Metabolic Hormone FGF21 Is Induced in Ground Squirrels during Hibernation but Its Overexpression Is Not Sufficient to Cause Torpor

<div><p>Hibernation is a natural adaptation that allows certain mammals to survive physiological extremes that are lethal to humans. Near freezing body temperatures, heart rates of 3–10 beats per minute, absence of food consumption, and depressed metabolism are characteristic of hibernation torpor bouts that are periodically interrupted by brief interbout arousals (IBAs). The molecular basis of torpor induction is unknown, however starved mice overexpressing the metabolic hormone fibroblast growth factor 21 (FGF21) promote fat utilization, reduce body temperature, and readily enter torpor–all hallmarks of mammalian hibernation. In this study we cloned FGF21 from the naturally hibernating thirteen-lined ground squirrel (<em>Ictidomys tridecemlineatus</em>) and found that levels of FGF21 mRNA in liver and FGF21 protein in serum are elevated during hibernation torpor bouts and significantly elevated during IBAs compared to summer active animals. The effects of artificially elevating circulating FGF21 concentrations 50 to 100-fold via adenoviral-mediated overexpression were examined at three different times of the year. This is the first time that a transgenic approach has been used in a natural hibernator to examine mechanistic aspects of hibernation. Surgically implanted transmitters measured various metrics of the hibernation phenotype over a 7-day period including changes in motor activity, heart rate and core body temperature. In April fed-state animals, FGF21 overexpression decreased blood insulin and free fatty acid concentrations, effects similar to those seen in obese mice. However, elevated FGF21 concentrations did not cause torpor in these fed-state animals nor did they cause torpor or affect metabolic parameters in fasted-state animals in March/April, August or October. We conclude that FGF21 is strongly regulated during torpor and IBA but that its overexpression is not sufficient to cause torpor in naturally hibernating ground squirrels.</p> </div

FGF21 expression in thirteen-lined ground squirrels.

<p>(<b>A</b>) FGF21 mRNA was measured by qRT-PCR relative to 18S RNA levels in multiple tissues of the thirteen-lined ground squirrel. RNA from 6 squirrels with 2 each from August active, winter torpid, and winter IBA were used. (<b>B</b>) Relative liver FGF21 mRNA levels during indicated activity states. (<b>C</b>) Concentration of serum FGF21 protein during indicated activity states. In panels B and C, fold change are shown relative to August active levels and data bars that do not share the same letter above the bar are significantly different from each other using ANOVA followed by Tukey's HSD (<i>p</i><0.01). Errors bars show standard error of the mean. <i>Abbreviations</i> – n, number of animals; FC, fold change; AUG, August active; TOR, torpor; IBA, interbout arousal; MAR, March active; WAT, white adipose tissue.</p

Q-RT-PCR Primers.

<p>Q-RT-PCR Primers.</p

Serum Metabolites in Thirteen-lined Ground Squirrels with Increased FGF21 at 7 days following infusion (Values followed by SEM).

<p><i>Abbreviations</i> – Apr, April; Aug, August; Mar, March; Oct, October; BHB, D-beta-hydroxybutyrate; FA, fatty acids; TG, triglycerides.</p

Physiological parameters in fasted October thirteen-lined ground squirrels infused with AdFGF21 (n = 8) and control adenovirus AdRR5 (n = 7).

<p>The squirrels were kept at 5°C, in 24 h darkness, without food for 7 days. Serum concentration of FGF21 and changes in body weight were measured 7 days after infusion with either AdFGF21 (FGF21) or AdRR5 (Control). Body temperatures and heart rate were measured continuously over 7 days using implanted transmitters. Statistical significance (*) was evaluated by Student's t-test. (<b>A</b>) Serum concentration of FGF21 (*, <i>p</i> = 0.004). (<b>B</b>) Mean body temperatures (*, <i>p</i><0.05). (<b>C</b>) Mean minimum body temperature. (<b>D</b>) Mean heart rate (*, <i>p</i><0.05). (<b>E</b>) Mean minimum heart rate. (<b>F</b>) Change in weight. Error bars show standard error of the mean.</p

Relative levels of FGF21 signaling component mRNAs in ground squirrel tissues.

<p>The relative levels of mRNA were measured in eight tissues from thirteen-lined ground squirrels with 2 squirrels in August active state, 2 squirrels in winter torpid state, and 2 squirrels in IBA state for a total of 6 squirrels. (<b>A</b>) βKlotho mRNA was detected predominantly in liver and white adipose tissue (WAT). (<b>B</b>) FGFR1 mRNA was detected in all tissues at a low level with the highest expression in WAT. Error bars show standard error of the mean.</p

Physiological parameters in fasted August thirteen-lined ground squirrels infused with AdFGF21 (n = 8) and control adenovirus AdRR5 (n = 7).

<p>The squirrels were kept at 5°C, in 24 h darkness, without food for 7 days. Serum concentration of FGF21 and changes in body weight were measured 7 days after infusion with either AdFGF21 (FGF21) or AdRR5 (Control). Body temperatures and heart rate were measured continuously over 7 days using implanted transmitters. Statistical significance (*) was evaluated by Student's t-test. (<b>A</b>) <b>S</b>erum concentration of FGF21 (*, <i>p</i><0.0001). (<b>B</b>) Mean body temperatures. (<b>C</b>) Mean minimum body temperature. (<b>D</b>) Mean heart rate (HR). (E) Mean minimum heart rate. (<b>F</b>) Change in weight. Error bars show standard error of the mean.</p

Adenoviral-mediated expression of cloned thirteen-lined ground squirrel FGF21 is capable of signaling in mouse adipocytes.

<p>Following FGF21 activation of FGFR, the MAPK pathway is initiated involving phosphorylation of ERK 1 and 2. The phosphorylation of ERK1/2 (P-ERK1/2) resulting from thirteen-lined ground squirrel FGF21 (AdsqFGF21) is similar to the positive control using mouse FGF21 (AdmFGF21). Adenovirus not encoding FGF21 (AdRR5) was used as a control. Each adenoviral assay was done in duplicate.</p

Physiological parameters in fed April thirteen-lined ground squirrels infused with AdFGF21 (n = 8) and control adenovirus AdRR5 (n = 8).

<p>The squirrels were kept at 23°C with a 12∶12 light:dark cycle with food <i>ad libitum</i> for 7 days. Serum concentration of FGF21 and changes in body weight were measured 7 days after infusion with either AdFGF21 (FGF21) or AdRR5 (Control) adenoviruses. Body temperatures and heart rate were measured continuously over 7 days using implanted transmitters. Statistical significance (*) was evaluated by Student's t-test. (<b>A</b>) Serum concentration of FGF21 (*, <i>p</i><0.0001). (<b>B</b>) Mean body temperatures. (<b>C</b>) Mean minimum body temperature (*, <i>p</i> = 0.012). (<b>D</b>) Mean heart rate (HR). (<b>E</b>) Mean minimum heart rate. (<b>F</b>) Change in weight. Error bars show standard error of the mean.</p