Introduction

The PPAR nuclear receptor family and their function as transcription factors

The peroxisome proliferator-activated receptors (PPARs) comprise a subfamily of the nuclear hormone receptor (NHR) superfamily. Among the NHR superfamily are receptors for steroid, thyroid, and retinoid hormones, which function as nuclear trans-acting transcriptional regulatory factors. In vertebrates, the PPAR proteins regulate diverse biological processes such as pattern formation, cellular differentiation, and metabolic homeostasis. The name PPAR derives from the initial cloning of one isoform as a target of various xenobiotic (nonendogenous) compounds that were observed to induce proliferation of peroxisomes in the liver.¹ This protein was named the peroxisome proliferator-activated receptor, now known as PPARalfa (PPAR). Within a few years, the family of PPARs was expanded to include PPARgamma (PPAR) and PPARdelta (PPAR).

PPAR is highly expressed in the liver, kidney, heart, and muscle, and is activated by peroxisome proliferators, a structurally diverse collection of compounds including hypolipidaemic drugs, herbicides, and phthalate ester plasticizers that cause hepatomegaly accompanied by dramatic increases in the size and number of peroxisomes in the livers of rodents. In humans PPAR is the target for the fibrate class of drugs, which are medically used to lower triglycerides and raise high-density lipoprotein (HDL) cholesterol in dyslipidaemic patients without causing peroxisome proliferation and hepatomegaly.² Thus, the PPAR is assumed to be a major player in liver lipid metabolism.

PPAR is expressed at comparable levels in virtually all tissues, but the role of PPAR has remained rather elusive until recently. Recent data suggest that PPAR is mainly involved in fatty-acid-controlled differentiation of preadipocyte cells,^3,4 and may affect the adaptive response of white adipose tissue to nutritional changes.⁵ There is also evidence that PPAR is involved in the regulation of cholesterol metabolism.^6,7

The PPAR protein is predominantly expressed in adipose tissue. The gene gives rise to three mRNAs (PPAR1, PPAR2 and PPAR3), with the PPAR1 and 3 mRNA ending up as the same protein. The PPAR2 mRNA yields a protein containing an additional 28 amino acids at the N-terminus. Until now there has been no evidence of any functional differences between the PPAR isoforms, but recent evidence suggests that the PPAR2 transcript is the critical player for adipogenesis.⁸ Initial studies in lean C57BL/6J mice showed that the PPAR1 and PPAR2 mRNA were abundantly expressed in adipose tissue. PPAR1 expression was also detected at lower levels in liver, spleen, and heart, whereas PPAR1 and 2 mRNA were both expressed at low levels in skeletal muscle.⁹ Subsequently, the expression of PPAR1 and PPAR2 was investigated in liver, heart, skeletal muscle, and subcutaneous fat tissue from human subjects. Both 1 and 2 mRNAs were abundantly expressed in adipose tissue, PPAR1 was detected at lower levels in liver and heart, whereas both 1 and 2 were expressed at low levels in the skeletal muscle.¹⁰ These results were corroborated in other studies in humans.^11,12,13 In addition to adipose tissue, PPAR is expressed at rather high levels in the large intestine.¹¹

Somewhat different results were reported by Loviscach et al,¹⁴ who found that the amount of PPAR protein in the muscle was on average two-thirds of that present in fat, suggesting that PPAR expression is also abundant in the skeletal muscle.

PPAR DNA-binding properties

All three PPAR subtypes bind to DNA as obligate heterodimers with the nuclear receptors for 9-cis retinoic acid (RXR, RXR or RXR), having the RXR receptor as the preferential partner for the PPAR receptor. The preferred DNA-binding site (called peroxisome proliferator-activated receptor response element, PPRE) for each of the PPAR/RXR heterodimers is a direct repeat of the consensus sequence, AGGTCA, separated by a single nucleotide spacer, a so-called DR-1 motif. In addition to this consensus sequence, there is evidence that the optimal response element differs slightly for each of the PPARs. Also, the PPAR DNA-binding activity is modulated by the isotype of the RXR heterodimeric partner. These subtle differences in binding specificity together with the differences in tissue expression patterns undoubtedly contribute to the distinct physiological roles of the three PPAR subtypes.

Natural endogenous and dietary PPAR ligands

In 1995, Forman et al¹⁵ reported that the arachidonate metabolite 15-deoxy-^12,24-prostanglandin J₂ and related metabolites were PPAR ligands and induced adipogenesis. A somewhat similar finding was reported by Kliewer et al.¹⁶ In addition, several other fatty acids were shown to be agonists of the PPAR receptor, including the dietary polyunsaturated fatty acid eicosapentanoic acid (Figure 1).¹⁷ Also two of the major oxidized lipid components of oxidized low-density lipoprotein particles (oxLDL) 9-HODE and 13-HODE were identified as endogenous ligands and activators of PPAR.¹⁸ From the evaluation of the studies performed so far, it seems likely that the PPAR serves as a receptor for many different fatty acids, with similar affinities in the K_D=2–50 M range, which actually is well below the published affinities of most of the other nuclear hormone receptors for their ligands. Many of these fatty acids also bind to the PPAR and PPAR, and in general saturated fatty acids are poor PPAR ligands compared to unsaturated fatty acids (for a more extensive list of natural and synthetic PPAR ligands and their affinity towards the respective PPAR receptors, see review by Desvergne and Wahli¹⁹). Thus, PPAR seems to bind all its endogenous ligands with rather low affinity, and hence to function as a generalized fatty acid sensor; however, it is likely that the amount of intracellular fatty-acid-binding proteins (FABPs) within the cells and their binding of the free fatty acids (FFA) is important for the actual PPAR activity. X-ray crystallography studies of the PPAR ligand-binding domain have shown that it has a ligand-binding pocket, which is at least twice the size of the corresponding pocket seen in the other nuclear receptors. It is significant that all of the identified natural ligands are lipophilic carboxylic acids. The structural features of the ligand-binding site suggest that the receptor has evolved to recognize acidic ligands that can bind to the cluster of polar residues involved in receptor activation. This characteristic is complemented by the large volume of the binding site, which can accommodate a range of lipo- philic ligands through relatively nonspecific hydrophobic interactions. Thus, the molecular structure of PPAR is consistent with its proposed physiological role as a fatty acid sensor.²⁰

Figure 1.

Chemical structures of PPAR ligands: synthetic ligands (Rosiglitazone, GI262570) and naturally occurring ligands (dietary polyunsaturated fatty acid eicosapentanoic acid, prostaglandin derivative 15-deoxy-12,14-PGJ2, and components of oxidized lipoprotein particles 9-HODE and 13-HODE).

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This suggestion is corroborated by evidence showing that fatty acids and the Thiazoledinedione (TZD) drugs (PPAR agonists) have the same effects on the expression of genes encoding proteins involved in fatty acid metabolism in preadipocytes.²¹

Artificial PPAR ligands

The discovery that PPAR is the molecular target of theTZDs, for example, Rosiglitazone, provided the critical insight necessary for the rational design of new antidiabetic drugs. Hence, a series of agonists optimized against the PPAR receptor have been developed. These include GI262570 (Figure 1), GW1929, and GW7845. Temporary studies have shown that these compounds activate PPAR at nanomolar concentrations, and that they in vivo reduce plasma glucose and triglyceride levels, accompanied with a rise in HDL cholesterol.²⁰ Recently, the novel PPAR antagonists GW0072,²² bisphenol A diglycidyl ether (BADGE),²³ PD068235^24,25 and LG100641,^24,25 have been identified, and they are all able to antagonize PPAR agonist-induced adipogenesis.

Adipogenesis—general concepts

In vivo studies of preadipocyte differentiation are troublesome. Fat tissue within animals consists of approximately one-third adipocytes. The remaining two-thirds consists of small blood vessels, nerve tissue, fibroblasts, and preadipocytes in various stages of development. In addition, the distinction between preadipocytes and fibroblasts is difficult to make. Few studies use preadipocyte primary cultures (eg preadipocytes isolated from the stromal–vascular fraction of dissociated fat pads), whereas most studies use tissue culture models of well-characterized cell lines. The 3T3-L1 preadipocytic cell line is the most well-characterized cell line, and hence it is the most commonly used. It faithfully displays in vivo characteristics, which includes morphological changes, cessation of cell growth, expression of many lipogenic enzymes, extensive lipid accumulation, and the establishment of sensitivity to most or all of the key hormones that impact on this cell type, including insulin. In 'nonadipogenic' growth medium (usually foetal bovine serum, FBS) the fibroblastic-like 3T3-L1 cells proliferate until confluence occurs. After reaching a state of density-induced growth arrest, the preadipocytic cell line can be differentiated synchronously by a defined adipogenic mixture (ie a combination of mitogenic and hormonal agents). Maximal differentiation is achieved upon addition of a combination of insulin, the glucocorticoid dexamethasone and the cAMP-phosphodiesterase inhibitor methyl-isobutylxanthine.²⁶

The structural and functional morphogenesis associated with adipocyte differentiation is quite well understood, involving a complex interaction between many transcription factors and cofactors as shown in Figure 2.²⁷ However, in spite of the indication in the figure of the involvement of a endogenously produced ligand for PPAR as suggested by Kim et al,²⁸ and also the indicated critical importance of STAT, among other uncertain aspects these issues need to be finally resolved. Recently, using the oligonucleotide microarray technique, the patterns of gene expression in preadipocytes and adipocytes in vitro and in vivo were studied.²⁹ The abundance of 11 000 genes and expressed sequence tags was measured at 10 different time points during in vitro 3T3-L1 adipocyte differentiation. The abundance of the same 11 000 genes was also measured in adipocytes and stromal cells (including preadipocytes) isolated from wild-type and ob/ob mouse white adipose tissue. Overall, the study corroborated many previously reported patterns of gene expression including changes of PREF1, AEBP1, C/EBP, C/EBP, C/EBP, aP2, adipsin, Acrp30/AdipoQ/adiponectin, LPL, HSL, SCD1, A2COL6, and others, but also showed major regulation of a set of heretofore uncharacterized genes.²⁹

Figure 2.

Transcriptional events in adipocyte differentiation. A summary (present view) of the molecular processes of adipocyte differentiation, focusing only on transcriptional events. Direct or indirect transcriptional events are indicated by solid lines. Broken lines represent interactions that are less well understood. Specific transcription factors are denoted with square boxes; unknown factors are indicated with question marks. Abbreviations: ADD1, adipocyte determination- and differentiation-dependent factor 1; C-EBP, CCAAT-enhancer binding protein-; RXR, retinoid X receptor; SREBP, sterol regulatory element binding proteins; STAT, signal transducers and activators of transcription. Adapted from Morrison and Farmer.²⁷ Reprinted with permission from The Journal of Nutrition Volume 130© 2000 by the American Society for Nutritional Sciences.

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Adipogenesis and the target phase of PPAR transcriptional activation

The target phase of PPAR activation has been studied by evaluating the effect of the PPAR agonist Pioglitazone on every stage during the course of adipocyte differentiation by Takamura et al.³⁰ They found that Pioglitazone did not stimulate proliferation of preadipocytes before confluence. In 3T3-L1 cells, proliferation and differentiation are not thought to occur simultaneously. The results are in accordance with the following concept: cell proliferation (and increased DNA synthesis) occurs until confluence. Induction with an adipogenic mixture (and Pioglitazone) induces growth arrest (decreases DNA synthesis) and enhances differentiation (increased protein synthesis). Subsequent protein synthesis, glucose transport, and triglyceride accumulation are also enhanced by the adipogenic mixture and Pioglitazone. Without Pioglitazone, PPAR mRNA levels reach maximum levels in mature adipocytes, whereas in Pioglitazone—treated cells, the level of PPAR mRNA reaches its maximum at day 6 (ie before maturity), whereafter continued Pioglitazone treatment decreases the PPAR mRNA level, although with an apparently continued enhanced triglyceride accumulation. In a recent study using mouse embryonic stem cells, Vernochet et al³¹ clearly distinguish between the role of PPAR in preadipocyte formation and preadipocyte differentiation, showing that lack of PPAR does not prevent commitment of stem cells into the adipose lineage, but leads to an arrest at the preadipose stage of the developmental programme. Actually, the activation of PPAR during the fibroblastic cell stage seems to be antimitotic.²⁶ Conversely, the activation of PPAR at the fibroblast stage is necessary and sufficient to trigger mitotic expansion of 3T3-L1 cells, which takes place before any PPAR expression and therefore its activation.³ In conclusion, these studies suggest that PPAR activity is predominantly involved in fat cell differentiation and not proliferation.

Is PPAR an essential or a major regulator of adipogenesis?

From the afore-mentioned studies of gene expression during adipogenesis, it is clear that the actions of PPAR activation are clearly and highly involved in the enhancement of adipogenesis. However, additional studies have tried to evaluate, whether PPAR is an essential regulator of adipogenesis, or whether it may be dispensable. Gain-of-function experiments with ectopic expression of PPAR in NIH-3T3 fibroblasts have shown that PPAR is sufficient to induce adipogenesis, and induces an adipocytic phenotype.³² Addition of PPAR and C/EBP genes together with a PPAR agonist to cultured myoblasts causes these cells to accumulate lipid and to express markers specific to adipocytes (ie trans-differentiation).³³ Definite proof of the major regulatory effect was thought to be provided with the generation of PPAR knockout mice. However, this has proved difficult because homozygous null mice do not survive past the mid-embryonic stage and the embryos succumb before they develop any adipose tissue.^34,35 However, the necessity of PPAR in adipogenesis was clearly established by Rosen et al.³⁶ In embryonic stem cells lacking PPAR, it was shown that, despite normal amounts of the transcriptional regulatory C/EBP and C/EBP proteins, no PPAR expression or C/EBP was found, and the cells were unable to undergo adipogenesis. Chimeric mice were generated by injecting PPAR^-/- cells into wild-type blastocysts at day 4. These blastocysts were then reimplanted into pseudopregnant wild-type mice. By isolating DNA from several tissues and performing appropriate procedures to distinguish between wild-type and -/- cells, it was found that in the heart, spleen, and small intestine, for example, an almost perfect 1 : 1 ratio of wild-type and -/- cells were found. In contrast, in isolated adipocytes, the ratio of wild-type to -/- cells was in the range of 3.5 or more. However, the authors could not fully rule out that a small number of fat cells could be formed in the absence of PPAR in vivo, though they considered it to be very unlikely.³⁶

These findings seem to be in line with the study by Vernochet et al,³¹ who showed that lack of PPAR does not prevent commitment of stem cells into the adipose lineage, but lead to an arrest at the preadipose stage of the developmental programme.

With the availability of PPAR antagonists another approach could be used to evaluate whether adipogenesis was essentially controlled by the PPAR receptor. The compound GW0072 was identified as a high-affinity PPAR ligand that was a weak partial agonist of PPAR transactivation. In cell culture, GW0072 was a potent antagonist of TZD-induced adipocyte differentiation.²² Another compound, bisphenol A diglycidylether (BADGE), was identified as a ligand for the PPAR receptor, and it was shown to antagonize the effects of agonists, and hence to block PPAR mediated-transcriptional and adipogenic activity. In a recent study, it was observed that addition of the PPAR antagonist PD068235 to 3T3-L1 preadipocytes significantly inhibited both PPAR-agonist-induced and hormone-induced adipogenesis in a dose-dependent manner. However, when added to terminally differentiated 3T3-L1 cells, PD068235 did not induce any morphological changes and had no effect on basal lipolysis rates, suggesting that PPAR activity may not be required for the maintenance of PPAR target gene expression in fully differentiated adipocytes, whereas the C/EBP transcription factors were assumed to be more important in the maintenance of the adipocyte phenotype.²⁴ Another novel PPAR antagonist LG100641 did not activate PPAR, but selectively and competitively blocked TZD-induced PPAR activation and adipocyte conversion in 3T3-L1 cells. However, it did not block adipocyte differentiation induced by a ligand for the retinoid X receptor (RXR) or by a standard differentiation 'hormone' cocktail, which suggests that it cannot be excluded that there are PPAR-independent pathways for adipocyte differentiation. Surprisingly, in addition LG100641 did also increase glucose uptake in 3T3-L1 adipocytes.²⁵ After mutation of highly conserved residues of the ligand-binding domain (AF2) in the PPAR receptor, normal ligand and DNA binding, but markedly reduced transactivation, were observed. When expressed in primary human preadipocytes, this PPAR mutant blocked TZD-induced differentiation, providing direct evidence that PPAR mediates adipogenesis.³⁷

In most studies, and for convenience, it has been anticipated that the known PPAR agonists (eg Troglitazone) are pure and selective agonists. However, it should be remembered that different PPAR ligands may have distinct downstream effects, where the exact response may be determined by the transcriptional cofactors available, differences in PPAR response elements, or the presence of different PPAR isoforms. In summary, even though there are some report showing that PPAR is not inherently essential for adipogenesis, it seems quite clear that PPAR-activated transcription is a strong regulator of adipogenesis. Also, the rank order of binding affinities of the PPAR agonists (RosiglitazonePioglitazoneTroglitazone) is consistent with their dose requirements for in vitro stimulation of glucose transport. This dose correlates with their antihyperglycaemic activity in ob/ob mice, which likely reflect their ability to enhance adipogenesis in vivo.³⁸

Regulation of PPAR protein activity

NHRs are transcription factors whose activity can be induced by ligand binding. Many of these receptors have ligands with long half-lives. Several mechanisms can be proposed to attenuate or terminate the actions of hormones through this class of receptors.

Werman et al³⁹ suggested that the relative expression of PPAR1/PPAR2 isoforms determines transcriptional activity. Also, growth or proliferation can decrease PPAR activity by PPAR phosphorylation via MAP kinase.^40,41,42 Insulin has been shown to enhance the transcriptional activity of the PPAR2.³⁹

Also, low cellular ATP/AMP levels may increase the phosphorylation of p300 (a transcriptional coactivator of PPAR) by AMP-activated protein kinase (AMP-kinase), and hence reduce its interaction with the PPAR receptor, leading to decreased PPAR activity.⁴³

Finally, excess PPAR stimulation seems to induce negative PPAR autoregulation.^30,44,45

In summary, PPAR is a major regulator of adipogenesis via stimulation by fatty acids, and under circumstances of plenty substrate access the PPAR activity is increased, whereas under circumstances of substrate depletion, for example, fasting, exercise or perhaps growth, the expression and/or activity of PPAR seems to be attenuated.

PPAR activation induces transcription of genes involved in lipid metabolism

If, as much evidence suggests, the PPAR should be the major controller of adipogenesis and white adipose tissue lipid accumulation and storage, one should expect that peroxisome proliferator-activated responsive elements (PPREs) would be found in many, or maybe all, of the promotor regions of the genes encoding proteins involved in white adipose tissue lipid storage.

Consequently, accumulation of triglycerides in white adipose tissue would require activation of genes encoding the accomplishment of (a) fatty acid release from lipoprotein-bound trilgycerides (eg LPL), (b) fatty acid transfer across the cell membrane (eg FATP), (c) intracellular fatty acid binding (eg FABP/Ap2), (d) intracellular acyl-CoA binding (eg ACBP), (e) glucose transport across the cell membrane (eg GLUT proteins), (f) glucose breakdown and synthesis of glycerol-3-phosphate (glycolytic enzymes and GPDH), (g) enzymes involved in the handling/modification of structurally different fatty acid isomers (eg SCD-1), and (h) genes involved in ATP or NADH generating pathways necessary for energizing the triglyceride synthesis in the fat cell.

In accordance with these assumptions, PPREs are found in many genes involved in lipid metabolism.¹⁹ However, although PPREs are found in many genes, they may not inherently be transcriptionally activated only by PPAR. The PPAR protein, and probably many of its transcriptional coactivators are mainly found in fat cells, but the PPAR protein, which is mainly and highly expressed in liver and muscle tissue, may be the major activator of PPRE-containing genes in these nonadipose tissues. Peroxisome proliferator-induced activation of PPAR in the liver leads to profound transcriptional activation of genes encoding the classical peroxisomal -oxidation system and cytochrome P450 CYP 4A isoforms, among others. There is strong evidence that very long fatty acids activate PPAR and mediates the upregulation of fatty acyl-CoA oxidase and other enzymes necessary for peroxisomal -oxidation, hence activating their own metabolism. Thus, any gene containing a PPRE may be regulated by different PPARs, depending on the tissue in question, the structural properties of the ligand, and the cofactor recruitment.

PPAR expression in obese and diabetic animals and humans

Studies of two murine models of obesity showed that adipose tissue levels of PPAR1 and 2 were not altered compared to lean mice, but were modestly increased in mice with toxigene-induced brown fat ablation uncoupling protein diphtheria toxin A mice.⁹

In humans, adipose expression of PPAR2 mRNA was increased in obese subjects, whereas no differences were found for PPAR1, and there was a strong positive correlation between the ratio of PPAR 2/1 and the body mass index (BMI).¹⁰

On the contrary, other studies found no correlation between subcutaneous or omental adipose tissue PPAR mRNA and BMI.^11,13,46 Also, in Rhesus monkeys there was no correlation between total PPAR adipose tissue mRNA expression and obesity, but the ratio of PPAR2/total PPAR was positively correlated with obesity and fasting insulin concentration.⁴⁷ In addition, in obese human subjects, the ratio of PPAR expression in omental/subcutaneous adipose tissue was increased compared to normal weight subjects, suggesting that the relative PPAR expression is increased in omental fat in obesity.⁴⁶ In the liver, the PPAR mRNA levels are substantially increased in ob/ob and db/db mouse models of obesity.^48,49 Also in skeletal muscle, the expression of PPAR mRNA was increased in both human obese nondiabetic and type II diabetic subjects in direct relation to BMI and fasting insulinaemia,⁵⁰ in relation to insulin resistance,¹⁴ or in relation to percent body fat.¹³ However, Rieusset et al⁵¹ did not find any correlation between BMI and muscle PPAR expression. In muscle samples from 14 male subjects there was up to three-fold variation in PPAR expression between subjects and the expression of LPL, muscle CPT1, and FABP all correlated significantly with PPAR expression. However, there was no correlation between the expression of PPAR and carnitine acyl carnitine transferase (CACT) or GLUT4.⁵²

The authors speculated that these findings were in favour of the hypothesis that PPAR regulates lipid metabolism genes also in skeletal muscle in a coordinated fashion, which would serve to increase the oxidation of fatty acids in muscle tissue.⁵² However, increased muscle PPAR levels would presumably be associated with increased levels of plasma lipids, flux of fatty acids into muscle tissues, and concomitant PPAR-activated fatty acid oxidation.

Nutritional regulation of PPAR expression

In different mice models, fasting for 12–48 h was associated with an 80% fall in PPAR2 and a 50% fall in PPAR1 mRNA levels in adipose tissue. Also, PPAR protein levels were markedly reduced after fasting. In contrary, high-fat feeding increased adipose tissue expression of PPAR (in normal mice) and induced PPAR2 mRNA expression in the liver of obese mice.⁹ In rat puppies, the level of PPAR2 increased in white adipose tissue during suckling (diet with high-fat milk), but rapidly reached a plateau after weaning. During the weaning period, however, the PPAR2 levels were not influenced by diet (high or low fat), but the increasing levels of PPAR2 expression during the suckling period correlated well with adipocyte hypertrophy.⁵³

In human obese subjects, after a 10% weight loss, subcutaneous adipose PPAR2 expression was significantly decreased, but stabilized after weight maintenance.¹⁰ Similarly, a 6% weight loss decreased PPAR2 expression by 30% in obese subjects.⁵⁴ After a 5 h infusion of intralipid + heparin (increases blood levels of fatty acids) in nine normal weight human subjects, the level of PPAR2 in subcutaneous fat increased two-fold, whereas no changes were observed after saline infusion.⁵⁵

Overall, the data available suggest that the levels of PPAR expression to a large extent correlates positively with the nutritional availability, whereas expression levels in white adipose tissue are not always correlated to obesity. In contrast, the level of skeletal muscle PPAR expression is to some extent correlated to variations in obesity. Whether this correlation to skeletal muscle levels reflects PPAR expression in real myocytes or more 'adipocyte-like' cells within the muscle tissue remains to be determined.

PPAR, involvement in physiological function—studies of PPAR agonist treatment

The mechanism by which PPAR agonist treatment relieves insulin resistance has been intensively discussed. Whereas most studies indicate that the PPAR is mainly expressed in adipose tissue, the fact that treatment with these compounds improves insulin sensitivity in skeletal muscle and liver (including the upregulation of insulin sensitive GLUT4 protein in muscle) has questioned whether PPAR activation in muscle could also contribute to the beneficial effects of PPAR treatment. One study reported that PPAR was expressed in myocytes within skeletal muscle, but preincubation of isolated soleus muscles from lean or ob/ob mice for 5 h with a TZD did not affect insulin-stimulated 2-deoxyglucose uptake. However, after prolonged TZD treatment with TZD, the in vivo insulin sensitivity was significantly improved.⁵⁶ Also, the possibility of increased muscle glucose utilization under TZD treatment, secondary to local adipose tissue differentiation (ie adipocyte differentiation within muscle tissue), was rejected.⁵⁷

A recent study investigated the changes in the expression of a large selection of genes in Zucker diabetic fatty rats, before and after 7 days of i.v. injection of the potent PPAR ligand GW1929.⁵⁸ Expression analyses were carried out for white adipose tissue, brown adipose tissue, muscle, and liver. It was found that: (1) PPAR regulates many more genes in white adipose tissue than in either liver or muscle; (2) in adipose tissue, GW1929 treatment resulted in a coordinate increase in the expression of a number of genes involved in fatty acid metabolism, for example, lipoprotein lipase (LPL), fatty acid transport protein (FATP), FABP, and stearyl-CoA desaturase. Also, the expression of the transcription factor ADD1/SREBP1c, which is highly involved in adipocyte differentiation, was increased. All these effects were consistent with a fat accumulating effect in adipocytes, stimulated by the PPAR agonist. However, also proteins involved in the oxidation of fat, for example, carnitine palmitoyl transferase-1 (CPT1) and uncoupling protein 3 (UCP3), gluconeogenesis (PEPCK) and glycogen storage (glycogen synthase) were upregulated in adipose tissue, which at first sight may not be expected from PPAR activation; (3) in skeletal muscle, the expression of the same adipogenic genes was consistently decreased. In addition, pyruvate dehydrogenase kinase 4 (PDK4) was significantly decreased. PDK4 phosphorylates and inactivates the pyruvate dehydrogenase complex that catalyses the first irreversible step in oxidative glucose metabolism. Hence, oxidative glucose metabolism was probably increased; (4) in the liver, the expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase (PC) and glucose-6-phosphatase (G6P) was decreased.

These data are consistent with a decreased reliance on fatty acids and an increase in glucose oxidation in muscle, a higher flux of fatty acids into adipose tissue and a decrease of gluconeogenesis in the liver, which is consistent with a lower flux of fatty acids to the liver. Based on the study, the authors proposed that insulin sensitivity was increased according to the illustrations in Figure 3. The data from that study does not determine whether PPAR agonists directly regulate gene expression in muscle and liver or whether the changes found could be secondary to lowered plasma FFA levels. However, in the study, it was observed that the decrease in FFA levels preceded the drop in glucose and triglyceride levels, strongly suggesting that decreases in FFA levels may be important for the insulin-sensitizing actions of the PPAR agonist. This time-dependent pattern was corroborated in a recent study showing that circulating FFA levels decreased concomitantly with insulin levels, but prior to the decrease in circulating triglycerides and also prior to indices of enhanced insulin sensitivity in the liver.⁵⁹ This hypothesis seems plausible, since it is well established that increased plasma FFA levels are highly responsible for insulin resistance.⁶⁰

Figure 3.

Physiological effects of 7 days of PPAR agonist treatment to Zucker diabetic fa/fa rats. PPAR activation either stimulates (+) or represses (-) key metabolic pathways in adipose, muscle, and liver tissue and in macrophages, and promotes the flux of FFAs from muscle and liver to adipose tissue. In addition to its metabolic effects, PPAR activation also increases macrophage-mediated cholesterol (CH) efflux via upregulation of ABC transporter protein-A1 (ABCA1). Adapted from Willson et al.²⁰ Reprinted with permission from the Annual Review of Biochemistry, Volume 70© 2001 by Annual Reviews www.AnnualReviews.org.

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The same conclusion was drawn from a study of 7 days treatment with another PPAR agonist (TZD300512) to rats. Decreases in triglycerides, FFA, glucose, and insulin were observed. Insulin sensitivity was enhanced and insulin-induced suppression of hepatic glucose production was increased during an euglycaemic hyperinsulinaemic clamp. Also, after TZD-treatment, an infusion with a lipid emulsion for 24 h clearly attenuated whole-body glucose uptake and increased hepatic glucose production,⁶¹ hence suggesting an increased insulin sensitivity because of decreased FFA levels. However, direct effects from PPAR stimulation in muscle and liver cannot be totally ruled out.

In addition to its lipogenic effects and the concomitant decrease in plasma FFA levels, the stimulation of PPAR in adipocytes may also decrease the expression of endocrine signalling molecules that can potentially influence glucose metabolism in peripheral tissues. These signalling molecules are, for example, leptin, tumour necrosis factor (TNF), adiponectin, adipsin, and plasminogen-activator inhibitor type 1 (PAI-1), collectively known as adipocytokines. Plasma levels of these peptides have all been associated with insulin sensitivity. The plasma levels of leptin, TNF and PAI-1 have been positively associated with insulin resistance, whereas adipsin and adiponectin have been negatively correlated with insulin resistance. However, for most of these novel adipocyte cytokines, it is not fully established whether the in vivo effects of these proteins are autocrine, paracrine, or systemic. The addition of TNF to fully differentiated 3T3-L1 adipocytes induces lipolysis in a dose-dependent manner and this effect can be reversed by addition of the PPAR agonist Rosiglitazone.²⁴ There is also evidence that TNF and leptin levels are decreased by PPAR agonists in vivo. Administration of Troglitazone to obese Zucker rats for 15 days decreased blood levels of TNF and Leptin, accompanied by normalization of hyperglycaemia, hyperinsulinaemia, and hypertriglyceridemia.³⁴ In addition there was a significant shift in the ratio of small vs large adipocytes in favour of small adipocytes (Figure 4), and a significant increase in apoptotic cells was observed during Troglitazone treatment (not shown). This redistribution of adipocyte size may also be responsible for the reduction in FFA levels because the ability to accumulate triglyceride is higher in small adipocytes than in larger and mature adipocytes. Also, smaller fat cells have been shown to secrete less TNF and leptin.

Figure 4.

Redistribution of adipocyte size after 15 days of Troglitazone treatment. Haematoxylin and eosin staining of retroperitoneal white adipose tissue. The size of adipocytes from obese Zucker rats was larger than from lean rats. Troglitazone caused a significant decrease in the size of adipocytes only in obese rats. Adapted from Okuno et al.⁷¹ Reproduced with permission from the Journal of Clinical Investigation, Copyright 1998 by Journal of Clinical Investigation.

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However, even though levels of TNF, leptin and other adipocyte endocrine factors may be associated with insulin resistance these findings may not necessarily be functionally related, and the expression of these adipocytokines may more likely reflect the nutritional and environmental state of the adipocytes.

PPAR agonist administration induces adipocyte proliferation and weight gain in vivo

Treating diabetic animals with PPAR agonists induces weight gain in most studies.^61,62,63,64 The extent of the weight gain depends on the dose given, but probably also on the pretreatment level of urinary glucosuria, plasma FFA, and the extent of hypertriglyceridemia (ie substrate availability for adipocyte differentiation). In accordance with this hypothesis, it is observed that TZD administration to lean animals is associated with substantially less weight gain.⁶¹ Also, the age (developmental stage) of the animal, the animal model, the length of the study, and the potency of the PPAR agonist used may determine the degree of weight gain.

In one study, 19 days of treatment with Pioglitazone to obese Zucker (fa/fa) rats substantially lowered the plasma levels of glucose, insulin, FFA, and triglycerides, but also increased the amount of adipose tissue throughout the body (though except subcutaneous fat!) compared to nontreated animals (Figure 5).⁶² In addition, the feed efficiency (weight gain/food consumption) was increased by TZD treatment, and concomitant increases in citrate synthase activity (Krebs' cycle) and fatty acid synthase activity (lipid synthesis) were observed in adipose tissue. However, it was not determined whether the weight gain induced a sedentary behaviour, which could explain the increased feed efficiency. Also, Rosiglitazone has been reported to induce hyperphagia and weight gain in rats,⁶⁵ and in both chow-fed (ie nonobese) and dietary obese rats, the size of the weight gain showed a linear correlation to the lowering in plasma FFA levels, but without changes in insulin or leptin levels.⁶⁶ Hence, TZD-induced improvement of insulin sensitivity seems to increase appetite and perhaps increase feed efficiency.

Figure 5.

Effects of 19 days of treatment with Pioglitazone on body weight gain (a) and daily food consumption (b). Obese animals administered Pioglitazone (), obese nontreated controls () and lean nontreated controls (). Feed efficiency (inset) was calculated as the total weight gain over the first 10-day treatment period divided by the total amount of food consumed during that period. P<0.05 vs control (cont) (*) and lean (#). Adapted from de Souza et al.⁶² Copyright © 2001 American Diabetes Association. From Diabetes, 2001; 50: 1863–1871. Reprinted with permission from The American Diabetes Association.

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The concept of adipose tissue functioning as a 'sink' for fatty acids to prevent an overload of lipid supply to other tissues has been suggested,⁶⁷ and this hypothesis may seem reasonable. Consequently, to prevent hyperlipidaemia, a continual capability of adipocyte differentiation is required. Accordingly, probably fat cells (or noncommitted fibroblast cells) may be necessary for the antidiabetic effects of PPAR agonists, and it is supported by studies of lipoatrophic A-ZIP/F-1 mice that suffer from a pronounced degree of diabetes. These mice have a severe lack of fat, marked insulin resistance, hyperglycaemia, hyperlipidaemia, and fatty liver. Treatment with Rosiglitazone or Troglitazone did not reduce glucose and insulin levels, though it did lower circulating triglycerides and increased whole body fatty acid oxidation in these mice. However, concomitantly the triglyceride content of the steatotic livers of these mice was increased.⁶³

Likewise, a study in lipoatrophic human diabetic patients treated with Troglitazone for 6 months increased the amount of subcutaneous adipose tissue and lowered FFA, triglycerides, and HbA1c. However, it was noticed that the oxidation of fat was increased, and that one subject experienced hepatotoxicity.⁶⁸ On the contrary, a study of aP2/DTA mice, whose white and brown fat is virtually eliminated by fat-specific expression of diphtheria toxin A chain, came to a somewhat different conclusion.⁶⁹ These hyperlipidaemic, hyperglycaemic, and insulin-resistant animals were treated with Troglitazone, and the serum level of glucose, insulin, FFA, and triglycerides were significantly lowered, indicative of improved insulin sensitivity. However, an increased level of PPAR expression in liver and a somewhat lowered glycogen synthesis in liver and muscle was also observed, suggesting some nonbeneficial effects on these tissues. In conclusion, the authors suggested that the Troglitazone treatment of these fat-tissue-depleted animals strongly indicated that Troglitazone enhances insulin sensitivity independent of adipose tissue. However, it may be relevant to consider whether Troglitazone also influences PPAR- activity in liver and muscle and/or accumulate triglycerides in nonadipose tissues.

Enhanced adipocyte proliferation has been reported in several animal studies.^62,64,70,71 All studies found a redistribution of the amounts of adipocytes in favour of more small adipocytes and fewer large adipocytes. Even though it is yet not known how this PPAR-induced adipose tissue redistribution occurs, and even though the proliferative pattern of adipocyte differentiation in two studies is not fully concordant (the new adipocytes were either homogeneously distributed,⁷¹ or formed as distinct patches of new fat cells⁶²), it seems evident that highly selective PPAR agonists do, on a morphological level, have strong white adipose tissue proliferative properties. However, when considering the observations from PPAR agonist treatment of 3T3-L1 cell cultures, where increased PPAR activity is associated with preadipocyte commitment and differentiation (and not proliferation), it may be reconsidered whether in vivo observations of adipocyte proliferation actually is a marker of enhanced preadipocyte recruitment.

The paradox of the insulin sensitive PPAR heterozygous mice

In a study of heterozygous PPAR (+/-) mice administered a high-fat diet, these mice exhibited resistance to adipocyte hypertrophy and insulin resistance, compared to WT mice.³⁴ When treating (+/-) mice with TZD, they became more insulin resistant.³⁴ However, in this study, leptin levels were increased in the (+/-) mice and the food intake and hence body weight and amount of fat tissue were significantly lower in (+/-) mice compared to the WT mice, which may have biased the study to some extent. However, the surprising phenotype of the heterozygous PPAR mice was subsequently corroborated.^64,72 In untreated (+/-) mice, serum levels of FFA are intermediate of WT- and TZD-treated mice, and thus there is an increased flux of FFA towards the liver and muscle, and the oxidation of FFA in muscle and liver is increased, compared to TZD-treated animals. The decreased weight gain in (+/-) mice may possibly induce a nonbeneficial accumulation of triglycerides in muscle and liver (also associated with the increased fat oxidation), compared to lean wild-type animals. However, in comparison to WT mice, these PPAR (+/-) mice have a higher insulin sensitivity.⁶⁴

To summarize, there seems to be a dose–response relation between PPAR activation and adipogenesis, that is, PPAR (+/-) mice have the smallest amounts of fat, WT mice have intermediate amounts and mice treated with PPAR agonists have increased adipose stores. Serum FFA levels are roughly correlated with the size distribution of fat cells (small cells are associated with low FFA levels), and the ability to lower glucose levels during a glucose tolerance test is roughly associated with FFA levels (high insulin sensitivity associated with low FFA levels).

These surprising characteristics may suggest a potential beneficial effect of PPAR antagonist treatment of human diabetics, since lower PPAR agonist activity seems to be associated with resistance to weight gain and enhancement of insulin sensitivity. However, it must be emphasized that these studies are carried out in (+/-) animals during growth, and caution must be taken when applying these results to obese human weight stable type II diabetics.

Human PPAR mutations—implications for T2D and obesity

Owing to its importance for fat tissue formation, it is anticipated that the PPAR gene is a susceptibility gene for the development of obesity. Also, with the findings that this receptor is the drug target of the TZD antidiabetic drugs, it could have the potential to affect the development of type II diabetes. So far, in humans four different single nucleotide polymorphisms have been reported:

Pro12Ala (5% in Asians, 13% in Caucasians), Pro115Gln (Rare), Val290Leu (Rare, centre of ligand-binding domain), and Pro467Leu (Rare, conserved part of the C-terminal ligand-binding domain (AF-2). The common Pro12Ala substitution has been studied by several groups. In vitro studies have reported that the Ala variant has a somewhat lower DNA-binding capacity and a lower transactivating capacity when transfected into several cell lines compared to the Pro variant.⁷³ Also, the ability of the Ala variant to mediate transcription and to induce adipogenesis is slightly reduced compared to the Pro variant.⁷⁴ So far, several studies have reported that the Ala variant is associated with a lower frequency of insulin resistance,^75,73,76 and although several other studies have failed to find any association with insulin sensitivity, most studies show a tendency for a protective effect of the Ala variant. Meta-analyses indicate that the Ala variant do, though not profoundly, protect against insulin resistance.⁷⁷

However, as it is generally accepted that T2D is strongly facilitated by obesity, it would be expected that the less active PPAR2 Ala12 variant would protect against obesity. One study found that this substitution was not associated with the development of juvenile obesity, and that in an obese subgroup Ala12Ala carriers had a higher tendency to gain weight, whereas in a lean subgroup Ala12Ala carriers were associated with resistance to weight gain.⁷⁸ In two independent Caucasian populations the opposite association was found, indicating that the Pro variant was somewhat protective against obesity.⁷⁹

The mutations Val290Leu and Pro467Leu were identified in three nonobese persons from a population of 85 unrelated subjects with severe insulin resistance and dyslipidaemia.⁸⁰ All subjects were heterozygous carriers, but these mutations that are positioned in the ligand-binding domain of PPAR did, in a dominant negative manner, inhibit PPAR coactivator recruitment and transcriptional activity, both in the ligand-bounded and nonligand-bounded state.

Another study reported that the Pro115Gln substitution was associated with obesity. This mutation is located immediately adjacent to the putative serine phosphorylation site at serine residue 115. Hence, this mutation was suggested to impair the MAPK-activated downregulation of PPAR transcriptional activity. In support of this, overexpression of the mutant gene Gln115 in murine fibroblasts led to a defective phosphorylation of serine 114, as well as to an accelerated differentiation of the cells into adipocytes and greater cellular accumulation of triglyceride than with the Pro115 variant.⁸¹

Medical use of PPAR agonists—beneficial effects and potential adverse events

The TZDs are a relatively new class of oral antidiabetic drugs, and they are often referred to as 'insulin sensitizers'. The first of this type of compounds was Ciglitazone, which was synthesized in 1982, and subsequently Pioglitazone, Englitazone, Troglitazone, Rosiglitazone, and Darglitazone were synthesized. Only Troglitazone, Pioglitazone, and Rosiglitazone were evaluated in clinical studies, and Troglitazone ('Rezulin' from Warner-Lambert) was approved for clinical use by the US Federal Drug Administration (FDA) in 1997, but were subsequently withdrawn from the market in March 2000 because of idiosyncratic liver toxicity. Rosiglitazone ('Avandia' from GlaxoSmithKline) and Pioglitazone ('Actos' from Eli Lilly) were approved by FDA in 1999, and at the moment approximately three million type II diabetics are being treated by these two drugs in the US.⁸²

These three PPAR agonists display distinct characteristics since, although they all contain the same active TZD ring, they have different side chains. As a consequence, these PPAR agonists differ in their pharmacological potency; the PPAR-binding affinity of Rosiglitazone is 100-fold greater than that of Troglitazone and over 30 times that of Pioglitazone.³⁸ The rank order of binding affinities of the PPAR agonists (RosiglitazonePioglitazoneTroglitazone) is consistent with their dose requirements for in vitro stimulation of glucose transport and their antihyperglycaemic activity in ob/ob mice.³⁸ Their respective effectivity likely reflect their ability to induce adipocyte differentiation, and hence to increase FFA uptake in WAT.

Despite their different degrees of potency, all the PPAR agonists are shown to be effective in relieving insulin resistance. The beneficial metabolic effects of PPAR agonist treatment of human type II diabetics include⁸³: (1) reduction in postprandial glucose, fasting plasma glucose and HbA1c, (2) increased insulin sensitivity and improved pancreatic island -cell function; (3) increase in HDL levels and (variable) lowering of LDL levels; (4) lowering of diastolic blood pressure, decreased microalbuminuria, and increased levels of the fibrinolytic substances plasminogen activator inhibitor 1 (PAI-1) and tissue plasminogen activator (tPA).

Many of these beneficial effects are presumably interconnected. Lowering of plasma FFA levels likely decreases the intracellular triglyceride accumulation within muscle cells, cardiac cells, and pancreatic cells, hence relieving this 'lipotoxicity',⁸⁴ and thus improves the glucose metabolism in liver, muscle, and adipose tissue, contributing to the overall improved glycaemic control. In addition, by enhancing insulin sensitivity and thereby reducing the burden on the pancreas, the PPAR agonists may also have the potential to improve -cell function and thereby delay disease progression. The powerful combination of improvement in -cell function and reduction in insulin resistance is key to the success of controlling type II diabetes. However, many patients do not achieve a large enough response to eliminate the use of insulin or other insulinotropic drugs, and many patients are nonresponsive to this type of drugs.

Despite all the beneficial effects of the use of PPAR agonists, there are some major considerations to take into account when considering the use of these compounds. So far, apart from the withdrawal of Troglitazone because of cases of hepatotoxicity, which is not considered to be a class effect, the PPAR agonists in use seem to show good tolerability, the major adverse events being weight gain, oedema, upper respiratory tract infection and headache.

However, diabetic KKA^y mice have been shown to exhibit increased hepatic PPAR1 gene expression after Rosiglitazone treatment. They developed severe microvesicular periacinar steatosis upon chronic treatment with TZDs, whereas lean control mice did not develop any pathological liver changes. However, these changes were not associated with alterations in hepatic triglyceride levels.⁸⁵ Hence, it may be suggested that PPAR agonists may also, depending on their specificity against, for example, the PPAR receptor (or interaction with other liver enzymes), increase liver fat oxidation and/or triglyceride content. In favour of this, it was reported that the TZDs Troglitazone and Rosiglitazone exhibited markedly different effects on cofactor recruitment, PPAR transcriptional activity and changes in expression of different genes.⁸⁶ Troglitazone and Rosiglitazone regulated distinct but overlapping sets of genes in several cell lines, indicating that TZDs may differentially activate PPAR (and perhaps PPAR) in a manner dependent on the cellular environment. However, in the same study, it was observed that the expression of the adipocyte-specific protein Ap2 was increased to a similar degree by both drugs.⁸⁶

Hence, it can be hypothesized that PPAR effects on adipose tissue from different TZDs may be similar, whereas in other cells/tissues the effects may be dissimilar.

If strong PPAR stimulation is necessary and sufficient to induce adipocyte differentiation in vivo, as seems to be the case in in vitro and in vivo studies, then it cannot be rejected that PPAR stimulation may promote the induction of 'adipocyte-like' cells in tissues other than adipose tissue. The significance of such, presumably small changes, is at present unknown.

To summarize, there is substantial evidence that the mechanism of action of the TZDs is based on their capability to enhance the flux of plasma FFA and triglycerides into adipose tissue. In vitro TZDs enhance adipocyte differentiation, and in vivo there is a time-dependent association between drug administration, decreasing levels of plasma FFA and increasing insulin sensitivity.

PPAR stimulation increases fat deposition and body weight gain also in humans

Apart from the hepatotoxic effects previously observed with Troglitazone, the major side-effect of TZD use is a substantial weight gain. In clinical trials Rosiglitazone caused a dose-dependent increase in body weight ranging from 0.7 to 3.5 kg, whereas placebo recipients had a mean weight loss of 1 kg.⁸⁷ Likewise, clinically effective doses of Pioglitazone dose dependently increased body weight.⁸⁸ Also, on a long-term basis the TZDs seem to promote weight gain (Figure 6). Long-term treatment with Pioglitazone monotherapy is accompanied by substantial weight gain,⁸⁹ and this weight gain may likely be even higher when provided in combination with sulphonylureas or insulin, and it may be speculated whether this weight gain will increase with continuous treatment.

Figure 6.

Weight changes during long-term treatment with Pioglitazone as monotherapy. Adapted from Belcher and Matthews⁸⁹ Copyright © 2000 Thieme Medical Publishers. Reproduced with permission from Thieme Medical Publishers.

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The cause of the weight gain in humans is unclear, but is presumably because of increased fat deposition, the inherent mechanism of PPAR activation. However, the weight gain observed in human diabetics may be substantially less than that observed in many animal obesity models treated with PPAR agonists. Animal models are often given access to ad libitum diet, whereas dietary prescriptions including energy restriction are often advised to human diabetics as a complement to the medical treatment.

Weight gain can be ascribed to either increased feeding efficiency or to increased hunger. Increased adipogenic efficiency because of PPAR stimulation, lowered hepatic gluconeogenesis and decreased protein synthesis because of lowering of plasma insulin levels, and possibly a slight decrease in urinary glucose excretion in some individuals are all likely to contribute to an increased feeding efficiency.

PPAR agonist treatment has been reported to increase hunger.⁹⁰ This may be explained by lowered blood levels of nutritional substrates such as FFA and glucose, lowered insulin levels, or perhaps lowering of leptin levels as reported by Shimizu et al,⁹⁰ or by other hypothalamic changes secondary to an improved glucose homeostasis. In accordance with this, a relatively reduced insulin secretion (ie higher insulin sensitivity) have been associated with subsequent increased body weight gain.⁹¹

The excess weight gain in humans has consistently been ascribed to an increase in the amount of subcutaneous fat, whereas the amount of visceral fat is unchanged or decreased.^{92,93,94,95,96,97} This fat redistribution may partly explain the insulin-sensitizing effect of PPAR agonist treatment, considering the well-established association between visceral adiposity and insulin resistance.⁹⁸ The cause of this apparent fat redistributing effect of TZD treatment remains unknown, but it is doubtful whether this may be caused by differential expression of the amount of PPAR in subcutaneous vs visceral fat. Earlier results showed no difference in expression levels between these fat tissues,^99,100 but recent results suggest that both PPAR1 and PPAR2 are more highly expressed in subcutaneous than omental preadipocytes.¹⁰¹ Also, the differentiating capacity of subcutaneous adipocyte precursor cells is higher than that of intra-abdominal fat precursor cells.^100,102 Despite the major concomitant weight gain, fasting plasma FFA levels and FFA levels during OGTT are substantially decreased during TZD treatment and hence increase the whole-body insulin sensitivity.¹⁰³ Whatever the exact mechanistic cause of the PPAR-induced weight gain, and despite the apparent beneficial redistribution of fat mass, it may seem counterintuitive to treat insulin resistance with a compound that induces weight gain, knowing that the degree of obesity is highly associated with insulin resistance. Thus, it is speculated that the negative effects of the weight gain may compromise the positive effects of the treatment. From current and present studies, it seems as if the beneficial effects of treatment with TZDs require a minimum of 8–12 weeks to lower fasting plasma glucose levels, and another few weeks to lower fasting plasma HbA1c levels. The size of the weight gain is generally correlated to the decrease in HbA1c. As in the animal studies, higher baseline BMI and the extent of hypertriglyceridemia predict a better improvement in HbA1c during Troglitazone treatment.¹⁰⁴ Also, the amount of subcutaneous fat and a gender effect (female better than male) have been shown to correlate positively with the TZD-induced improvement in HbA1c.^96,105 Hence, higher baseline BMI is likely to predict a higher weight gain during treatment. In agreement with this, it has also been shown that lean normoglycaemic subjects have no benefit of Troglitazone treatment for 12 weeks, and actually tend (ie nonsignificant) to have a nonbeneficial effect on plasma FFA and TG levels.¹⁰⁶

The weight gain during PPAR treatment is highest during the first few weeks of treatment and tend to stabilize thereafter. After the first 3–6 months, it seems as if glucose levels does not decrease any further, and it is likely that the adipogenic efficiency of PPAR treatment has reached its maximum, that is, that a steady-state level is accomplished, where TZD treatment cannot lower plasma FFA and TG levels any further. Beyond this time point, one might fear that the long-term efficacy of PPAR stimulation may be insufficient to sustain lowered FFA levels, and that FFA levels may rise again. Another potential concern is the effect on adipocyte precursor recruitment. From animal studies, it seems as if PPAR treatment increases the recruitment of adipocyte precursors, followed by a concomitant apoptosis of mature adipocytes. It is not known whether these observations are relevant for TZD treatment of humans, and hence it is not known whether the beneficial effect of PPAR treatment depends on these mechanisms, and whether this adipocyte recruitment effect can be sustained in the long term. A recent report with a novel PPAR compound with insulin-sensitizing activity with lower stimulation of adipogenesis, probably because of a new interaction mode with a 2 : 1 agonist : receptor binding relation,¹⁰⁷ and reports of non-TZD compounds showing hypoglycaemic effects without effects on body fat weight¹⁰⁸ remain to be clinically corroborated.

Conclusion and perspectives

The PPAR nuclear hormone receptor is primarily expressed in adipose tissue where it is a major player in the regulation of adipocyte differentiation (in vitro). The natural agonists of this receptor are a variety of different fatty acids that are either absorbed from the diet or partly metabolized compounds originating from the diet, presumably including fatty acids originating from de novo lipogenesis. However, these agonists are apparently all rather low-ffinity ligands for the PPAR receptor. This 'set-up' allows for a very appropriate regulation of fat tissue accumulation. Irrespective of the type of diet consumed and the exact structure of the fatty acids consumed, any positive energy balance will eventually end up in adipose tissue, at least in part, via stimulation of the PPAR receptor.

During chronic overfeeding and adipocyte hypertrophy, the activity of the PPAR receptor is presumably attenuated, and hence the adipocytes have a lowered fat-depositioning capacity, giving rise to increased plasma levels of FFAs, which subsequently may lead to insulin resistance in adipose tissue, liver, and muscle. The TZDs are a group of PPAR receptor agonists which in adipose tissue strongly stimulate PPAR-mediated transcription of the genes encoding proteins involved in adipose tissue lipid metabolism, and these drugs are probably able to recruit and convert noncommitted preadipocytes into phenotypical adipocytes. Thus, selective PPAR agonists are novel drugs used to treat hyperglycaemia and insulin resistance. However, in addition to their beneficial effects on glucose homeostasis, they 'inherently' increase fat cell differentiation and body fat accumulation. Since obesity is highly associated with the progression of insulin resistance, any weight gain must be considered unfavourable in the treatment of type II diabetes if the increased body weight appears to compromise the positive effects of the treatment. The weight gain accompanying the use of PPAR agonists occurs usually early in the treatment, and tends to stabilize. However, the PPAR agonists have only been approved for clinical use since 1997 and the tolerability and long-term effects have not been extensively investigated. Hence, we strongly advise to monitor not only possible hepatic complications, but also the extent of the concomitant weight gain and the glucoregulatory ability in diabetic patients when PPAR agonists are used on a chronic basis.

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Acknowledgements

We acknowledge the help from Gian Francesco Alberto M.D., Department of Internal Medicine, University of Turin.