Several secreted polypeptides, including insulin, glucagon-like peptide–1 (GLP-1), adiponectin, and others are ultimately involved in the regulation of glucose homeostasis, which thus makes them clinically relevant pharmacological agents or attractive candidates for novel medicines for the treatment of diabetes mellitus (27
). Recent reports on bone morphogenic protein–9 (BMP-9) (28
) and FGF-19 (12
), and our findings with FGF-21, have shown that the other proteins also constitute this list of promising biomolecules.
FGF-21 bioactivity was discovered through a cell-based functional screen aimed at identifying novel secreted molecules that affect glucose uptake on mouse 3T3-L1 adipocytes and was found to be very potent in this assay (EC50, ~0.5 nM) (Figure A). With comparable potency, FGF-21 was active on differentiated human primary adipocytes (Figure B), which indicates that FGF-21 bioactivity is not limited to murine adipocytes.
The follow-up analysis on the initial observation in the glucose uptake assay revealed what we believe to be a novel and unique mechanism of the FGF-21 mode of action. FGF-21 effects appeared to be insulin independent and additive to insulin activity upon coadministration (Figure C). FGF-21 needed to be present on cells for several hours to produce a robust response in glucose uptake, and the effect was significantly diminished by the protein synthesis inhibitor cycloheximide (Figure D). While insulin is known to work in a rapid, hormone-like manner, we hypothesized that FGF-21 activity is likely to be mediated through changes in gene expression. Indeed, we showed that FGF-21 induced a significant upregulation of the insulin-independent glucose transporter GLUT1 in 3T3-L1 adipocytes (Figure , E and F) and further confirmed this finding in vivo when we administered FGF-21 to ob/ob
mice (Figure G). Thus, in contrast to insulin, which is known to function via GLUT4 translocation (29
), FGF-21 may primarily act through upregulation of cellular GLUT1 in these cells.
In addition to the changes in GLUT1, we observed a modest reduction in GLUT4 levels after 48 and 72 hours of cell stimulation with FGF-21 (Figure F). While FGF-21 stimulation caused a decrease in GLUT4 levels in vitro, it did not appear to negatively impact glucose uptake (Figure C), which suggests either that the decrease is not sufficient to affect glucose transport or that other compensatory mechanisms are at work in 3T3-L1 adipocytes. Moreover, we observed improved insulin sensitivity in FGF-21–administered diabetic rodents even under enforced overexpression conditions in FGF-21–transgenic mice or after 8 weeks of chronic constant infusion of FGF-21 in db/db mice (Figure G and data not shown). Thus, the reduction observed in GLUT4 in 3T3-L1 adipocytes does not appear to be functionally relevant.
FGF-21 is a typical FGF molecule with respect to its ability to stimulate MAPK activation and FRS-2 phosphorylation (25
) (Figure A). In contrast, while heparin or heparin-like molecules are considered to be essential for the biological activity of proteins of the FGF family (30
), none of the FGF-21–induced in vitro responses observed in 3T3-L1 or human adipocytes were heparin regulated. Importantly, FGF-21 appears to be mitogenically inactive in vitro when tested on several otherwise FGF-sensitive cell lines and primary cells (Figure ). This further establishes FGF-21 as a unique protein within the FGF family, as FGFs are well known to induce proliferation (1
). Whereas the exact cause of the nonmitogenic character of the in vitro action of FGF-21 is currently unknown, these data help significantly in validating FGF-21 as a potential protein therapeutic.
The fact that FGF-21 induced tyrosine phosphorylation of FGFR-1 and FGFR-2 (Figure B) suggests that these molecules may function as FGF-21 receptors. Although they carry readily detectable and functional FGFR-1 and/or FGFR-2 molecules, it is, however, currently unclear why several FGF-sensitive cells that were tested for FGF-21 bioactivity do not respond to FGF-21 stimulation. Moreover, in preliminary experiments with all commercially available FGFR extracellular domain–Fc fusion proteins (R&GD Systems), we were unable to demonstrate direct interaction between any of these FGFR variants and FGF-21, despite the fact that we clearly observed binding for both FGF-1 and FGF-2 (data not shown). Thus, FGF-21 may be physically interacting with different splice variants of FGFR-1 and FGFR-2 that are induced upon adipocyte differentiation. Alternatively, FGF-21–dependent activation of these receptors may require a fat cell–specific modification or additional cofactor.
FGF-21 did not stimulate glucose uptake on insulin-sensitive liver clone 9 and muscle L6-GLUT-4myc cells and on 3T3-L1 fibroblasts. Also, we were unable to detect FGF-21 activity in proliferation assays on several cell lines and primary cells of a different nature, which further indicates that FGF-21 effects might be adipocyte specific. Nevertheless, we recently observed a clear FGF-21 response on cells of nonfat origin. Unexpectedly, FGF-21 (1 μg/ml) showed efficacy in modulating glucagon secretion from isolated rat islets (Figure ), while no effect on insulin secretion was observed. Thus, the specificity of FGF-21 bioactivity remains to be further studied.
Figure 7 FGF-21 inhibits glucagon secretion in isolated rat pancreatic islets. The values (± SE) shown are the average of 6 measurements. *P < 0.05 and **P < 0.02 compared with vehicle controls. Islets were stimulated (more ...)
The administration of FGF-21 to diabetic ob/ob and db/db mice and obese ZDF rats led to significant lowering of circulating glucose and triglycerides, as well as a reduction in fasted insulin levels and improved glucose clearance during an OGTT (Figure , A–H). All these effects were observed after at least 3 days of injections and were more pronounced after 7 days of administration. Since no changes in levels of fed and fasted glucose, circulating lipid levels, insulin levels, and glucose disposal during OGTT were observed after a single s.c. injection of FGF-21, it appears that beneficial FGF-21–dependent effects require that animals be exposed to the protein multiple times. However, once the reduction in circulating glucose was achieved, FGF-21–induced changes were sustained for at least 24 hours (Figure F). Thus, despite its short elimination half-life, FGF-21 induced an extended pharmacodynamic effect in these diabetic animals. Taken together, these observations are remarkable in highlighting the difference in time action between FGF-21 and insulin.
Although potent in correcting elevated glucose levels in ob/ob and db/db mice, FGF-21 did not induce hypoglycemia in normal or diabetic rodents in either fasted or fed states (Figure , A, C–F, and H) at efficacious or significantly higher doses, and no hypoglycemia was seen in fasted FGF-21–transgenic mice (Table ). This further distinguishes the effects of FGF-21 from those of insulin, which induced a significant reduction of blood glucose in lean animals (Figure E). Moreover, FGF-21 did not affect food intake or body weight/composition of diabetic or lean mice and rats over the course of 2 weeks of administration (doses ranging from 25 μg/kg/d to 8 mg/kg/d).
Further insights into the FGF-21 mechanism of action can be gleaned from the phenotype of FGF-21–transgenic mice. These animals are viable and are not metabolically distinguishable from wild-type littermates at 2 months of age. However, they appeared to be resistant to the age-related impairment of glucose metabolism since they had lower plasma glucose levels at 9 months (Table ). Moreover, when challenged on HFHC diet for 15 weeks, FGF-21–transgenic mice were resistant to diet-induced weight gain and fat accumulation (Figure , A and B), even though they consumed more food when the amounts were normalized to body weights (Table ). We also observed lower levels of circulating leptin (Table ). The reduction in leptin is consistent with lower adiposity in the transgenic animals and may be a primary cause of the increased food intake in the transgenic mice (31
). However, these changes in feeding behavior are unlikely to have been induced by a direct effect of FGF-21, since no impact on food intake in rodents administered the protein was observed. There was also a decrease in circulating glucagon levels (Table ), which is consistent with the in vitro observations made with rat pancreatic islets (Figure ).
There was no evidence of poor nutrient absorption in FGF-21–transgenic animals. Thus, another potential reason for the observed resistance to diet-induced obesity may be an effect of FGF-21 on energy expenditure. However, if this is true, it was not reflected in any changes in rectal body temperatures (Table ). We speculate that FGF-21 overexpression may increase brown adipose tissue (BAT) activity, as we observed retention of BAT in the transgenic mice compared with wild-type controls (Figure A). Whether or not BAT activation may contribute to the effects of FGF-21 remains to be evaluated in future studies.
The FGF family member most closely related to FGF-21 is FGF-19, with 31% amino acid sequence identity between these 2 molecules (21
). The phenotype of transgenic mice overexpressing FGF-19 (12
) is strikingly reminiscent of that of mice overexpressing FGF-21. However, we have uncovered a fundamental in vivo difference that clearly distinguishes these 2 molecules from each other. While transgenic mice overexpressing FGF-19 were resistant to high-fat diet–induced obesity, they also developed histologically detectable liver tumors. Furthermore, wild-type mice that were injected with FGF-19 for 6 days had an increase in hepatocellular proliferation (26
). In contrast to FGF-19–overexpressing animals, FGF-21–transgenic mice did not form tumors in liver or show histological evidence of hyperplasia in any other tissue after 10 months of age (Figure C). Nevertheless, in order to further determine the potential of FGF-21 to stimulate liver mitogenicity in vivo, we administered FGF-21 to db/db
mice via ALZET pumps at an efficacious 11 μg/kg/h dose (Figure C) for 8 weeks. Again, no evidence of hepatocellular proliferation or tumor formation was seen in FGF-21–infused mice as determined by H&GE staining or proliferative cell nuclear antigen (PCNA) (32
) immunohistochemistry (Figure D). Coupled with the lack of proliferation observed in vitro, the absence of tissue hyperplasia and activation of proliferative markers in FGF-21–treated and transgenic mice strongly suggests that FGF-21 is not mitogenic.
While our injection studies with FGF-21 in diabetic rodents clearly demonstrate that FGF-21 can lower glucose and lipids in circulation without inducing mitogenicity, hypoglycemia, or weight gain, the molecular mechanism by which FGF-21 functions is currently unclear. Despite the fact that FGF-21 and insulin induce similar functional outcomes in 3T3-L1 adipocytes (Figures –) and FGF-21 has a positive impact on insulin sensitivity when administered to diabetic rodents or transgenic mice (Figure G and data not shown), it is unlikely that FGF-21 directly affects insulin-dependent pathways. Signaling and mitogenic profiles for these 2 proteins are different in vitro; no FGF-21–dependent tyrosine phosphorylation of insulin receptor was observed in 3T3-L1 adipocytes (data not shown); and there is no apparent interference between FGF-21 and insulin actions in glucose uptake assay (Figure C). Moreover, the pharmacology observed in FGF-21–treated animals (absence of hypoglycemia and weight gain; extended time-action) further distinguishes this molecule from insulin, which thus rules out a possibility that FGF-21 functions as an insulin mimetic and/or sensitizer.
The beneficial in vivo effects of FGF-21 may be mediated through changes in circulating levels of endocrine-relevant hormones, in particular, adipokines, since adipocytes appear to be a target for FGF-21 bioactivity. To explore this possibility, we dosed ob/ob mice s.c. once daily with 125 μg/kg/day of FGF-21 for 7 days and measured the levels of several secreted polypeptides in circulation (Table ). While we were able to achieve a clear glucose-lowering effect in the study (Figure A), only insulin and glucagon levels were changed in a statistically significant manner. The reduction of insulin levels is consistent with our observation in ob/ob mice during OGTT (Figure G) and is suggestive of improvements in insulin sensitivity in FGF-21–treated mice.
Administration of FGF-21 in ob/ob mice affects serum levels of glucagon and insulin but not of other secreted polypeptides
GLUT1 and glucagon potentially mediate the mode of action of FGF-21. We were able to detect FGF-21–dependent upregulation of GLUT1 message specifically in white fat upon bolus injection into ob/ob
mice (Figure G), which thus confirms our in vitro observations on 3T3-L1 adipocytes. The increase in GLUT1 may mechanistically be linked to FGF-21–dependent glucose lowering in diabetic rodents. Alternatively, or in concert, the glucose lowering effect of FGF-21 is likely to result from reduced glucagon secretion from pancreatic α cells, since FGF-21 inhibits glucagon release in vitro (Figure ) and is lowered in FGF-21–transgenic mice (Table ). The observation of glucagon lowering in FGF-21–injected ob/ob
mice (Table ) further strengthens the hypothesis that this hormone is an important mediator of FGF-21 in vivo effects. There is accumulating evidence supporting a pathophysiological role of glucagon in the development and progression of type 2 diabetes. Basal glucagon is inappropriately elevated and its suppression is impaired following food consumption, which leads to increased hepatic glucose production and aggravation of the hyperglycemia associated with the disease (33
). Interestingly, attenuation of signaling through the glucagon receptor leads to normalization of plasma glucose and triglyceride levels in diabetic animals (35
Despite substantial progress in understanding the pathophysiology of diabetes mellitus and the development of new drugs to treat diabetic patients, this disease remains a major health problem (36
). New treatments are required that will allow an efficacious regulation of glycemia and reduce the risk of the side effects associated with current therapies (27
). Here we demonstrate that FGF-21, as a single agent, can be used to provide efficient and durable glucose control and triglyceride lowering in diabetic animals, without apparent mitogenicity, hypoglycemia, or weight gain. FGF-21 thus holds promise as an effective therapeutic agent for the treatment of diabetes.