Human fibroblast growth factor 21 (hFGF21) has become a candidate drug for regulating blood glucose and lipid metabolism. The poor stability and short half-life of hFGF21 resulted in low target tissue availability, which hampers its clinical application. In this study, the hFGF21 was fused with a recombinant human serum albumin (HSA), and the resulted fusion protein rHSA-hFGF21 was expressed in Pichia pastoris. After codon optimization, the recombinant gene fragment rHSA-hFGF21 was inserted into two different vectors (pPIC9k and pPICZαA) and transformed into three different strains (X33, GS115 and SMD1168), respectively. We investigated the rHSA-hFGF21 expression levels in three different strains and screened an engineered strain X33-pPIC9K-rHSA-hFGF21 with the highest expression level. To improve the production efficiency of rHSA-hFGF21, we optimized the shake flask fermentation conditions, such as the OD value, methanol concentration and induction time. After purification by hollow fiber membrane separation, Blue affinity chromatography and Q ion exchange chromatography, the purity of the rHSA-hFGF21 protein obtained was 98.18%. Compared to hFGF21, the biostabilities of rHSA-hFGF21, including their resistance to temperature and trypsinization were significantly enhanced, and its plasma half-life was extended by about 27.6 times. Moreover, the fusion protein rHSA-hFGF21 at medium and high concentration showed a better ability to promote glucose uptake after 24 h of stimulation in vitro. In vivo animal studies showed that rHSA-hFGF21 exhibited a better long-term hypoglycemic effect than hFGF21 in type 2 diabetic mice. Our results demonstrated a small-scale production of rHSA-hFGF21, which is important for large-scale production and clinical application in the future.
Animals ; Blood Glucose/metabolism* ; Diabetes Mellitus, Experimental ; Fibroblast Growth Factors ; Humans ; Hypoglycemic Agents/metabolism* ; Methanol/metabolism* ; Mice ; Pichia/metabolism* ; Recombinant Fusion Proteins ; Recombinant Proteins/metabolism* ; Saccharomycetales ; Serum Albumin/metabolism* ; Serum Albumin, Human/metabolism*

OBJECTIVE: To observe the effect of Ronghuang granule on serum fibroblast growth factor 23 (FGF23), fibroblast growth factor receptor (FGFRs) and Klotho protein levels in non-dialysis patients with chronic kidney disease-mineral and bone disorder (CKD-MBD) and kidney deficiency and damp heat syndrome. METHODS: Seventy non-dialysis CKD-MBD patients with kidney deficiency and dampness-heat syndrome were randomized into control group (=35) and treatment group (=35). All the patients were given routine treatment combined with traditional Chinese medicine retention enema, and the patients in the treatment group received additional Ronghuang granule treatment (3 times a day). After the 12-week treatments, the patients were examined for changes of TCM syndromes. Serum levels of Ca, P, parathyroid hormone (iPTH), FGF23, FGFRs and Klotho proteins were detected before and after treatment. These parameters were also examined in 20 healthy volunteers. RESULTS: Sixty-five patients completed the study, including 33 in the control group and 32 in the treatment group. The patients in the treatment group showed significantly better treatment responses than those in the control group ( < 0.05 or 0.01). At 4, 8, and 12 weeks of treatment, the patients in the treatment group had significantly lowered scores of TCM syndromes compared with the score before treatment ( < 0.05 or 0.01), while in the control group, significant reduction of the scores occurred only at 12 weeks ( < 0.05); at each of the time points, the treatment group had significantly greater reductions in the score than the control group ( < 0.01). Significant improvements in serum Ca, P and iPTH levels were observed at 4, 8, and 12 weeks in the treatment group ( < 0.05) but only at 12 weeks in the control group ( < 0.05). The patients in the control and treatment groups all showed elevated serum levels of FGF23, FGFRs and Klotho protein compared with the normal subjects ( < 0.01); FGF23, FGFRs and Klotho levels were significantly reduced in the treatment group ( < 0.05) but remained unchanged in the control group (>0.05), showing significant differences between the two groups. CONCLUSIONS Ronghuang granule improves the clinical symptoms of non-dialysis CKD-MBD patients with kidney deficiency and dampness heat syndrome by reducing serum levels of FGF23, FGFRs and Klotho, improving calcium and phosphorus metabolism disorder, and inhibiting secondary hyperparathyroidism.
Calcium ; blood ; Chronic Kidney Disease-Mineral and Bone Disorder ; blood ; therapy ; Drugs, Chinese Herbal ; pharmacology ; Enema ; Fibroblast Growth Factors ; blood ; Glucuronidase ; blood ; Humans ; Parathyroid Hormone ; blood ; Phosphorus ; blood ; Receptors, Fibroblast Growth Factor ; blood ; Renal Insufficiency, Chronic ; blood ; therapy ; Sweating Sickness ; blood ; therapy ; Syndrome

PURPOSE: Fibroblast growth factor 21 (FGF21) is a crucial metabolic regulator, with multiple favorable effects on glucose homeostasis and lipid metabolism. Since serum FGF21 level has been implicated as a potential marker for the early identification of metabolic syndrome (MetS), we investigated the association between serum FGF21 level and the development of MetS in a population-based prospective study. MATERIALS AND METHODS: We conducted a prospective study of 221 randomly sampled adults without MetS from a general population-based cohort study who were examined from 2005–2008 (baseline) and from 2008–2011 (follow-up). Baseline serum FGF21 levels were analyzed using enzyme-linked immunosorbent assay. RESULTS: During the average 2.8-year follow-up period, 82 participants (36.6%) developed new-onset MetS. Serum FGF21 levels were significantly higher in patients with new-onset MetS than in those without MetS (209.56±226.80 vs. 110.09±81.10, p < 0.01). In multivariate adjusted models, the odds for MetS development were greater in patients with serum FGF21 levels in the highest quartile, compared to those in the lowest quartile (3.84, 95% confidence interval: 1.59–9.28). CONCLUSION: Serum FGF21 level was an independent predictor for new-onset MetS in a population-based prospective study.
Biomarkers/blood ; Female ; Fibroblast Growth Factors/*blood ; Humans ; Male ; Metabolic Syndrome/*blood ; Middle Aged ; Multivariate Analysis ; Odds Ratio ; Prospective Studies

Fibroblast growth factor 21 (FGF21) is a metabolic hormone with pleiotropic effects on energy metabolism and insulin sensitivity. Besides its antiobese and antidiabetic activity, FGF21 also possesses the protective effects against atherosclerosis. Circulating levels of FGF21 are elevated in patients with atherosclerosis, macrovascular and microvascular complications of diabetes, possibly due to a compensatory upregulation. In apolipoprotein E-deficient mice, formation of atherosclerotic plaques is exacerbated by genetic depletion of FGF21, but is attenuated upon replenishment with recombinant FGF21. However, the blood vessel is not the direct target of FGF21, and the antiatherosclerotic activity of FGF21 is attributed to its actions in adipose tissues and liver. In adipocytes, FGF21 promotes secretion of adiponectin, which in turn acts directly on blood vessels to reduce endothelial dysfunction, inhibit proliferation of smooth muscle cells and block conversion of macrophages to foam cells. Furthermore, FGF21 suppresses cholesterol biosynthesis and attenuates hypercholesterolemia by inhibiting the transcription factor sterol regulatory element-binding protein-2 in hepatocytes. The effects of FGF21 on elevation of adiponectin and reduction of hypercholesterolemia are also observed in a phase-1b clinical trial in patients with obesity and diabetes. Therefore, FGF21 exerts its protection against atherosclerosis by fine-tuning the interorgan crosstalk between liver, brain, adipose tissue, and blood vessels.
Adipocytes ; Adiponectin ; Adipose Tissue ; Animals ; Apolipoproteins ; Atherosclerosis* ; Blood Vessels ; Brain ; Cholesterol ; Energy Metabolism ; Fibroblast Growth Factors* ; Fibroblasts* ; Foam Cells ; Hepatocytes ; Humans ; Hypercholesterolemia ; Insulin Resistance ; Liver ; Macrophages ; Mice ; Myocytes, Smooth Muscle ; Obesity ; Plaque, Atherosclerotic ; Transcription Factors ; Up-Regulation ; Vascular Diseases

OBJECTIVETo investigate the role and mechanism of action of fibroblast growth factor-21 (FGF-21) in reducing triglyceride (TG) in the in vitro and in vivo models of nonalcoholic fatty liver disease (NAFLD).

METHODS(1) A mixture of free fatty acids was used to establish a model of steatosis in L02 cells, and the cells were treated with various concentrations of FGF-21 or fenofibrate. Twenty-four hours later, oil red O staining was performed to observe the degree of steatosis, and intracellular TG content was determined. RT-PCR and Western blot were applied to measure the mRNA and protein expression of sterol regulatory element-binding protein-1c (SREBP-1c). (2) High-fat diet was used to establish a mouse model of steatosis, and these mice were intraperitoneally injected with FGF-21 or fenofibrate. Eight weeks later, whole blood and liver samples were collected, and HE staining was performed to observe steatosis. Meanwhile, the serum levels of TG, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured, and TG content in the liver was also measured. One-way analysis of variance was used for comparison of data between multiple groups, and the least significant difference t-test was used for comparison between any two groups.

RESULTS(1) Compared with the control group, the model group showed significant steatosis, with significant increases in intracellular lipid droplets and TG content (t = -20.57, P < 0.01), while FGF-21 reduced the number of intracellular lipid droplets and TG content (F = 98.16, P < 0.01) in a dose-dependent manner. In addition, the model group had significantly increased mRNA and protein expression of SREBP-1c compared with the control group (t = -10.73 and -0.1006, both P < 0.01), while FGF-21 down-regulated the mRNA and protein expression of SREBP-1c (F = 161.35 and 36.72, both P < 0.01). (2) Compared with the mice in the control group, those in the model group showed significant steatosis and had significant increases in serum TG level and TG content in the liver (t = -18.84 and 15.71, both P < 0.01). FGF-21 relieved hepatic steatosis and reduced the serum TG level and TG content in the liver (t = 18.11 and 9.46, both P < 0.01). Moreover, FGF-21 reduced the serum levels of ALT and AST in NAFLD mice (t = 25.93 and 12.50, both P < 0.01).

CONCLUSIONFGF-21 can inhibit the synthesis of TG through suppressing the expression of SREBP-1c, which further confirms the potential therapeutic effect of FGF-21 in the treatment of NAFLD. This may provide new ideas for the treatment of NAFLD.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Cell Line ; Diet, High-Fat ; Disease Models, Animal ; Fenofibrate ; pharmacology ; Fibroblast Growth Factors ; pharmacology ; Mice ; Non-alcoholic Fatty Liver Disease ; blood ; drug therapy ; Sterol Regulatory Element Binding Protein 1 ; metabolism ; Triglycerides ; blood

The aim of this study was to evaluate the association of plasma fibroblast growth factor (FGF)-21 with angiographically significant coronary artery disease (CAD) in patients with type 2 diabetes mellitus. Serum FGF-21 was measured in 120 patients undergoing coronary angiography. Patients were divided into 4 groups based on the presence/absence of type 2 diabetes mellitus and of significant CAD. The atherosclerotic burden was obtained by two angiographic scores: Gensini score (GS) and Extent score (ES). FGF-21 levels were higher in type 2 diabetes mellitus than in non-diabetic patients (P = 0.014). FGF-21 levels were significantly correlated with GS (r = 0.358, P < 0.001) and ES (r = 0.324, P < 0.001) in univariate analysis with all patients. After adjusting for several confounding factors, both GS and ES were associated with FGF-21 in all patients (r = 0.271, P = 0.014; r = 0.217, P = 0.041, respectively). However, FGF-21 lost significant correlation with both GS and ES with type 2 diabetes mellitus in the final model. The patients with type 2 diabetes mellitus and CAD feature had elevated FGF-21 levels. Despite of a limited role in diabetic patients, FGF-21 levels are independently associated with angiographic severity and extent of CAD.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Coronary Angiography ; Coronary Artery Disease/complications/*diagnosis/pathology ; Diabetes Mellitus, Type 2/complications/*diagnosis ; Female ; Fibroblast Growth Factors/*blood ; Humans ; Male ; Middle Aged ; Regression Analysis ; Severity of Illness Index ; Young Adult

OBJECTIVETo investigate the effect of topical propranolol gel on the levels of plasma vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (bFGF) and matrix metalloproteinases-9 (MMP-9) in proliferating infantile hemangiomas (IHs) of superficial type.

METHODS33 consecutive children with superficial IHs were observed pre-treatment, 1 and 3 months after application of topical propranolol gel for the levels of plasma VEGF, MMP-9 and bFGF by enzyme-linked immunosorbent assay (ELISA) in Department of General Surgery of Dongfang Hospital from February 2013 to February 2014. The plasma results of IHs were compared with those of 30 healthy infants. The clinical efficacy in IHs was evaluated by Achauer system. Differences of plasma results between the healthy group and the IHs group pre-treatment were analyzed using Mann-Whitney U-test. Paired sample comparisons of any two time points of pre-treatment, 1 month and 3 months after treatment in IHs were evaluated by Wilcoxon signed-rank test.

RESULTSThe clinical efficiency of topical propranolol gel at 1, 3 months after application were 45.45%, 81.82% respectively. The levels of plasma VEGF and MMP-9 in patients pre- treatment were higher than those in healthy infants [(362.16 ± 27.29) pg/ml vs (85.63 ± 8.14) pg/ml, (1376.41 ± 42.15) pg/ml vs (687.27 ± 44.1) pg/ml, P < 0.05], but the level of bFGF did not show significant difference [(176.03 ± 13.60 ) pg/ml vs (235.94 ± 35.43 ) pg/ml, P > 0. 05 ]. The concentrations of VEGF and bFGF at 1, 3 months after treatment decreased obviously [(271.51 ± 18.59) pg/ml vs (362.16 ± 27.29 ) pg/ml, (135.85 ± 12.66) pg/ml vs (176.03 ± 13.60) pg/ml], 1 month after treatment vs pre-treatment, P < 0.05; (240.80 ± 19.89) pg/ml vs (362.16 ± 27.29) pg/ml, (107.31 ± 5.82) pg/ml vs (176.03 ± 13.60) pg/ml, 3 month after treatment vs pre-treatment, P < 0.05, whereas the levels of plasma MMP-9 declined slightly [(1321.18 ± 48.74) pg/ml vs (1376.41 ± 42.15 ) pg/ml, (1468.68 ± 32.78) pg/ml vs (1376.41 ± 42 2.15 ) pg/ml, P > 0.05 ].

CONCLUSIONSPropranolol gel may suppress the proliferation of superficial infantile bemangiomas by reducing VEGF and bFGF.

Administration, Topical ; Case-Control Studies ; Child ; Enzyme-Linked Immunosorbent Assay ; Fibroblast Growth Factor 2 ; blood ; Gels ; Hemangioma ; blood ; drug therapy ; Humans ; Infant ; Matrix Metalloproteinase 9 ; blood ; Propranolol ; pharmacology ; Time Factors ; Vascular Endothelial Growth Factor A ; blood

BACKGROUNDThis study was to investigate the relationship among aortic artery calcification (AAC), cardiac valve calcification (CVC), and mortality in maintenance hemodialysis (MHD) patients.

METHODSAll MHD patients in Shanghai Ruijin Hospital in July 2011 were included. To follow up for 42 months, clinical data, predialysis blood tests, echocardiography, and lateral lumbar X-ray plain radiography results were collected. Plasma FGF23 level was measured using a C-terminal assay.

RESULTSTotally, 110 MHD patients were involved in this study. Of which, 64 (58.2%) patients were male, the mean age was 55.2 ± 1.4 years old, and the median dialysis duration was 29.85 (3.0-225.5) months. About 25.5% of the 110 MHD patients had CVC from echocardiography while 61.8% of the patients had visible calcification of aorta from lateral lumbar X-ray plain radiography. After 42 months follow-up, 25 (22.7%) patients died. Kaplan-Meier analysis showed that patients with AAC or CVC had a significant greater number of all-cause and cardiovascular deaths than those without. In multivariate analyses, the presence of AAC was a significant factor associated with all-cause mortality (hazard ratio [HR]: 3.149, P = 0.025) in addition to lower albumin level and lower 25-hydroxy Vitamin D (25(OH)D) level. The presence of CVC was a significant factor associated with cardiovascular mortality (HR: 3.800, P = 0.029) in addition to lower albumin level and lower 25(OH)D level.

CONCLUSIONLateral lumbar X-ray plain radiography and echocardiography are simple methods to detect AAC and CVC in dialysis patients. The presence of AAC and CVC was independently associated with mortality in MHD patients. Regular follow-up by X-ray and echocardiography could be a useful method to stratify mortality risk in MHD patients.

Aortic Diseases ; blood ; complications ; Calcinosis ; blood ; complications ; China ; Female ; Fibroblast Growth Factors ; blood ; Follow-Up Studies ; Heart Valve Diseases ; blood ; complications ; Heart Valves ; pathology ; Humans ; Male ; Middle Aged ; Proportional Hazards Models ; Renal Dialysis ; mortality

This study is to evaluate the therapeutic effect of fibroblast growth factor 21 (FGF21) on type 2 diabetic mice model and to provide mechanistic insights into its therapeutic effect. Type 2 diabetic animal model was established with high calorie fat diet and low dose streptozotocin (STZ) injection. Mice were then randomized into 5 groups: model control, FGF21 0.25 and 0.05 μmol x kg(-1) x d(-1) groups, insulin treatment group. Ten age-matched normal KM mouse administered with saline were used as normal controls. Serum glucose, insulin, lipid products and the change of serum and liver tissue inflammation factor levels between five groups of mouse were determined. The results showed that blood glucose, insulin, free fatty acids (FFAs), triglycerides, and inflammatory factor average FGF-21 of type 2 diabetes model group and normal control group were significantly higher (P < 0.01), while compared with insulin group, no difference was significant. Average blood glucose, insulin, blood lipid and inflammatory factor of FGF-21 treatment group compared with type 2 diabetes group was significantly lower (P < 0.01) and insulin group has no difference with the model control group. The results of OGTT and HOMA-IR showed that insulin resistance state was significantly relieved in a dose-dependent manner. Thus, this study demonstrates that FGF-21 significantly remits type 2 diabetic mice model's insulin resistance state and participates in the regulation of inflammatory factor levels and type 2 diabetes metabolic disorders.
Animals ; Blood Glucose ; Diabetes Mellitus, Experimental ; drug therapy ; Diabetes Mellitus, Type 2 ; drug therapy ; Diet, High-Fat ; Fatty Acids, Nonesterified ; blood ; Fibroblast Growth Factors ; pharmacology ; Insulin ; blood ; Insulin Resistance ; Mice ; Streptozocin ; Triglycerides ; blood

Previous studies proposed that the synergistic effect of fibroblast growth factor-21 (FGF-21) and insulin may be due to the improvement of insulin sensitivity by FGF-21. However, there is no experimental evidence to support this. This study was designed to elucidate the mechanism of synergistic effect of FGF-21 and insulin in the regulation of glucose metabolism. The synergistic effect of FGF-21 and insulin on regulating glucose metabolism was demonstrated by investigating the glucose absorption rate by insulin resistance HepG2 cell model and the blood glucose chances in type 2 diabetic db/db mice after treatments with different concentrations of FGF-21 or/and insulin; The synergistic metabolism was revealed through detecting GLUT1 and GLUT4 transcription levels in the liver by real-time PCR method. The experimental results showed that FGF-21 and insulin have a synergistic effect on the regulation of glucose metabolism. The results of real-time PCR showed that the effective dose of FGF-21 could up-regulate the transcription level of GLUT1 in a dose-dependent manner, but had no effect on the transcription level of GLUT4. Insulin (4 u) alone could up-regulate the transcription level of GLUT4, yet had no effect on that of GLUT1. Ineffective dose 0.1 mg kg(-1) FGF-21 alone could not change the transcription level of GLUT1 or GLUT4. However, when the ineffective dose 0.1 mg x kg(-1) FGF-21 was used in combination with insulin (4 u) significantly increased the transcription levels of both GLUT1 and GLUT4, the transcription level of GLUT1 was similar to that treated with 5 time concentration of FGF-21 alone; the transcription level of GLUT4 is higher than that treated with insulin (4 u) alone. In summary, in the presence of FGF-21, insulin increases the sensitivity of FGF-21 through enhancing GLUT1 transcription. Vice versa, FGF-21 increases the sensitivity of insulin by stimulating GLUT4 transcription in the presence of insulin. FGF-21 and insulin exert a synergistic effect on glucose metabolism through mutual sensitization.
Animals ; Blood Glucose ; Diabetes Mellitus, Experimental ; metabolism ; Drug Synergism ; Fibroblast Growth Factors ; pharmacology ; Glucose ; metabolism ; Glucose Transporter Type 1 ; metabolism ; Glucose Transporter Type 4 ; metabolism ; Hep G2 Cells ; Humans ; Insulin ; pharmacology ; Insulin Resistance ; Liver ; metabolism ; Mice