methods IMO, although slightly different than other 1-test cycles so far.

    BTW popping milk thistle and potassium like pez

  5. About 12 days in and so far....

    up 6-7 lbs and leaner.....hell yeah baby!!

    I think if I would've done more cardio I would be even leaner, but damn I'm impressed.

    Sides:
    Hair - fine, no shedding
    Libido - up and down, lol nothing extreme though I think
    Restlessness - very ancy, definitely feel "on", but I like that, lol
    Pumps - damn nice, lean pumped feeling
    Lifts - all are up slightly, some extra reps some additional weight
    •   
       


  6. Originally posted by jweave23
    About 12 days in and so far....

    up 6-7 lbs and leaner.....hell yeah baby!!

    I think if I would've done more cardio I would be even leaner, but damn I'm impressed.

    Sides:
    Hair - fine, no shedding
    Libido - up and down, lol nothing extreme though I think
    Restlessness - very ancy, definitely feel &quot;on&quot;, but I like that, lol
    Pumps - damn nice, lean pumped feeling
    Lifts - all are up slightly, some extra reps some additional weight
    DAMN 6lbs in 12 days. Good sht man.

  7. Can you tell us what kind off work out split you are doing and the volume per body part?

  8. Originally posted by Molehonea
    Can you tell us what kind off work out split you are doing and the volume per body part?
    4 day split

    Day 1 - Chest/Tri's 3 exercises each, 3 sets of 6-8

    Day 2 - Back/Bicep 3 exercises, 3 sets of 6-8

    Day&nbsp;3 - Shoulder/Traps - 3 exercises, 3 sets of 6-8

    Day 4 - Legs/Abs - 5 exercises for legs of 6-8 reps (Squats, standing calf raise, leg extension, seated ham curl, seated calf raise) 2 exerices (weighted crunches and oblique twists) around 20 reps

    &nbsp;

  9. I should add I've been off for almost a week now, totalling up at about a 7-8 lb gain in 2 weeks while slightly leaner, definitely a body composition change. Can't wait till next cycle in a week

    As for the Anarchy/UCP-1/Gugg stack - down about 2lbs this week, not too bad

  10. Originally posted by jweave23


    4 day split

    Day 1 - Chest/Tri's 3 exercises each, 3 sets of 6-8

    Day 2 - Back/Bicep 3 exercises, 3 sets of 6-8

    Day&amp;nbsp;3 - Shoulder/Traps - 3 exercises, 3 sets of 6-8

    Day 4 - Legs/Abs - 5 exercises for legs of 6-8 reps (Squats, standing calf raise, leg extension, seated ham curl, seated calf raise) 2 exerices (weighted crunches and oblique twists) around 20 reps

    &amp;nbsp;
    When you say: 3 exercises each, 3 sets of 6-8 - does that mean you do one set for each of the 3 exercises or 3 sets for each exercise?

  11. 3 sets for each exercise

    i.e flat DB bench 3 sets of 6-8 reps

  12. Ok so I took a few&nbsp;random pics tonight and decided to put them up. I was quickly disgusted by my own bodyfat level, so this is all you'll get right now

    &nbsp;

  13. What are your stats bro, you look like a big boy.

  14. Age: 25
    Height: 6'
    Weight: 265

    I haven't measured lately but arms are around 19", as with calves. Chest is around 51" and quads are about 28" now. Waist unfortunately is around 38 but hopefully I can cure the bigorexia at some point soon, lol

  15. J,
    what was your most resent bf%? and what is your goal?

    Looking swole bro, looking swole.

    db

  16. Most recent bf was at 20%, done with calipers I'm hoping to0 half that. If I can get to around 10% and maintain that bf%, I'll be happy

  17. Lookin good Jweave! Those are some big guns you're sportin there..19's wow. If I had 19's I'd officially be a freak. Hang in there man, I'm sure with your dedication you can hit that 10% no problem.

  18. How many days off between splits?

  19. Killer results weave, good **** bro!

  20. Originally posted by jweave23
    Most recent bf was at 20%, done with calipers I'm hoping to0 half that. If I can get to around 10% and maintain that bf%, I'll be happy
    Good luck getting to your goal.

    One suggestion when you decide to really cut, even though you may have already done this or planning to do this. I would try cutting completely natural for a few weeks. By this I mean nothing except for diet change and lifting and cardio. This will allow you to get accustomed to see how well you can cut just thru diet and cardio alone. Then if needed add the supps you want.

    I have found this to be effective, especially when trying to lose that last bit of fat b/c if you go clean the whole time once you start to get frustrated then add the supps and the loses/gains start to come again.

  21. Originally posted by Molehonea
    How many days off between splits?

    I do:

    1 on, 1 off, 2 on, 2 off, repeat

    Originally posted by size


    Good luck getting to your goal.

    One suggestion when you decide to really cut, even though you may have already done this or planning to do this. I would try cutting completely natural for a few weeks. By this I mean nothing except for diet change and lifting and cardio. This will allow you to get accustomed to see how well you can cut just thru diet and cardio alone. Then if needed add the supps you want.

    I have found this to be effective, especially when trying to lose that last bit of fat b/c if you go clean the whole time once you start to get frustrated then add the supps and the loses/gains start to come again.
    Yeah I have a few things running around in my head right now. I wasn't even going to try the M1T at first, then I couldn't resist, lol. I'm thinking I'll finish this up, then either try cutting natty or run a test/EQ/winny cycle then start natty cutting in the spring.

  22. J-What is your Diet Like?

  23. Originally posted by wildman536
    J-What is your Diet Like?
    Basically 40/40/20, although aiming for more 50/30/20 usually. I've done a pretty good job of limiting high GI carbs this time around.

    Update:

    End of week 2 of 2nd cycle. This **** rocks!! I'm up about 6 more lbs, although the fat loss has stopped some, so it's time to cut back harder on the diet and increase cardio. I feel lean and frickin big, lifts are the best ever, just feel like a damn machine! Muscles are pumped to hell, feel jacked all day.

    Sides: I have to say really not many at ll. I felt a slight back ache a few days ago so I skipped deads this week, but that's about it. No hair loss, acne, libido problems to speak of. Couldn't possibly be any happier with it

    &nbsp;
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