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Restoring Homeostasis in the Face of Overnutrition

 

By: Carol A. Rouzer, VICB Communications
Published:  October 23, 2015

 

 

Chemical biology combined with genetic approaches defines the role of fibroblast growth factor-1 in β cell differentiation under conditions of overnutrition.

 

Insulin is one of the most important metabolic regulatory hormones. Following a meal, levels of glucose and other nutrients increase in the blood, stimulating the secretion of insulin by β cells in the Islets of Langerhans of the pancreas (Figure 1). Insulin promotes the absorption and utilization of nutrients, particularly glucose, by muscle and adipose tissue. When this regulatory mechanism fails, diabetes mellitus results, leading to hyperglycemia (increased blood glucose levels) and eventually, multiple deleterious health effects. A major cause of diabetes in modern western cultures is overnutrition, which stresses glucose homeostasis in two ways. The first results from the excessive availability of glucose from food, and the second from insulin resistance, a response to overnutrition-related obesity. Both of these conditions place high demands on β cells for increased insulin production. A physiological response to this stress is an increase in β cell number, which occurs via both proliferation of existing β cells and the differentiation of precursor cells to form new β cells (neogenesis). This response helps to maintain glucose homeostasis under stress conditions, and its failure contributes to the development of diabetes. The signaling pathways that contribute to β cell proliferation in response to overnutrition have been extensively studied, but little is known regarding the triggers of β cell neogenesis. Now, Vanderbilt Institute of Chemical Biology member Wenbiao Chen and his laboratory reveal the role of fibroblast growth factor-1 (FGF1) in triggering the differentiation of precursor cells into new β cells (M. Li, et al. (2015) Diabetes, published online September 25, DOI: 10.2337/db15-0085).

 

 




Figure 1. Mechanism of insulin secretion. In going from the fasted state (A) to the fed state (B), the levels of glucose and other nutrients in the blood increase. Glucose enters the β cell through a glucose transporter. Metabolism via the glycolytic pathway produces pyruvate, which enters the mitochondrion, leading to further metabolism and production of ATP. ATP blocks the ATP-sensitive potassium channel, preventing the efflux of potassium ions from the cell. This results in membrane depolarization, which activates a voltage-dependent calcium channel. The influx of calcium through this channel promotes the movement of insulin-containing vesicles to the plasma membrane, resulting in the exocytosis of insulin. Figure provided by Carol Rouzer.


 

To learn more about control of β cell neogenesis, the Chen lab investigators used a zebrafish model of overnutrition. They discovered that exposure of larval zebrafish to medium containing 5% chicken egg yolk for 8 hours led to a 25% increase in insulin-producing β cells as compared to the number of β cells in larvae exposed to standard embryo medium containing no added nutrients. A similar increase in β cell number occurred in response to glibenclamide, an inhibitor of ATP-sensitive potassium channels that play a role in insulin secretion (Figure 1). Using lineage-tracing experiments, the researchers found that the precursor cells that gave rise to the increase in β cells were in the ventral bud, an embryonic structure that eventually becomes part of the pancreas. They also found that the progenitor cells express mnx1 (motor neuron and pancreas homeobox 1), a transcription factor known to be involved in β cell development.

 

These initial findings led the investigators to search for the signals leading to β cell neogenesis under conditions of overnutrition. To address this question, they screened small molecule modulators of various signaling pathways in their zebrafish model. They exposed larvae to medium with or without 5% egg yolk in the presence or absence of modulators of the IGF-1 (insulin-like growth factor-1), AMPK (5´ AMP-activated protein kinase), Hedgehog, PKA (protein kinase A), EGF (epidermal growth factor), VEGF (vascular endothelial growth factor), PDGF (platelet-derived growth factor), FGF (fibroblast growth factor), TGF-β (transforming growth factor-β), and Notch signaling pathways. The results of the screen yielded two compounds of particular interest due to their ability to block β cell neogenesis in the larvae. These were SU5402, an antagonist of the receptors for FGF, VEGF, and PDGF, and U0126, an inhibitor of MEK (mitogen activated protein kinase kinase). SU5402 also blocked β cell neogenesis in response to glibenclamide.

 

As SU5402 has multiple effects, the researchers sought to narrow the possible signaling pathways that could be involved in β cell neogenesis. Further studies revealed that specific inhibitors of the receptors for VEGF and PDGF had no effect on β cell number in zebrafish larvae exposed to overnutrition, leading the researchers to focus on the FGF signaling pathway. FGFs comprise a family of growth factors known to play a role in numerous processes, including development, angiogenesis, adipogenesis, and wound healing through their action at four distinct receptors (FGFRs). Among the large FGF family, one member, FGF1, caught the investigators’ attention. Although FGF1 is known to play a role in multiple developmental and homeostatic processes, genetically engineered mice lacking the fgf1 gene appear to have no deficits under normal conditions. However, when placed on a high fat diet, fgf1-/- mice develop insulin resistance, resulting in an aggressive form of diabetes. Consistently, injections of recombinant FGF1 reduce glucose levels in diabetic mice. These observations suggest that FGF1 may play an important role in glucose homeostasis, particularly under stress conditions, leading the Chen lab investigators to hypothesize that SU5402 blocks overnutrition-mediated β cell neogenesis by blocking FGF1-dependent signaling.

 

To test their hypothesis, the researchers created genetically engineered zebrafish lacking fgf1. As in the case of the mouse mutants, fgf1-/- zebrafish were viable and fertile, and they possessed a normal number of β cells when grown under standard conditions. However, these fish failed to exhibit β cell neogenesis, and their blood glucose levels were elevated relative to those of wild-type mice in response to overnutrition. This was also the case in four week-old juvenile zebrafish, indicating that the observations did not only apply to larvae.

 

The results obtained with fgf1-/- zebrafish were consistent with the hypothesis that FGF1 is important to β cell neogenesis, but they did not immediately explain how the MEK inhibitor U0126 could affect the neogenesis pathway. The answer to this puzzle was found, however, through an understanding of FGF1 secretion. FGF1 is unusual among secreted proteins in that it does not possess a signal peptide that would enable it to be processed through the common endoplasmic reticulum (ER)- and Golgi-dependent pathway for exocytosis. Instead, FGF1 is packaged as an inactive dimer into a multiprotein complex that enables transfer of FGF1 across the plasma membrane. Although the mechanism of this transfer is not completely understood, it clearly occurs in response to stress, and it requires Cu2+ ions. The researchers noted that U0126 is a Cu2+ chelator, so they hypothesized that this might be the mechanism by which it could affect FGF1-mediated β cell neogenesis. Consistent with this hypothesis, other MEK inhibitors had no effect on β cell number in zebrafish larvae responding to overnutrition. However, neocuproine, a potent Cu2+ chelator, exhibited the same effects as U0126.

 

Prior studies had shown that the signal responsible for triggering β cell neogenesis under stress conditions comes exclusively from mature β cells. This led the investigators to hypothesize that β cells were primarily responsible for FGF1 secretion during overnutrition. They tested this hypothesis by creating fgf1-/- mice transgenic for fgf1 expression under the control of the insulin promotor. In these mice, the only cells capable of producing FGF1 under any conditions would be β cells. These mice responded normally to overnutrition with increased numbers of β cells. The investigators carried this experiment one step further by creating transgenic zebrafish that express fgf1 containing a signal peptide sequence. The resulting zebrafish produced FGF1 constitutively via the ER-Golgi-dependent exocytotic pathway, and as a result, they exhibited elevated numbers of β cells even under normal growth conditions. These results further confirmed that FGF1 is the primary stimulatory signal for β cell neogenesis, and that mature β cells are the source of FGF1 during overnutrition.

 

Having established a role for FGF1 in β cell homeostasis in zebrafish, the Chen lab next turned to mammalian cells. They established a stable FGF1-expressing mammalian β cell line that was capable of producing high levels of the growth factor, but only in the presence of an appropriate stimulus. For this purpose, they used prolonged (8 hours) exposure to glibenclamide. Consistent with their zebrafish results, U0126 and neocuproine blocked FGF1 secretion in glibenclamide-treated mammalian β cells, confirming a role for Cu2+ in the FGF1 secretory pathway. As FGF1 secretion occurs in response to various forms of cellular stress, the Chen lab researchers hypothesized that the source of stress in β cells treated with glibeclamide might be the ER, due to the high demand for insulin biosynthesis and secretion. In support of this hypothesis, they found that tauroursodeoxycholic acid (TUDCA, a chemical chaparone that reduces ER stress) suppressed, while tunicamycin (an inducer of ER stress) promoted FGF1 release by glibeclamide-treated β cells. Also consistent with their hypothesis was the presence of dilated ER, a physical sign of ER stress, in the β cells of zebrafish larvae exposed to overnutrition.

 

From their studies, the Chen lab investigators were able to construct a picture of overnutrition-stimulated β cell neogenesis (Figure 2). They concluded that the high blood glucose levels resulting from overnutrition induce hyperstimulation of insulin biosynthesis and secretion. This leads to ER stress, which serves as a trigger for FGF1 secretion. FGF1 then acts on mnx1-expressing precursors, leading them to differentiate into new β cells, thereby increasing the insulin-producing capacity of the organism. An adequate response can return the organism to metabolic homeostasis, but failure will lead to diabetes. Clearly, understanding this key mechanism by which organisms respond to metabolic stress is important if we are to address the growing problem of diabetes associated with the current obesity epidemic.

 

 

 

Figure 2. Mechanism of FGF1-mediated β cell neogenesis during overnutrition. Overnutrition results in increased blood glucose levels, resulting in overstimulation of the insulin secretion pathway (see Figure 1). A result is ER stress (giving rise to ER swelling) due to the increased requirement for insulin biosynthesis and secretion. ER stress triggers the Cu2+-mediated secretion of FGF1, which stimulates precursor cells in the ventral bud to differentiate into new insulin-producing β cells. The enlarged β cell population increases the insulin secretory capacity of the islet, leading to an improvement in metabolic homeostasis. Figure provided by Carol Rouzer.


 

 

Click here to view W. Chen Diabetes article.

 

 

 

 

 

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