Biological and pharmacological functions of the FGF19- and FGF21-coreceptor beta klotho.

Beta klotho (KLB) is a fundamental component in fibroblast growth factor receptor (FGFR) signaling as it serves as an obligatory coreceptor for the endocrine hormones fibroblast growth factor 19 (FGF19) and fibroblast growth factor 21 (FGF21). Through the development of FGF19- and FGF21 mimetics, KLB has emerged as a promising drug target for treating various metabolic diseases, such as type 2 diabetes (T2D), non-alcoholic fatty liver disease (NAFLD), and cardiovascular disease. While rodent studies have significantly increased our understanding of KLB function, current clinical trials that test the safety and efficacy of KLB-targeting drugs raise many new scientific questions about human KLB biology. Although most KLB-targeting drugs can modulate disease activity in humans, individual patient responses differ substantially. In addition, species-specific differences in KLB tissue distribution may explain why the glucose-lowering effects that were observed in preclinical studies are not fully replicated in clinical trials. Besides, the long-term efficacy of KLB-targeting drugs might be limited by various pathophysiological conditions known to reduce the expression of KLB. Moreover, FGF19/FGF21 administration in humans is also associated with gastrointestinal side effects, which are currently unexplained. A better understanding of human KLB biology could help to improve the efficacy and safety of existing or novel KLB/FGFR-targeting drugs. In this review, we provide a comprehensive overview of the current understanding of KLB biology, including genetic variants and their phenotypic associations, transcriptional regulation, protein structure, tissue distribution, subcellular localization, and function. In addition, we will highlight recent developments regarding the safety and efficacy of KLB-targeting drugs in clinical trials. These insights may direct the development and testing of existing and future KLB-targeting drugs.

Pharmacological inhibition of MEK1/2 signaling disrupts bile acid metabolism through loss of Shp and enhanced Cyp7a1 expression.

The RAS-MAPK signaling pathway is one of the most frequently dysregulated pathways in human cancer. Small molecule inhibitors directed against this pathway have clinical activity in patients with various cancer types and can improve patient outcomes. However, the use of these drugs is associated with adverse effects, which can result in dose reduction or treatment interruption. A better molecular understanding of on-target, off-tumor effects may improve toxicity management. In the present study, we aimed to identify early initiating biological changes in the liver upon pharmacological inhibition of the RAS-MAPK signaling pathway. To this end, we tested the effect of MEK inhibitor PD0325901 using mice and human hepatocyte cell lines. Male C57BL/6 mice were treated with either vehicle or PD0325901 for six days, followed by transcriptome analysis of the liver and phenotypic characterization. Pharmacological MEK inhibition altered the expression of 423 genes, of which 78 were upregulated and 345 were downregulated. We identified Shp, a transcriptional repressor, and Cyp7a1, the rate-limiting enzyme in converting cholesterol to bile acids, as the top differentially expressed genes. PD0325901 treatment also affected other genes involved in bile acid regulation, which was associated with changes in the composition of plasma bile acids and composition and total levels of fecal bile acids and elevated predictive biomarkers of early liver toxicity. In conclusion, short-term pharmacological MEK inhibition results in profound changes in bile acid metabolism, which may explain some of the clinical adverse effects of pharmacological inhibition of the RAS-MAPK pathway, including gastrointestinal complications and hepatotoxicity.

Autocrine FGF1 signaling promotes glucose uptake in adipocytes.

Fibroblast growth factor 1 (FGF1) is an autocrine growth factor released from adipose tissue during over-nutrition or fasting to feeding transition. While local actions underlie the majority of FGF1's anti-diabetic functions, the molecular mechanisms downstream of adipose FGF receptor signaling are unclear. We investigated the effects of FGF1 on glucose uptake and its underlying mechanism in murine 3T3-L1 adipocytes and in ex vivo adipose explants from mice. FGF1 increased glucose uptake in 3T3-L1 adipocytes and epididymal WAT (eWAT) and inguinal WAT (iWAT). Conversely, glucose uptake was reduced in eWAT and iWAT of FGF1 knockout mice. We show that FGF1 acutely increased adipocyte glucose uptake via activation of the insulin-sensitive glucose transporter GLUT4, involving dynamic crosstalk between the MEK1/2 and Akt signaling proteins. Prolonged exposure to FGF1 stimulated adipocyte glucose uptake by MEK1/2-dependent transcription of the basal glucose transporter GLUT1. We have thus identified an alternative pathway to stimulate glucose uptake in adipocytes, independent from insulin, which could open new avenues for treating patients with type 2 diabetes.

A novel role for GalNAc-T2 dependent glycosylation in energy homeostasis.

OBJECTIVE: GALNT2, encoding polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2), was initially discovered as a regulator of high-density lipoprotein metabolism. GalNAc-T2 is known to exert these effects through post-translational modification, i.e., O-linked glycosylation of secreted proteins with established roles in plasma lipid metabolism. It has recently become clear that loss of GALNT2 in rodents, cattle, nonhuman primates, and humans should be regarded as a novel congenital disorder of glycosylation that affects development and body weight. The role of GALNT2 in metabolic abnormalities other than plasma lipids, including insulin sensitivity and energy homeostasis, is poorly understood. METHODS: GWAS data from the UK Biobank was used to study variation in the GALNT2 locus beyond changes in high-density lipoprotein metabolism. Experimental data were obtained through studies in Galnt2-/- mice and wild-type littermates on both control and high-fat diet. RESULTS: First, we uncovered associations between GALNT2 gene variation, adiposity, and body mass index in humans. In mice, we identify the insulin receptor as a novel substrate of GalNAc-T2 and demonstrate that Galnt2-/- mice exhibit decreased adiposity, alterations in insulin signaling and a shift in energy substrate utilization in the inactive phase. CONCLUSIONS: This study identifies a novel role for GALNT2 in energy homeostasis, and our findings suggest that the local effects of GalNAc-T2 are mediated through posttranslational modification of the insulin receptor.

Mice with a deficiency in Peroxisomal Membrane Protein 4 (PXMP4) display mild changes in hepatic lipid metabolism.

Peroxisomes play an important role in the metabolism of a variety of biomolecules, including lipids and bile acids. Peroxisomal Membrane Protein 4 (PXMP4) is a ubiquitously expressed peroxisomal membrane protein that is transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα), but its function is still unknown. To investigate the physiological function of PXMP4, we generated a Pxmp4 knockout (Pxmp4-/-) mouse model using CRISPR/Cas9-mediated gene editing. Peroxisome function was studied under standard chow-fed conditions and after stimulation of peroxisomal activity using the PPARα ligand fenofibrate or by using phytol, a metabolite of chlorophyll that undergoes peroxisomal oxidation. Pxmp4-/- mice were viable, fertile, and displayed no changes in peroxisome numbers or morphology under standard conditions. Also, no differences were observed in the plasma levels of products from major peroxisomal pathways, including very long-chain fatty acids (VLCFAs), bile acids (BAs), and BA intermediates di- and trihydroxycholestanoic acid. Although elevated levels of the phytol metabolites phytanic and pristanic acid in Pxmp4-/- mice pointed towards an impairment in peroxisomal α-oxidation capacity, treatment of Pxmp4-/- mice with a phytol-enriched diet did not further increase phytanic/pristanic acid levels. Finally, lipidomic analysis revealed that loss of Pxmp4 decreased hepatic levels of the alkyldiacylglycerol class of neutral ether lipids, particularly those containing polyunsaturated fatty acids. Together, our data show that while PXMP4 is not critical for overall peroxisome function under the conditions tested, it may have a role in the metabolism of (ether)lipids.

Potential of therapeutic bile acids in the treatment of neonatal Hyperbilirubinemia.

Neonatal hyperbilirubinemia or jaundice is associated with kernicterus, resulting in permanent neurological damage or even death. Conventional phototherapy does not prevent hyperbilirubinemia or eliminate the need for exchange transfusion. Here we investigated the potential of therapeutic bile acids ursodeoxycholic acid (UDCA) and obeticholic acid (OCA, 6-α-ethyl-CDCA), a farnesoid-X-receptor (FXR) agonist, as preventive treatment options for neonatal hyperbilirubinemia using the hUGT1*1 humanized mice and Ugt1a-deficient Gunn rats. Treatment of hUGT1*1 mice with UDCA or OCA at postnatal days 10-14 effectively decreased bilirubin in plasma (by 82% and 62%) and brain (by 72% and 69%), respectively. Mechanistically, our findings indicate that these effects are mediated through induction of protein levels of hUGT1A1 in the intestine, but not in liver. We further demonstrate that in Ugt1a-deficient Gunn rats, UDCA but not OCA significantly decreases plasma bilirubin, indicating that at least some of the hypobilirubinemic effects of UDCA are independent of UGT1A1. Finally, using the synthetic, non-bile acid, FXR-agonist GW4064, we show that some of these effects are mediated through direct or indirect activation of FXR. Together, our study shows that therapeutic bile acids UDCA and OCA effectively reduce both plasma and brain bilirubin, highlighting their potential in the treatment of neonatal hyperbilirubinemia.

Pegbelfermin (BMS-986036): an investigational PEGylated fibroblast growth factor 21 analogue for the treatment of nonalcoholic steatohepatitis.

Introduction: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide and is strongly associated with obesity and insulin resistance. NAFLD refers to a spectrum of disorders ranging from asymptomatic hepatic steatosis (nonalcoholic fatty liver, NAFL) to nonalcoholic steatohepatitis (NASH), which increases the risk of developing more severe forms of liver disease such as progressive fibrosis, cirrhosis, and liver cancer. Currently, there are no food and drug administration (FDA) approved drugs to treat NASH. Pegbelfermin (BMS-986036) is a PEGylated fibroblast growth factor 21 (FGF21) analogue that is under investigation for the treatment of NASH.Areas covered: We reviewed the (pre)clinical pegbelfermin studies and compared these with other studies that assessed FGF21 and FGF21 analogues in the treatment of NASH.Expert opinion: With no FDA approved treatments available for NASH, there is an urgent need for novel therapies. Pegbelfermin is a systemic treatment with pleiotropic effects on various tissues. Short-term adverse effects are limited, but more research is required to study potential long-term safety issues. In a phase 2a trial, pegbelfermin has shown promising improvements in several NASH related outcomes. However, clinical trials demonstrating long-term benefits on hard outcomes such as liver histology, cirrhosis development, or survival are required for further validation.

Fibroblast growth factors in control of lipid metabolism: from biological function to clinical application.

PURPOSE OF REVIEW: Several members of the fibroblast growth factor (FGF) family have been identified as key regulators of energy metabolism in rodents and nonhuman primates. Translational studies show that their metabolic actions are largely conserved in humans, which led to the development of various FGF-based drugs, including FGF21-mimetics LY2405319, PF-05231023, and pegbelfermin, and the FGF19-mimetic NGM282. Recently, a number of clinical trials have been published that examined the safety and efficacy of these novel therapeutic proteins in the treatment of obesity, type 2 diabetes (T2D), nonalcoholic steatohepatitis (NASH), and cholestatic liver disease. In this review, we discuss the current understanding of FGFs in metabolic regulation and their clinical potential. RECENT FINDINGS: FGF21-based drugs induce weight loss and improve dyslipidemia in patients with obesity and T2D, and reduce steatosis in patients with NASH. FGF19-based drugs reduce steatosis in patients with NASH, and ameliorate bile acid-induced liver damage in patients with cholestasis. In contrast to their potent antidiabetic effects in rodents and nonhuman primates, FGF-based drugs do not appear to improve glycemia in humans. In addition, various safety concerns, including elevation of low-density lipoprotein cholesterol, modulation of bone homeostasis, and increased blood pressure, have been reported as well. SUMMARY: Clinical trials with FGF-based drugs report beneficial effects in lipid and bile acid metabolism, with clinical improvements in dyslipidemia, steatosis, weight loss, and liver damage. In contrast, glucose-lowering effects, as observed in preclinical models, are currently lacking.

Dietary Sargassum fusiforme improves memory and reduces amyloid plaque load in an Alzheimer's disease mouse model.

Activation of liver X receptors (LXRs) by synthetic agonists was found to improve cognition in Alzheimer's disease (AD) mice. However, these LXR agonists induce hypertriglyceridemia and hepatic steatosis, hampering their use in the clinic. We hypothesized that phytosterols as LXR agonists enhance cognition in AD without affecting plasma and hepatic triglycerides. Phytosterols previously reported to activate LXRs were tested in a luciferase-based LXR reporter assay. Using this assay, we found that phytosterols commonly present in a Western type diet in physiological concentrations do not activate LXRs. However, a lipid extract of the 24(S)-Saringosterol-containing seaweed Sargassum fusiforme did potently activate LXRß. Dietary supplementation of crude Sargassum fusiforme or a Sargassum fusiforme-derived lipid extract to AD mice significantly improved short-term memory and reduced hippocampal Aß plaque load by 81%. Notably, none of the side effects typically induced by full synthetic LXR agonists were observed. In contrast, administration of the synthetic LXRα activator, AZ876, did not improve cognition and resulted in the accumulation of lipid droplets in the liver. Administration of Sargassum fusiforme-derived 24(S)-Saringosterol to cultured neurons reduced the secretion of Aß42. Moreover, conditioned medium from 24(S)-Saringosterol-treated astrocytes added to microglia increased phagocytosis of Aß. Our data show that Sargassum fusiforme improves cognition and alleviates AD pathology. This may be explained at least partly by 24(S)-Saringosterol-mediated LXRß activation.

The Modulating Role of Sex and Anabolic-Androgenic Steroid Hormones in Cannabinoid Sensitivity.

Cannabis is the most commonly used illicit drug worldwide. Although its use is associated with multiple adverse health effects, including the risk of developing addiction, recreational and medical cannabis use is being increasing legalized. In addition, use of synthetic cannabinoid drugs is gaining considerable popularity and is associated with mass poisonings and occasional deaths. Delineating factors involved in cannabis use and addiction therefore becomes increasingly important. Similarly to other drugs of abuse, the prevalence of cannabis use and addiction differs remarkably between males and females, suggesting that sex plays a role in regulating cannabinoid sensitivity. Although it remains unclear how sex may affect the initiation and maintenance of cannabis use in humans, animal studies strongly suggest that endogenous sex hormones modulate cannabinoid sensitivity. In addition, synthetic anabolic-androgenic steroids alter substance use and further support the importance of sex steroids in controlling drug sensitivity. The recent discovery that pregnenolone, the precursor of all steroid hormones, controls cannabinoid receptor activation corroborates the link between steroid hormones and the endocannabinoid system. This article reviews the literature regarding the influence of endogenous and synthetic steroid hormones on the endocannabinoid system and cannabinoid action.

Distribution of Non-Persistent Endocrine Disruptors in Two Different Regions of the Human Brain.

Non-persistent endocrine disrupting chemicals (npEDCs) can affect multiple organs and systems in the body. Whether npEDCs can accumulate in the human brain is largely unknown. The major aim of this pilot study was to examine the presence of environmental phenols and parabens in two distinct brain regions: the hypothalamus and white-matter tissue. In addition, a potential association between these npEDCs concentrations and obesity was investigated. Post-mortem brain material was obtained from 24 individuals, made up of 12 obese and 12 normal-weight subjects (defined as body mass index (BMI) > 30 and BMI < 25 kg/m², respectively). Nine phenols and seven parabens were measured by isotope dilution TurboFlow-LC-MS/MS. In the hypothalamus, seven suspect npEDCs (bisphenol A, triclosan, triclocarban and methyl-, ethyl-, n-propyl-, and benzyl paraben) were detected, while five npEDCs (bisphenol A, benzophenone-3, triclocarban, methyl-, and n-propyl paraben) were found in the white-matter brain tissue. We observed higher levels of methylparaben (MeP) in the hypothalamic tissue of obese subjects as compared to controls (p = 0.008). Our findings indicate that some suspected npEDCs are able to cross the blood-brain barrier. Whether the presence of npEDCs can adversely affect brain function and to which extent the detected concentrations are physiologically relevant needs to be further investigated.

The anabolic steroid nandrolone alters cannabinoid self-administration and brain CB1 receptor density and function.

Clinical and pre-clinical observations indicate that anabolic-androgenic steroids can induce neurobiological changes that alter the rewarding effects of drugs of abuse. In this study, we investigated the effect of the anabolic steroid nandrolone on the rewarding properties of the cannabinoid CB1 receptor agonist WIN55,212-2 (WIN) in rats. Lister Hooded male rats were treated intramuscularly with nandrolone (15mg/kg) or vehicle for 14 consecutive days, and then allowed to self-administer WIN (12.5µg/kg/infusion) intravenously. After reaching stable drug intake, self-administration behavior was extinguished to examine drug- and cue-induced reinstatement of cannabinoid-seeking behavior. Other behavioral parameters presumed to influence drug-taking and drug-seeking behaviors were examined to gain more insight into the behavioral specificity of nandrolone treatment. Finally, animals were sacrificed for analysis of CB1 receptor density and function in selected brain areas. We found that nandrolone-treated rats self-administered up to 2 times more cannabinoid than vehicle-treated rats, but behaved similarly to control rats when tested for drug- and cue-induced reinstatement of cannabinoid-seeking behavior. Enhanced cannabinoid intake by nandrolone-treated rats was not accompanied by changes in locomotor activity, sensorimotor gating, or memory function. However, our molecular data show that after chronic WIN self-administration nandrolone-treated rats display altered CB1 receptor density and function in selected brain areas. We hypothesize that increased cannabinoid self-administration in nandrolone-treated rats results from a nandrolone-induced decrease in reward function, which rats seem to compensate by voluntarily increasing their cannabinoid intake. Altogether, our findings corroborate the hypothesis that chronic exposure to anabolic-androgenic steroids induces dysfunction of the reward pathway in rats and might represent a potential risk factor for abuse of cannabis and other drugs in humans.

Effective treatment of steatosis and steatohepatitis by fibroblast growth factor 1 in mouse models of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder and is strongly associated with obesity and type 2 diabetes. Currently, there is no approved pharmacological treatment for this disease, but improvement of insulin resistance using peroxisome proliferator-activated receptor-γ (PPARγ) agonists, such as thiazolidinediones (TZDs), has been shown to reduce steatosis and steatohepatitis effectively and to improve liver function in patients with obesity-related NAFLD. However, this approach is limited by adverse effects of TZDs. Recently, we have identified fibroblast growth factor 1 (FGF1) as a target of nuclear receptor PPARγ in visceral adipose tissue and as a critical factor in adipose remodeling. Because FGF1 is situated downstream of PPARγ, it is likely that therapeutic targeting of the FGF1 pathway will eliminate some of the serious adverse effects associated with TZDs. Here we show that pharmacological administration of recombinant FGF1 (rFGF1) effectively improves hepatic inflammation and damage in leptin-deficient ob/ob mice and in choline-deficient mice, two etiologically different models of NAFLD. Hepatic steatosis was effectively reduced only in ob/ob mice, suggesting that rFGF1 stimulates hepatic lipid catabolism. Potentially adverse effects such as fibrosis or proliferation were not observed in these models. Because the anti-inflammatory effects were observed in both the presence and absence of the antisteatotic effects, our findings further suggest that the anti-inflammatory property of rFGF1 is independent of its effect on lipid catabolism. Our current findings indicate that, in addition to its potent glucose-lowering and insulin-sensitizing effects, rFGF1 could be therapeutically effective in the treatment of NAFLD.

Fibroblast Growth Factor Signaling in Metabolic Regulation.

The prevalence of obesity is a growing health problem. Obesity is strongly associated with several comorbidities, such as non-alcoholic fatty liver disease, certain cancers, insulin resistance, and type 2 diabetes, which all reduce life expectancy and life quality. Several drugs have been put forward in order to treat these diseases, but many of them have detrimental side effects. The unexpected role of the family of fibroblast growth factors in the regulation of energy metabolism provides new approaches to the treatment of metabolic diseases and offers a valuable tool to gain more insight into metabolic regulation. The known beneficial effects of FGF19 and FGF21 on metabolism, together with recently discovered similar effects of FGF1 suggest that FGFs and their derivatives carry great potential as novel therapeutics to treat metabolic conditions. To facilitate the development of new therapies with improved targeting and minimal side effects, a better understanding of the molecular mechanism of action of FGFs is needed. In this review, we will discuss what is currently known about the physiological roles of FGF signaling in tissues important for metabolic homeostasis. In addition, we will discuss current concepts regarding their pharmacological properties and effector tissues in the context of metabolic disease. Also, the recent progress in the development of FGF variants will be reviewed. Our goal is to provide a comprehensive overview of the current concepts and consensuses regarding FGF signaling in metabolic health and disease and to provide starting points for the development of FGF-based therapies against metabolic conditions.