Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states

Mice fed a high-fat, low-carbohydrate ketogenic diet (KD) exhibit marked changes in hepatic metabolism and energy homeostasis. Here, we identify liver-derived fibroblast growth factor 21 (FGF21) as an endocrine regulator of the ketotic state. Hepatic expression and circulating levels of FGF21 are induced by both KD and fasting, are rapidly suppressed by refeeding, and are in large part downstream of PPARα. Importantly, adenoviral knockdown of hepatic FGF21 in KD-fed mice causes fatty liver, lipemia, and reduced serum ketones, due at least in part to altered expression of key genes governing lipid and ketone metabolism. Hence, induction of FGF21 in liver is required for the normal activation of hepatic lipid oxidation, triglyceride clearance, and ketogenesis induced by KD. These findings identify hepatic FGF21 as a critical regulator of lipid homeostasis and identify a physiological role for this hepatic hormone.     

Fibroblast growth factor 21 is induced upon cardiac stress and alters cardiac lipid homeostasis

Fibroblast growth factor 21 (FGF21) is a PPARα-regulated gene elucidated in the liver of PPARα-deficient mice or PPARα agonist-treated mice. Mice globally lacking adipose triglyceride lipase (ATGL) exhibit a marked defect in TG catabolism associated with impaired PPARα-activated gene expression in the heart and liver, including a drastic reduction in hepatic FGF21 mRNA expression. Here we show that FGF21 mRNA expression is markedly increased in the heart of ATGL-deficient mice accompanied by elevated expression of endoplasmic reticulum (ER) stress markers, which can be reversed by reconstitution of ATGL expression in cardiac muscle. In line with this assumption, the induction of ER stress increases FGF21 mRNA expression in H9C2 cardiomyotubes. Cardiac FGF21 expression was also induced upon fasting of healthy mice, implicating a role of FGF21 in cardiac energy metabolism. To address this question, we generated and characterized mice with cardiac-specific overexpression of FGF21 (CM-Fgf21). FGF21 was efficiently secreted from cardiomyocytes of CM-Fgf21 mice, which moderately affected cardiac TG homeostasis, indicating a role for FGF21 in cardiac energy metabolism. Together, our results show that FGF21 expression is activated upon cardiac ER stress linked to defective lipolysis and that a persistent increase in circulating FGF21 levels interferes with cardiac and whole body energy homeostasis.     

Impact of dietary protein content on uncoupling protein mRNA abundance in swine

The present study was designed to determine if dietary protein can alter uncoupling protein (UCP) expression in swine, as has been shown in rats, and attempt to identify the mechanism. Eight pigs (~ 50 kg body mass) were fed an 18% crude protein (CP) diet while another eight pigs were switched to a diet containing 12% crude protein (CP) and fed these diets until 110 kg body mass. The outer (OSQ) and middle (MSQ) subcutaneous adipose tissues, liver, leaf fat, longissimus (LM), red portion of the semitendinosus (STR) and the white portion of the ST (STW) were analyzed for gene expression by real-time PCR. Feeding of 12% CP did not alter growth or carcass composition, relative to 18% CP (P > 0.05). Serum growth hormone, non-esterified fatty acids, triglycerides and urea nitrogen were reduced with the feeding of 12% CP (P < 0.05). The UCP2 mRNA abundance was reduced in LM, STR, MSQ and OSQ with feeding of 12% CP (P < 0.05), as was UCP3 mRNA abundance in MSQ and STW (P < 0.01). Peroxisome proliferation activated receptor α (PPARα) and PPARγ were reduced in MSQ and STR (P < 0.05) with feeding 12% CP as was the PPARα regulated protein, acyl CoA oxidase (ACOX, P < 0.05). These data suggest that feeding 12% CP relative to 18% CP reduces serum NEFA, which reduces PPARα and PPARγ expression and consequently reduces UCP2 lipoperoxidation in OSQ and STR and also reduced UCP3 associated fatty acid transport in MSQ and STW.     

Selective PPARδ agonist seladelpar suppresses bile acid synthesis by reducing hepatocyte CYP7A1 via the fibroblast growth factor 21 signaling pathway

Peroxisome proliferator–activated receptor delta (PPARδ) agonists have been shown to exert beneficial effects in liver disease and reduce total bile acid levels. The mechanism(s) whereby PPARδ agonism reduces bile acid levels are, however, unknown, and therefore the aim of the present study was to investigate the molecular pathways responsible for reducing bile acid synthesis in hepatocytes, following treatment with the selective PPARδ agonist, seladelpar. We show that administration of seladelpar to WT mice repressed the liver expression of cholesterol 7 alpha-hydroxylase (Cyp7a1), the rate-limiting enzyme for bile acid synthesis, and decreased plasma 7α-hydroxy-4-cholesten-3-one (C4), a freely diffusible metabolite downstream of Cyp7a1. In primary mouse hepatocytes, seladelpar significantly reduced the expression of Cyp7a1 independent of the nuclear bile acid receptor, Farnesoid X receptor. In addition, seladelpar upregulated fibroblast growth factor 21 (Fgf21) in mouse liver, serum, and in cultured hepatocytes. We demonstrate that recombinant Fgf21 protein activated the c-Jun N-terminal kinase (JNK) signaling pathway and repressed Cyp7a1 gene expression in primary hepatocytes. The suppressive effect of seladelpar on Cyp7a1 expression was blocked by a JNK inhibitor as well as in the absence of Fgf21, indicating that Fgf21 plays an indispensable role in PPARδ-mediated downregulation of Cyp7a1. Finally, reduction of CYP7A1 expression by seladelpar was confirmed in primary human hepatocytes. In conclusion, we show that seladelpar reduces bile acid synthesis via an FGF21-dependent mechanism that signals at least partially through JNK to repress CYP7A1.     

Activation of a HIF1α-PPARγ Axis Underlies the Integration of Glycolytic and Lipid Anabolic Pathways in Pathologic Cardiac Hypertrophy

Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1α and PPARγ, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1α activates glycolytic genes and PPARγ, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1α in mice prevents hypertrophy-induced PPARγ activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1α-PPARγ axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.     

FGF21 attenuates pulmonary arterial hypertension via downregulation of miR‐130, which targets PPARγ

The proliferation, migration and apoptotic resistance of pulmonary artery smooth muscle cells (PASMCs) are central to the progression of pulmonary arterial hypertension (PAH). Our previous study identified that fibroblast growth factor 21 (FGF21) regulates signalling pathway molecules, such as peroxisome proliferator‐activated receptor gamma (PPARγ), to play an important role in PAH treatment. However, the biological roles of miRNAs in these effects are not yet clear. In this study, using miRNA sequencing and real‐time PCR, we found that FGF21 treatment inhibited miR‐130 elevation in hypoxia‐induced PAH in vitro and in vivo. Dual luciferase reporter gene assays showed that miR‐130 directly negatively regulates PPARγ expression. Inhibition of miR‐130 expression suppressed abnormal proliferation, migration and apoptotic resistance in hypoxic PASMCs, and this effect was corrected upon PPARγ knockdown. Both the ameliorative effect of FGF21 on pulmonary vascular remodelling and the inhibitory effect on proliferation, migration and apoptotic resistance in PASMCs were observed following exogenous administration of miR‐130 agomir. In conclusion, this study revealed the protective effect and mechanism of FGF21 on PAH through regulation of the miR‐130/PPARγ axis, providing new ideas for the development of potential drugs for PAH based on FGF21.     

Carnitine synthesis and uptake into cells are stimulated by fasting in pigs as a model of nonproliferating species

In rodents, fasting increases the carnitine concentration in the liver by an up-regulation of enzymes of hepatic carnitine synthesis and novel organic cation transporter (OCTN) 2, mediated by activation of peroxisome proliferator-activated receptor (PPAR) α. This study was performed to investigate whether such effects occur also in pigs which like humans, as nonproliferating species, have a lower expression of PPARα and are less responsive to treatment with PPARα agonists than rodents. An experiment with 20 pigs was performed, which were either fed a diet ad-libitum or fasted for 24 h. Fasted pigs had higher relative mRNA concentrations of the PPARα target genes carnitine palmitoyltransferase 1 and acyl-CoA oxidase in liver, heart, kidney, and small intestinal mucosa than control pigs, indicative of PPARα activation in these tissues (P<.05). Fasted pigs had a higher activity of γ-butyrobetaine dioxygenase (BBD), enzyme that catalyses the last step of carnitine biosynthesis in liver and kidney, and higher relative mRNA concentrations of OCTN2, the most important carnitine transporter, in liver, kidney, skeletal muscle, and small intestinal mucosa than control pigs (P<.05). Fasted pigs moreover had higher concentrations of free and total carnitine in liver and kidney than control pigs (P<.05). This study shows for the first time that fasting increases the activity of BBD in liver and kidney and up-regulates the expression of OCTN2 in various tissues of pigs, probably mediated by PPARα activation. It is concluded that nonproliferating species are also able to cover their increased demand for carnitine during fasting by an increased carnitine synthesis and uptake into cells.     

The circulating metabolic regulator FGF21 is induced by prolonged fasting and PPARalpha activation in man

FGF21 is a critical metabolic regulator, pivotal for fasting adaptation and directly regulated by PPARα in rodents. However, the physiological role of FGF21 in man is not yet defined and was investigated in our study. Serum FGF21 varied 250-fold among 76 healthy individuals and did not relate to age, gender, body mass index (BMI), serum lipids, or plasma glucose. FGF21 levels had no diurnal variation and were unrelated to bile acid or cholesterol synthesis. Ketosis induced by a 2 day fast or feeding a ketogenic diet (KD) did not influence FGF21 levels, whereas a 74% increase occurred after 7 days of fasting. Hypertriglyceridemic nondiabetic patients had 2-fold elevated FGF21 levels, which were further increased by 28% during fenofibrate treatment. FGF21 circulates in human plasma and increases by extreme fasting and PPARα activation. The wide interindividual variation and the induction of ketogenesis independent of FGF21 levels indicate that the physiological role of FGF21 in humans may differ from that in mice.     

Metabolomics reveal 1-palmitoyl lysophosphatidylcholine production by peroxisome proliferator-activated receptor α

PPARα is well known as a master regulator of lipid metabolism. PPARα activation enhances fatty acid oxidation and decreases the levels of circulating and cellular lipids in obese diabetic patients. Although PPARα target genes are widely known, little is known about the alteration of plasma and liver metabolites during PPARα activation. Here, we report that metabolome analysis-implicated upregulation of many plasma lysoGP species during bezafibrate (PPARα agonist) treatment. In particular, 1-palmitoyl lysophosphatidylcholine [LPC(16:0)] is increased by bezaf­ibrate treatment in both plasma and liver. In mouse primary hepatocytes, the secretion of LPC(16:0) increased on PPARα activation, and this effect was attenuated by PPARα antagonist treatment. We demonstrated that Pla₂g7 gene expression levels in the murine hepatocytes were increased by PPARα activation, and the secretion of LPC(16:0) was suppressed by Pla₂g7 siRNA treatment. Interestingly, LPC(16:0) activates PPARα and induces the expression of PPARα target genes in hepatocytes. Furthermore, we showed that LPC(16:0) has the ability to recover glucose uptake in adipocytes induced insulin resistance. These results reveal that LPC(16:0) is induced by PPARα activation in hepatocytes; LPC(16:0) contributes to the upregulation of PPARα target genes in hepatocytes and the recovery of glucose uptake in insulin-resistant adipocytes.     

Pigment Epithelium-Derived Factor 34-mer Peptide Prevents Liver Fibrosis and Hepatic Stellate Cell Activation through Down-Regulation of the PDGF Receptor

Pigment epithelium-derived factor (PEDF) has been shown previously to prevent liver fibrosis and hepatic stellate cell (HSC) activation. By investigating the functional domains in PEDF, we identified a 34-mer peptide (residues Asp44-Asn77) that harbors the same function as the full-length PEDF protein. Not only did the 34-mer suppress the development of fibrosis in carbon tetrachloride (CCl₄)-treated mouse liver but it also upregulated peroxisome proliferator-activated receptor-gamma (PPARγ) expression in HSCs in vivo. Platelet-derived growth factor (PDGF) plays a crucial role on the process of HSC activation in response to liver damage. The 34-mer suppressed PDGF-induced cell proliferation and expression of myofibroblastic marker proteins in primary rat HSC culture, increased the levels of PPARγ mRNA and protein in a dose-dependent manner and markedly reduced the level of active β-catenin protein, an HSC activating factor, in HSC-T6 cells. Similarly, IWR-1, an inhibitor of the Wnt response, displayed the same effect as the 34-mer in preventing HSC-T6 activation. The Wnt signaling-mediated PPARγ suppression was abolished by both the IWR-1 inhibitor and a small interfering RNA (siRNA) targeting β-catenin and the Wnt coreceptor, LRP6. Both PEDF and the 34-mer down-regulated PDGF receptor-α/β expression and blocked the PDGF-induced phosphorylation of Akt and ERK. Moreover, the inhibitory effect on PDGF receptor expression was abolished by PPARγ antagonists and PPARγ siRNA. Our observations indicate that the PEDF-derived 34-mer peptide can mimic PEDF in attenuating HSC activation. Investigation of this 34-mer peptide led to the identification of a signaling mechanism involving PPARγ induction, suppression of Wnt/β-catenin signaling and down-regulation of the PDGF receptor-α/β.     

Fibroblast Growth Factor 21 (FGF21) Protects against High Fat Diet Induced Inflammation and Islet Hyperplasia in Pancreas

Fibroblast growth factor 21 (FGF21) is an important endocrine metabolic regulator expressed in multiple tissues including liver and adipose tissue. Although highest levels of expression are in pancreas, little is known about the function of FGF21 in this tissue. In order to understand the physiology of FGF21 in the pancreas, we analyzed its expression and regulation in both acinar and islet tissues. We found that acinar tissue express 20-fold higher levels than that observed in islets. We also observed that pancreatic FGF21 is nutritionally regulated; a marked reduction in FGF21 expression was noted with fasting while obesity is associated with 3–4 fold higher expression. Acinar and islet cells are targets of FGF21, which when systemically administered, leads to phosphorylation of the downstream target ERK 1/2 in about half of acinar cells and a small subset of islet cells. Chronic, systemic FGF21 infusion down-regulates its own expression in the pancreas. Mice lacking FGF21 develop significant islet hyperplasia and periductal lymphocytic inflammation when fed with a high fat obesogenic diet. Inflammatory infiltrates consist of TCRb+ Thy1+ T lymphocytes with increased levels of Foxp3+ regulatory T cells. Increased levels of inflammatory cells were coupled with elevated expression of cytokines such as TNFα, IFNγ and IL1β. We conclude that FGF21 acts to limit islet hyperplasia and may also prevent pancreatic inflammation.     

FGF21 alleviates acute liver injury by inducing the SIRT1‐autophagy signalling pathway

Liver injury can lead to different hepatic diseases, which are the mainly causes of high global mortality and morbidity. Autophagy and Sirtuin type 1 (SIRT1) have been shown protective effects in response to liver injury. Previous studies have showed that Fibroblast growth factor 21 (FGF21) could alleviate acute liver injury (ALI), but the mechanism remains unclear. Here, we verified the relationship among FGF21, autophagy and SIRT1 in carbon tetrachloride (CCl₄)‐induced ALI. We established CCl₄‐induced ALI models in C57BL/6 mice and the L02 cell line. The results showed that FGF21 was robustly induced in response to stress during the development of ALI. After exogenous FGF21 treatment in ALI models, liver damage in ALI mice was significantly reduced, as well as serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Consistently, FGF21 also greatly reduced the levels of ALT, AST, pro‐inflammatory cytokines interleukin 6 (IL6) and tumour necrosis factor‐alpha (TNFα) in ALI cell lines. Mechanistically, exogenous FGF21 treatment efficiently upregulated the expression of autophagy marker microtubule‐associated protein light chain‐3 beta (LC3 II) and autophagy key molecule coiled‐coil myosin‐like BCL2‐interacting protein (Beclin1), which was accompanied by alleviating hepatotoxicity in CCl₄‐treated wild‐type mice. Then, we examined how FGF21 induced autophagy expression and found that SIRT1 was also upregulated by FGF21 treatment. To further verify our results, we constructed an anti‐SIRT1 lentit‐RNAi to inhibit SIRT1 expression in mice and L02 cells, which reversed the protective effect of FGF21 on ALI. In summary, these results indicate that FGF21 alleviates ALI by enhancing SIRT1‐mediated autophagy.     

Stress-related hormones modulate cytokine expression in the head kidney of gilthead seabream (Sparus aurata)

Neuro-endocrine and immune systems closely interact in fish, and their regulation is crucial for the maintenance of good health of cultured fish. We have used the seabream head kidney to study whether stress-related hormones can modulate the immune response. For this purpose, the effects of adrenaline, adrenocorticotropic hormone (ACTH) and cortisol on the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and the anti-inflammatory cytokine TGF-β1 were determined by means of quantitative real-time PCR on isolated head kidney cells. ACTH (150 ng mL⁻¹) caused an acute increase of TNF-α and IL-6 mRNA levels as well as an inhibition of IL-1β expression. The expression of the anti-inflammatory cytokine TGF-β1 was also increased, although in a lower extent. Adrenaline (1 μM) early effects were only clear inhibiting IL-1β expression but not TNF-α, IL-6 or TGF-β1 mRNA levels, while a longer exposure to the hormone inhibited all cytokines. Moreover, cortisol (50 and 100 ng mL⁻¹) reduced the expression of all cytokines in a dose-dependent manner. Bacterial lipopolysaccharide (LPS) stimulated IL-1β expression and inhibited that of the anti-inflammatory TGF-β1, although it was ineffective on TNF-α and IL-6. In addition, adrenaline and cortisol decreased the LPS-stimulated IL-1β expression, further demonstrating their previously reported anti-inflammatory effects. The combination of ACTH and LPS, on the other hand, did not affect LPS-stimulated IL-1β expression but was effective increasing TNF-α expression. Taking all these results in consideration, we conclude that the expression of pro- and anti-inflammatory cytokines in the seabream head kidney is highly influenced by stress-related hormones, thus indicating an important role for the endocrine system in the modulation of the immune response in teleost fish.