FGF 21 Mouse, His

What is the physiological role of FGF21 in mice?

FGF21 functions as an endocrine factor that regulates whole-body energy homeostasis in mice. It plays essential roles in glucose and lipid metabolism, particularly during nutritional challenges. FGF21 is primarily expressed in the liver but also in pancreas, skeletal muscle, and brown adipose tissue, where it signals through FGFR1c and co-receptor beta-klotho . Studies with FGF21-knockout mice have demonstrated its importance in ketogenesis during ketogenic diet feeding, as these mice develop hepatosteatosis and show marked impairments in ketone production when challenged with such diets . FGF21 also mediates increased water intake during ketogenic dieting and contributes to alcohol preference .

How does FGF21 expression change under different metabolic conditions in mice?

FGF21 expression is dynamically regulated in mice based on nutritional status. It is induced during fasting and promotes ketogenesis as an alternative energy source . When mice are fed a ketogenic diet, hepatic FGF21 expression increases significantly to facilitate metabolic adaptation . FGF21 knockout mice fed a ketogenic diet gain weight instead of losing it (as wild-type mice do), develop fatty liver, and show impaired glucose control, underscoring FGF21's role in adapting to dietary changes .

What phenotypes are observed in FGF21 knockout mice?

FGF21 knockout (FGF21KO) mice show mild weight gain and slightly impaired glucose homeostasis with age, indicating FGF21's role in long-term energy balance . While these mice can tolerate short-term (24-hour) fasting without severe complications, they demonstrate dramatically impaired adaptation to ketogenic diets. Unlike wild-type mice that become ketotic and lose substantial weight on ketogenic diets, FGF21KO mice have impaired ketosis, gain weight rather than lose it, and develop hepatosteatosis . At the molecular level, these effects correlate with reduced expression of key metabolic regulators PGC1α and PGC1β in FGF21KO mice .

What are the optimal conditions for expressing His-tagged FGF21 in bacterial systems?

For bacterial expression of His-tagged FGF21, the protein has been successfully purified using Escherichia coli with a pET30a vector system . Optimal expression typically requires induction at lower temperatures (16-18°C) to enhance protein solubility. The His-tag placement (N-terminal versus C-terminal) can significantly impact protein functionality, with N-terminal His-tags generally preferred as they appear to maintain better biological activity. Purification should employ immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography to remove aggregates that can form during the expression process.

How can researchers verify the biological activity of purified His-tagged FGF21?

Verification of His-tagged FGF21 biological activity can be performed through several functional assays. In vitro, researchers should assess the protein's ability to activate FGFR1c/β-klotho signaling in appropriate cell lines, measuring phosphorylation of downstream targets such as ERK. Glucose uptake assays in adipocytes provide a direct measurement of one of FGF21's key metabolic effects . For in vivo validation, acute administration to ob/ob mice should improve glucose tolerance in glucose tolerance tests (GTTs) . Additionally, chronic administration to diet-induced obese (DIO) mice should result in weight reduction and metabolic improvements, including decreased hepatic triglyceride content .

What are the available FGF21 knockout mouse models and how do they differ?

Several FGF21 knockout mouse models have been developed. One established model uses the pGTN29 vector to replace part of exon 1 (30 bp downstream of the ATG), all of exon 2, and the 5' region of exon 3 with a neomycin resistance gene, deleting approximately 1200 bp of the genomic FGF21 sequence . This model is available from Eleftheria Maratos-Flier. Another knockout line replaces part of exon 1 and all of exons 2 and 3 with an IRES-LacZ-polyA/PGK-neo cassette, available from Nobuyuki Itoh . Both models are viable and fertile. Additionally, adenovirus vectors encoding shRNA against FGF21 have been developed for specific knockdown studies .

What considerations should be made when designing studies with FGF21 knockout mice?

When designing studies with FGF21 knockout mice, researchers should consider several factors. First, the background strain is crucial, as C57BL/6 mice (the common background after backcrossing) have specific metabolic characteristics that may influence results. Age is also important, as the metabolic phenotype in FGF21KO mice becomes more pronounced with age . Environmental factors, particularly housing temperature, significantly affect brown adipose tissue (BAT) and white adipose tissue (WAT) biology, which are major targets of FGF21's metabolic effects . This may explain some divergent findings between studies. Additionally, diet challenges (standard chow vs. high-fat diet vs. ketogenic diet) produce markedly different phenotypes in FGF21KO mice and should be selected based on the specific research question .

How should researchers account for compensatory mechanisms in FGF21 knockout models?

FGF21 knockout mice may develop compensatory mechanisms that mask or alter phenotypes. To account for these, researchers should consider conditional knockout models (using Cre-lox systems) for tissue-specific and time-specific deletion of FGF21. Alternatively, acute knockdown using adenoviral shRNA delivery can circumvent long-term adaptations . Comprehensive metabolic phenotyping should include measures beyond the primary expected outcomes, such as examining changes in related growth factors (FGF19/15, FGF23) that might compensate for FGF21 deficiency. Researchers should also assess changes in FGF receptors and β-klotho co-receptor expression across tissues, as receptor upregulation may occur to increase sensitivity to remaining FGF signals.

How does FGF21 mediate its effects through both peripheral and central nervous system mechanisms?

FGF21 exerts its metabolic effects through both peripheral tissues and the central nervous system. In diet-induced obese (DIO) mouse models, FGF21's direct effects on the nervous system are essential for chronic benefits on body weight, insulin sensitivity, and hepatic triglyceride content . This primarily reflects the importance of weight loss in driving other metabolic improvements. The FGF21 receptor complex is expressed both within the hypothalamus (inside the blood-brain barrier) and in the hindbrain (outside the blood-brain barrier) . While FGF21 can cross the blood-brain barrier, it does so with low efficiency , suggesting that central and peripheral effects may be differentially regulated depending on circulating levels and pathophysiological conditions.

What are the key differences between mouse and human FGF21 biology that impact translational research?

Despite robust beneficial effects of FGF21 on glucose metabolism in mice, significant species differences exist that complicate translation to humans. Most notably, while FGF21 analogs dramatically improve glucose control in mouse models, they fail to lower blood glucose in human clinical trials . This discrepancy may result from species-specific adaptations related to metabolism, as mice and humans differ significantly in lipoprotein metabolism, susceptibility to atherosclerosis, and inflammatory responses . Housing temperatures for laboratory mice also dramatically affect brown adipose tissue (BAT) and white adipose tissue (WAT) biology, which are major targets of FGF21 . Since BAT is more prominent in mice than humans, this may explain differing effects on glucose metabolism between species. Researchers must consider these species differences when extrapolating mouse findings to human applications.

How can FGF21 be glyco-engineered to improve its pharmacological properties?

Glyco-engineering of FGF21 can dramatically enhance its pharmaceutical properties. One approach involves N-glycan engineering to increase protease resistance and improve solubility, combined with Fc fusion for half-life extension . A specific example is the development of Fc-FGF21[R19V][N171] (PF-06645849), which incorporates a single point mutation (R19V) to improve manufacturability in Chinese Hamster Ovary cells . This modified FGF21 demonstrated substantially improved solubility and stability compatible with subcutaneous administration, low systemic clearance (0.243 mL/hr/kg), and a long terminal half-life (approximately 200 hours for intact protein) in cynomolgus monkeys . The improved pharmacokinetic properties translated to robust glucose tolerance improvements lasting 14 days after a single subcutaneous dose in ob/ob mice, and greater body weight loss in DIO mice at lower and less frequent doses compared to previous FGF21 analogs .

How should researchers interpret contradictory findings between mouse and human FGF21 studies?

When interpreting contradictory findings between mouse and human FGF21 studies, researchers should consider several factors. First, examine the specific endpoints measured, as some effects (like improved lipid profiles) translate between species while others (like glucose lowering) do not . Second, consider the models used—genetic mouse models of obesity (ob/ob, db/db) differ fundamentally from human obesity. Third, methodological factors matter significantly, including the age of mice, housing temperature (which affects BAT activity), and dietary conditions . Finally, consider pharmacological versus physiological dosing—many mouse studies use supraphysiological doses that may engage pathways not relevant at endogenous levels. Rather than dismissing contradictions as species differences, researchers should use these discrepancies to generate hypotheses about the underlying mechanisms and pathway dependencies that might differ between species .

What biomarkers can be used to compare FGF21 activity across species?

To compare FGF21 activity across species, researchers should employ multiple biomarkers reflecting both direct signaling and downstream physiological effects. For direct signaling, measuring phosphorylation of ERK1/2 and other MAPK pathway components in FGF21-responsive tissues provides insight into immediate receptor activation. For downstream effects, changes in gene expression of key FGF21-regulated genes (PGC1α, PGC1β) in liver and adipose tissue offer cross-species comparability . Metabolic parameters like serum ketone levels during ketogenic diet challenge can assess functional responses . Additionally, changes in energy expenditure, body weight, and adiposity are conserved responses to FGF21 across species, though with different magnitudes . Finally, lipidomic profiles (particularly triglyceride and cholesterol levels) respond similarly to FGF21 treatment across species and provide reliable comparative biomarkers .

What are the optimal methods for measuring FGF21 levels in mouse samples?

For accurate measurement of FGF21 levels in mouse samples, enzyme-linked immunosorbent assay (ELISA) is commonly employed . When selecting commercial kits, researchers should verify specificity for mouse FGF21 versus human FGF21, as cross-reactivity varies between assays. Sample handling is crucial—serum or plasma should be collected at consistent times of day to account for diurnal variations in FGF21 levels. Samples should be processed quickly and stored at -80°C to prevent degradation. For tissue-specific FGF21 expression, quantitative PCR for mRNA expression provides valuable information, particularly for liver, where FGF21 is predominantly expressed. Western blot analysis using specific antibodies (such as goat polyclonal anti-FGF21 from R&D Systems) can detect protein levels in tissue lysates, though sensitivity may be limited for tissues with lower expression.

What controls are essential when studying genetically modified FGF21 mouse models?

When studying genetically modified FGF21 mouse models, several controls are essential. First, appropriate wild-type littermates should be used to control for genetic background effects. For FGF21 knockout studies, heterozygote breeding pairs should be used to generate experimental knockouts and wild-type littermate controls, ensuring identical environmental conditions during development . In transgenic overexpression models, non-transgenic littermates serve as primary controls, but researchers should also consider using "empty vector" transgenic controls to account for insertion effects . When studying metabolic phenotypes, pair-feeding controls are crucial to distinguish direct FGF21 effects from those secondary to changes in food intake. For studies involving specific diets (particularly ketogenic diets), both standard chow and dietary intervention groups should be included for knockout and wild-type animals to properly assess diet-genotype interactions .

How can researchers address issues with FGF21 knockout mouse fertility and viability?

While FGF21 knockout mice are reported to be viable and fertile , researchers may occasionally encounter reduced fertility or viability issues. To address these, several approaches are recommended. First, consider the genetic background, as backcrossing to certain strains may introduce or exacerbate fertility issues. Maintaining the colony through heterozygous breeding pairs can help mitigate reduced homozygous fertility. For colonies with reduced viability, adjusting housing conditions, particularly temperature, may help, as FGF21 plays roles in thermoregulation. Dietary interventions should be carefully introduced—particularly, ketogenic diets should be gradually implemented in FGF21KO mice rather than abruptly switched, as these mice have impaired adaptive responses to such diets . Finally, if persistent issues occur, consider alternative approaches such as conditional knockouts or acute knockdown using adenoviral-delivered shRNA against FGF21 .

What are the common pitfalls when interpreting metabolic phenotypes in FGF21 mouse models?

Several pitfalls can complicate interpretation of metabolic phenotypes in FGF21 mouse models. First, age-dependency—FGF21KO mice develop more pronounced phenotypes with age, so timing of experiments is crucial . Second, environmental factors, particularly housing temperature, dramatically affect BAT/WAT biology and consequently FGF21's metabolic effects . Standard mouse housing (22°C) represents a chronic cold stress for mice, potentially exaggerating certain FGF21 phenotypes. Third, the baseline metabolic state (fed vs. fasted) significantly impacts results, as FGF21 function differs between these states . Fourth, compensatory mechanisms may develop in constitutive knockout models, potentially masking phenotypes. Finally, strain-specific metabolism can influence outcomes—C57BL/6 mice (common background) are relatively resistant to insulin resistance, potentially blunting some FGF21-related phenotypes. Researchers should account for these factors through appropriate controls, careful experimental design, and comprehensive phenotyping beyond primary outcome measures.