human fgf21 Search Results


recombinant human fgf21  (R&D Systems)
96
R&D Systems recombinant human fgf21
FIGURE 2 | Activating effects of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and <t>FGF21</t> (20 nmol L−1), in the presence of PD173074 (FGFR1 inhibitor, 50 nmol L−1) or palmitic acid (PA, 500 µmol L−1), on FGF21 signaling in HepG2 human hepatocytes. Coffee by-product’s bioactive compounds interacted in silico with the subunits of the FGF21 receptor (FGFR) (A), exhibiting different binding (Continued)
Recombinant Human Fgf21, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 1 article reviews
recombinant human fgf21 - by Bioz Stars, 2026-03
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fgf21 levels  (Sino Biological)
94
Sino Biological fgf21 levels
FIGURE 2 | Activating effects of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and <t>FGF21</t> (20 nmol L−1), in the presence of PD173074 (FGFR1 inhibitor, 50 nmol L−1) or palmitic acid (PA, 500 µmol L−1), on FGF21 signaling in HepG2 human hepatocytes. Coffee by-product’s bioactive compounds interacted in silico with the subunits of the FGF21 receptor (FGFR) (A), exhibiting different binding (Continued)
Fgf21 Levels, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 1 article reviews
fgf21 levels - by Bioz Stars, 2026-03
94/100 stars
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fgf21  (R&D Systems)
99
R&D Systems fgf21
FIGURE 2 | Activating effects of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and <t>FGF21</t> (20 nmol L−1), in the presence of PD173074 (FGFR1 inhibitor, 50 nmol L−1) or palmitic acid (PA, 500 µmol L−1), on FGF21 signaling in HepG2 human hepatocytes. Coffee by-product’s bioactive compounds interacted in silico with the subunits of the FGF21 receptor (FGFR) (A), exhibiting different binding (Continued)
Fgf21, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 1 article reviews
fgf21 - by Bioz Stars, 2026-03
99/100 stars
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human fgf21  (R&D Systems)
90
R&D Systems human fgf21
FIGURE 1. Nonesterified unsaturated fatty acids and chylomicron remnants increase hepatic <t>FGF21</t> mRNA abundance. A, nutritional regulation of hepatic FGF21 expression in rats. Sprague-Dawley rats were fed a chow diet, a standard purified diet, or an HF-LC ketogenic diet for 7 days. The composition of the diets is described under “Experimental Procedures.” A fourth group of animals was fed the standard purified diet for 6 days and then starved for 30 h. Animals were killed 6 h after the start of the dark cycle, total RNA was isolated from liver, and the abundance of FGF21 mRNA was measured by quantitative real time PCR. Hepatic FGF21 mRNA abundance was also measured in a fifth group of animals fed a standard purified diet for 6 days followed by starvation for 30 h and refeeding for 5 h. The level of FGF21 mRNA in animals fed the chow diet was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of six animals. An asterisk indicates that the mean is significantly (p 0.05) higher compared with that of animals fed the standard (Std) purified diet. B–F, effect of nutrients and hormones on FGF21 expression in primary cultures of rat hepatocytes. Rat hepatocytes were prepared as described under “Experimental Procedures” and incubated in serum-free Medium 199. B, time course of the effect of nonesterified fatty acids (250 M) on FGF21 mRNA abundance. Hepatocytes were incubated with the indicated fatty acids complexed to BSA. Hepatocytes receiving no additions (NA) were incubated in medium containing an equivalent concentration of BSA. The level of FGF21 mRNA in cells incubated with fatty acids for 0 h was set at 1, and the other values were adjustedproportionately.ValuesaremeansS.E.(errorbars)offourexperiments.C,effectofdifferentoleatetreatmentprotocolsonFGF21mRNAabundance. Hepatocytes were incubated with or without oleate for 2 and 6 h without a medium change. A third treatment group (designated 4 2) was incubated with oleate for 6 h with a medium change 2 h prior to the end of the treatment period. The level of FGF21 mRNA in hepatocytes incubated without oleate for 2 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. Different superscript letters indicate that means are significantly (p 0.05) different. D, time course of the effect of GW7647 on FGF21 mRNA abundance. Hepatocytes were incubated with or without GW7647 (1 M) for the indicated time periods. The level of FGF21 mRNA in cells incubated with GW7647 for 0 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated in the absence of GW7647 for the same time period. E, effect of chylomicrons and chylomicron remnants on FGF21 mRNA abundance. Hepatocytes were incubated with chylomicrons and chylomicron remnants prepared from rats fed safflower oil. FGF21 mRNA was measured after 2 h of treatment. The level of FGF21 mRNA in cells incubated with no additions was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of five experiments. The asterisk indicates that the mean is significantly (p 0.05) different. F, effects of glucagon, corticosterone, GW0742, TCPOBOP, glucose, insulin, T3, and leptin on FGF21 mRNA abundance. Rat hepatocytes were incubated in serum-free Medium 199 containing low glucose (5.5 mM) or high glucose (25 mM) with or without glucagon (100 nM), corticosterone (1 M), GW0742 (1 M), TCPOBOP (1 M), insulin (50 nM), T3 (150 nM), or leptin (125 nM). FGF21 mRNA abundance was measured after 6 h of treatment. The level of FGF21 mRNA incubated with 5.5 mM glucose and vehicle was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of three experiments. Different superscript letters indicate that means are significantly (p 0.05) different.
Human Fgf21, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
human fgf21 - by Bioz Stars, 2026-03
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serum fgf21  (R&D Systems)
93
R&D Systems serum fgf21
FIGURE 1. Nonesterified unsaturated fatty acids and chylomicron remnants increase hepatic <t>FGF21</t> mRNA abundance. A, nutritional regulation of hepatic FGF21 expression in rats. Sprague-Dawley rats were fed a chow diet, a standard purified diet, or an HF-LC ketogenic diet for 7 days. The composition of the diets is described under “Experimental Procedures.” A fourth group of animals was fed the standard purified diet for 6 days and then starved for 30 h. Animals were killed 6 h after the start of the dark cycle, total RNA was isolated from liver, and the abundance of FGF21 mRNA was measured by quantitative real time PCR. Hepatic FGF21 mRNA abundance was also measured in a fifth group of animals fed a standard purified diet for 6 days followed by starvation for 30 h and refeeding for 5 h. The level of FGF21 mRNA in animals fed the chow diet was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of six animals. An asterisk indicates that the mean is significantly (p 0.05) higher compared with that of animals fed the standard (Std) purified diet. B–F, effect of nutrients and hormones on FGF21 expression in primary cultures of rat hepatocytes. Rat hepatocytes were prepared as described under “Experimental Procedures” and incubated in serum-free Medium 199. B, time course of the effect of nonesterified fatty acids (250 M) on FGF21 mRNA abundance. Hepatocytes were incubated with the indicated fatty acids complexed to BSA. Hepatocytes receiving no additions (NA) were incubated in medium containing an equivalent concentration of BSA. The level of FGF21 mRNA in cells incubated with fatty acids for 0 h was set at 1, and the other values were adjustedproportionately.ValuesaremeansS.E.(errorbars)offourexperiments.C,effectofdifferentoleatetreatmentprotocolsonFGF21mRNAabundance. Hepatocytes were incubated with or without oleate for 2 and 6 h without a medium change. A third treatment group (designated 4 2) was incubated with oleate for 6 h with a medium change 2 h prior to the end of the treatment period. The level of FGF21 mRNA in hepatocytes incubated without oleate for 2 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. Different superscript letters indicate that means are significantly (p 0.05) different. D, time course of the effect of GW7647 on FGF21 mRNA abundance. Hepatocytes were incubated with or without GW7647 (1 M) for the indicated time periods. The level of FGF21 mRNA in cells incubated with GW7647 for 0 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated in the absence of GW7647 for the same time period. E, effect of chylomicrons and chylomicron remnants on FGF21 mRNA abundance. Hepatocytes were incubated with chylomicrons and chylomicron remnants prepared from rats fed safflower oil. FGF21 mRNA was measured after 2 h of treatment. The level of FGF21 mRNA in cells incubated with no additions was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of five experiments. The asterisk indicates that the mean is significantly (p 0.05) different. F, effects of glucagon, corticosterone, GW0742, TCPOBOP, glucose, insulin, T3, and leptin on FGF21 mRNA abundance. Rat hepatocytes were incubated in serum-free Medium 199 containing low glucose (5.5 mM) or high glucose (25 mM) with or without glucagon (100 nM), corticosterone (1 M), GW0742 (1 M), TCPOBOP (1 M), insulin (50 nM), T3 (150 nM), or leptin (125 nM). FGF21 mRNA abundance was measured after 6 h of treatment. The level of FGF21 mRNA incubated with 5.5 mM glucose and vehicle was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of three experiments. Different superscript letters indicate that means are significantly (p 0.05) different.
Serum Fgf21, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews
serum fgf21 - by Bioz Stars, 2026-03
93/100 stars
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human fgf21 dy2539 assay kit  (R&D Systems)
94
R&D Systems human fgf21 dy2539 assay kit
FIGURE 1. Nonesterified unsaturated fatty acids and chylomicron remnants increase hepatic <t>FGF21</t> mRNA abundance. A, nutritional regulation of hepatic FGF21 expression in rats. Sprague-Dawley rats were fed a chow diet, a standard purified diet, or an HF-LC ketogenic diet for 7 days. The composition of the diets is described under “Experimental Procedures.” A fourth group of animals was fed the standard purified diet for 6 days and then starved for 30 h. Animals were killed 6 h after the start of the dark cycle, total RNA was isolated from liver, and the abundance of FGF21 mRNA was measured by quantitative real time PCR. Hepatic FGF21 mRNA abundance was also measured in a fifth group of animals fed a standard purified diet for 6 days followed by starvation for 30 h and refeeding for 5 h. The level of FGF21 mRNA in animals fed the chow diet was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of six animals. An asterisk indicates that the mean is significantly (p 0.05) higher compared with that of animals fed the standard (Std) purified diet. B–F, effect of nutrients and hormones on FGF21 expression in primary cultures of rat hepatocytes. Rat hepatocytes were prepared as described under “Experimental Procedures” and incubated in serum-free Medium 199. B, time course of the effect of nonesterified fatty acids (250 M) on FGF21 mRNA abundance. Hepatocytes were incubated with the indicated fatty acids complexed to BSA. Hepatocytes receiving no additions (NA) were incubated in medium containing an equivalent concentration of BSA. The level of FGF21 mRNA in cells incubated with fatty acids for 0 h was set at 1, and the other values were adjustedproportionately.ValuesaremeansS.E.(errorbars)offourexperiments.C,effectofdifferentoleatetreatmentprotocolsonFGF21mRNAabundance. Hepatocytes were incubated with or without oleate for 2 and 6 h without a medium change. A third treatment group (designated 4 2) was incubated with oleate for 6 h with a medium change 2 h prior to the end of the treatment period. The level of FGF21 mRNA in hepatocytes incubated without oleate for 2 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. Different superscript letters indicate that means are significantly (p 0.05) different. D, time course of the effect of GW7647 on FGF21 mRNA abundance. Hepatocytes were incubated with or without GW7647 (1 M) for the indicated time periods. The level of FGF21 mRNA in cells incubated with GW7647 for 0 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated in the absence of GW7647 for the same time period. E, effect of chylomicrons and chylomicron remnants on FGF21 mRNA abundance. Hepatocytes were incubated with chylomicrons and chylomicron remnants prepared from rats fed safflower oil. FGF21 mRNA was measured after 2 h of treatment. The level of FGF21 mRNA in cells incubated with no additions was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of five experiments. The asterisk indicates that the mean is significantly (p 0.05) different. F, effects of glucagon, corticosterone, GW0742, TCPOBOP, glucose, insulin, T3, and leptin on FGF21 mRNA abundance. Rat hepatocytes were incubated in serum-free Medium 199 containing low glucose (5.5 mM) or high glucose (25 mM) with or without glucagon (100 nM), corticosterone (1 M), GW0742 (1 M), TCPOBOP (1 M), insulin (50 nM), T3 (150 nM), or leptin (125 nM). FGF21 mRNA abundance was measured after 6 h of treatment. The level of FGF21 mRNA incubated with 5.5 mM glucose and vehicle was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of three experiments. Different superscript letters indicate that means are significantly (p 0.05) different.
Human Fgf21 Dy2539 Assay Kit, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 1 article reviews
human fgf21 dy2539 assay kit - by Bioz Stars, 2026-03
94/100 stars
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polypeptide  (R&D Systems)
93
R&D Systems polypeptide
FIGURE 1. Nonesterified unsaturated fatty acids and chylomicron remnants increase hepatic <t>FGF21</t> mRNA abundance. A, nutritional regulation of hepatic FGF21 expression in rats. Sprague-Dawley rats were fed a chow diet, a standard purified diet, or an HF-LC ketogenic diet for 7 days. The composition of the diets is described under “Experimental Procedures.” A fourth group of animals was fed the standard purified diet for 6 days and then starved for 30 h. Animals were killed 6 h after the start of the dark cycle, total RNA was isolated from liver, and the abundance of FGF21 mRNA was measured by quantitative real time PCR. Hepatic FGF21 mRNA abundance was also measured in a fifth group of animals fed a standard purified diet for 6 days followed by starvation for 30 h and refeeding for 5 h. The level of FGF21 mRNA in animals fed the chow diet was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of six animals. An asterisk indicates that the mean is significantly (p 0.05) higher compared with that of animals fed the standard (Std) purified diet. B–F, effect of nutrients and hormones on FGF21 expression in primary cultures of rat hepatocytes. Rat hepatocytes were prepared as described under “Experimental Procedures” and incubated in serum-free Medium 199. B, time course of the effect of nonesterified fatty acids (250 M) on FGF21 mRNA abundance. Hepatocytes were incubated with the indicated fatty acids complexed to BSA. Hepatocytes receiving no additions (NA) were incubated in medium containing an equivalent concentration of BSA. The level of FGF21 mRNA in cells incubated with fatty acids for 0 h was set at 1, and the other values were adjustedproportionately.ValuesaremeansS.E.(errorbars)offourexperiments.C,effectofdifferentoleatetreatmentprotocolsonFGF21mRNAabundance. Hepatocytes were incubated with or without oleate for 2 and 6 h without a medium change. A third treatment group (designated 4 2) was incubated with oleate for 6 h with a medium change 2 h prior to the end of the treatment period. The level of FGF21 mRNA in hepatocytes incubated without oleate for 2 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. Different superscript letters indicate that means are significantly (p 0.05) different. D, time course of the effect of GW7647 on FGF21 mRNA abundance. Hepatocytes were incubated with or without GW7647 (1 M) for the indicated time periods. The level of FGF21 mRNA in cells incubated with GW7647 for 0 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated in the absence of GW7647 for the same time period. E, effect of chylomicrons and chylomicron remnants on FGF21 mRNA abundance. Hepatocytes were incubated with chylomicrons and chylomicron remnants prepared from rats fed safflower oil. FGF21 mRNA was measured after 2 h of treatment. The level of FGF21 mRNA in cells incubated with no additions was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of five experiments. The asterisk indicates that the mean is significantly (p 0.05) different. F, effects of glucagon, corticosterone, GW0742, TCPOBOP, glucose, insulin, T3, and leptin on FGF21 mRNA abundance. Rat hepatocytes were incubated in serum-free Medium 199 containing low glucose (5.5 mM) or high glucose (25 mM) with or without glucagon (100 nM), corticosterone (1 M), GW0742 (1 M), TCPOBOP (1 M), insulin (50 nM), T3 (150 nM), or leptin (125 nM). FGF21 mRNA abundance was measured after 6 h of treatment. The level of FGF21 mRNA incubated with 5.5 mM glucose and vehicle was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of three experiments. Different superscript letters indicate that means are significantly (p 0.05) different.
Polypeptide, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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biotin goat anti human fgf 21 igg pab  (R&D Systems)
94
R&D Systems biotin goat anti human fgf 21 igg pab
FIGURE 1. Nonesterified unsaturated fatty acids and chylomicron remnants increase hepatic <t>FGF21</t> mRNA abundance. A, nutritional regulation of hepatic FGF21 expression in rats. Sprague-Dawley rats were fed a chow diet, a standard purified diet, or an HF-LC ketogenic diet for 7 days. The composition of the diets is described under “Experimental Procedures.” A fourth group of animals was fed the standard purified diet for 6 days and then starved for 30 h. Animals were killed 6 h after the start of the dark cycle, total RNA was isolated from liver, and the abundance of FGF21 mRNA was measured by quantitative real time PCR. Hepatic FGF21 mRNA abundance was also measured in a fifth group of animals fed a standard purified diet for 6 days followed by starvation for 30 h and refeeding for 5 h. The level of FGF21 mRNA in animals fed the chow diet was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of six animals. An asterisk indicates that the mean is significantly (p 0.05) higher compared with that of animals fed the standard (Std) purified diet. B–F, effect of nutrients and hormones on FGF21 expression in primary cultures of rat hepatocytes. Rat hepatocytes were prepared as described under “Experimental Procedures” and incubated in serum-free Medium 199. B, time course of the effect of nonesterified fatty acids (250 M) on FGF21 mRNA abundance. Hepatocytes were incubated with the indicated fatty acids complexed to BSA. Hepatocytes receiving no additions (NA) were incubated in medium containing an equivalent concentration of BSA. The level of FGF21 mRNA in cells incubated with fatty acids for 0 h was set at 1, and the other values were adjustedproportionately.ValuesaremeansS.E.(errorbars)offourexperiments.C,effectofdifferentoleatetreatmentprotocolsonFGF21mRNAabundance. Hepatocytes were incubated with or without oleate for 2 and 6 h without a medium change. A third treatment group (designated 4 2) was incubated with oleate for 6 h with a medium change 2 h prior to the end of the treatment period. The level of FGF21 mRNA in hepatocytes incubated without oleate for 2 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. Different superscript letters indicate that means are significantly (p 0.05) different. D, time course of the effect of GW7647 on FGF21 mRNA abundance. Hepatocytes were incubated with or without GW7647 (1 M) for the indicated time periods. The level of FGF21 mRNA in cells incubated with GW7647 for 0 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated in the absence of GW7647 for the same time period. E, effect of chylomicrons and chylomicron remnants on FGF21 mRNA abundance. Hepatocytes were incubated with chylomicrons and chylomicron remnants prepared from rats fed safflower oil. FGF21 mRNA was measured after 2 h of treatment. The level of FGF21 mRNA in cells incubated with no additions was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of five experiments. The asterisk indicates that the mean is significantly (p 0.05) different. F, effects of glucagon, corticosterone, GW0742, TCPOBOP, glucose, insulin, T3, and leptin on FGF21 mRNA abundance. Rat hepatocytes were incubated in serum-free Medium 199 containing low glucose (5.5 mM) or high glucose (25 mM) with or without glucagon (100 nM), corticosterone (1 M), GW0742 (1 M), TCPOBOP (1 M), insulin (50 nM), T3 (150 nM), or leptin (125 nM). FGF21 mRNA abundance was measured after 6 h of treatment. The level of FGF21 mRNA incubated with 5.5 mM glucose and vehicle was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of three experiments. Different superscript letters indicate that means are significantly (p 0.05) different.
Biotin Goat Anti Human Fgf 21 Igg Pab, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 1 article reviews
biotin goat anti human fgf 21 igg pab - by Bioz Stars, 2026-03
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goat anti human fgf 21 igg polyclonal antibody  (R&D Systems)
94
R&D Systems goat anti human fgf 21 igg polyclonal antibody
FIGURE 1. Nonesterified unsaturated fatty acids and chylomicron remnants increase hepatic <t>FGF21</t> mRNA abundance. A, nutritional regulation of hepatic FGF21 expression in rats. Sprague-Dawley rats were fed a chow diet, a standard purified diet, or an HF-LC ketogenic diet for 7 days. The composition of the diets is described under “Experimental Procedures.” A fourth group of animals was fed the standard purified diet for 6 days and then starved for 30 h. Animals were killed 6 h after the start of the dark cycle, total RNA was isolated from liver, and the abundance of FGF21 mRNA was measured by quantitative real time PCR. Hepatic FGF21 mRNA abundance was also measured in a fifth group of animals fed a standard purified diet for 6 days followed by starvation for 30 h and refeeding for 5 h. The level of FGF21 mRNA in animals fed the chow diet was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of six animals. An asterisk indicates that the mean is significantly (p 0.05) higher compared with that of animals fed the standard (Std) purified diet. B–F, effect of nutrients and hormones on FGF21 expression in primary cultures of rat hepatocytes. Rat hepatocytes were prepared as described under “Experimental Procedures” and incubated in serum-free Medium 199. B, time course of the effect of nonesterified fatty acids (250 M) on FGF21 mRNA abundance. Hepatocytes were incubated with the indicated fatty acids complexed to BSA. Hepatocytes receiving no additions (NA) were incubated in medium containing an equivalent concentration of BSA. The level of FGF21 mRNA in cells incubated with fatty acids for 0 h was set at 1, and the other values were adjustedproportionately.ValuesaremeansS.E.(errorbars)offourexperiments.C,effectofdifferentoleatetreatmentprotocolsonFGF21mRNAabundance. Hepatocytes were incubated with or without oleate for 2 and 6 h without a medium change. A third treatment group (designated 4 2) was incubated with oleate for 6 h with a medium change 2 h prior to the end of the treatment period. The level of FGF21 mRNA in hepatocytes incubated without oleate for 2 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. Different superscript letters indicate that means are significantly (p 0.05) different. D, time course of the effect of GW7647 on FGF21 mRNA abundance. Hepatocytes were incubated with or without GW7647 (1 M) for the indicated time periods. The level of FGF21 mRNA in cells incubated with GW7647 for 0 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated in the absence of GW7647 for the same time period. E, effect of chylomicrons and chylomicron remnants on FGF21 mRNA abundance. Hepatocytes were incubated with chylomicrons and chylomicron remnants prepared from rats fed safflower oil. FGF21 mRNA was measured after 2 h of treatment. The level of FGF21 mRNA in cells incubated with no additions was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of five experiments. The asterisk indicates that the mean is significantly (p 0.05) different. F, effects of glucagon, corticosterone, GW0742, TCPOBOP, glucose, insulin, T3, and leptin on FGF21 mRNA abundance. Rat hepatocytes were incubated in serum-free Medium 199 containing low glucose (5.5 mM) or high glucose (25 mM) with or without glucagon (100 nM), corticosterone (1 M), GW0742 (1 M), TCPOBOP (1 M), insulin (50 nM), T3 (150 nM), or leptin (125 nM). FGF21 mRNA abundance was measured after 6 h of treatment. The level of FGF21 mRNA incubated with 5.5 mM glucose and vehicle was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of three experiments. Different superscript letters indicate that means are significantly (p 0.05) different.
Goat Anti Human Fgf 21 Igg Polyclonal Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/goat anti human fgf 21 igg polyclonal antibody/product/R&D Systems
Average 94 stars, based on 1 article reviews
goat anti human fgf 21 igg polyclonal antibody - by Bioz Stars, 2026-03
94/100 stars
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hfgf21  (R&D Systems)
92
R&D Systems hfgf21
FIGURE 1. Nonesterified unsaturated fatty acids and chylomicron remnants increase hepatic <t>FGF21</t> mRNA abundance. A, nutritional regulation of hepatic FGF21 expression in rats. Sprague-Dawley rats were fed a chow diet, a standard purified diet, or an HF-LC ketogenic diet for 7 days. The composition of the diets is described under “Experimental Procedures.” A fourth group of animals was fed the standard purified diet for 6 days and then starved for 30 h. Animals were killed 6 h after the start of the dark cycle, total RNA was isolated from liver, and the abundance of FGF21 mRNA was measured by quantitative real time PCR. Hepatic FGF21 mRNA abundance was also measured in a fifth group of animals fed a standard purified diet for 6 days followed by starvation for 30 h and refeeding for 5 h. The level of FGF21 mRNA in animals fed the chow diet was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of six animals. An asterisk indicates that the mean is significantly (p 0.05) higher compared with that of animals fed the standard (Std) purified diet. B–F, effect of nutrients and hormones on FGF21 expression in primary cultures of rat hepatocytes. Rat hepatocytes were prepared as described under “Experimental Procedures” and incubated in serum-free Medium 199. B, time course of the effect of nonesterified fatty acids (250 M) on FGF21 mRNA abundance. Hepatocytes were incubated with the indicated fatty acids complexed to BSA. Hepatocytes receiving no additions (NA) were incubated in medium containing an equivalent concentration of BSA. The level of FGF21 mRNA in cells incubated with fatty acids for 0 h was set at 1, and the other values were adjustedproportionately.ValuesaremeansS.E.(errorbars)offourexperiments.C,effectofdifferentoleatetreatmentprotocolsonFGF21mRNAabundance. Hepatocytes were incubated with or without oleate for 2 and 6 h without a medium change. A third treatment group (designated 4 2) was incubated with oleate for 6 h with a medium change 2 h prior to the end of the treatment period. The level of FGF21 mRNA in hepatocytes incubated without oleate for 2 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. Different superscript letters indicate that means are significantly (p 0.05) different. D, time course of the effect of GW7647 on FGF21 mRNA abundance. Hepatocytes were incubated with or without GW7647 (1 M) for the indicated time periods. The level of FGF21 mRNA in cells incubated with GW7647 for 0 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated in the absence of GW7647 for the same time period. E, effect of chylomicrons and chylomicron remnants on FGF21 mRNA abundance. Hepatocytes were incubated with chylomicrons and chylomicron remnants prepared from rats fed safflower oil. FGF21 mRNA was measured after 2 h of treatment. The level of FGF21 mRNA in cells incubated with no additions was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of five experiments. The asterisk indicates that the mean is significantly (p 0.05) different. F, effects of glucagon, corticosterone, GW0742, TCPOBOP, glucose, insulin, T3, and leptin on FGF21 mRNA abundance. Rat hepatocytes were incubated in serum-free Medium 199 containing low glucose (5.5 mM) or high glucose (25 mM) with or without glucagon (100 nM), corticosterone (1 M), GW0742 (1 M), TCPOBOP (1 M), insulin (50 nM), T3 (150 nM), or leptin (125 nM). FGF21 mRNA abundance was measured after 6 h of treatment. The level of FGF21 mRNA incubated with 5.5 mM glucose and vehicle was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of three experiments. Different superscript letters indicate that means are significantly (p 0.05) different.
Hfgf21, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/hfgf21/product/R&D Systems
Average 92 stars, based on 1 article reviews
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mouse monoclonal anti fgf21 antibody  (R&D Systems)
90
R&D Systems mouse monoclonal anti fgf21 antibody
(A) Cold-induced increase in serum <t>FGF21</t> levels was blunted by injection of miR-32-ASO as measured by ELISA (n = 7). (B) qRT-PCR showed that cold stress decreased FGF21 mRNA expression in liver but increased it in BAT. Injection of miR-32-ASO significantly repressed the increase in FGF21 expression in BAT during cold exposure when normalized to PPIA . (C) FGF21 immunostaining showed that cold stress decreased FGF21 level in liver but increased it in BAT. Injection of miR-32-ASO significantly repressed the increase in FGF21 expression in BAT during cold exposure. (D) Quantification of FGF21 immunostaining in BAT showed that FGF21 levels within BAT increased greatly after cold exposure but were significantly lower in miR-32-ASO-treated mice compared to control-ASO-treated mice. (E) Quantification of FGF21 immunostaining in liver showed that FGF21 levels within liver decreased greatly after cold exposure but were similar between miR-32-ASO-treated mice and control-ASO-treated mice. (F) Western blot showing cold-induced increases in BAT FGF21 protein levels were blunted by injection of miR-32-ASO. Calnexin served as a loading control. (G) Western blot showing cold-induced decreases in liver FGF21 protein levels were unaffected by injection of miR-32-ASO. Calnexin served as a loading control. (H) Quantification of the western blot results in (F) and (G) using ImageJ. Average intensities were normalized to that of Calnexin. (I) FGF21 mRNA expression in WT-1 cells increased or decreased when transfected with miR-32 mimic or miR-32-ASO, respectively. (J) Western blotting showed that FGF21 protein levels in WT-1 cells increased or decreased when transfected with miR-32 mimic or miR-32-ASO, respectively. Data represent mean ± SEM. *p < 0.05 and ** p < 0.01. See also .
Mouse Monoclonal Anti Fgf21 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse monoclonal anti fgf21 antibody/product/R&D Systems
Average 90 stars, based on 1 article reviews
mouse monoclonal anti fgf21 antibody - by Bioz Stars, 2026-03
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FIGURE 2 | Activating effects of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and FGF21 (20 nmol L−1), in the presence of PD173074 (FGFR1 inhibitor, 50 nmol L−1) or palmitic acid (PA, 500 µmol L−1), on FGF21 signaling in HepG2 human hepatocytes. Coffee by-product’s bioactive compounds interacted in silico with the subunits of the FGF21 receptor (FGFR) (A), exhibiting different binding (Continued)

Journal: Frontiers in nutrition

Article Title: Activating Effects of the Bioactive Compounds From Coffee By-Products on FGF21 Signaling Modulate Hepatic Mitochondrial Bioenergetics and Energy Metabolism in vitro .

doi: 10.3389/fnut.2022.866233

Figure Lengend Snippet: FIGURE 2 | Activating effects of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and FGF21 (20 nmol L−1), in the presence of PD173074 (FGFR1 inhibitor, 50 nmol L−1) or palmitic acid (PA, 500 µmol L−1), on FGF21 signaling in HepG2 human hepatocytes. Coffee by-product’s bioactive compounds interacted in silico with the subunits of the FGF21 receptor (FGFR) (A), exhibiting different binding (Continued)

Article Snippet: Recombinant human FGF21 was obtained from R&D Systems (Minneapolis, MN, USA), whereas the FGFR Frontiers in Nutrition | www.frontiersin.org 2 March 2022 | Volume 9 | Article 866233 inhibition, PD173074, was purchased from AdoQ Bioscience (Irvine, CA, USA).

Techniques: In Silico, Binding Assay

FIGURE 4 | Role of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and FGF21 (20 nmol L−1), in the presence of palmitic acid (PA, 500 µmol L−1) on lipo/cytotoxicity and inflammation markers in HepG2 human hepatocytes. Coffee by-products’ bioactive compounds preserved cell viability (A) and diminished lactate dehydrogenase (LDH) release (B). PA-triggered tumor necrosis factor α (TNF-α) (C), interleukin (IL)-6 (D), and IL-1β (E) release and nitric oxide synthase (NOS) activity (F) were reduced. Hierarchical cluster analysis and heat map [from the lowest (■red) to the highest (■green) value for each parameter] (G) and an integrative diagram illustrating the effects of the bioactive compounds from coffee by-products on lipo/cytotoxicity and inflammation (H). The results are expressed as mean ± SD (n = 3). Bars with different letters significantly (p < 0.05) differ according to ANOVA and Tukey’s multiple range test. NT, non-treated cells; CAF, caffeine; CGA, chlorogenic acid; CA, caffeic acid; PCA, protocatechuic acid; GA, gallic acid; KMP, kaempferol; FGF21, fibroblast growth factor 21.

Journal: Frontiers in nutrition

Article Title: Activating Effects of the Bioactive Compounds From Coffee By-Products on FGF21 Signaling Modulate Hepatic Mitochondrial Bioenergetics and Energy Metabolism in vitro .

doi: 10.3389/fnut.2022.866233

Figure Lengend Snippet: FIGURE 4 | Role of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and FGF21 (20 nmol L−1), in the presence of palmitic acid (PA, 500 µmol L−1) on lipo/cytotoxicity and inflammation markers in HepG2 human hepatocytes. Coffee by-products’ bioactive compounds preserved cell viability (A) and diminished lactate dehydrogenase (LDH) release (B). PA-triggered tumor necrosis factor α (TNF-α) (C), interleukin (IL)-6 (D), and IL-1β (E) release and nitric oxide synthase (NOS) activity (F) were reduced. Hierarchical cluster analysis and heat map [from the lowest (■red) to the highest (■green) value for each parameter] (G) and an integrative diagram illustrating the effects of the bioactive compounds from coffee by-products on lipo/cytotoxicity and inflammation (H). The results are expressed as mean ± SD (n = 3). Bars with different letters significantly (p < 0.05) differ according to ANOVA and Tukey’s multiple range test. NT, non-treated cells; CAF, caffeine; CGA, chlorogenic acid; CA, caffeic acid; PCA, protocatechuic acid; GA, gallic acid; KMP, kaempferol; FGF21, fibroblast growth factor 21.

Article Snippet: Recombinant human FGF21 was obtained from R&D Systems (Minneapolis, MN, USA), whereas the FGFR Frontiers in Nutrition | www.frontiersin.org 2 March 2022 | Volume 9 | Article 866233 inhibition, PD173074, was purchased from AdoQ Bioscience (Irvine, CA, USA).

Techniques: Activity Assay

FIGURE 5 | Protective effects of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and FGF21 (20 nmol L−1), in the presence of palmitic acid (PA, 500 µmol L−1) against oxidative stress in HepG2 human hepatocytes. Coffee by-products’ bioactive compounds diminished the production of reactive oxygen species (ROS) (A) and mitochondrial O•− 2 (B), preserving the mitochondrial membrane potential (19m) (C) in PA-challenged HepG2 cells. The enzymatic activity NADPH oxidase (NOX) (D), superoxide dismutase (SOD) (E), and catalase (F) activities, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) phosphorylation (G) were regulated, thereby diminishing oxidative stress. Hierarchical cluster analysis and heat map [from the lowest (■red) to the highest (■green) value for each parameter] (H) and an integrative diagram illustrating the effects of the bioactive compounds from coffee by-products on oxidative stress (I). The results are expressed as mean ± SD (n = 3). Bars with different letters significantly (p < 0.05) differ according to ANOVA and Tukey’s multiple range test. NT, non-treated cells; CAF, caffeine; CGA, chlorogenic acid; CA, caffeic acid; PCA, protocatechuic acid; GA, gallic acid; KMP, kaempferol; FGF21, fibroblast growth factor 21.

Journal: Frontiers in nutrition

Article Title: Activating Effects of the Bioactive Compounds From Coffee By-Products on FGF21 Signaling Modulate Hepatic Mitochondrial Bioenergetics and Energy Metabolism in vitro .

doi: 10.3389/fnut.2022.866233

Figure Lengend Snippet: FIGURE 5 | Protective effects of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and FGF21 (20 nmol L−1), in the presence of palmitic acid (PA, 500 µmol L−1) against oxidative stress in HepG2 human hepatocytes. Coffee by-products’ bioactive compounds diminished the production of reactive oxygen species (ROS) (A) and mitochondrial O•− 2 (B), preserving the mitochondrial membrane potential (19m) (C) in PA-challenged HepG2 cells. The enzymatic activity NADPH oxidase (NOX) (D), superoxide dismutase (SOD) (E), and catalase (F) activities, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) phosphorylation (G) were regulated, thereby diminishing oxidative stress. Hierarchical cluster analysis and heat map [from the lowest (■red) to the highest (■green) value for each parameter] (H) and an integrative diagram illustrating the effects of the bioactive compounds from coffee by-products on oxidative stress (I). The results are expressed as mean ± SD (n = 3). Bars with different letters significantly (p < 0.05) differ according to ANOVA and Tukey’s multiple range test. NT, non-treated cells; CAF, caffeine; CGA, chlorogenic acid; CA, caffeic acid; PCA, protocatechuic acid; GA, gallic acid; KMP, kaempferol; FGF21, fibroblast growth factor 21.

Article Snippet: Recombinant human FGF21 was obtained from R&D Systems (Minneapolis, MN, USA), whereas the FGFR Frontiers in Nutrition | www.frontiersin.org 2 March 2022 | Volume 9 | Article 866233 inhibition, PD173074, was purchased from AdoQ Bioscience (Irvine, CA, USA).

Techniques: Preserving, Membrane, Activity Assay, Derivative Assay, Phospho-proteomics

FIGURE 6 | Regulative role of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and FGF21 (20 nmol L−1), in the presence of palmitic acid (500 µmol L−1) on the mitochondrial bioenergetics of HepG2 human hepatocytes. Coffee (Continued)

Journal: Frontiers in nutrition

Article Title: Activating Effects of the Bioactive Compounds From Coffee By-Products on FGF21 Signaling Modulate Hepatic Mitochondrial Bioenergetics and Energy Metabolism in vitro .

doi: 10.3389/fnut.2022.866233

Figure Lengend Snippet: FIGURE 6 | Regulative role of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and FGF21 (20 nmol L−1), in the presence of palmitic acid (500 µmol L−1) on the mitochondrial bioenergetics of HepG2 human hepatocytes. Coffee (Continued)

Article Snippet: Recombinant human FGF21 was obtained from R&D Systems (Minneapolis, MN, USA), whereas the FGFR Frontiers in Nutrition | www.frontiersin.org 2 March 2022 | Volume 9 | Article 866233 inhibition, PD173074, was purchased from AdoQ Bioscience (Irvine, CA, USA).

Techniques:

FIGURE 7 | Modulatory effects of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and FGF21 (20 nmol L−1), in the presence of palmitic acid (500 µmol L−1) on lipid metabolism in HepG2 human hepatocytes. Palmitic acid-treated (Continued)

Journal: Frontiers in nutrition

Article Title: Activating Effects of the Bioactive Compounds From Coffee By-Products on FGF21 Signaling Modulate Hepatic Mitochondrial Bioenergetics and Energy Metabolism in vitro .

doi: 10.3389/fnut.2022.866233

Figure Lengend Snippet: FIGURE 7 | Modulatory effects of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and FGF21 (20 nmol L−1), in the presence of palmitic acid (500 µmol L−1) on lipid metabolism in HepG2 human hepatocytes. Palmitic acid-treated (Continued)

Article Snippet: Recombinant human FGF21 was obtained from R&D Systems (Minneapolis, MN, USA), whereas the FGFR Frontiers in Nutrition | www.frontiersin.org 2 March 2022 | Volume 9 | Article 866233 inhibition, PD173074, was purchased from AdoQ Bioscience (Irvine, CA, USA).

Techniques:

FIGURE 8 | Regulatory role of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and FGF21 (20 nmol L−1), in the presence of palmitic acid (500 µmol L−1) on glucose metabolism in HepG2 human hepatocytes. Hepatocytes exhibited a modulation on glucose uptake (A), glucokinase (GK) activity (B), glucose production (C), phosphoenolpyruvate carboxykinase (PEPCK) activity (D), insulin receptor (Continued)

Journal: Frontiers in nutrition

Article Title: Activating Effects of the Bioactive Compounds From Coffee By-Products on FGF21 Signaling Modulate Hepatic Mitochondrial Bioenergetics and Energy Metabolism in vitro .

doi: 10.3389/fnut.2022.866233

Figure Lengend Snippet: FIGURE 8 | Regulatory role of standard solutions of the bioactive compounds from coffee by-products (50 µmol L−1), aqueous extracts (CSE and CHE, 100 µg mL−1), and FGF21 (20 nmol L−1), in the presence of palmitic acid (500 µmol L−1) on glucose metabolism in HepG2 human hepatocytes. Hepatocytes exhibited a modulation on glucose uptake (A), glucokinase (GK) activity (B), glucose production (C), phosphoenolpyruvate carboxykinase (PEPCK) activity (D), insulin receptor (Continued)

Article Snippet: Recombinant human FGF21 was obtained from R&D Systems (Minneapolis, MN, USA), whereas the FGFR Frontiers in Nutrition | www.frontiersin.org 2 March 2022 | Volume 9 | Article 866233 inhibition, PD173074, was purchased from AdoQ Bioscience (Irvine, CA, USA).

Techniques: Activity Assay

FIGURE 9 | Integrative hierarchical cluster analysis and heat map (from the lowest (■red) to the highest (■green) value for each parameter) unifying all parameters demonstrates that chlorogenic and protocatechuic acids are the bioactive compounds exhibiting the highest NAFLD-protecting effects (A). Diagram illustrating the molecular mechanisms from the effects of the bioactive compounds in coffee by-products on hepatic FGF21 signaling, oxidative stress, mitochondrial bioenergetics, and energy metabolism (B). NT, non-treated cells; PA, palmitic acid; CAF, caffeine; CGA, chlorogenic acid; CA, caffeic acid; PCA, protocatechuic acid; GA, gallic acid; KMP, kaempferol; FGF21, fibroblast growth factor 21.

Journal: Frontiers in nutrition

Article Title: Activating Effects of the Bioactive Compounds From Coffee By-Products on FGF21 Signaling Modulate Hepatic Mitochondrial Bioenergetics and Energy Metabolism in vitro .

doi: 10.3389/fnut.2022.866233

Figure Lengend Snippet: FIGURE 9 | Integrative hierarchical cluster analysis and heat map (from the lowest (■red) to the highest (■green) value for each parameter) unifying all parameters demonstrates that chlorogenic and protocatechuic acids are the bioactive compounds exhibiting the highest NAFLD-protecting effects (A). Diagram illustrating the molecular mechanisms from the effects of the bioactive compounds in coffee by-products on hepatic FGF21 signaling, oxidative stress, mitochondrial bioenergetics, and energy metabolism (B). NT, non-treated cells; PA, palmitic acid; CAF, caffeine; CGA, chlorogenic acid; CA, caffeic acid; PCA, protocatechuic acid; GA, gallic acid; KMP, kaempferol; FGF21, fibroblast growth factor 21.

Article Snippet: Recombinant human FGF21 was obtained from R&D Systems (Minneapolis, MN, USA), whereas the FGFR Frontiers in Nutrition | www.frontiersin.org 2 March 2022 | Volume 9 | Article 866233 inhibition, PD173074, was purchased from AdoQ Bioscience (Irvine, CA, USA).

Techniques:

FIGURE 1. Nonesterified unsaturated fatty acids and chylomicron remnants increase hepatic FGF21 mRNA abundance. A, nutritional regulation of hepatic FGF21 expression in rats. Sprague-Dawley rats were fed a chow diet, a standard purified diet, or an HF-LC ketogenic diet for 7 days. The composition of the diets is described under “Experimental Procedures.” A fourth group of animals was fed the standard purified diet for 6 days and then starved for 30 h. Animals were killed 6 h after the start of the dark cycle, total RNA was isolated from liver, and the abundance of FGF21 mRNA was measured by quantitative real time PCR. Hepatic FGF21 mRNA abundance was also measured in a fifth group of animals fed a standard purified diet for 6 days followed by starvation for 30 h and refeeding for 5 h. The level of FGF21 mRNA in animals fed the chow diet was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of six animals. An asterisk indicates that the mean is significantly (p 0.05) higher compared with that of animals fed the standard (Std) purified diet. B–F, effect of nutrients and hormones on FGF21 expression in primary cultures of rat hepatocytes. Rat hepatocytes were prepared as described under “Experimental Procedures” and incubated in serum-free Medium 199. B, time course of the effect of nonesterified fatty acids (250 M) on FGF21 mRNA abundance. Hepatocytes were incubated with the indicated fatty acids complexed to BSA. Hepatocytes receiving no additions (NA) were incubated in medium containing an equivalent concentration of BSA. The level of FGF21 mRNA in cells incubated with fatty acids for 0 h was set at 1, and the other values were adjustedproportionately.ValuesaremeansS.E.(errorbars)offourexperiments.C,effectofdifferentoleatetreatmentprotocolsonFGF21mRNAabundance. Hepatocytes were incubated with or without oleate for 2 and 6 h without a medium change. A third treatment group (designated 4 2) was incubated with oleate for 6 h with a medium change 2 h prior to the end of the treatment period. The level of FGF21 mRNA in hepatocytes incubated without oleate for 2 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. Different superscript letters indicate that means are significantly (p 0.05) different. D, time course of the effect of GW7647 on FGF21 mRNA abundance. Hepatocytes were incubated with or without GW7647 (1 M) for the indicated time periods. The level of FGF21 mRNA in cells incubated with GW7647 for 0 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated in the absence of GW7647 for the same time period. E, effect of chylomicrons and chylomicron remnants on FGF21 mRNA abundance. Hepatocytes were incubated with chylomicrons and chylomicron remnants prepared from rats fed safflower oil. FGF21 mRNA was measured after 2 h of treatment. The level of FGF21 mRNA in cells incubated with no additions was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of five experiments. The asterisk indicates that the mean is significantly (p 0.05) different. F, effects of glucagon, corticosterone, GW0742, TCPOBOP, glucose, insulin, T3, and leptin on FGF21 mRNA abundance. Rat hepatocytes were incubated in serum-free Medium 199 containing low glucose (5.5 mM) or high glucose (25 mM) with or without glucagon (100 nM), corticosterone (1 M), GW0742 (1 M), TCPOBOP (1 M), insulin (50 nM), T3 (150 nM), or leptin (125 nM). FGF21 mRNA abundance was measured after 6 h of treatment. The level of FGF21 mRNA incubated with 5.5 mM glucose and vehicle was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of three experiments. Different superscript letters indicate that means are significantly (p 0.05) different.

Journal: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE 1. Nonesterified unsaturated fatty acids and chylomicron remnants increase hepatic FGF21 mRNA abundance. A, nutritional regulation of hepatic FGF21 expression in rats. Sprague-Dawley rats were fed a chow diet, a standard purified diet, or an HF-LC ketogenic diet for 7 days. The composition of the diets is described under “Experimental Procedures.” A fourth group of animals was fed the standard purified diet for 6 days and then starved for 30 h. Animals were killed 6 h after the start of the dark cycle, total RNA was isolated from liver, and the abundance of FGF21 mRNA was measured by quantitative real time PCR. Hepatic FGF21 mRNA abundance was also measured in a fifth group of animals fed a standard purified diet for 6 days followed by starvation for 30 h and refeeding for 5 h. The level of FGF21 mRNA in animals fed the chow diet was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of six animals. An asterisk indicates that the mean is significantly (p 0.05) higher compared with that of animals fed the standard (Std) purified diet. B–F, effect of nutrients and hormones on FGF21 expression in primary cultures of rat hepatocytes. Rat hepatocytes were prepared as described under “Experimental Procedures” and incubated in serum-free Medium 199. B, time course of the effect of nonesterified fatty acids (250 M) on FGF21 mRNA abundance. Hepatocytes were incubated with the indicated fatty acids complexed to BSA. Hepatocytes receiving no additions (NA) were incubated in medium containing an equivalent concentration of BSA. The level of FGF21 mRNA in cells incubated with fatty acids for 0 h was set at 1, and the other values were adjustedproportionately.ValuesaremeansS.E.(errorbars)offourexperiments.C,effectofdifferentoleatetreatmentprotocolsonFGF21mRNAabundance. Hepatocytes were incubated with or without oleate for 2 and 6 h without a medium change. A third treatment group (designated 4 2) was incubated with oleate for 6 h with a medium change 2 h prior to the end of the treatment period. The level of FGF21 mRNA in hepatocytes incubated without oleate for 2 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. Different superscript letters indicate that means are significantly (p 0.05) different. D, time course of the effect of GW7647 on FGF21 mRNA abundance. Hepatocytes were incubated with or without GW7647 (1 M) for the indicated time periods. The level of FGF21 mRNA in cells incubated with GW7647 for 0 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated in the absence of GW7647 for the same time period. E, effect of chylomicrons and chylomicron remnants on FGF21 mRNA abundance. Hepatocytes were incubated with chylomicrons and chylomicron remnants prepared from rats fed safflower oil. FGF21 mRNA was measured after 2 h of treatment. The level of FGF21 mRNA in cells incubated with no additions was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of five experiments. The asterisk indicates that the mean is significantly (p 0.05) different. F, effects of glucagon, corticosterone, GW0742, TCPOBOP, glucose, insulin, T3, and leptin on FGF21 mRNA abundance. Rat hepatocytes were incubated in serum-free Medium 199 containing low glucose (5.5 mM) or high glucose (25 mM) with or without glucagon (100 nM), corticosterone (1 M), GW0742 (1 M), TCPOBOP (1 M), insulin (50 nM), T3 (150 nM), or leptin (125 nM). FGF21 mRNA abundance was measured after 6 h of treatment. The level of FGF21 mRNA incubated with 5.5 mM glucose and vehicle was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of three experiments. Different superscript letters indicate that means are significantly (p 0.05) different.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Expressing, Purification, Isolation, Real-time Polymerase Chain Reaction, Incubation, Concentration Assay

FIGURE 3. Bile acids increase hepatic FGF21 secretion and plasma FGF21 concentration. The effect of bile acids on FGF21 accumulation in the culture medium was investigated using primary rat hepatocyte cultures (A) and human HepG2cells(B).Cellswereincubatedwithorwithouttheindicatedbileacids(100 M) in serum-free medium. After 6 and 12 h of treatment, the level of FGF21 and albumin in the culture medium was measured by Western analysis. Top panels, Western analysis of FGF21 from a representative experiment. Bottom panels, sig- nals for FGF21 protein were quantitated. The level of FGF21 protein in the medium of cells incubated with no additions (NA) for 6 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. C, effect of consuming a diet containing cholate on plasma FGF21 concentration. Rats were starved for 24 h and then fed a standard (Std) purifieddietsupplementedwithorwithout1%cholatefor5h.TheplasmaFGF21 concentrationwasmeasuredusinganELISA.Valuesarethemeansofsixanimals. An asterisk indicates that the mean is significantly (p 0.05) different compared withthatofcellsoranimalstreatedwithoutofbileacidforthesametimeperiod. T-MCA, tauro--muricholic acid.

Journal: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE 3. Bile acids increase hepatic FGF21 secretion and plasma FGF21 concentration. The effect of bile acids on FGF21 accumulation in the culture medium was investigated using primary rat hepatocyte cultures (A) and human HepG2cells(B).Cellswereincubatedwithorwithouttheindicatedbileacids(100 M) in serum-free medium. After 6 and 12 h of treatment, the level of FGF21 and albumin in the culture medium was measured by Western analysis. Top panels, Western analysis of FGF21 from a representative experiment. Bottom panels, sig- nals for FGF21 protein were quantitated. The level of FGF21 protein in the medium of cells incubated with no additions (NA) for 6 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. C, effect of consuming a diet containing cholate on plasma FGF21 concentration. Rats were starved for 24 h and then fed a standard (Std) purifieddietsupplementedwithorwithout1%cholatefor5h.TheplasmaFGF21 concentrationwasmeasuredusinganELISA.Valuesarethemeansofsixanimals. An asterisk indicates that the mean is significantly (p 0.05) different compared withthatofcellsoranimalstreatedwithoutofbileacidforthesametimeperiod. T-MCA, tauro--muricholic acid.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Clinical Proteomics, Concentration Assay, Western Blot, Incubation

FIGURE 4. FGF19 increases hepatic FGF21 secretion and plasma FGF21 concentration. A and B, effect of FGF19 and GLP-1 on FGF21 mRNA abundance and FGF21 secretion. Primary rat hepatocyte cultures (A) and human HepG2 cells (B) were incubated with or without FGF19 (100 ng/ml) or GLP-1 (100 nM) in serum-free medium for the indicated treatment times. Cells and culture medium were harvested, total RNA was isolated, and FGF21 mRNA abundance and FGF21 protein levels were measured as described under “Experimental Procedures.” Left panels, effect of FGF19 and GLP-1 on FGF21 mRNA abundance. The level of FGF21 mRNA in cells incubated with vehicle for 2 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) offourexperiments.Rightpanels,effectofFGF19andGLP-1onFGF21secretionintotheculturemedium.TheFGF21proteinlevelintheculturemediumofcells incubated with vehicle for 1 (rat hepatocytes) or 2 h (HepG2) was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated with vehicle for the same timeperiod.C,effectofFGF19onplasmaFGF21concentration.MicewereinjectedintravenouslywithvehicleorrecombinantFGF19(0.4mg/kg).Animalswere killed 4 h after injection. Hepatic FGF21 mRNA abundance (left panel) and plasma FGF21 concentration (right panel) were measured. D, interaction between FGF19andCDCAintheregulationofFGF21mRNAabundanceandFGF21secretion.PrimaryrathepatocytecultureswereincubatedwithorwithoutCDCA(100 M), FGF19 (100 ng/ml), or CDCA plus FGF19 for 6 h. The level of FGF21 mRNA (left panel) and the level of FGF21 protein in the culture medium (right panel) of cells incubated with vehicle was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of three experiments. *, mean is significantly (p 0.05) different from that of cells or mice treated with vehicle for the same time period. **, mean is significantly (p 0.05) higher than that of any other treatment of the same time period.

Journal: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE 4. FGF19 increases hepatic FGF21 secretion and plasma FGF21 concentration. A and B, effect of FGF19 and GLP-1 on FGF21 mRNA abundance and FGF21 secretion. Primary rat hepatocyte cultures (A) and human HepG2 cells (B) were incubated with or without FGF19 (100 ng/ml) or GLP-1 (100 nM) in serum-free medium for the indicated treatment times. Cells and culture medium were harvested, total RNA was isolated, and FGF21 mRNA abundance and FGF21 protein levels were measured as described under “Experimental Procedures.” Left panels, effect of FGF19 and GLP-1 on FGF21 mRNA abundance. The level of FGF21 mRNA in cells incubated with vehicle for 2 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) offourexperiments.Rightpanels,effectofFGF19andGLP-1onFGF21secretionintotheculturemedium.TheFGF21proteinlevelintheculturemediumofcells incubated with vehicle for 1 (rat hepatocytes) or 2 h (HepG2) was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated with vehicle for the same timeperiod.C,effectofFGF19onplasmaFGF21concentration.MicewereinjectedintravenouslywithvehicleorrecombinantFGF19(0.4mg/kg).Animalswere killed 4 h after injection. Hepatic FGF21 mRNA abundance (left panel) and plasma FGF21 concentration (right panel) were measured. D, interaction between FGF19andCDCAintheregulationofFGF21mRNAabundanceandFGF21secretion.PrimaryrathepatocytecultureswereincubatedwithorwithoutCDCA(100 M), FGF19 (100 ng/ml), or CDCA plus FGF19 for 6 h. The level of FGF21 mRNA (left panel) and the level of FGF21 protein in the culture medium (right panel) of cells incubated with vehicle was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of three experiments. *, mean is significantly (p 0.05) different from that of cells or mice treated with vehicle for the same time period. **, mean is significantly (p 0.05) higher than that of any other treatment of the same time period.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Clinical Proteomics, Concentration Assay, Incubation, Isolation, Injection

FIGURE 5. Activation of FXR increases hepatic FGF21 mRNA abundance and FGF21 secretion. A and B, effect of the FXR-selective agonist GW4064 on FGF21 mRNA abundance and FGF21 secretion in primary rat hepatocyte cultures (A) and human HepG2 cells (B). Cells were incubated with or without GW4064 (3 M) for the indicated time periods. Cells and culture medium were harvested, total RNA was isolated, and FGF21 mRNA abundance and FGF21 protein levels were measured as described under “Experimental Procedures.” Left panels, FGF21 mRNA abundance in cells incubated with GW4064 for 0 h was set at 1, and the other values were adjusted proportionately. Right panels, the FGF21 protein level in the culture medium of cells incubated with vehicle (DMSO) for 6 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated with vehicle for the same time period. C, effect of GW4064 administration on FGF21 and BSEP mRNA levels in intact mice. Wild-type mice (Fxr/) and FXR knock-out mice (Fxr/) were orally administered GW4064 (30 mg/kg twice a day) or vehicle (2-hydroxypropyl--cyclodextrin) for 7 days, and FGF21 and BSEP mRNA levels were measured in liver. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of animals of the same genotype treated with vehicle.

Journal: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE 5. Activation of FXR increases hepatic FGF21 mRNA abundance and FGF21 secretion. A and B, effect of the FXR-selective agonist GW4064 on FGF21 mRNA abundance and FGF21 secretion in primary rat hepatocyte cultures (A) and human HepG2 cells (B). Cells were incubated with or without GW4064 (3 M) for the indicated time periods. Cells and culture medium were harvested, total RNA was isolated, and FGF21 mRNA abundance and FGF21 protein levels were measured as described under “Experimental Procedures.” Left panels, FGF21 mRNA abundance in cells incubated with GW4064 for 0 h was set at 1, and the other values were adjusted proportionately. Right panels, the FGF21 protein level in the culture medium of cells incubated with vehicle (DMSO) for 6 h was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of four experiments. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of cells incubated with vehicle for the same time period. C, effect of GW4064 administration on FGF21 and BSEP mRNA levels in intact mice. Wild-type mice (Fxr/) and FXR knock-out mice (Fxr/) were orally administered GW4064 (30 mg/kg twice a day) or vehicle (2-hydroxypropyl--cyclodextrin) for 7 days, and FGF21 and BSEP mRNA levels were measured in liver. An asterisk indicates that the mean is significantly (p 0.05) different compared with that of animals of the same genotype treated with vehicle.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Activation Assay, Incubation, Isolation, Knock-Out

FIGURE 6. Identification of an FXRE in the FGF21 gene. A, hepatocytes were transiently transfected with a series of plasmids containing fragments of the rat FGF21 gene linked to the luciferase (Luc) gene as described under “Experimental Procedures.” After transfection, cells were treated with or without GW4064 for 24 h. Cells were harvested, extracts were prepared, and luciferase assays were performed. Left, the constructs used in these experiments. The number at the left of each construct is the 5-end of FGF21 DNA in nucleotides relative to the transcription initiation site. The 3-end of each construct is 68 bp. The location of the FXRE (1222 to 1210 bp) is indicated by a vertical line. A mutation of the FXRE (FXRE Mut) is indicated by an X through the vertical line. Right, luciferase activity of cells transfected with the 2949 to 68 bp FGF21 construct and treated with vehicle was set at 1, and all other activities were adjusted proportion- ately. The -fold stimulation by GW4064 was calculated by dividing the luciferase activity for cells treated with GW4064 by that for cells treated with vehicle. The -fold responses were calculated for individual experiments and then averaged. The results are the means S.E. (error bars) of three experiments. B, the sequence of the rat FGF21 gene between 1231 and 1196 bp. The hexameric half-sites comprising the FXRE are indicated by arrows. The sequence of a mutation of the FXRE (FXRE Mut) is shown underneath. Mutated sequences are boxed. C, gel mobility shift assays were performed using recombinant FXR and/or RXR and an oligonucleotide probe containing the FGF21 FXRE (1231 to 1196 bp). In lanes 4–10, competition analyses were performed with a 2.5-, 5-, and 10-fold molar excess of unlabeled competitor DNA. The sequences of the probe and competitor DNAs are shown in B. In lanes 12 and 14, the receptor preparations were incubated with antibodies against FXR or nuclear factor 1 (NF1) prior to the addition of the probe. Positions of FXR/RXR and supershifted complexes are indicated by arrows.

Journal: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE 6. Identification of an FXRE in the FGF21 gene. A, hepatocytes were transiently transfected with a series of plasmids containing fragments of the rat FGF21 gene linked to the luciferase (Luc) gene as described under “Experimental Procedures.” After transfection, cells were treated with or without GW4064 for 24 h. Cells were harvested, extracts were prepared, and luciferase assays were performed. Left, the constructs used in these experiments. The number at the left of each construct is the 5-end of FGF21 DNA in nucleotides relative to the transcription initiation site. The 3-end of each construct is 68 bp. The location of the FXRE (1222 to 1210 bp) is indicated by a vertical line. A mutation of the FXRE (FXRE Mut) is indicated by an X through the vertical line. Right, luciferase activity of cells transfected with the 2949 to 68 bp FGF21 construct and treated with vehicle was set at 1, and all other activities were adjusted proportion- ately. The -fold stimulation by GW4064 was calculated by dividing the luciferase activity for cells treated with GW4064 by that for cells treated with vehicle. The -fold responses were calculated for individual experiments and then averaged. The results are the means S.E. (error bars) of three experiments. B, the sequence of the rat FGF21 gene between 1231 and 1196 bp. The hexameric half-sites comprising the FXRE are indicated by arrows. The sequence of a mutation of the FXRE (FXRE Mut) is shown underneath. Mutated sequences are boxed. C, gel mobility shift assays were performed using recombinant FXR and/or RXR and an oligonucleotide probe containing the FGF21 FXRE (1231 to 1196 bp). In lanes 4–10, competition analyses were performed with a 2.5-, 5-, and 10-fold molar excess of unlabeled competitor DNA. The sequences of the probe and competitor DNAs are shown in B. In lanes 12 and 14, the receptor preparations were incubated with antibodies against FXR or nuclear factor 1 (NF1) prior to the addition of the probe. Positions of FXR/RXR and supershifted complexes are indicated by arrows.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Transfection, Luciferase, Construct, Mutagenesis, Activity Assay, Sequencing, Mobility Shift, Recombinant, Incubation

FIGURE7.DeletionofFXRsuppressesthestimulatoryeffectofHF-LCcon- sumption on hepatic FGF21 mRNA abundance and plasma FGF21 con- centration. FXR knock-out mice (Fxr/) and wild-type mice (Fxr/) were fed a standard (Std) purified diet or an HF-LC ketogenic diet for 7 days. A third group was fed a standard purified diet for 6 days and then starved for 24 h. Animals were killed 6 h after the start of the dark cycle, and the FGF21 mRNA abundance (A) and plasma FGF21 concentration (B) were measured. The level of FGF21 mRNA in Fxr/ mice fed the standard purified diet was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of seven animals. The asterisk indicates that the mean is signifi- cantly (p 0.05) lower compared with that of Fxr/ mice fed the HF-LC ketogenic diet. Carb, carbohydrate.

Journal: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE7.DeletionofFXRsuppressesthestimulatoryeffectofHF-LCcon- sumption on hepatic FGF21 mRNA abundance and plasma FGF21 con- centration. FXR knock-out mice (Fxr/) and wild-type mice (Fxr/) were fed a standard (Std) purified diet or an HF-LC ketogenic diet for 7 days. A third group was fed a standard purified diet for 6 days and then starved for 24 h. Animals were killed 6 h after the start of the dark cycle, and the FGF21 mRNA abundance (A) and plasma FGF21 concentration (B) were measured. The level of FGF21 mRNA in Fxr/ mice fed the standard purified diet was set at 1, and the other values were adjusted proportionately. Values are means S.E. (error bars) of seven animals. The asterisk indicates that the mean is signifi- cantly (p 0.05) lower compared with that of Fxr/ mice fed the HF-LC ketogenic diet. Carb, carbohydrate.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Clinical Proteomics, Knock-Out, Purification, Concentration Assay

FIGURE 8. Proposed model for how consumption of an HF-LC ketogenic diet increases hepatic FGF21 gene expression and secretion. Consumption of an HF-LC ketogenic diet enhances the hepatic delivery of multiple signaling molecules that stimulate FGF21 secretion. These signaling factors include dietary unsaturated fatty acids derived from the hepatic hydrolysis of triacylglycerols in chylomicron remnants and the extrahepatic hydrolysis of triacylglycerols in chylomicrons (i.e. nonesterified fatty acid spillover). HF-LC consumption also increases the enterohepatic circulation of bile acids and intestinal secretion of FGF19. Unsaturated fatty acids and bile acids increase hepatic FGF21 gene transcription and secretion by activating FXR and PPAR, respectively. Unsaturated fatty acids and bile acids may also act through FXR- and PPAR-independent pathways to increase FGF21 gene expression. FGF19 activates fibroblast growth factor receptor 4 (FGFR4)/-Klotho causing an increase in FGF21 secretion via a translational and/or posttranslational mechanism. NEFA, non-esterified fatty acid; LPL, lipoprotein lipase.

Journal: Journal of Biological Chemistry

Article Title: Activation of the Farnesoid X Receptor Induces Hepatic Expression and Secretion of Fibroblast Growth Factor 21

doi: 10.1074/jbc.m112.375907

Figure Lengend Snippet: FIGURE 8. Proposed model for how consumption of an HF-LC ketogenic diet increases hepatic FGF21 gene expression and secretion. Consumption of an HF-LC ketogenic diet enhances the hepatic delivery of multiple signaling molecules that stimulate FGF21 secretion. These signaling factors include dietary unsaturated fatty acids derived from the hepatic hydrolysis of triacylglycerols in chylomicron remnants and the extrahepatic hydrolysis of triacylglycerols in chylomicrons (i.e. nonesterified fatty acid spillover). HF-LC consumption also increases the enterohepatic circulation of bile acids and intestinal secretion of FGF19. Unsaturated fatty acids and bile acids increase hepatic FGF21 gene transcription and secretion by activating FXR and PPAR, respectively. Unsaturated fatty acids and bile acids may also act through FXR- and PPAR-independent pathways to increase FGF21 gene expression. FGF19 activates fibroblast growth factor receptor 4 (FGFR4)/-Klotho causing an increase in FGF21 secretion via a translational and/or posttranslational mechanism. NEFA, non-esterified fatty acid; LPL, lipoprotein lipase.

Article Snippet: Goat polyclonal antibodies against mouse FGF21 (AF3057), human FGF21 (TA303289), and albumin (sc-46293) were obtained from R&D Systems, Origene, and Santa Cruz Biotechnology, respectively.

Techniques: Gene Expression, Derivative Assay

(A) Cold-induced increase in serum FGF21 levels was blunted by injection of miR-32-ASO as measured by ELISA (n = 7). (B) qRT-PCR showed that cold stress decreased FGF21 mRNA expression in liver but increased it in BAT. Injection of miR-32-ASO significantly repressed the increase in FGF21 expression in BAT during cold exposure when normalized to PPIA . (C) FGF21 immunostaining showed that cold stress decreased FGF21 level in liver but increased it in BAT. Injection of miR-32-ASO significantly repressed the increase in FGF21 expression in BAT during cold exposure. (D) Quantification of FGF21 immunostaining in BAT showed that FGF21 levels within BAT increased greatly after cold exposure but were significantly lower in miR-32-ASO-treated mice compared to control-ASO-treated mice. (E) Quantification of FGF21 immunostaining in liver showed that FGF21 levels within liver decreased greatly after cold exposure but were similar between miR-32-ASO-treated mice and control-ASO-treated mice. (F) Western blot showing cold-induced increases in BAT FGF21 protein levels were blunted by injection of miR-32-ASO. Calnexin served as a loading control. (G) Western blot showing cold-induced decreases in liver FGF21 protein levels were unaffected by injection of miR-32-ASO. Calnexin served as a loading control. (H) Quantification of the western blot results in (F) and (G) using ImageJ. Average intensities were normalized to that of Calnexin. (I) FGF21 mRNA expression in WT-1 cells increased or decreased when transfected with miR-32 mimic or miR-32-ASO, respectively. (J) Western blotting showed that FGF21 protein levels in WT-1 cells increased or decreased when transfected with miR-32 mimic or miR-32-ASO, respectively. Data represent mean ± SEM. *p < 0.05 and ** p < 0.01. See also .

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) Cold-induced increase in serum FGF21 levels was blunted by injection of miR-32-ASO as measured by ELISA (n = 7). (B) qRT-PCR showed that cold stress decreased FGF21 mRNA expression in liver but increased it in BAT. Injection of miR-32-ASO significantly repressed the increase in FGF21 expression in BAT during cold exposure when normalized to PPIA . (C) FGF21 immunostaining showed that cold stress decreased FGF21 level in liver but increased it in BAT. Injection of miR-32-ASO significantly repressed the increase in FGF21 expression in BAT during cold exposure. (D) Quantification of FGF21 immunostaining in BAT showed that FGF21 levels within BAT increased greatly after cold exposure but were significantly lower in miR-32-ASO-treated mice compared to control-ASO-treated mice. (E) Quantification of FGF21 immunostaining in liver showed that FGF21 levels within liver decreased greatly after cold exposure but were similar between miR-32-ASO-treated mice and control-ASO-treated mice. (F) Western blot showing cold-induced increases in BAT FGF21 protein levels were blunted by injection of miR-32-ASO. Calnexin served as a loading control. (G) Western blot showing cold-induced decreases in liver FGF21 protein levels were unaffected by injection of miR-32-ASO. Calnexin served as a loading control. (H) Quantification of the western blot results in (F) and (G) using ImageJ. Average intensities were normalized to that of Calnexin. (I) FGF21 mRNA expression in WT-1 cells increased or decreased when transfected with miR-32 mimic or miR-32-ASO, respectively. (J) Western blotting showed that FGF21 protein levels in WT-1 cells increased or decreased when transfected with miR-32 mimic or miR-32-ASO, respectively. Data represent mean ± SEM. *p < 0.05 and ** p < 0.01. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Immunostaining, Control, Western Blot, Transfection

(A) High mirSVR score and favorable binding energy from bioinformatic analysis predicted that miR-32 directly targets the 3′ UTR of Tob1 . (B) The complementary sequence in the Tob1 3′ UTR and the seed region of miR-32 (red letters) are conserved among mammalian species. (C) Tob1 mRNA level in WT-1 cells decreased or increased 24 hr after transfection of miR-32 mimic or miR-32-ASO, respectively. (D) Tob1 protein level decreased or increased in WT-1 cells when transfected with miR-32 mimic or miR-32-ASO, respectively. (E and F) Activities of a luciferase reporter gene linked to the Tob1 3′ UTR decreased or increased 24 hr after transfection of miR-32 mimic (E) or miR-32-ASO (F), respectively. (G and H) Site-directed mutagenesis of the Tob1 3′ UTR sequence complimentary to the miR-32 seed sequence abolished the effects of miR-32 mimic (G) and miR-32-ASO (H) transfection on the luciferase activity. (I) Knockdown of Tob1 by siTob1 suppressed the increase in Tob1 expression and rescued the decrease in FGF21 expression caused by miR-32-ASO transfection. (J) Western blots showed that siTob1 suppressed the increase in Tob1 expression and rescued the decreases of phospho-p38, phospho-ATF2, and FGF21 expression caused by miR-32-ASO transfection. Numbers indicate protein levels quantified using ImageJ relative to control-ASO. (K) qRT-PCR showed miR-32-ASO-treated mice failed to fully downregulate Tob1 level in response to cold stimulation. (L) Western blots showed miR-32-ASO-treated mice fail to downregulate Tob1 levels and activate p38 and ATF2 phosphorylation in BAT in response to cold stimulation. (M) Quantification of relative protein expression in BAT from the western blots showed in (L) using ImageJ. Average intensities were normalized to that of Calnexin. (N) Cold-exposure time course showing miR-32 and Tob1 expression in BAT at various time points after cold exposure. (O) Cold-exposure time course showing FGF21 expression in BAT at various time points after cold exposure. (P) Cold-exposure time course showing Tob1, UCP1, and FGF21 protein expression in BAT at various time points after cold exposure. (Q) Cold-exposure time course showing UCP1 protein expression in iWAT at various time points after cold exposure. Data represent mean ± SEM. *p < 0.05 and ** p < 0.01. See also .

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) High mirSVR score and favorable binding energy from bioinformatic analysis predicted that miR-32 directly targets the 3′ UTR of Tob1 . (B) The complementary sequence in the Tob1 3′ UTR and the seed region of miR-32 (red letters) are conserved among mammalian species. (C) Tob1 mRNA level in WT-1 cells decreased or increased 24 hr after transfection of miR-32 mimic or miR-32-ASO, respectively. (D) Tob1 protein level decreased or increased in WT-1 cells when transfected with miR-32 mimic or miR-32-ASO, respectively. (E and F) Activities of a luciferase reporter gene linked to the Tob1 3′ UTR decreased or increased 24 hr after transfection of miR-32 mimic (E) or miR-32-ASO (F), respectively. (G and H) Site-directed mutagenesis of the Tob1 3′ UTR sequence complimentary to the miR-32 seed sequence abolished the effects of miR-32 mimic (G) and miR-32-ASO (H) transfection on the luciferase activity. (I) Knockdown of Tob1 by siTob1 suppressed the increase in Tob1 expression and rescued the decrease in FGF21 expression caused by miR-32-ASO transfection. (J) Western blots showed that siTob1 suppressed the increase in Tob1 expression and rescued the decreases of phospho-p38, phospho-ATF2, and FGF21 expression caused by miR-32-ASO transfection. Numbers indicate protein levels quantified using ImageJ relative to control-ASO. (K) qRT-PCR showed miR-32-ASO-treated mice failed to fully downregulate Tob1 level in response to cold stimulation. (L) Western blots showed miR-32-ASO-treated mice fail to downregulate Tob1 levels and activate p38 and ATF2 phosphorylation in BAT in response to cold stimulation. (M) Quantification of relative protein expression in BAT from the western blots showed in (L) using ImageJ. Average intensities were normalized to that of Calnexin. (N) Cold-exposure time course showing miR-32 and Tob1 expression in BAT at various time points after cold exposure. (O) Cold-exposure time course showing FGF21 expression in BAT at various time points after cold exposure. (P) Cold-exposure time course showing Tob1, UCP1, and FGF21 protein expression in BAT at various time points after cold exposure. (Q) Cold-exposure time course showing UCP1 protein expression in iWAT at various time points after cold exposure. Data represent mean ± SEM. *p < 0.05 and ** p < 0.01. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Binding Assay, Sequencing, Transfection, Luciferase, Mutagenesis, Activity Assay, Knockdown, Expressing, Western Blot, Control, Quantitative RT-PCR, Phospho-proteomics

(A) miR-32 expression in BAT but not in liver was blunted in BAT-specific miR-32-ASO-injected mice (n = 8) compared to control-ASO-injected mice (n = 7). (B) miR-32-ASO-BS mice (n = 8) showed lower core body temperatures during cold exposure when compared to control-ASO-BS mice (n = 7). (C) Total energy expenditure was significantly reduced after 7 days’ cold stress in miR-32-ASO-BS mice (n = 6) as compared with control-ASO-BS mice (n = 6). Energy expenditure was normalized to lean body mass. (D) Average total energy expenditure was significantly lower in miR-32-ASO-BS mice (n = 6). (E) Percentage BAT mass was significantly lower in miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). (F) In BAT, mRNA levels of Tob1 were higher in miR-32-ASO-BS mice (n = 8), whereas expression of thermogenic genes was significantly lower in miR-32-ASO-BS mice. (G) Protein levels of Tob1 were higher in miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). However, p-p38, p-ATF2, FGF21, UCP1, and PGC1α were lower in miR-32-ASO-BS mice (n = 8). Total p38 and total ATF2 expression were not significantly different between the two groups. (H) Quantification of relative protein expression using ImageJ showed that protein level of Tob1 was upregulated, but p-p38, p-ATF2, FGF21, UCP1, and PGC1α were significantly lower in BAT of miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). Total p38 and total ATF2 expression were comparable between the two groups. Average intensities were normalized to that of Calnexin. (I) Serum FGF21 levels were decreased in miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). (J) mRNA levels of thermogenic genes in iWAT were significantly reduced in miR-32-ASO-BS mice. Data were normalized to PPIA . (K) Immunoblots showed that miR-32-ASO-BS mice (n = 8) had significantly reduced UCP1 and PGC1α protein levels in iWAT compared to control-ASO-BS mice (n = 7). (L) Quantification of relative UCP1 and PGC1α protein levels using ImageJ. Average intensities were normalized to that of Calnexin. Data represent mean ± SEM. *p < 0.05, **p < 0.01, and *** p < 0.001. See also .

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) miR-32 expression in BAT but not in liver was blunted in BAT-specific miR-32-ASO-injected mice (n = 8) compared to control-ASO-injected mice (n = 7). (B) miR-32-ASO-BS mice (n = 8) showed lower core body temperatures during cold exposure when compared to control-ASO-BS mice (n = 7). (C) Total energy expenditure was significantly reduced after 7 days’ cold stress in miR-32-ASO-BS mice (n = 6) as compared with control-ASO-BS mice (n = 6). Energy expenditure was normalized to lean body mass. (D) Average total energy expenditure was significantly lower in miR-32-ASO-BS mice (n = 6). (E) Percentage BAT mass was significantly lower in miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). (F) In BAT, mRNA levels of Tob1 were higher in miR-32-ASO-BS mice (n = 8), whereas expression of thermogenic genes was significantly lower in miR-32-ASO-BS mice. (G) Protein levels of Tob1 were higher in miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). However, p-p38, p-ATF2, FGF21, UCP1, and PGC1α were lower in miR-32-ASO-BS mice (n = 8). Total p38 and total ATF2 expression were not significantly different between the two groups. (H) Quantification of relative protein expression using ImageJ showed that protein level of Tob1 was upregulated, but p-p38, p-ATF2, FGF21, UCP1, and PGC1α were significantly lower in BAT of miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). Total p38 and total ATF2 expression were comparable between the two groups. Average intensities were normalized to that of Calnexin. (I) Serum FGF21 levels were decreased in miR-32-ASO-BS mice (n = 8) compared to control mice (n = 7). (J) mRNA levels of thermogenic genes in iWAT were significantly reduced in miR-32-ASO-BS mice. Data were normalized to PPIA . (K) Immunoblots showed that miR-32-ASO-BS mice (n = 8) had significantly reduced UCP1 and PGC1α protein levels in iWAT compared to control-ASO-BS mice (n = 7). (L) Quantification of relative UCP1 and PGC1α protein levels using ImageJ. Average intensities were normalized to that of Calnexin. Data represent mean ± SEM. *p < 0.05, **p < 0.01, and *** p < 0.001. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Expressing, Injection, Control, Western Blot

(A) miR-32 expression in BAT but not in liver or iWAT was increased in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). (B) miR-32-AAV-BS mice (n = 8) showed higher core body temperatures during cold exposure when compared to control-AAV-BS mice (n = 7). (C) Total energy expenditure was significantly increased after 7 days’ cold stress in miR-32-AAV-BS mice (n = 6) as compared with control-AAV-BS mice (n = 6). Energy expenditure was normalized to lean body mass. (D) Average total energy expenditure was significantly higher in miR-32-AAV-BS mice (n = 6). (E) Percentage BAT mass was significantly higher in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). (F) In BAT, mRNA levels of Tob1 were lower in miR-32-AAV-BS mice, whereas expression of thermogenic genes was significantly higher in miR-32-AAV-BS mice. (G) Protein levels of Tob1 were lower in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). However, p-p38, p-ATF2, FGF21, UCP1, and PGC1α were higher in miR-32-AAV-BS mice (n = 8). Total p38 and total ATF2 expression were not significantly different between the two groups. (H) Quantification of relative protein expression using ImageJ showed that protein level of Tob1 was reduced, but p-p38, p-ATF2, FGF21, UCP1, and PGC1α were significantly higher in BAT of miR-32-AAV-BS mice compared to control. Total p38 and total ATF2 expression were comparable between the two groups. Average intensities were normalized to that of Calnexin. (I) Serum FGF21 levels were increased in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). (J) mRNA levels of thermogenic genes in iWAT were significantly increased in miR-32-AAV-BS mice. Data were normalized to PPIA . (K) Immunoblots showed that miR-32-AAV-BS mice (n = 8) had significantly increased UCP1 and PGC1α protein levels in iWAT compared to control-AAV-BS mice (n = 7). (L) Quantification of relative UCP1 and PGC1α protein levels using ImageJ. Average intensities were normalized to that of Calnexin. Data represent mean ± SEM. *p < 0.05, **p < 0.01, and *** p < 0.001. See also .

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) miR-32 expression in BAT but not in liver or iWAT was increased in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). (B) miR-32-AAV-BS mice (n = 8) showed higher core body temperatures during cold exposure when compared to control-AAV-BS mice (n = 7). (C) Total energy expenditure was significantly increased after 7 days’ cold stress in miR-32-AAV-BS mice (n = 6) as compared with control-AAV-BS mice (n = 6). Energy expenditure was normalized to lean body mass. (D) Average total energy expenditure was significantly higher in miR-32-AAV-BS mice (n = 6). (E) Percentage BAT mass was significantly higher in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). (F) In BAT, mRNA levels of Tob1 were lower in miR-32-AAV-BS mice, whereas expression of thermogenic genes was significantly higher in miR-32-AAV-BS mice. (G) Protein levels of Tob1 were lower in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). However, p-p38, p-ATF2, FGF21, UCP1, and PGC1α were higher in miR-32-AAV-BS mice (n = 8). Total p38 and total ATF2 expression were not significantly different between the two groups. (H) Quantification of relative protein expression using ImageJ showed that protein level of Tob1 was reduced, but p-p38, p-ATF2, FGF21, UCP1, and PGC1α were significantly higher in BAT of miR-32-AAV-BS mice compared to control. Total p38 and total ATF2 expression were comparable between the two groups. Average intensities were normalized to that of Calnexin. (I) Serum FGF21 levels were increased in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). (J) mRNA levels of thermogenic genes in iWAT were significantly increased in miR-32-AAV-BS mice. Data were normalized to PPIA . (K) Immunoblots showed that miR-32-AAV-BS mice (n = 8) had significantly increased UCP1 and PGC1α protein levels in iWAT compared to control-AAV-BS mice (n = 7). (L) Quantification of relative UCP1 and PGC1α protein levels using ImageJ. Average intensities were normalized to that of Calnexin. Data represent mean ± SEM. *p < 0.05, **p < 0.01, and *** p < 0.001. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Expressing, Control, Western Blot

(A) FGF21 mRNA expression in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6). (B) FGF21 protein expression in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6). (C) Quantification of relative protein expression using ImageJ showed that protein level of FGF21 in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6). (D) Serum FGF21 levels were decreased in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6) compared to wild-type mice (n = 4). (E) miR-32-AAV-BS+Cre mice (n = 6) showed higher core body temperatures only during first 48 hr of cold exposure when compared to control-AAV-BS+Cre mice (n = 6). (F) Total energy expenditure was similar after 7 days’ cold stress in miR-32-AAV-BS+Cre mice (n = 6) as compared with control-AAV-BS+Cre mice (n = 6). Energy expenditure was normalized to lean body mass. (G) Average total energy expenditure was slightly higher in miR-32-AAV-BS+Cre mice (n = 6) than control-AAV-BS+Cre mice (n = 6) but not statistically significant. (H) Percentage BAT mass was slightly higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6). (I) In BAT, mRNA levels of Tob1 were lower in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6), whereas expression of several thermogenic genes including UCP1 was higher in miR-32-AAV-BS+Cre mice. (J) Protein levels of PGC1α and UCP1 were higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6). (K) Quantification of relative protein expression using ImageJ showed that protein levels of PGC1 and UCP1 were higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6). Average intensities were normalized to that of Calnexin. (L) mRNA levels of thermogenic genes in iWAT were similar in both groups of mice (both n = 6). Data were normalized to PPIA . (M) Immunoblots showed that miR-32-AAV-BS+Cre mice (n = 6) had similar UCP1 and PGC1α protein levels in iWAT compared to control-AAV-BS+Cre mice (n = 6). (N) Quantification of relative UCP1 and PGC1α protein levels using ImageJ. Average intensities were normalized to that of Calnexin. (O) Proposed mechanism by which miR-32 promotes BAT thermogenesis by inhibiting Tob1, activating p38/MAPK signaling and driving UCP1, PGC1α, and FGF21 expression in BAT. The BAT secreted FGF21 functions in a paracrine fashion to promote further thermogenic gene expression in BAT as well as in an endocrine fashion to promote iWAT browning. Data represent mean ± SEM. *p < 0.05, **p < 0.01, and *** p < 0.001. See also .

Journal: Cell reports

Article Title: miRNA-32 Drives Brown Fat Thermogenesis and Trans-activates Subcutaneous White Fat Browning in Mice

doi: 10.1016/j.celrep.2017.04.035

Figure Lengend Snippet: (A) FGF21 mRNA expression in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6). (B) FGF21 protein expression in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6). (C) Quantification of relative protein expression using ImageJ showed that protein level of FGF21 in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6). (D) Serum FGF21 levels were decreased in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6) compared to wild-type mice (n = 4). (E) miR-32-AAV-BS+Cre mice (n = 6) showed higher core body temperatures only during first 48 hr of cold exposure when compared to control-AAV-BS+Cre mice (n = 6). (F) Total energy expenditure was similar after 7 days’ cold stress in miR-32-AAV-BS+Cre mice (n = 6) as compared with control-AAV-BS+Cre mice (n = 6). Energy expenditure was normalized to lean body mass. (G) Average total energy expenditure was slightly higher in miR-32-AAV-BS+Cre mice (n = 6) than control-AAV-BS+Cre mice (n = 6) but not statistically significant. (H) Percentage BAT mass was slightly higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6). (I) In BAT, mRNA levels of Tob1 were lower in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6), whereas expression of several thermogenic genes including UCP1 was higher in miR-32-AAV-BS+Cre mice. (J) Protein levels of PGC1α and UCP1 were higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6). (K) Quantification of relative protein expression using ImageJ showed that protein levels of PGC1 and UCP1 were higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6). Average intensities were normalized to that of Calnexin. (L) mRNA levels of thermogenic genes in iWAT were similar in both groups of mice (both n = 6). Data were normalized to PPIA . (M) Immunoblots showed that miR-32-AAV-BS+Cre mice (n = 6) had similar UCP1 and PGC1α protein levels in iWAT compared to control-AAV-BS+Cre mice (n = 6). (N) Quantification of relative UCP1 and PGC1α protein levels using ImageJ. Average intensities were normalized to that of Calnexin. (O) Proposed mechanism by which miR-32 promotes BAT thermogenesis by inhibiting Tob1, activating p38/MAPK signaling and driving UCP1, PGC1α, and FGF21 expression in BAT. The BAT secreted FGF21 functions in a paracrine fashion to promote further thermogenic gene expression in BAT as well as in an endocrine fashion to promote iWAT browning. Data represent mean ± SEM. *p < 0.05, **p < 0.01, and *** p < 0.001. See also .

Article Snippet: Thermo Scientific 96-well, clear, flat-bottom plates (Nunc) were coated overnight at 4°C with a mouse monoclonal anti-FGF21 antibody (MAB25371, R&D Systems) at 1:1,500 dilution in Na 2 CO 3 /NaHCO 3 buffer at pH 9.6.

Techniques: Expressing, Control, Western Blot, Gene Expression