Fibroblast Growth Factor 21 and Browning of White Adipose Tissue

1Fibroblast growth factor 21 (FGF21) has important effects on energy balance, glucose metabolism, and lipid metabolism. Initial reports identified the liver as its main source. On secretion, its most important target is white adipose tissue (WAT), where FGF21 increases expression of GLUT1 and consequently glucose uptake. During fasting or starvation, lipolysis is triggered, with a subsequent increment in circulating free fatty acids (FFAs). FFAs induce the activation of the peroxisome proliferator-activated receptor (PPAR)-alfa in the liver, resulting in the synthesis and release of FGF21. Since carbohydrate ingestion is absent during starvation, FGF21 induces ketone body formation in the liver as an additional energy source. FGF21 may also be considered an adipokine, since it is also synthesized and released from WAT. It shows complementary actions to adiponectin, increasing insulin sensitivity, improving the lipid profile, reducing glucose levels without causing hypoglycemia, and ensuring energy availability during starvation. In human insulin resistance states, FGF21 levels have a positive correlation with the number of metabolic syndrome traits, the severity of oxidative stress, and the presence of type 2 diabetes. Other human stress-related conditions that increase circulating FGF21 levels are lactation, exercise, growth hormone treatment, and anorexia nervosa. Interestingly, FGF21 is now considered an important mediator for decreasing oxidative stress, and possibly, preventing microvascular diseases such as diabetic nephropathy.

2The role of FGF21 as a therapeutic option in human metabolic diseases is of increasing importance. Currently, there are multiple recombinant FGF21 analogs in phase 2 and phase 3 clinical trials. Interest has increased even more so after the discovery of the crucial role of FGF21 in inducing the proliferation of brown adipose tissue (BAT). This review describes the mechanisms by which FGF21 induces “browning” of adipose tissue and how it may have a role in the treatment of human metabolic diseases, including obesity and type 2 diabetes.

3WAT and BAT are the two main subtypes of adipose tissue in humans. WAT has important endocrine functions in addition to its role as a reservoir of energy in the form of triglycerides. The hormones released from WAT, namely adipokines, are a well-recognized group of bioactive factors with endocrine actions that act through specific cell-membrane receptors. Adipokines trigger certain intracellular signaling pathways, which modulate human metabolism. The most important are leptin and adiponectin, but visfatin, chemerin, omentin, hepcidin, apelin, and vaspin have also been described.

4BAT is also an important endocrine organ. It consists of a group of specialized cells with abundant expression of uncoupling protein 1 (UCP1) in the inner mitochondrial membrane. The BAT hormones are named “batokines”. The principle function of BAT is to dissipate stored energy in the form of heat by uncoupling energy oxidation from ATP synthesis. Initially, BAT was only considered as an energy-producing organ in rodents and human infants. However, after the development of 18F-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET-CT), BAT has also been identified in human adults. Nevertheless, the origin of BAT is still under debate, although originally it was thought to be derived from skeletal muscle-like lineage (Myf5+). In human adults, a type of adipose tissue showing characteristics between that of white and brown adipocytes has been identified; this kind of adipose tissue is known as beige adipose tissue (brite, brown-in-white). It appears that beige and white adipocytes arise from both Myf5+ and Myf5- progenitor cells. These findings confirm that the skeletal muscle-like lineage is not the only source of BAT.

5The location of the different types of adipose tissue varies. Beige and WAT have mainly visceral (mesenteric, perigonadal or omental adipose tissue surrounding organs) and subcutaneous (under skin) locations. BAT is located only in axillary, subscapular, interscapular, and periaortic regions in rodents; in cervical, supraclavicular, paravertebral, mediastinal regions in humans; and in perirenal regions in both. Brite or beige adipocytes have basal metabolic actions similar to those seen in white adipocytes, and with the enough stimulus, they are able to transform into thermogenic adipocytes with higher UCP1 expression similar to BAT. This process is referred as “browning” and it describes the capacity of white adipocytes to acquire a phenotype similar to that of BAT, leading to increased thermogenesis. It is achieved when white adipocytes are exposed to cold or to beta 3-adrenoreceptor agonists. Browning occurs mainly in subcutaneous white adipose fat depots. The underlying molecular mechanisms for this trans-differentiation are currently under intensive research. In addition, there are important structural differences among WAT, brite, and BAT. WAT is a large lipid droplet, with a peripheral nucleus and a small amount of cytoplasm, whereas BAT has a central nucleus with more cytoplasm but smaller lipid droplets. In between these is the brite or beige tissue; this has the mixed structural characteristics of both. Sometimes the different structures are found together; for example, the ectopic expression of UCP1 and the presence of the PR domain containing 16 (PRDM16) suggests that brite adipocytes are mixed with white adipocyte depots. The balance between WAT and BAT, and their endocrine regulation, are key elements to better understand the development of weight gain and human metabolic diseases.

6Since the discovery of FGF21, it has been appreciated that its synthesis is strongly related to cold exposure. In mice, during hypothermia, FGF21 induces torpor, a short-term hibernation state in which animals can save energy by reducing body temperature and physical activity. More recently, studies have shown a higher expression of FGF21 in inguinal WAT after cold exposure. The role of FGF21 produced in WAT includes both paracrine and autocrine actions; this results in the local upregulation of peroxisome proliferator-activated receptor gamma co-activator (PGC)-1-alfa and thus an increase in thermogenesis. PGC1-alfa is a protein involved in modulating several effects in post-exercise skeletal muscle, including the improvement of energy and glucose metabolism. Interestingly, PGC1-alfa is also induced after irisin or insulin exposure, both hormones showing a clear interaction with FGF21 post-exercise. Irisin-induced phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-related kinase (ERK) show a positive correlation with shivering intensity. FGF21 also shows a direct relationship with exercise intensity. The consequence of these PGC1-alfa inducers is to promote adaptive thermogenesis with “browning” of WAT. The main mechanism following FGF21 action is PPAR-gamma activation in WAT, together with the irisin effect inducing MAPK and ERK pathways. This results in differentiation of pre-adipocytes to mature white adipocytes, which are then available for “browning”.

7Some animal models have reported findings consistent with these actions. For example, FGF21 deficiency in mice results in increased body weight with excessive adiposity, higher serum cholesterol, insulin resistance, and hyperglycemia. The finding of a 30–40% lower nuclear content of PGC1-alfa at the hepatic mitochondrial level in Fgf21 KO mice compared with WT mice, is a potential explanation for these results. FGF21 induces palmitate oxidation and β-hydroxyacyl-CoA dehydrogenase (β-HAD) activity. In the Fgf21 KO model, these enzymatic activities are decreased, indicating lower lipid oxidation, a reduction in glucose metabolism, and a lower degree of energy waste. In contrast, overexpression of FGF21 effectively decreases weight, adiposity, levels of FFAs, triglycerides, glucose, and insulin, all due to the normalization of mitochondrial oxidation and the improvement in insulin sensitivity. Interestingly, exercise, irisin, and noradrenaline were necessary to restore PGC1-alfa content in the liver despite overexpression of FGF21, emphasizing the key interaction of such inducers with FGF21. Insulin, the most important regulator of energy and glucose metabolism, enhanced differentiation of WAT to brite and brown adipocytes through pro-opiomelanocortin (POMC) neurons (Table 1 and Figure 1);. Therefore, multiple mechanisms may be inter-connected to improve metabolism in humans, with FGF21 functioning as an important link between them.

8The exogenous administration of an FGF21 analog in animal models has been associated with a thermogenic effect, an improvement in glucose homeostasis, lipid profile, and a reduction in body weight. However, in humans, recombinant FGF21 analogs have not shown these effects. FGF21 is paradoxically increased in insulin-resistant states such as obesity or type 2 diabetes; this suggests either a resistance to FGF21 effects or a compensatory response to these metabolic disarrangements. Nevertheless, the key metabolic role of FGF21 is clear. Firstly, WAT increases FGF21 expression in response to feeding, which has an autocrine action on PPAR-gamma activity; PPAR-gamma is an important inducer of insulin sensitivity pathways, adipocyte maturation, and function. Secondly, FGF21 is significantly induced after moderate to intensive physical activity, and interestingly, its release has been correlated with sympathetic nervous system activation and lipolysis markers, such as noradrenaline and serum free fatty acids, respectively. Thirdly, FGF21 regulates metabolism through increasing insulin sensitivity at WAT and BAT, but this may happen in an acute and a chronic manner. Acute and chronic exogenous administration of FGF21 induce insulin sensitivity independently of adiponectin action. However, the chronic effect of FGF21 is not necessarily via a direct effect on WAT since it also induces signaling in the central nervous system via POMC neurons, and on brown adipocytes, enhancing thermogenesis and insulin-sensitivity in mice (Figure 1). These effects require functional insulin signaling in adipose tissue, otherwise the FGF21-benefit is lost. In addition, the action of FGF21 on the central nervous system is important to induce energy expenditure, mainly by increasing noradrenaline release (Figure 1). Noradrenaline plays an important role in the regulation of “browning.” It is the most studied activator of thermogenesis, increasing UCP1 transcription, enhancing lipolysis and mitochondrial oxidation (Figure 1;. After cold-induced shivering or physical activity stress, noradrenergic pathways increase thermogenic gene expression through c-AMP-mediated mechanisms. Lipolysis and FFAs increase following noradrenaline release. With acute intensive exercise, the increase in beta 3-receptor activity also causes an increment in circulating FGF21. Increased circulating FFAs induce PPAR-alfa expression in the liver, increasing FGF21 synthesis and release into circulation. FGF21 can also increase insulin sensitivity by promoting the expansion of subcutaneous WAT. Fgf21 knockout mice show less subcutaneous WAT and a greater degree of insulin resistance. After treatment with recombinant FGF21, subcutaneous adipose tissue was restored with a subsequent improvement in insulin sensitivity. The expression of co-factor beta klotho is necessary to accomplish the FGF21-related expansion of subcutaneous fat. Finally, chronic pharmacologic administration of FGF21 in obese mice has been shown to suppress growth hormone (GH) and the insulin growth factor-1 (IGF1) signaling axis in the liver, increasing lifespan through an improvement in insulin sensitization, normalization of glycemia, and a reduction in body weight.

9Under normal conditions, FGF21 is synthetized and released from the liver. However, in certain circumstances, such as adaptive thermogenesis induced by cold exposure or exercise, BAT expresses and releasees FGF21. The production of FGF21 by BAT is not negligible; it significantly contributes to systemic FGF21 levels. Following exposure to cold, the production of FGF21 by BAT is greater than that of the liver, enhancing thermogenesis, confirming the key roles of BAT in regulating FGF21 levels. Moreover, a dramatic rise in Fgf21 expression in BAT has also been reported in Ucp1-null mice or after genetic inactivation of UCP1 protein. In these situations, there is an increase in serum FGF21 levels without changes in FGF21 gene expression in the liver. This suggests thermogenic regulation of FGF21 through both UCP1-dependent as well as UCP1-independent mechanisms.

10Taken together, adipose tissue, liver, and skeletal muscle respond to multiple stimuli in order to increase adaptive thermogenesis and induce the browning of WAT (Figure 1). Expression and release of FGF21 by the liver, BAT, and skeletal muscle is induced by shivering, physical activity, protein synthesis after growth hormone treatment, and as a consequence of experimental or clinical mitochondrial dysfunction following DNA mutations. FGF21, together with irisin, insulin, and noradrenaline, provokes metabolically healthy effects that are concomitantly associated with the browning of WAT. The higher BAT activity and increased heat production may benefit human health, reducing weight, preventing hyperglycemia, and hyperlipidemia, and protecting against obesity through enhancement of energy waste. These effects explain the association of FGF21 and explain its key role in the “browning” of WAT. This was probably aimed to allow the adaptation of human metabolism to obesity, diabetes, dyslipidemia, metabolic syndrome, and other insulin resistance states (Figure 1).

11The beneficial metabolic consequences of “browning” may be useful to treat metabolic diseases in humans. There are multiple medical drugs or nutritional inducers that may help to stimulate browning (Table 1). Recently, the role of FGF21 in the browning of WAT has been evaluated with the development of recombinant FGF21-analogs. Initially, clinical trials with analogs LY2405319 and PF05231023 were focused on treating human metabolic diseases including obesity, metabolic syndrome and type 2 diabetes. However, resistance to their actions, resulting in an inadequate clinical effect, has been a problem; in humans, only a slight glucose, weight or triglyceride reduction was achieved. The subcutaneous depots of WAT are small cells with greater potential to differentiate. Therefore, it is feasible to hypothesize a positive effect if “browning” can be induced, following a sufficient stimulus using FGF21-analogs. In addition, the FGF receptor type 1 together with beta klotho cofactor, called the FGFR1/KLB complex, is the functional target for FGF21. The use of FGF21 agonist antibodies that specifically activate this complex largely mimic the action of recombinant FGF21 in mice. These include the agonist antibodies BFKB8488 and NGM313, which are currently under clinical research (ClinicalTrials.gov, NCT02593331, NCT02708576, and NCT03060538). Both FGF21 recombinant analogs and the FGFR1/KLB complex agonist antibodies induce higher thermogenesis and browning of BAT through UCP1-dependent pathways. Although UCP1 has traditionally been thought of as indispensable for browning and thermogenesis, recent research suggests a role of a UCP1-independent pathway. In Ucp1 KO mice, higher thermogenesis with weight loss and beneficial changes in cardiometabolic markers have been reported. The origin of this UCP1-independent thermogenesis is still controversial, with different reports suggesting the opposite. Further investigation is warranted to clarify if higher thermogenesis can be obtained without overexpression of UCP1.

12In addition to FGF21-recombinant analogs or the FGFR1-KLB complex agonist antibodies, other drugs have been used with similar aims; however, most have shown little clinical utility. For example, PPAR-alfa agonists (fibrates), adrenergic beta-3-receptor stimulators, thyroid hormones, and more recently irisin have been tested (Table 1). There are also certain nutritional inducers of “browning” of WAT that may be considered as therapeutic options. The most important hormones, drugs and nutritional inducers of browning are summarized on Table 1. It is important to mention that although nicotine has been associated with body weight reduction, mainly due to the associated decreased appetite, greater lipolysis, and increased energy waste, it has never been confirmed that smoking cigarettes can induce browning.

13FGF21 is a key regulator of the differentiation of WAT to brown adipocytes, resulting in enhanced thermogenesis and energy waste. The main action seems to be through UCP1-dependent and -independent mechanisms. In addition, after cold exposure or exercising, FGF21-induced upregulation of local peroxisome proliferator-activated receptor gamma co-activator (PGC)-1-alfa increases thermogenesis in adipose tissue and skeletal muscle. Potential mechanisms involve higher noradrenaline levels that act on the WAT-beta-3 adrenergic receptor, inducing lipolysis, and higher serum free fatty acids, which in turn increase PPAR-alfa agonism at liver, and higher FGF21 synthesis and then release into circulation. This effect contributes to other FGF21-related mechanisms that integrate metabolic pathways to regulate human energy balance, glucose and lipid levels. Therefore, the development of better recombinant FGF21 analogs as a potential treatment for metabolic diseases in humans is necessary.

14All authors made substantial contributions to the conception and/or design of the work; acquisition, analysis, and interpretation of data for the work; drafting the work or revising it critically for important intellectual content; and approved the final version of the article to be published.

15The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.