MBB317 Dr D MANGNALL OBESITY Lecture 2 When the structure of the insulin receptor was first discovered it was assumed that the active beta subunit tyrosine kinase would phosphorylate some intracellular protein substrate, and a lot of time and energy was spent looking for intracellular substrates, and it took several years before any were clearly identified. To my knowledge there are only a few such substrates known so far. These are known as IRS1, IRS2 and IRS3, etc, where IRS means insulin receptor substrate. Part of insulin s actions depend on the phosphorylation of these intracellular substrates, although what happens after that is not totally clear. However, that is only part of the insulin action story. The receptor itself autophosphorylates, that is the receptor phosphorylates itself on tyrosine residues within the beta subunit. This phosphorylated form of the receptor is then recognised by other proteins within the cell, which then dock onto it, and form a complex, and these complexes are part of the signalling pathway. The key features as far as these lectures are concerned are that the receptor becomes active as a tyrosine kinase when insulin binds, and in the test tube the activated receptor will phosphorylate artificial proteins made as a polymer of glutamate and tyrosine. Thus, in vitro one can incubate the receptor with the glutamate: tyrosine polymer, and 32 P labelled ATP, and in the presence of insulin the activated tyrosine kinase will transfer 32 P from the ATP to tyrosine residues in the artificial acceptor protein, and this forms the basis of an assay to determine the ability of insulin to switch on the kinase, and to initiate the insulin signalling pathway. The other way of measuring the ability of insulin to switch on the tyrosine kinase activity is to incubate the receptors with 32 P- ATP as before, but to leave out the artificial tyrosine: glutamate polymer, so that the receptor itself becomes radioactively labelled with 32 P on tyrosine residues, and to measure the amount of labelling on the receptor. Both assay systems have been used successfully. The next overhead shows the insulin binding and insulin induced tyrosine kinase activity of receptors isolated from human muscle using the assay system, which looks at autophosphorylation of the receptor.
Figure 8 muscle. Insulin binding and autophosphorylation of receptors from human The panel on the left shows the binding of I 125 - labelled insulin. Here receptors have been incubated with a fixed amount of I 125 -labelled insulin and increasing amounts of non-labelled insulin. Results from receptors from lean controls are shown in the open symbols, and the filled symbols show data from receptors prepared from obese subjects. There is no difference in the ability of either group of receptors to bind insulin, thus the alteration in the insulin sensitivity is not due to any change in ability of the alpha subunits to form the binding domain for the insulin, and the insulin binds equally well to both sets of receptors. The right hand panel shows the ability of insulin to induce receptor autophosphorylation. The top line (with open symbols ) is the response for receptors from non-obese control subjects, and the bottom 3 sets are from 3 preparations of receptors from obese subjects. The asterisks indicate that the upper values are significantly higher than those of the lower group, and suggest that receptors from muscle obese subjects are inherently defective in their ability to autophosphorylate, and since this is part of the way in which insulin signalling is initiated, suggests that muscle from obese subjects is relatively insulin resistant. The next overhead shows similar data, but from mice rather than humans. Figure 9. Autophosphorylation of receptors from mouse muscle.
This again shows that receptors from obese mice are insulin resistant relative to receptors from lean controls. The next overhead (Figure 10) shows that with receptors from adipocytes (rather than muscle ), the situation seems to be reversed, ie, the ability of receptors from obese animals to phosphorylate a glutamate: tyrosine artificial acceptor protein is greater than that of receptors from lean controls. Figure 10. Phosphorylation of poly Glut:Tyr by receptors from rat adipocytes.
Collectively, this sort of data suggests that at least part of the reason for the altered insulin sensitivities of tissues in obese humans and rodents can be attributed to alterations at the level of the insulin receptor, and suggests that for obese subjects, insulin signalling via receptor auto phosphorylation is reduced in the muscle, but that signalling via the phosphorylation of intracellular proteins is enhanced in the adipose tissue. It should be clear from these kinds of studies that the obese human and the obese rodent are metabolically different from their lean, non-obese counterparts. There are lots of studies of this kind in the literature, and conflicting reports are not unknown, so the story is not one which is yet set in stone. However, one can construct a story around this kind of data which is consistent at least with the notion that in the obese situation skeletal muscle becomes less insulin sensitive, and adipose tissue changes in its insulin sensitivities. Thus the capacity for insulin stimulated oxidation of glucose to CO2 is reduced in adipose tissue in the obese, whilst the insulin stimulated uptake for 2-deoxyglucose is only slightly affected, and in the absence of insulin the basal level of 2 deoxyglucose uptake is increased. In skeletal muscle insulin binding is unaffected, but insulin signalling, at least via receptor autophosphorylation is reduced, In adipose tissue insulin signalling, as measured by the ability of insulin to stimulate the receptor to phosphorylate an artificial protein substrate, is increased., but not all insulin sensitive functions are stimulated (glucose oxidation to CO2 is clearly reduced ), but none the less some insulin signalling mechanism is potentially at least increased. This may be related to increased lipoprotein lipase activity, and together with the decreased capacity for
glucose oxidation, may indicate an increase in the ability of the adipose tissue to synthesise and store fatty acids as triglyceride. An unanswered question is 'how is altered kinase activity of the receptor achieved?' This is unclear at present, but it may be that alternative splicing of the receptor gene exons leads to alterations in receptor activity. Whist these kinds of study highlight metabolic differences between the obese and non-obese individuals they don't really say too much about why the obese subject has all that extra adipose tissue. So one can ask what accounts for the large amount of adipose tissue? ie what do we know about control of adipose tissue growth? ADIPOSE TISSUE GROWTH AND DEVELOPMENT. Although there have been considerable advances in recent years in understanding how adipocytes are formed, particularly an explosive increase in the identification of transcription factors which promote adipogenesis, there remain major gaps in our knowledge and only a speculative understanding of how it may relate to the obese state. (At present the work seems to be at the stage of establishing the general principles of adipogenesis rather than at the level of saying how this relates to the development of obesity.) The salient features of what is currently known are highlighted in the box below and subsequently discussed a little more fully.
ADIPOSE TISSUE DEVELOPMENT In both rodents and humans, adipose tissue develops during late gestation and first few weeks after birth. Adipose tissue comprises a mixture of cell types Precursor cells called preadipocytes fibroblast-like little of the enzymatic make up of the mature adipocyte can divide Mature Adipocytes Are fully differentiated do not divide Mild obesity results from existing adipocytes enlarging Severe obesity involves an increase in the number of adipocytes as well Much of our understanding of the differentiation process comes from studies of cells in culture eg 3T3-L1 ) 3T3-F442A ) from embryonic tissue Ob17 from adult tissue when treated in culture with a cocktail of stimulants these cells form fully differentiated adipocytes ( 3T3 lines need camp dexamethasone and insulin, Ob17 needs fatty acids ) generally a mixture of insulin, T3, glucocorticoids, GH, prostaglandins have been used. Transformation occurs in a step wise fashion in which a number of transcription factors are activated sequentially. The main ones are PPARγ and δ ( Peroxisome Proliferator-Activated Receptor ) C/EBP ( CCAAT/ Enhancer Binding Protein ) ADD-1 (Adipocyte Determination and Differentiation factor-1) In both rodents and humans adipose tissue development occurs during late gestation and the first few weeks after birth. It is now clear that adipocytes arise from a population of pre-adipocyte stem cells, which are lipid-free mesenchymal cells, which, in contrast to the mature adipocyte, are fibroblast-like and have little of the enzymatic capacity that characterises the mature adipocyte. The preadipocytes are able to divide and increase in numbers whilst the mature, fully differentiated adipocyte does not divide. The adipose tissue
thus comprises a mixture of cell types, but it is the presence of the preadipocytes that explains why the adipose tissue can increase in mass. In humans, mild obesity results from the enlargement of the adipocyte that accompanies increased triglyceride accumulation, whilst severe obesity involves an increase in the number of adipocytes as well. In rodents, and probably in humans also, high fat and high carbohydrate diets lead to increases in adipose tissue mass involving the appearance of new fat cells. This occurs firstly by the pre-adipocytes proliferating, which occurs within days, followed by a terminal differentiation step which takes weeks during which time there is lipid deposition within the adipocytes. Much of our current understanding of the molecular events of the differentiation process comes from studies of cell cultures. Some of the lines are derived from embryonic tissue (eg. 3T3-L1 and 3T3-F442A cells) and some from adult animals (eg the Ob17 cell line ). When these cells are treated in culture with a cocktail of stimulants they will convert from pre-adipocytes to the fully differentiated adipocyte, a process that involves the very ordered activation of a whole range of genes. The stimulants needed vary depending on the cell line ( perhaps reflecting the stage of development of the animal from which they were first isolated) so that the 3T3 cell lines respond to mixture of high cyclic AMP, dexamethasone and insulin, whist Ob17 cells will differentiate on expose to high fatty acid levels. Generally some combination of hormones ( insulin, triiodothyronine, glucocorticoids and growth hormone ) and prostaglandins has been used to promote the in vitro conversion of the preadipocytes in culture to the mature adipocyte. This transformation occurs in stepwise, sequential fashion, and involves the sequential activation of a number of transcription factors, the predominant ones being PPARγ and δ ( Peroxisome Proliferator-Activated Receptor ), the C/EBP (CCAAT/Enhancer Binding Protein ) family members and ADD-1 ( Adipocyte Differentiation and Determination factor-1). The PPAR proteins are found on the nuclear membrane, and when complexed to the appropriate ligand will act as transcription factors by moving into the nucleus and interacting with promoter regions of genes and switching on these genes specifically. PPARγ exists in 2 forms, PPARγ1 and PPARγ2 formed by alternative splicing. PPARγ2 is expressed at high levels in adipose tissue, whilst PPARγ1 is found at low levels in many other tissues. PPARγ2 is induced early in the adipocyte differentiation and appears to be a crucial regulator of many fat-specific genes as well as a 'master
regulator' capable of triggering the whole of the differentiation program. Interestingly, the adipogenic activity of PPARγ is markedly enhanced by insulin. PPARγ is not the only transcription factor of importance in adipocyte differentiation. C/EBPα,β and δ are all markedly increased relatively late in the process of adipogenesis, at least in cells in culture. However, in C/EBPα knockout mice, although there is a major reduction in the amount of fat in the adipose tissue, fat cell differentiation still occurs. The available evidence suggests that C/EBPα and PPARγ cooperate dramatically in vivo, and when expressed together the differentiation inducing effect is markedly stimulated. The promoter region for PPARγ 2 contains 2 C/EBP binding regions, and C/EBPβ, which is increased early in adipogenesis, increases the expression of PPARγ. Another factor, which interacts with PPARγ is ADD-1. Under conditions, which are not conducive to adipogenesis, this transcription factor induces 2 key enzymes of fatty acid metabolism, fatty acid synthetase and lipoprotein lipase, without stimulating fat cell differentiation, but under conditions, which favour adipogenesis ADD-1 increases the number of cells undergoing differentiation. This is at least in part due to an increase in the activity of PPARγ, ADD-1 is also involved in the control of key genes involved in cholesterol metabolism. A simplified scheme is shown in Figure 11 below. (Fig 1from Grimaldi review). FACTORS INFLUENCING PREADIPOCYTE TO ADIPOCYTE DIFFERENTIATION Insulin ADD-1 Long Chain Fatty acids Prostaglandin I2 Glucocorticoids camp, LIF, GH PPARδ C/EBPβ C/EBPδ PPARγ C/EBPα Terminal Differentiation Related Genes DIFFERENTIATING PREADIPOCYTE ADIPOCYTE ADIPOCYTE (From Grimaldi PA. The roles of PPARs in adipocyte differentiation (2001). Prpgress in Lipid Research. 40. 269-281) Exposure of the pre-adipocytes to glucocorticoids, camp, growth hormone (GH) and leukaemia inhibitory factor (LIF) promotes the activation of C/EBPδ and C/EBPβ. In the presence of long chain fatty acid and prostaglandin I2 this leads to the activation of PPARδ. These changes occur at the point that the cultures reach confluence and
result in the cells becoming committed to differentiation. An early marker enzyme activity appearing at this stage is lipoprotein lipase. The second stage is a proliferation stage during which there is a clonal expansion of these committed cells, which divide and multiply. During this stage the pattern of activated transcription factors changes. C/EBPαbecomes the predominant C/EBP form, and is induced by the action of the β and δ forms. C/EBPα, β and δ also combine with PPARδ to activate PPARγ. If the necessary hormones and PPARγ ligands are available, the transcriptional activity of PPARγ activates the gene cascade that leads to adipogenesis. Activation of PPARγ is also promoted by ADD-1, which is activated by the presence of insulin. The appearance of PPARγ and C/EBPα leads to a terminal differentiation stage associated with the expression of a number of adipose tissue specific genes. ADD-1 is needed for the expression of the fatty acid synthetase, and ADD-1 expressing cells produce lipid molecules that bind to and activate PPARγ. Thus, late on in the process, PPARγ activates C/EBPα expression and PPARγ and C/EBPα co-operation brings about maximal differentiation and full lipogenic capacity. Almost certainly this scheme of things will be shown to be much more complex as more research is done. There are several issues which are clearly yet to be resolved, not least of which is the nature of the PPARγ ligand, and which are the important hormones. Despite these recent advances in understanding how adipogenesis and differentiation are achieved at the molecular level, there is very little hard data currently available which relates directly to how this process is accelerated or exaggerated in the obese subject. However, one may speculate that the ingestion, digestion and absorption of nutrients, which is generally believed to increase during the time at which obesity develops, gives rise not only to nutrients which might stimulate insulin release from the pancreas, but also produces a range of signals in the form of hormones from the gut, and signals from the brain. These gut signals may be part of the hormonal environment which promotes adipogenesis. Insulin is known to enhance the adipogenic activity of PPARγ, possibly through a change in phosphorylation state of PPARγ. The abundance of PPARγ falls in starvation and in diabetes. The insulin stimulation of PPARγ may be due to an increase in C/EBPβ and C/EBPδ which are induced by insulin in cells in culture. ADD1/SREBP1 activity is also increased by insulin. Glucocorticoids are also important in this scheme, since induction of PPARγ by C/EBPβ and C/EBPδ. is dependent upon the presence of a glucocorticoid.
Although precisely how this relates to the development of an obese state remains to be elucidated, THE OBESITY GENE AND LEPTIN Regulation of adipose tissue mass Since total body mass can be kept constant (+/- 1% over the course of many years) despite a widely fluctuating food intake and energy expenditure, it has been postulated that there must be a powerful, slow, feedback pathway operating. It has been proposed that it is the total amount of fat which is sensed. It is postulated that when mammals overeat the resultant extra fat signals to the brain that the body is obese and the animal then responds to signals from the brain by eating less and/or burning more fuel. The key points, (derived mainly from animal studies, but probably applying to mammals generally) are: a). The hypothalamus, ( more specifically the ventromedial nucleus of the hypothalamus, VMN ) is probably the main control centre for satiety and energy expenditure. Damage can result in obesity, similar to that in ob/ob mice, whilst stimulation reduces eating and increases energy expenditure. b). Rats forced to overeat lay down excess fat, but when offered a normal eating routine they eat less until a normal body weight Is obtained. c). Surgical removal of substantial amounts of fat is followed by increased eating and an increase in the remaining fat stores. (Presumably this has implications for surgical interventionist approaches such as 'apronectomy' or liposuction ). d). Overfeeding one of a pair of parabiotic mice, (ie. mice joined surgically to have some interchange of blood) causes reduced food intake and loss of weight in the partner mouse, suggesting transfer of a circulating hormone. e). Hypothalamic lesions on one of a pair of parabiotic mice leads to obesity in that mouse, but reduced food intake and loss of adipose tissue in the other mouse, again suggesting transfer of a proposed satiety hormone from the obese mouse to the lean mouse. Ie. the 'fed' signal is produced by both mice but not recognised by the mouse with the hypothalamic lesion, and the normal mouse gets an overdose of the 'stop eating ' signal.
f). If an ob/ob mouse is parabiotically joined to a normal mouse it eats less and looses weight. This suggests the obesity of the ob/ob mouse is due the loss of a satiety hormone, which is then provided by the normal animal of the parabiotic pair. g). Mice homozygous for another mutation, db (diabetes), are also obese. The db/db phenotype reflects a defect in the action of the Ob protein (leptin) due to a defect in the receptor for leptin. Normal mice parabiotically joined to db/db mice reject food and die of starvation. When ob/ob mice are joined to db/db mice the ob/ob mouse reduces food intake and looses adipose tissue. The ob/ob mouse appears to react to a putative excess of normal Ob protein produced by the db/db partner. All of this suggests that fat produces a factor, which acts as a satiety factor, and that when normal animals overeat the resultant extra fat somehow signals to the brain that the body is obese, with the result that less food is consumed and/ or energy expenditure is increased. In 1994 J.M. Friedman and colleagues reported the cloning of a gene from ob/ob mice ( the ob gene ), which had been recognised 40 years earlier to lead to profound obesity and type ll diabetes in mice homozygous for the mutation, resulting in a state resembling morbid obesity in man. The ob gene was only expressed to any extent by adipose tissue The gene consists of 3 exons and 2 introns, and encodes a 4.5 kilobase mrna derived from coding sequences in exons 2 and 3. The predicted protein product, which is now called Leptin, ( from the Greek Leptos, meaning 'thin' ), was derived from a 167 amino acid open reading frame which included a 21 amino acid secretory signal sequence, suggesting that the product was a secreted protein. They suggested mice homozygous for ob gene fail to secrete the normal protein, and that leptin may represent a 'satiety' factor. It is now recognised that the original strain of ob/ob mice used by Friedman's group had a non-sense mutation at codon 105, and so produced a mutated form of leptin. The protein is now known to be 84 % homologous to the human protein, and similar proteins have now been described for several other mammals, chickens and eels ( but not fruit flies!). In 1995, it was shown that injections of purified leptin caused mice to lose weight and maintain their weight loss.
Mice lost 40% of their body weight after a month of daily injections. Compared to pair fed untreated obese mice, the injected group lost 50% more weight than the untreated group, suggesting reduced food intake alone cannot explain the weight loss. Leptin also had an effect on energy expenditure, sluggish ob/ob mice became more active, and their slow metabolism was stimulated, thermogenesis was stimulated. Similar results were obtained using a strain of mouse, which, like the human, becomes obese when its diet contains too much fat, a condition called dietary induced obesity.. These mice, like the ob/ob mice, ate less of the high fat food and lost weight in response to injections of the Ob protein. In mice, which stay lean, when young but become fat as they get older, also lost weight when injected with the Ob protein. Thus, this kind of obesity (not due to a mutation in the ob gene) is correctable by the Ob protein. Ob also causes weight loss in mice that are not fat. Lean mice receiving a relatively high dose of Ob lost 12% of body weight and virtually all body fat after 4 days of injections and maintained the new weight for a further 2 weeks whilst they continued to receive injections. End of Lecture 2