Effects of PTG over-expression on glucose-6-phosphate (G-6-P) and phosphorylation of Akt in starved mice.

<p>Intracellular G-6-P (A) and p-Ser-473-Akt protein (B) was determined in excised, snap-frozen IBAT from WT and Tg male 5–7 week-old mice sacrificed in the Fed, S, or SR 2 states as described in “Experimental Procedures.” N = 5 mice for G-6-P in each condition and genotype. N = 6 for p-Ser-473-Akt in each condition and genotype spread across 3 western blots (2 biological replicates from each condition, time point, and genotype per blot, 30 ug protein loaded per well as determined by BCA assay). Error bars are ±SD. Statistics: A Mann-Whitney rank sum test was performed within each genotype against the Fed state (stars directly over individual bars), as well as between genotypes in each condition (stars over lines indicating comparisons). *p<0.05, **p<0.01.</p

Effects of Protein Targeting to Glycogen over-expression on interscapular brown adipose tissue (IBAT) glycogen.

<p>IBAT was excised and snap-frozen from 8-week old mice either <i>ad libitum</i> chow-fed (Fed), 24-hr starved (S), or starved for 24 hr then allowed to refeed for 1, 2, or 4 hr (SR 1, SR 2, SR 4, respectively) before sacrifice. IBAT glycogen (6 mice per condition and time point) (A), and relative gene expressions for genes of interest (5 mice per condition and time point) (B-F) were determined. Error bars are ±SD. Statistics: A Mann-Whitney Rank Sum test was performed between genotypes within each condition (significance indicated above each line indicating the comparison) as well as between the Fed state and each other condition within each genotype (stars directly over the bar). For gene expressions, comparisons were made between Fed and S conditions for each transcript *p<0.05, **p<0.01.</p

Effects of nutritional perturbations and PTG over-expression on GP.

<p>Phospho-glycogen phosphorylase (GP_a) <i>(A)</i> and total glycogen phosphorylase (tGP) <i>(B)</i> protein levels were measured in snap-frozen IBAT excised from 5–7 week-old WT and Tg mice sacrificed in the Fed, S, or SR 2 states as described in “Experimental Procedures.” N = 6 per condition and genotype, spread out across 3 western blots (2 biological replicated from each condition, time point, and genotype per blot, 30 ug protein loaded per well as determined by BCA assay). Relative signal was quantified by densitometry and normalized to total Akt signal. Representative blots are shown above each densitometry chart. Error bars are ±SD. Statistics: A Mann-Whitney rank sum test was performed within each genotype against the Fed state (stars directly over individual bars), as well as between genotypes in each condition (stars over lines indicating comparisons) *p<0.05, **p<0.01.</p

Quantitative, real-time PCR primer sequences.

<p>Quantitative, real-time PCR primer sequences.</p

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

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

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

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

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

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