Dissertation
FGF21 enhances GLUT4 translocation in primary brown adipocytes and the hunt for new hepatokines
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2025
DOI: 10.25820/etd.008108
Abstract
Fibroblast growth factor 21 (FGF21) is a liver-derived hormone that regulates several key factors influencing bodyweight – including energy expenditure, food intake, and sucrose/sweet-taste preference. FGF21’s tissue targets are conferred by β-Klotho (KLB), it’s obligate co-receptor thar forms a complex with FGFR1c to initiate downstream signaling. KLB is found in several regions of the brain, adipose tissue, the pancreas, and the liver. Acute administration of FGF21 to lean and obese rodents increases insulin sensitivity, quantified by glucose uptake, by driving glucose to brown adipose tissue (BAT). Despite the numerous FGF21 analogues and mimetics that have made it to clinical trials, little is still known about the downstream signaling that is elicited when FGF21 is applied to BAT. FGF21, like other FGFs, activates MAPK signaling via MEK -> ERK1/2, a kinase with a plethora of targets. To explore how FGF21 signaling increases insulin sensitivity, we employed phosphoproteomics to analyze FGF21 treated primary brown adipocytes (PBAs) to probe it’s downstream signaling targets at acute timepoints (5 and 15 minutes). We found several proteins involved in glucose transporter type 4 (GLUT4) translocation were phosphorylated in response to FGF21 treatment, most of which overlap with insulin treated PBAs. Ribosomal Kinase 2 (RSK2) was identified as a possible mediator of the phosphorylation of GLUT4 machinery in FGF21-treated samples, due to a lack of complete Protein Kinase B (AKT) activation in the absence of insulin and known phosphorylation activity against Akt substrate of 160 kDa (AS160). To confirm our findings, we utilized a mouse model that allowed us to kinetically read GLUT4 translocation in PBAs to investigate FGF21’s effects on insulin-stimulated GLUT4 translocation.
At a molecular level, our phosphoproteomics results revealed FGF21 treatment in PBAs increases the phosphorylation of proteins involved in GLUT4 translocation: AS160, DENND4c, and TUG. Functionally, FGF21 increases GLUT4 translocation and glucose uptake when co-administered with insulin. We propose this is mediated through a well-characterized signaling cascade involving ERK1/2’s activation of RSK2. Given FGF21’s endocrine capability, hepatic origin, and on-going developments of FGF21 analogues to treat liver disease, we also set out to explore how other liver-derived hormones might be sending signals to the rest of the body.
In an effort to identify novel hepatokines, we employed TurboID, a BirA biotin ligase derivative, to label secreted proteins from the liver. To determine find liver-secreted proteins that were signaling to other organs, we utilized TurboID in vivo with liver-specific expression and analyzed other peripheral organs’ level of biotin labeled proteins. We found increased biotin signal in both the adipose tissues and muscle depots. Given FGF21’s important actions in the brain, we next utilized proteomics, in conjunction with streptavidin immunoprecipitation, to analyze proteins that were enriched by the streptavidin purification. The nucleus tractus solitarius (NTS) and hypothalamus are important areas in the brain for bodyweight regulation and energy homeostatsis and key targets for FGF21 action, thus we isolated these two areas for whole proteome analysis. Several carboxylesterase proteins were found enriched in our TurboID samples, enzymes important for drug and lipid metabolism. These proteins are predicted to be liver-derived and potentially secreted, although there is some expression in the intestines as well.
Notably, we find these proteins are enriched in both our NTS and hypothalamus samples, indicating these proteins’ can pass the blood-brain barrier and land in at least these 2 brain areas. Further studies will be needed to assess what other areas these proteins are reaching after secretion from the liver. Recently, CES2a and CES2c were found to be secreted from the liver in response to exercise. The same report also demonstrates overexpression of these proteins has an anti-obesity effect, slowing high-fat diet induced weight gain. Our data indicates these CES2 family members, along with others including: CES2e, CES3a, CES3b, and CES1, are capable of reaching the brain after secretion from the liver, an important potential avenue to influence bodyweight.
In summation, utilizing TurboID in conjunction with peripheral blotting, immunoprecipitation, and proteomics demonstrates the potential for interrogating liver-periphery and liver-brain crosstalk with this method. Importantly, this system can be utilized completely in-vivo, representing the endogenous physiology, as opposed to in vitro studies. Further studies will be needed to elucidate if these carboxylesterase enzymes are still active upon reaching the NTS and the hypothalamus to determine how they might affect the various cell types in the brain. Increased secreted CES2a/c proteins have anti-obesity effects, but it is yet to be determined what other tissues they might travel to outside the brain to mediate this.
Details
- Title: Subtitle
- FGF21 enhances GLUT4 translocation in primary brown adipocytes and the hunt for new hepatokines
- Creators
- Matthew C. Juber
- Contributors
- Matthew J. Potthoff (Advisor)Chad Grueter (Committee Member)Brian O'Neill (Committee Member)Renata Pereira Alambert (Committee Member)Bhagirath Chaurasia (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biomedical Science (Molecular Medicine)
- Date degree season
- Summer 2025
- DOI
- 10.25820/etd.008108
- Publisher
- University of Iowa
- Number of pages
- xi, 81 pages
- Copyright
- Copyright 2025 Matthew C. Juber
- Language
- English
- Date submitted
- 06/12/2025
- Description illustrations
- Illustrations, graphs, charts, tables
- Description bibliographic
- Includes bibliographical references (pages 68-81).
- Public Abstract (ETD)
There is a hormone in the body, fibroblast growth factor 21 (FGF21), that can increase insulin sensitivity in rodents with just a single dose. FGF21 has been studied for its potential to treat obesity, diabetes, liver disease, and even alcohol use disorder. When administered, it enhances insulin s ability to lower blood glucose by driving glucose into brown adipose tissue, a type of fat that burns energy. The first goal of this work was to understand what FGF21 does to brown fat cells to cause this effect. Using primary brown adipocytes and phosphoproteomics, we found that FGF21 activates several proteins involved in glucose uptake, many of which overlap with insulin signaling. We propose that FGF21 acts through a complementary but distinct pathway to enhance insulin action.
The second goal was to identify other liver-derived proteins that might reach the brain, like FGF21. To do this, we used TurboID, a tool that tags proteins as they are secreted from the liver. We then purified tagged proteins from brain tissue and performed proteomics to see which liver proteins were present. We focused on two brain regions involved in bodyweight regulation: the hypothalamus and the nucleus tractus solitarius (NTS). Among the proteins enriched in these areas were several carboxylesterases enzymes involved in fat and drug metabolism. This work demonstrates a strategy for identifying liver-derived proteins in the brain and sets the stage for future studies on their function.
- Academic Unit
- Biomedical Science Program; Craniofacial Anomalies Research Center
- Record Identifier
- 9984948540302771
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