Histological visualization of glycogen storage in IBAT (top row) and liver (bottom row) using Periodic Acid Schiff staining.

<p>Male, wild-type, CD1 mice were either fed ad libitum (<b>A</b>, <b>E</b>), fed 60% of their normal daily intake of chow once daily for 5 days and then sacrificed (<b>B</b>, <b>F</b>), or fed 60% of their normal daily intake of chow once daily for 5 days and then allowed to refeed <i>ad libitum</i> 4 hr (<b>C</b>, <b>G</b>) or 48 hr (<b>D</b>, <b>H</b>) before sacrifice. IBAT (<b>A–D</b>) and liver (<b>E–H</b>) were harvested and immediately placed in formalin fixative solution. Fixed samples were mounted in paraffin, sectioned, and stained using Periodic Acid Schiff staining with Hematoxylin and Eosin counter-staining by the University of Chicago Human Tissue Resources Center.</p

Fed-mouse tissue glycogen concentrations.

<p>Group-housed, fed, male, wild-type, CD1 mice were sacrificed at 19∶30, 23∶30, or 07∶30 and tissues were harvested and snap-frozen in liquid nitrogen. Tissue glycogen concentrations were measured in epididymal adipose tissue (EPI) (<b>A</b>), interscapular brown adipose tissue (IBAT) (<b>B</b>), and liver (<b>C</b>). Error bars are ±SEM. Statistical comparisons for each tissue were made between time points using a 2-tailed Student’s t-test. ^#, p<0.001. 10–30 mice were used for each time point.</p

Effects of catecholamine-synthesis inhibition on tissue glycogen and serum parameters during starvation and refeeding.

<p>Male, wild-type CD1 mice were used for these experiments. 24H-SR mice were starved for 24 hr and either sacrificed (0) or allowed to refeed <i>ad libitum</i> 2–4 hours and then sacrificed (2, 4). 24H-SAR mice were starved for 24 hr, but also received an IP injection of 300 mg/kg AMPT in 0.9% NaCl 2 hr prior to refeeding. 24H-SAR mice were either sacrificed following the full 24 hr starvation (0), or allowed to refeed 2 or 4 hr prior to sacrifice (2, 4 respectively). Upon sacrifice, tissues were snap-frozen in liquid nitrogen and serum was collected. Glycogen was measured in IBAT (<b>A</b>) and liver (<b>B</b>), and circulating glucose (<b>C</b>) and insulin (<b>D</b>) were also measured. Error bars are ±SEM. Statistical comparisons were made between experimental conditions using a 2-tailed Student’s t-test. ^#, <i>p<0.001</i>. Measurements for each time point from AMPT-injected mice were obtained for 7–12 mice.</p

IBAT glycogen and serum parameters after 24 hr starvation and a hyperinsulinemic, hyperglycemic clamp.

<p>Male, wild-type, CD1 mice were starved for 24 hr and then subjected to a 2 hr hyperinsulinemic, hyperglycemic clamp (24H-SC) in which glucose was clamped at 300 mg/dL and insulin was infused at a rate of 3 mU/kg/min. At the end of the clamp, mice were sacrificed, serum was collected, and tissues were collected and snap-frozen in liquid nitrogen. Final serum glucose (<b>A</b>), serum insulin (<b>B</b>), and IBAT glycogen (<b>C</b>) were compared to those of fed mice sacrificed at 19∶30 (Fed) and 24 hr starved/refed mice sacrificed 2 hr after the onset of refeeding (24H-SR). Error bars are ±SEM. Statistical comparisons were made between experimental conditions using a 2-tailed Student’s t-test. **, <i>p<0.01</i>; ^#, <i>p<0.001</i>; ^&, <i>p<0.0001</i>. All measurements represent 8–9 mice, except Fed 19∶30 and 24H-SR IBAT glycogen, obtained from 30 and 15 mice, respectively.</p

Mechanism by which glycogen stores may enhance free fatty acid esterification in adipose tissues during refeeding.

<p>During the first several hours of refeeding, hyperaccumulation of intracellular BAT glycogen occurs. The eventual glycogenolytic and subsequent glycolytic processing of glycogen stores may generate elevated levels of glycerol-3-phosphate, a precursor to monoglyceride formation. An increase in this precursor may enhance the rate of esterification of free fatty acids (FFA), both from <i>de novo</i> synthesized sources as well as FFA recently liberated from stored neutral lipids. This process may greatly enhance lipid repletion during refeeding in order to maximize recovery from the starvation state.</p

Tissue glycogen during refeeding after total starvation.

<p>Male, wild-type, CD1 mice were starved for either 72 hr (<b>A–C</b>) or 24 hr (<b>D,E</b>) and then sacrificed (0) or allowed to refeed <i>ad libitum</i> 1–48 hr before sacrifice (1–48). Tissues were harvested and snap-frozen in liquid nitrogen. Tissue glycogen was measured in EPI (<b>A</b>), IBAT (<b>B, D</b>), and liver (<b>C, E</b>). Error bars are ±SEM. Statistical comparisons were made between the starved time point (0) and each refeeding time point (1–48) for each tissue using a 2-tailed Student’s t-test. *, p<0.05; **, <i>p<0.01</i>; ^#, <i>p<0.001</i>; ^&, <i>p<0.0001.</i> Fasting time points utilized 3–5 mice, except 24 hr fasting BAT glycogen which utilized 19 mice. All refed measurements were taken from 3–9 mice except 24 hr fasted/refed 2 hr BAT time point, which was measured in 15 mice.</p

Tissue glycogen during 5 days of partial fasting and refeeding.

<p>Male, wild-type, CD1 mice were fed 60% of their normal daily intake of chow once daily for 5 days and then sacrificed (0) or allowed to refeed <i>ad libitum</i> 4–48 hr before sacrifice (4–48). Upon sacrifice, tissues were harvested and snap-frozen in liquid nitrogen. Tissue glycogen was measured in EPI (<b>A</b>), mesenteric adipose tissue (<b>B</b>), perirenal adipose tissue (<b>C</b>), subcutaneous adipose tissue (<b>D</b>), IBAT (<b>E</b>), and the liver (<b>F</b>). Error bars are ±SEM. Statistical comparisons were made between the partial-fasting time point (0) and each refeeding time point (4–48) for each tissue using a 2-tailed Student’s t-test. *, p<0.05; **, <i>p<0.01</i>; ^#, <i>p<0.001</i>; ^&, <i>p<0.0001.</i> Fasted time points utilized 2 mice. All other time points were obtained from 4–8 mice.</p