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OBJECTIVE: Fibroblast growth factor (FGF)-21 improves insulin sensitivity and lipid metabolism in obese or diabetic animal models, while human studies revealed increased FGF-21 levels in obesity and type 2 diabetes. Given that FGF-21 has been suggested to be a peroxisome proliferator-activator receptor (PPAR) alpha-dependent regulator of fasting metabolism, we hypothesized that free fatty acids (FFAs), natural agonists of PPARalpha, might modify FGF-21 levels. RESEARCH DESIGN AND METHODS: The effect of fatty acids on FGF-21 was investigated in vitro in HepG2 cells. Within a randomized controlled trial, the effects of elevated FFAs were studied in 21 healthy subjects (13 women and 8 men). Within a clinical trial including 17 individuals, the effect of insulin was analyzed using an hyperinsulinemic-euglycemic clamp and the effect of PPARgamma activation was studied subsequently in a rosiglitazone treatment trial over 8 weeks. RESULTS: Oleate and linoleate increased FGF-21 expression and secretion in a PPARalpha-dependent fashion, as demonstrated by small-interfering RNA-induced PPARalpha knockdown, while palmitate had no effect. In vivo, lipid infusion induced an increase of circulating FGF-21 in humans, and a strong correlation between the change in FGF-21 levels and the change in FFAs was observed. An artificial hyperinsulinemia, which was induced to delineate the potential interaction between elevated FFAs and hyperinsulinemia, revealed that hyperinsulinemia also increased FGF-21 levels in vivo, while rosiglitazone treatment had no effect. CONCLUSIONS: The results presented here offer a mechanism explaining the induction of the metabolic regulator FGF-21 in the fasting situation but also in type 2 diabetes and obesity.
Mai et al. Diabetes. 2009 Jul;58(7):1532-8.
Mai et al. Diabetes. 2009 Jul;58(7):1532-8.
Diabetes mellitus is a major health concern, affecting more than 5% of the population. Here we describe a potential novel therapeutic agent for this disease, FGF-21,
which was discovered to be a potent regulator of glucose uptake in mouse 3T3-L1 and primary human adipocytes. FGF-21–transgenic mice were viable and resistant
to diet-induced obesity. Therapeutic administration of FGF-21 reduced plasma glucose and triglycerides to near normal levels in both ob/ob and db/db mice. These effects persisted for
at least 24 hours following the cessation of FGF-21 administration. Importantly, FGF-21 did not induce mitogenicity, hypoglycemia, or weight gain at any dose
tested in diabetic or healthy animals or when overexpressed in transgenic mice. Thus, we conclude that FGF-21, which we have identified as a novel metabolic
factor, exhibits the therapeutic characteristics necessary for an effective treatment of diabetes.
Kharitonenkov et al. J Clin Invest. 2005 Jun;115(6):1627-35.
 
 
Tissue Sample |
Human liver and hepatocellular sarcinoma tissues |
Fixative |
10% formalin |
Embedding |
Paraffin |
Negative Control |
No primary antibody |
Pretreatment |
N/A |
Blocking |
3% H2O2, 2% Normal Goat Serum |
Primary Antibody |
Rabbit anti-FGF-21 (26-47) (Human) antibody (Cat. No.: H-002-46) |
Optimal Dilution |
1:500 |
Secondary Antibody |
Goat Anti-Rabbit IgG, Biotinylated (1:400), 30 min |
Amplification |
Streptavidin-HRP (Vector), 1:400, 30 min |
Detection System |
HRP |
Substrate |
DAB (Sigma), 3 min |
Counterstained |
Hematoxylin, 30 sec |
 
Tissue Sample |
Mouse Liver Tissue |
Fixative |
10% formalin |
Embedding |
Paraffin |
Negative Control |
No primary antibody |
Pretreatment |
N/A |
Blocking |
3% H2O2, 2% Normal Goat Serum |
Primary Antibody |
Rabbit anti-FGF-21 (184-209) (Human) antibody (Cat. No.: H-002-47)
Rabbit anti-FGF-21 (26-47) (Human) antibody (Cat. No.: H-002-46) |
Optimal Dilution |
1:500 |
Secondary Antibody |
Goat Anti-Rabbit IgG, Biotinylated (1:400), 30 min |
Amplification |
Streptavidin-HRP (Vector), 1:400, 30 min |
Detection System |
HRP |
Substrate |
DAB (Sigma), 3 min |
Counterstained |
Hematoxylin, 30 sec |
 
Fixative |
10% formalin |
Embedding |
Paraffin |
Negative Control |
No primary antibody |
Pretreatment |
N/A |
Blocking |
3% H2O2, 2% Normal Goat Serum |
Primary Antibody |
Rabbit anti-FGF-21 (98-121) (Human) antibody (Cat. No.: H-002-48) |
Optimal Dilution |
1:500 |
Secondary Antibody |
Goat Anti-Rabbit IgG, Biotinylated (1:400), 30 min |
Amplification |
Streptavidin-HRP (Vector), 1:400, 30 min |
Detection System |
HRP |
Substrate |
DAB (Sigma), 3 min |
Counterstained |
Hematoxylin, 30 sec |
|
(A) H&GE staining of brown fat.
Notice an increase in intensity of brown fat in the FGF-21–transgenic mouse compared with the wild-type mouse. (B) H&GE staining of
subcutaneous white fat. Notice the smaller adipocytes in the FGF-21 mouse compared with the wild type. (C) H&GE staining of livers from
FGF-21–transgenic and wild-type mice. There are no differences between the 2. Magnification, x200 (A–C). (D) PCNA staining of the livers from
FGF-21–infused and saline-treated db/db mice. PNCA immunostaining shows very low proliferation (less than 5%) of hepatocytes (brown staining, arrows) in both the
control and treated groups. Magnification, x400.
|
 |
The
values (± SE) shown are the average of at least 3 independent measurements. *P
< 0.02, **P < 0.001 compared with no stimulation or vehicle control.
FGF-21 dose response on 3T3-L1 (A) and human primary adipocytes
(B) in glucose uptake
assay. (C) FGF-21
augments insulin activity. Cells were pretreated with or without FGF-21 and then
stimulated with insulin as indicated. (D) Cycloheximide diminishes FGF-21
bioactivity in glucose uptake assay. 3T3-L1 adipocytes were stimulated with
FGF-21 for 24 hours in the presence or absence of cycloheximide. P < 0.001 at
all doses for FGF-21 versus FGF-21 plus cycloheximide stimulations. FGF-21
affects GLUT1 mRNA (E) and protein (F) levels and does not upregulate
GLUT4 protein (F) in 3T3-L1 adipocytes (immunoblot). Cells were starved and then stimulated with
FGF-21 or vehicle as indicated. Quantitative PCR and immunoblotting analyses
were used to measure mRNA and protein levels, respectively. (G) GLUT1 mRNA is upregulated in
adipose tissue of FGF-21–injected ob/ob mice. Two groups (5 animals each) of
8-week-old mice were injected s.c. with FGF-21 or vehicle. Quantitative PCR
analysis was used to measure mRNA |
 |
The values (± SE) shown are the average
of the measurements of at least 5 animals in a group. *P < 0.05, **P <
0.02, and #P < 0.001 compared with vehicle control. Fed blood glucose
(A) and triglyceride levels (B) in ob/ob
mice treated with FGF-21. FGF-21 was administered once daily, and blood glucose
and triglyceride levels were measured 1 hour after the last injection.
(C) Fed blood glucose levels in db/db mice at days 18 and 46 during 8-week constant-infusion study.
Mice were infused s.c. with 11 µg/kg/h FGF-21 using ALZET minipumps.
(D and E) FGF-21 lowers glucose in obese
ZDF rats and does not induce hypoglycemia in lean ZDF rats. Fed blood glucose
levels were measured in obese (D) and lean (E) ZDF rats that were administered
s.c. twice daily with FGF-21, Humulin, or vehicle at indicated total daily doses
for 1 week. (F) FGF-21 induces extended lowering of fed blood glucose in ob/ob mice. FGF-21 was
administered once daily for 7 days, and blood glucose levels were measured after
the last injection at indicated time points. (G and H) FGF-21 affects insulin levels
(G) and glucose disposal (H) during
OGTT in ob/ob mice. At indicated time points, blood samples were obtained for
glucose and insulin measurements. |
 |
(A) FGF-21 induces phosphorylation
of MAPK and FRS-2 in 3T3-L1 adipocytes. Upon stimulation, cells were lysed, and
phospho-specific antibodies were used to determine phosphorylation of MAPK and
FRS-2 in immunoblots. After immunoblots were stripped, anti-MAPK and anti–FRS-2
antibodies were used to confirm that protein loads were equal. For MAPK
experiment, cells were stimulated with FGF-21 for the indicated times. For FRS-2
experiment, cells were stimulated with FGF-21 or FGF-1 (positive control).
(B) FGF-21 stimulates
tyrosine phosphorylation of FGFR-1 and FGFR-2 in 3T3-L1 adipocytes. Cells were
stimulated with FGF-21 and lysed. FGFR-1 and FGFR-2 immunoprecipitates were
analyzed in immunoblots with anti-phosphotyrosine antibodies. After stripping,
anti–FGFR-1 and anti-FGFR-2 antibodies were used to confirm that protein loads
were equal. pErk, phospho-Erk; PY, phosphotyrosine. |
 |
FGF-21 does not induce proliferation and does not block FGF-7–dependent
mitogenicity on 4MBr5 cells. Cells were stimulated as indicated with different
concentrations of FGF-7, FGF-21, and FGF-21 in the presence of a constant
concentration of heparin and FGF-21 in the presence of a constant concentration
of FGF-7. |
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