FGF21 promotes metabolic homeostasis via white adipose and leptin in mice.

Lipodystrophy mice are resistant to anti-diabetic effects of recombinant FGF21 treatment.Recombinant muFGF21 (1 or 10 mg/kg, pink and red symbols respectively) or vehicle (green symbols) was injected BID in wildtype (WT - open bars) and aP2-nSrebp1c lipodystrophy (Tg - hatched bars) male mice as described in Animal Study Design I (Methodology). (A–B) Body weight was monitored daily. Dramatic drop in body weight on day 17 was caused by the 12 h fast for GTT. (C–D) Adipose and lean mass was recorded in WT and Tg mice at 0, 7 and 14 days of treatment. (E–F) Fed blood glucose (OneTouch Basic glucometer) and plasma insulin were measured in WT and Tg mice at the start (PRE) and the end (POST) of the treatment. (G–H) Glucose tolerance was measured on day 18 of treatment (OneTouch Basic glucometer). (I) Area under the curve (AUC) was calculated in WT and Tg mice. (J–L) Plasma lipids (cholesterol, triglycerides, NEFA – non-esterified fatty acids) were measured in WT and Tg mice at the start (PRE) and the end (POST) of the treatment. WT groups N = 8; Tg vehicle group N = 8; Tg FGF21 treatment group N = 7. * P<0.05, ** P<0.01, *** P<0.001 vs. vehicle-treated genotype-matched mice at same time point, ns – not statistically significant. ++ P<0.01, +++ P<0.001 vs. WT mice (T-Test).

Increased Fgf21 expression and normalβ-klotho protein levels in lipodystrophy mice.Recombinant muFGF21 (1 or 10 mg/kg, pink and red bars respectively) or vehicle (green) was injected BID in wildtype (WT - open bars) and aP2-nSrebp1c lipodystrophy (Tg – hatched bars) male mice for 21 days as described in Animal Study Design I (Methodology). (A) Fgf21, Klb or Fgfr1c expression levels were measured using semi-quantitative RT-PCR in RNA isolated from inguinal fat pads; N = 5/group. (B) Western analysis of β-klotho and β-actin was carried out on epididymal fat pad lysates from WT or Tg mice. * P<0.05 vs. vehicle-treated WT mice.

Impaired FGF21 signaling in adipose tissue from lipodystrophy mice.Wildtype (WT) and aP2-nSrebp1c lipodystrophy (Tg) male mice were treated with vehicle (−) or a single rhuFGF21 (+) injection at 1 mg/kg (Animal Study Design II – Methodology). White adipose tissue (WAT) samples were subjected to immunoblot analysis with antibodies directed against (A) ERK (extracellular signal-regulated kinase), (B) FRS2 (Fibroblast growth factor receptor substrate (C) SHP2 (Src homology domain 2 containing protein tyrosine phosphatase), 2), (D) ERK signaling was analyzed in liver samples. Antibodies directed against phosphorylated (p-) or total (T-) proteins of specific resides are noted alongside each panel.

Adipose tissue transplantation restores anti-diabetic effects of recombinant FGF21 treatment in lipodystrophy mice.Approximately 2 g of wildtype (WT) adipose tissue was transplanted subcutaneously into aP2-nSrebp1c lipodystrophy (Tg) male mice (Animal Study Design III – Methods). Following a 2-week recovery period, WT sham (green bars/symbols), Tg sham (blue bars/symbols) and Tg transplanted (red bars/symbols) mice were treated with vehicle (open bars/solid line) or 10 mg/kg rmuFGF21 (hatched bars/dashed line) BID for 21 days. (A) Adipose mass was measured in WT and Tg mice before (−3 days), immediately after (1 day) and 7, 14, 21 and 28 days following adipose tissue transplantation. Adipose tissue mass was increased in the Tg transplanted mice after implantation and although declined by MRI analysis was still intact at necropsy. Fourteen days following adipose tissue transplantation, mice were treated with vehicle or rmuFGF21. (B) Plasma leptin was measured prior to, and at necropsy. (C–E) Percent change in body weight was calculated daily from the start of rmuFGF21 treatment. Days 17–21 were excluded due to dramatic weight changes following 12 hr fast for the GTT. (F–H) Glucose tolerance was measured after 18 days of rmuFGF21 treatment (AlphaTRAK glucose monitor). (I–J) Insulin and adiponectin levels were measured 3 days before adipose tissue transplantation (Pre-transplant), 14 days following sham or implantation surgery (Post-transplant Pre-treatment) and 32 days following surgery including 14 days of rmuFGF21 or vehicle injections (Post-treatment). (K–L) Plasma lipids (cholesterol and triglycerides) were measured in the same samples described in (I). N = 6–7 group. * P<0.05, ** P<0.01, ** P<0.001 vs. vehicle-treated genotype and surgery-matched mice at same time point;. + P<0.05, ++ P<0.01, +++ P<0.001 vs. comparator groups as indicated. # P<0.05. vs. the same mice Post-transplant Pre-treatment (all analysis by T-Test); ns – not statistically significant.

Changes in Pparg and Fgf21 in adipose transplanted lipodystrophy mice.Relative quantitative RT-PCR was carried out on Fgf21 and Pparg genes in tissues collected from animals presented in Figure 4 at terminal necropsy (Animal Study Design III – Methods). (A) Fgf21 expression was increased in liver and white and brown adipose tissues (WAT and BAT) in Tg mice compared to WT mice. (B) Pparg expression was increased in the livers of Tg mice compared to WT mice and transplantation reduced liver Pparg expression in Tg mice in a similar manner to rmuFGF21 treatment, revealing a similar trend to Fgf21 expression. Pparg expression was approximately 50% lower in Tg adipose tissues compared to WT mice. Treatment with rmuFGF21 increased the expression of Pparg in transplanted adipose only. N = 5 group. * P<0.05, ** P<0.01, vs. vehicle-treated genotype and surgery-matched mice at same time point (T-Test), ns – not statistically significant;. + P<0.05, ++ P<0.01, +++ P<0.001 vs. comparator groups as indicated (ANOVA).

Leptin restores FGF21 resistance in lipodystrophy mice.Either rmuFGF21 (10 mg/kg, pink bars/symbols), muleptin (10 mg/kg/day, blue bars/symbols), both muFGF21+ muleptin (red bars/symbols), or vehicle (green bars/symbols) was IP injected BID in aP2-nSrebp1c lipodystrophy (Tg) male mice (Animal Study Design IV – Methodology). (A) Body weight was monitored daily, days 11–15 were omitted due to large changes at the time of GTT. (B) Blood glucose was measured prior to, and on the 8th day of treatment (AlphaTRAK glucose monitor). (C) Plasma insulin was measured prior to, and on the 15th day of treatment. (D) Glucose tolerance was measured on the 11th day of treatment (AlphaTRAK glucose monitor). Groups of N = 6. * P<0.05, ** P<0.01, *** P<0.0001 vs. saline and vehicle-treated mice at same time point, † P<0.05, ††† P<0.001 vs. leptin and vehicle-treated mice at the same time point.