Epinephrine-stimulated glycogen breakdown activates glycogen ...

Am J Physiol Endocrinol Metab 308: E231–E240, 2015. First published December 2, 2014; doi:10.1152/ajpendo.00282.2014.

Epinephrine-stimulated glycogen breakdown activates glycogen synthase and increases insulin-stimulated glucose uptake in epitrochlearis muscles Anders J. Kolnes,1 Jesper B. Birk,2 Einar Eilertsen,3 Jorid T. Stuenæs,3 Jørgen F. P. Wojtaszewski,2 and Jørgen Jensen2,4 1

Charles University Third Faculty of Medicine, Prague, Czech Republic; 2Molecular Physiology Group, The August Krogh Centre, Department of Nutrition, Exercise and Sports, Copenhagen University, Copenhagen, Denmark; 3National Institute of Occupational Health, Oslo, Norway; and 4Department of Physical Performance, Norwegian School of Sports Sciences, Oslo, Norway

Submitted 17 June 2014; accepted in final form 1 December 2014

Kolnes AJ, Birk JB, Eilertsen E, Stuenæs JT, Wojtaszewski JF, Jensen J. Epinephrine-stimulated glycogen breakdown activates glycogen synthase and increases insulin-stimulated glucose uptake in epitrochlearis muscles. Am J Physiol Endocrinol Metab 308: E231–E240, 2015. First published December 2, 2014; doi:10.1152/ajpendo.00282.2014.—Epinephrine increases glycogen synthase (GS) phosphorylation and decreases GS activity but also stimulates glycogen breakdown, and low glycogen content normally activates GS. To test the hypothesis that glycogen content directly regulates GS phosphorylation, glycogen breakdown was stimulated in condition with decreased GS activation. Saline or epinephrine (0.02 mg/100 g rat) was injected subcutaneously in Wistar rats (⬃130 g) with low (24-h-fasted), normal (normal diet), and high glycogen content (fasted-refed), and epitrochlearis muscles were removed after 3 h and incubated ex vivo, eliminating epinephrine action. Epinephrine injection reduced glycogen content in epitrochlearis muscles with high (120.7 ⫾ 17.8 vs. 204.6 ⫾ 14.5 mmol/kg, P ⬍ 0.01) and normal glycogen (89.5 ⫾ 7.6 vs. 152 ⫾ 8.1 mmol/kg, P ⬍ 0.01), but not significantly in muscles with low glycogen (90.0 ⫾ 5.0 vs. 102.8 ⫾ 7.8 mmol/kg, P ⫽ 0.17). In saline-injected rats, GS phosphorylation at sites 2⫹2a, 3a⫹3b, and 1b was higher and GS activity lower in muscles with high compared with low glycogen. GS sites 2⫹2a and 3a⫹3b phosphorylation decreased and GS activity increased in muscles where epinephrine decreased glycogen content; these parameters were unchanged in epitrochlearis from fasted rats where epinephrine injection did not decrease glycogen content. Incubation with insulin decreased GS site 3a⫹3b phosphorylation independently of glycogen content. Insulin-stimulated glucose uptake was increased in muscles where epinephrine injection decreased glycogen content. In conclusion, epinephrine stimulates glycogenolysis in epitrochlearis muscles with normal and high, but not low, glycogen content. Epinephrinestimulated glycogenolysis decreased GS phosphorylation and increased GS activity. These data for the first time document direct regulation of GS phosphorylation by glycogen content. insulin sensitivity; GSK3; AMPK; TBC1D4; diabetes; phosphorylation; liver; Cori cycle SKELETAL MUSCLE GLYCOGEN is an important energy substrate during exercise (24). In addition, skeletal muscle glycogen functions as major store for ingested carbohydrates and contributes to regulation of blood glucose (21). The glycogen content in skeletal muscles is limited, and high glycogen content inhibits glycogen synthase (GS) activity, whereas low glycogen content activates GS (9, 19, 29, 33). This inverse

Address for reprint requests and other correspondence: J. Jensen, Dept. of Physical Performance, Norwegian School of Sport Sciences, Sognsvann 220, PO Box 4014 Ullevål Stadion, 0806 Oslo, Norway (e-mail: jorgen. [email protected]). http://www.ajpendo.org

relationship between glycogen content and GS activation has been established by manipulating glycogen content by fastingrefeeding (19, 28 –30) and exercise-diet protocols (33). Epinephrine stimulates glycogen breakdown in skeletal muscles as muscle contraction does, but epinephrine also increases GS phosphorylation and decreases GS activity (5, 18, 38). The effect of epinephrine-stimulated glycogen breakdown on GS phosphorylation after removal of epinephrine has not been carefully characterized. GS is phosphorylated at at least nine sites, and increased phosphorylation decreases fractional activity (5, 20, 36). Epinephrine stimulates GS phosphorylation via ␤2-adrenergic receptors, cAMP, and activation of PKA; activated PKA phosphorylates GS at sites 2, 1a, and 1b (5, 36). In addition, PKA increases GS phosphorylation via phosphorylation of the protein phosphatase-1 (PP1) glycogen-targeting regulatory subunit RGL. Phosphorylation of RGL dissociates PP1 from the glycogen particle and increases GS phosphorylation (36). Increased glycogen content is associated with increased phosphorylation, whereas decreased glycogen content decreases GS phosphorylation at several sites (19, 25, 29, 35). Insulin increases GS activity via PI 3-kinase and reduces phosphorylation of GS at sites phosphorylated by GSK-3 (sites 3a, 3b, 3c, and 4) (2, 20, 36). Muscle contraction activates GS independently of GSK-3 but requires RGL to activate GS (1). Reduced glycogen content after exercise contributes to increase GS activity (33), but muscle contraction additionally increases GS activity independently of glycogen content (29). PP1 activity has been suggested to decrease as glycogen content increases and allow GS phosphorylation (20, 36). Favoring this idea, it has been shown that overexpression of RGL decreases GS phosphorylation and increases glycogen content, whereas the glycogen content is low in muscles where RGL is deleted (1). Glycogen content and muscle contraction are much stronger regulators of GS phosphorylation and activity than insulin (9, 19, 33), which may reflect a more active phosphatase activity and a more comprehensive dephosphorylation of GS than insulin-stimulated inhibition of GSK-3. Three accompanying papers entitled: “The mechanism of epinephrine action” (6 – 8) are the experimental background for the Cori cycle stating that “glycogen mobilized in the muscles is converted into liver glycogen with lactic acid as an intermediary stage.” (p. 317 in Ref. 6). The three papers consisted of experiments where epinephrine was injected into rats under different nutritional status, and it was shown that epinephrine directed glycogen to the liver at the expense of carcass glyco-

0193-1849/15 Copyright © 2015 the American Physiological Society

E231

E232

CORI CYCLE AND INSULIN SENSITIVITY IN SKELETAL MUSCLES

gen. Importantly, the effect of epinephrine-stimulated glycogen breakdown on GS phosphorylation and activation after removal of epinephrine has not been carefully characterized. The primary aim of the present study was to investigate GS phosphorylation and activation in epitrochlearis muscles following epinephrine-stimulated glycogen breakdown and after removal of epinephrine. The studies were performed in muscles with different glycogen content to investigate whether initial glycogen content influences the magnitude of epinephrine-stimulated glycogen breakdown and GS activation. We hypothesized that skeletal muscle GS phosphorylation and activation would adapt to the new glycogen content via autoregulatory feedback mechanisms after removal of epinephrine. The glycogen content in skeletal-muscles influences insulin-, contraction-, and pharmacological stimulated glucose uptakes. In rodents, fasting and refeeding (17, 19, 29, 30), acute exercise (10, 11), and training (26) have been used to modulate glycogen content and established an inverse relationship between glycogen content and glucose uptake. In humans, glucose uptake is also regulated by glycogen when glycogen content is modulated by exercise and diet (42). There is also evidence that decreasing glycogen content by epinephrine stimulation increases insulin-stimulated glucose uptake (22, 34). However, these studies do not allow separating the effect of epinephrine-stimulated glycogen breakdown from potential effects independent of glycogen content. The secondary aim was therefore to investigate the role of epinephrine-mediated glycogen breakdown on insulin-stimulated glucose uptake in muscles with different glycogen content. MATERIALS AND METHODS

Experimental protocol. Male Wistar rats were kept on a 12:12-h light-dark cycles at 21°C and ⱖ55% humidity with free access to chow and water for at least 1 wk before experiments. Prior to experiments, glycogen content in epitrochlearis muscle was manipulated by fasting and refeeding, as described previously (17). Briefly, rats with low glycogen (LG) were fasted for 24 h prior to experiments.

Rats with normal glycogen (NG) were kept on their normal diet until experiments. Rats with high glycogen (HG) were fasted for 24 h followed 24 h with free access to chow prior to experiments (Fig. 1). Fasting was started at 9:00 AM. On experimental days, rats were given a subcutaneous injection of saline (0.9% saline) or epinephrine (0.02 mg/100 g body wt of rats) and placed alone in new cages without food but access to water. Epinephrine [epinephrine (⫹) bitratrate, E4375; Sigma, St. Louis, MO] was dissolved in 0.9% saline (0.1 mg/ml), and 0.2 ml/100 g body wt was injected subcutaneously into the back of the rats. Muscles used for incubation were removed 3 h after saline/epinephrine injection. Additional muscles and livers were removed and frozen directly in liquid nitrogen from a group of rats without injection and with free access to food and water until experiments (untreated controls) and from groups of rats 1 h after epinephrine/saline injection. Blood glucose was also measured in these rats. The weight of the rats was 120 –150 g on the day of experiment. Rats were anesthetized with an intraperitoneal injection of 7.5 mg of pentobarbital sodium (50 mg/ml) per 100 g of rat 3 h after epinephrine/saline injection. Blood samples were taken from the tail ¨ ngelholm, Sweden) after for measuring glucose (HemoCue AB, A anesthetization. Epitrochlearis and soleus muscles were dissected for incubation. Additional muscles (data not included in this article) were dissected out and frozen in liquid nitrogen. Finally, the liver was dissected out and weighed, and a piece was frozen in liquid nitrogen. Muscles and liver tissue were stored at ⫺70°C until analyses. The experiments and procedures were approved by National Animal Research Authority and performed according to laws and regulations controlling experiments on live animals in Norway, and the European Convention for the Protection of Vertebrate Animals used in Experimental and Other Scientific Purposes. Incubations. Epitrochlearis muscles were immediately mounted on holders at their approximate resting length and preincubated ⬇45 min in 3.5 ml of Krebs-Henseleit buffer containing 5.5 mM glucose, 2 mM pyruvate, 0.1% bovine serum albumin (Fraction V), and 5 mM HEPES, as described (27). The incubations were performed at 30°C, and the buffers were gassed with 95% O2-5% CO2. After preincubation, glucose uptake was measured for 30 min as described previously (27). In brief, 0.25 ␮Ci/ml 2-deoxy-D-[1,23 H(N)]glucose (25.5 Ci/mmol; DuPont, NEN) and 0.1 ␮Ci/ml D-[114 C]mannitol (51.5 mCi/mmol; DuPont, NEN) were added to the

Fig. 1. Schematic overview of the study design.

AJP-Endocrinol Metab • doi:10.1152/ajpendo.00282.2014 • www.ajpendo.org

E233


Fasted-Saline Fasted-Adrenaline

16 14 12

a

10

a

8 6 4 2 0

C

18 16

a

14 12

a

10 8 6 4

Normal diet-Saline Normal diet-Adrenaline

2

Blood glucose (mM)

B

18

Blood glucose (mM)

Blood glucose (mM)

A

18

14 12

1

2

3

a

10

0 0

a

16

8 6 4

Fatsted/Refed-Saline Fasted/Refed-Adrenaline

2 0

0

1

Time (h)

2

3

0

1

Time (h)

2

3

Time (h)

Fig. 2. Blood glucose concentrations after injection of epinephrine or saline in rats under different nutritional conditions. Blood samples were taken from groups of rats before injection and 1 or 3 h after subcutaneous injection of epinephrine (0.02 mg/100 g rat) or saline. A: blood glucose concentrations in rats fasted for 24 h before and after injection of saline (open symbols) or epinephrine (filled symbols). B: blood glucose concentrations in rats maintained on their normal diet until injection of saline (open symbols) or epinephrine (filled symbols). C: blood glucose concentrations in fasted-refed rats before and after injection of saline (open symbols) or epinephrine (filled symbols). All groups were fasted after injection of epinephrine or saline. For all groups: before injection (0 h), n ⫽ 8; 1 h after injection, n ⫽ 4; 3 h after injection, n ⫽ 14. aP ⬍ 0.05 vs. saline.

buffer (containing 5.5 mM glucose), and glucose uptake was calculated from the intracellular accumulation of [3H]-2-deoxy-D-glucose as described previously (15). Insulin (Actrapid, Novo Nordisk) was added in concentrations of 100 ␮U/ml (physiological concentration) or 10,000 ␮U/ml, as indicated in figures. Muscles were, after the 30-min incubation, rapidly removed from the holders, avoiding unnecessary stretching, blotted on filter paper, and frozen in liquid nitrogen. For measurement of glucose uptake and glycogen, about one-half the muscle was freeze-dried and weighed and dissolved in 600 ␮l of 1 M KOH for 20 min at 70°C. Of the digest, 400 ␮l was added to 3 ml of scintillation solution (Hionic-Fluor, Packard), mixed, and counted for radioactivity (TRI-CARB 460C, Packard). Glycogen content in muscles. For analysis of glycogen, 100 ␮l of the KOH digest was acidified with 25 ␮l of 7 M acetic acid before 500 ␮l of 0.3 M acetate buffer (pH 4.8) containing 0.2 U/ml of amyloglucosidase (Boehringer-Mannheim) was added, and the glycogen was hydrolyzed at 37°C for 3 h. Glucose units were measured enzymatically with reactions (hexokinase and glucose-6-phosphate dehydrogenase) coupled to NADPH formation as described by Lowry and Passonneau (32). Reactions were allowed for 30 min at room temperature in 96-well plates, and fluorescence was measured on a BMG plate reader (BMG, Germany). Glycogen content in liver. Liver samples were freeze-dried and weighed, and glycogen was hydrolyzed in 1 M HCl (2.5 h at 100°C). Hydrolysate was neutralized with NaOH as described (23). Homogenization of muscles. Epitrochlearis muscle tissue (⬃10 mg) was homogenized in buffer containing 50 mM HEPES (pH 7.5), 10% glycerol, 20 mM Na-pyrophosphate, 150 mM NaCl, 1% NP-40, 20 mM ␤-glycerolphosphate, 10 mM NaF, 2 mM PMSF, 1 mM EDTA, 1 mM EGTA, 10 ␮g/l aprotinin, 10 ␮g/l leupeptin, 2 mM Na3VO4, and 3 mM benzamidine. Homogenates were rotated for 1 h at 4°C. Protein content was analysed in triplicates in 96-well plate with the bicinchoninic acid method (Pierce Chemical, Rockford, IL). GS activity. GS activity was measured with the filter paper method and [14C]UDP-glucose (PerkinElmer) as substrate as described by Thomas et al. (37), but modified to 96-well filter plates (Unifilter 350 plates; Watman, Cambridge, UK) as described (15). GS activity was measured with 1.5 mM UDP-glucose (high) and 0.03 mM UDPglucose (physiological) in assay buffer and different concentrations of glucose 6-phosphate. Total GS activity was measured with 8 mM glucose 6-phosphate. GS activity measured with 0.17 mM glucose 6-phosphate was used to calculate fractional velocity [FV ⫽ (activity with 0.17 mM G6P)/(activity with 8 mM G6P)]. GS activity measured with 0.01 mM glucose 6-phosphate was used to calculate GS I-form [I-form ⫽ (activity with 0.01 mM G6P)/(activity with 8 mM G6P)]. Western blot. Proteins were separated using 10% SDS gels and transferred (semidry) to polyvinylidene difluoride (PVDF) mem-

branes (Immobilion Transfer Membrane; Millipore, Copenhagen, Denmark). Membranes were probed with antibodies against GS sites 2⫹2a, 3a⫹3b, and 1b as described (15). The antibodies used to study phosphorylation of GSK-3␤, AMPK, and TBC1D4 were as follows: GKS-3␤ (BD Transduction Laboratories) GSK-3␣/␤ Ser21/9 (Cell Signaling Technology, Danvers, MA), AMPK Thr172 (Cell Signaling Technology), AMPK␣2 (Santa Cruz Biotechnology, Dallas, TX), TBC1D4 (Millipore, Billerica, MA), and TBC1D1 Thr642 (Symansis, New Zealand). Statistics. Data are presented as means ⫾ SE. Analysis of variance was performed to investigate differences, and Fisher’s least significant difference (LSD) was used as a post hoc test to compare different treatments. Liver glycogen content varied ⬃75-fold between fasted and fasted-refed rats, and effect of epinephrine injection on liver glycogen content was compared with a t-test within similar feeding protocol. P ⬍ 0.05 was considered significant. RESULTS

Blood glucose was ⬃4 mM in 24-h-fasted rats, and blood glucose increased to ⬃8 mM during epinephrine injection (Fig. 2A). Blood glucose was ⬃8 mM in rats on normal diet and in fasted-refed rats. It peaked at ⬃14 mM 1 h after epinephrine injection but stayed stable during saline injection (Fig. 2, B and C). Three hours after epinephrine injection blood glucose remained slightly higher in epinephrine-injected rats compared with saline-injected rats. Liver glycogen content in rats on normal diet, but fasted for 3 h after saline injection, was ⬃1,000 mmol/kg dry wt, and epinephrine injection did not influence glycogen content (Table 1). Liver glycogen content was reduced to very low Table 1. Effect of epinephrine injection on liver glycogen content in rats under different nutritional conditions Glycogen

Saline Epinephrine

Fasted

Normal Diet

Fasted/Refed

17.8 ⫾ 1.1 (9) 120.0 ⫾ 23.3 (9)a

978.1 ⫾ 45.7 (16) 964.2 ⫾ 43.9 (16)b b

1,284.9 ⫾ 55.7 (16)c 1,299.5 ⫾ 64.5 (16)c

Data are means ⫾ SE in mmol/kg dry wt. Rats were injected with saline or epinephrine (0.02 mg/100 g rat), and livers were removed 3 h later.Glycogen contents in livers from rats without injection were: Fasted: 26.2 ⫾ 2.9 (7); Normal diet 1,125.9 ⫾ 77.5 (8); Fasted-Refed 1,642.6 ⫾ 73.9 mmol/kg dry wt (8). Groups were compared with ANOVA; t-test was used for comparison of effect of epinephrine. aP ⬍ 0.01 vs. saline; bP ⬍ 0.01 vs. fasted; cP ⬍ 0.01 vs. normal diet.


E234


levels during 24-h fasting, and epinephrine injection increased glycogen content by ⬃100 mmol/kg dry wt (P ⬍ 0.01; Table 1). Fasting-refeding increased liver glycogen content to ⬃ 1,300 mmol/kg dry wt, and epinephrine injection did not influence glycogen content (Table 1). Glycogen content in epitrochlearis muscles from rats on normal diet was ⬃150 mmol/kg dry wt 3 h after saline infusion, whereas glycogen content 3 h after epinephrine infusion was decreased to ⬃100 mmol/kg dry wt (Fig. 3A). In epitrochlearis from fasted-refed rats, the glycogen content was higher than in epitrochlearis from rats kept on normal diet and was ⬃200 mmol/kg dry wt 3 h after saline infusion. Three

a

150

a 100

50

a b

b

a

40

b

a,b

Normal diet

a

100

50

0

0

Fasted

Normal diet

Fasted-refed

a

a

(Arbitrary units)

a

b

Normal diet

Fasted-refed

1.2 Saline Adrenaline

1.0

150

50

a (a)

Fasted

I

250

(Arbitrary units)

b

p-GS Site 1b

b

(b) 100

Fasted-refed

200

100

150

50

b

Saline Adrenaline

150

c

200

0

Fasted

200

Fasted-refed

250

(Arbitrary units)

60

20

Normal diet

Saline Adrenaline

a

H

Saline Adrenaline

(Arbitrary units)

Fasted

F

80

Fasted-refed

250

40

20

0

Normal diet

b

Fasted-refed

p-GS Site 2+2a

40

Fasted

p-GS Site 3a+3b

a 60

Saline Adrenaline

(FV0.03- %)

60

Normal diet

100

0

0.8 0.6 0.4 0.2 0.0

Fasted

Normal diet

Fasted-refed

Fasted

Normal diet

Fasted-refed

2.0 Saline Adrenaline

(Arbitrary units)

p-αAMPK Thr172

b

a

0

Fasted

E Saline Adrenaline

20

J

b

40

Fasted-refed

Glycogen synthase

(I-Form0.03- %)

Glycogen synthase

Normal diet

100

G

a

Saline Adrenaline 80

0

Fasted

80

60

20

0

D

a

100

(FV1.5- %)

b

80

Saline Adrenaline

p-GSK-3β Ser9

(mmol/kg dw)

200

C

100

c

Glycogen synthase

B

Saline Adrenaline

(I-Form1.5- %)

250

Glycogen synthase

Glycogen content

A

hours after epinephrine injection, glycogen content was reduced to ⬃120 mmol/kg dry wt in epitrochlearis muscles from fasted-refed rats, and glycogen content tended to be higher than in muscles from rats on a normal diet injected with epinephrine (P ⬍ 0.1). In epitrochlearis muscles from fasted rats glycogen contents was ⬃100 mmol/kg dry wt, and epinephrine injection did not decrease glycogen content significantly (Fig. 3A). GS activity was influenced by glycogen content in salineinjected rats, when measured with both physiological and high concentration of UDP-glucose (Fig. 3, B–E). GS fractional activities and GS I-form percent measured with 0.03 mM UDP-glucose (FV0.03 and I-form0.03) were, as expected, much

1.5

b 1.0

b

b b

0.5

0.0

Fasted

Normal diet

Fasted-refed

Fig. 3. Effect of epinephrine injection on glycogen content, glycogen synthase (GS) activation, and phosphorylation of GS, GSK-3␤, and AMPK in epitrochlearis muscles from rats under different nutritional conditions. Rats were diet manipulated as described in MATERIALS AND METHODS. Saline or epinephrine (0.02 mg/100 g rat) was injected subcutaneously, and epitrochlearis muscles were removed 3 h later and preincubated for 45 min and incubated for 30 min in control buffer. A: glycogen content in epitrochlearis muscles after injection of saline (open bars) or epinephrine (filled bars). B: GS %I-form1.5 (measured with 1.5 mM UDP-glucose) in epitrochlearis muscles after injection of saline (open bars) or epinephrine (filled bars). C: GS fractional velocity (FV)1.5 (measured with 1.5 mM UDP-glucose) in epitrochlearis muscles after injection of saline (open bars) or epinephrine (filled bars). D: GS %I-form0.03 (measured with 0.03 mM UDP-glucose) in epitrochlearis muscles after injection of saline (open bars) or epinephrine (filled bars). E: GS FV0.03 (measured with 0.03 mM UDP-glucose) in epitrochlearis muscles after injection of saline (open bars) or epinephrine (filled bars). F: GS site 2⫹2a phosphorylation in epitrochlearis muscles after injection of saline (open bars) or epinephrine (filled bars). G: GS site 3a⫹3b phosphorylation in epitrochlearis muscles after injection of saline (open bars) or epinephrine (filled bars). H: GS site 1b phosphorylation in epitrochlearis muscles after injection of saline (open bars) or epinephrine (filled bars). I: GSK-3␤ Ser9 phosphorylation in epitrochlearis muscles after injection of saline (open bars) or epinephrine (filled bars). J: AMPK Thr172 phosphorylation in epitrochlearis muscles after injection of saline (open bars) or epinephrine (filled bars). Data are means ⫾ SE; n ⫽ 7– 8 in all groups. aP ⬍ 0.05; (a)P ⬍ 0.1 vs. muscles from saline-treated rats; bP ⬍ 0.05; (b)P ⬍ 0.1 vs. muscles from fasted rats treated similarly; cP ⬍ 0.05 vs. normal diet with similar treatment. AJP-Endocrinol Metab • doi:10.1152/ajpendo.00282.2014 • www.ajpendo.org


lower than activities measured with 1.5 mM UDP-glucose, but the effect of glycogen content was comparable. GS activity was increased in muscles from epinephrine-injected rats on normal diet and fasted-refed (Fig. 3, B–E). In epitrochlearis muscles from fasted rats, GS activity was not increased in muscles from epinephrine-injected rats; of note, epinephrine did not decrease glycogen content in these muscles. Some epitrochlearis muscles were removed 1 h after epinephrine injection and frozen immediately to describe the acute effect of epinephrine injection; epinephrine injection decreased GS activity as expected in all groups, although not significantly in epitrochlearis from fasted-refed rats (Table 2). Phosphorylation of GS sites 2⫹2a and 3a⫹3b were influenced by modulation of glycogen content by fasting and fasting-refeeding (Fig. 3, F and G). GS phosphorylation at sites 2⫹2a and 3a⫹3b was reduced in epitrochlearis muscles from epinephrine-injected rats on normal diet and fasted-refed (Fig. 3, F and G); epinephrine injection decreased glycogen content in muscles from these groups of rats (Fig. 3A). GS site 1b phosphorylation was higher in epitrochlearis from fasted-refed rats compared with muscles from fasted rats (Fig. 3H). GS site 1b was not regulated by epinephrine injection (Fig. 3H). GSK-3␤ Ser9 phosphorylation was not influenced by glycogen content or epinephrine injection (Fig. 3I). AMPK Thr172 phosphorylation was higher in epitrochlearis from fasted rats compared with both rats on normal diet and from fasted-refed rats (Fig. 3J). AMPK Thr172 phosphorylation was similar in muscles from saline- and epinephrine-injected rats (Fig. 3J). Insulin increased GS activation in epitrochlearis from salineinjected rats (Fig. 4). However, insulin-stimulated GS activation was observed mainly when GS activities were measured with physiological concentration of UDP-glucose. GS FV0.03 and GS I-form0.03 were increased by insulin in epitrochlearis from rats on normal diet and from fasted rats injected with saline (Fig. 4, G, H, J, and K). Insulin only increased GS GS I-form1.5 in epitrochlearis muscles from rats on normal diet (Fig. 4B), and insulin did not increase FV1.5 significantly in any groups (Fig. 4, D–F). In epitrochlearis muscles from epinephrine-injected rats, insulin increased GS activation in all groups independently of glycogen content when measured with 0.03 mM UDP-glucose (Fig. 4, G–L). With a high concentration of UDP-glucose, insulin increased GS I-form percent in epitrochlearis from epinephrine-injected rats on normal diet (Fig. 4B). Insulin did not influence GS site 2⫹2a phosphorylation in any of the groups (Fig. 5, A–C). Insulin decreased GS site 3a⫹3b phosphorylation in epitrochlearis muscles with low and normal glycogen but not in epitrochlearis from fasted-refed rats injected with saline (Fig. 5, D–F). GS site 3a⫹3b phosphoryTable 2. Glycogen synthase I-form in epitrochlearis muscles directly frozen 1 h after epinephrine injection Glycogen Synthase I-Form1.5 (%)

Fasted Normal diet Fasted-refed

Control

Saline 1 h

Epinephrine 1 h

53.1 ⫾ 2.9 51.3 ⫾ 4.2 39.3 ⫾ 4.1c

63.0 ⫾ 1.8 65.5 ⫾ 4.7b 37.5 ⫾ 9.3c b

31.7 ⫾ 1.7a 33.5 ⫾ 4.5a 25.2 ⫾ 8.7

Data are means ⫾ SE. Epinephrine was injected subcutaneously, muscles were removed 1 h later, and GS I-form1.5 (measured with 1.5 mM UDP glucose). aP ⬍ 0.05 vs. Saline; bP ⬍ 0.05 vs. control; cP ⬍ 0.05 vs. fasted and normal diet. Control, n ⫽ 7– 8; saline and epinephrine 1 h, n ⫽ 4 for all groups.

E235

lation was lower in epitrochlearis from epinephrine-injected rats in all groups also during insulin stimulation except in muscles from fasted rats in the presence of 10,000 ␮U/ml insulin (5D). In epitrochlearis muscles from epinephrine-injected rats on normal diet or fasted rats, insulin stimulation tended to decrease GS site 3a⫹3b phosphorylation (P ⬍ 0.1; Fig. 5, D and E). Insulin did not decrease GS site 3a⫹3b phosphorylation significantly in epinephrine-injected epitrochlearis muscles from fasted-refed rats (Fig. 5F). GS site 1b phosphorylation was not regulated by insulin (Fig. 5, G–I). Insulin increased GSK-3␤ Ser9 phosphorylation similarly in epitrochlearis muscles from fasted, fasted-refed, and normaldiet rats independently of epinephrine injection (Fig. 5, J–L). Incubation with insulin did not influence AMPK Thr172 phosphorylation (data not shown). Basal and insulin-stimulated glucose uptake was higher in epitrochlearis muscles from fasted rats than in muscles from rats on normal diet and fasted-refed rats when saline was injected. Insulin increased glucose uptake in all groups (Fig. 6, B, E, and H). Insulin-stimulated glucose uptake was higher in epitrochlearis muscles from epinephrine-injected rats on normal diet and fasted-refeed rats (Fig. 6, E and H); epinephrine injection decreased glycogen content in these muscles (Fig. 6, D and G). In muscles from fasted rats, glucose uptake was similar in saline- and epinephrine-injected rats when measured with 0 (basal) and 10,000 ␮U/ml of insulin (Fig. 6B). Glucose uptake was higher in epitrochlearis muscles from epinephrineinjected measured with 100 ␮U/ml of insulin (Fig. 6B). Of note, glycogen content was lower in epitrochlearis muscles from fasted rats after epinephrine injection at this insulin concentration (Fig. 6A). Insulin increased TBC1D4 Thr642 phosphorylation in muscles independently of glycogen content (Fig. 6, C, F, and I). Furthermore, insulin-stimulated TBC1D4 Thr642 phosphorylation was similar in muscles from salineand epinephrine-injected rats despite the glucose uptake being higher when glycogen content was reduced by epinephrine injection. DISCUSSION

It is well documented that glycogen content influences GS phosphorylation and activity, but the mechanisms linking glycogen content to regulation of GS activity remain elusive. Most previous studies have manipulated glycogen content by a combination of exercise and diet or fasting-refeeding protocols. Therefore, to further understand the role of glycogen content on GS phosphorylation and activation, epinephrinestimulated glycogen breakdown was initiated in muscles with different glycogen content to test the hypothesis that GS phosphorylation adapts to reduced glycogen content when epinephrine is removed. GS phosphorylation was decreased and GS activation increased in epitrochlearis muscles with epinephrine-stimulated glycogen breakdown. Importantly, epinephrine did not decrease glycogen content in muscles with low glycogen content, and no effect on GS phosphorylation or activation was observed in these muscles, showing a condition during which epinephrine stimulation per se does not modulate GS phosphorylation and activation. The primary aim of the present study was to investigate the effect of epinephrine-mediated glycogen breakdown on GS phosphorylation and GS activity. Epinephrine activates PKA,


E236


a

60

40

20

80

20

Insulin (µU/ml) 0

F a

a

0

10,000

a

60

40

20

100

40

b

20

b

(I-Form0.03 - %)

Glycogen synthase

60

100

10,000


60

40

a,b,c a,b a

20

0

100

10,000

60

40

Insulin (µU/ml) 0

b

b

b

40

b

20

100

10,000

80


60

a,b

40

a

a

b

20

80

100

10,000

Fasted/Refed-Saline Fasted/Refed-Adrenaline

60

40

a

20

a

a,b

Insulin (µU/ml) 0

Insulin (µU/ml) 0

100

10,000

100

80


60

40

a

a

a,b

20

0

0

0

Insulin (µU/ml) 0

10,000

100

L

100

(FV0.03 - %)

60

Glycogen synthase

K Fasted-Saline Fasted-Adrenaline

100

0

Insulin (µU/ml) 0

100

a

a

20

0

Insulin (µU/ml) 0


a

I

100

80

10,000

0

Insulin (µU/ml) 0

H Fasted-Saline Fasted-Adrenaline

100

80

(I-Form0.03 - %)

10,000

Glycogen synthase

100

100

(I-Form0.03 - %)

Glycogen synthase

a

a

40

0

Insulin (µU/ml) 0

(FV0.03 - %)

a 60

20

(FV0.03 - %)

(FV1.5 - %)

40

100


80

(FV1.5 - %)

Glycogen synthase

Glycogen synthase

60

100

0

Glycogen synthase

40

Insulin (µU/ml) 0

E

80

80

b


80

(FV1.5 - %)

10,000


J

b

Glycogen synthase

100

100

80

a

60

100

0

Insulin (µU/ml) 0

G

a,b

20

0

D


Glycogen synthase

(I-Form1.5 - %)

80

C

100

(I-Form1.5 - %)

Glycogen synthase

Glycogen synthase


Glycogen synthase

B

100

(I-Form1.5 - %)

A

100

10,000

Insulin (µU/ml) 0

100

10,000

Fig. 4. Effects of epinephrine injection on insulin-stimulated GS activation in epitrochlearis muscles from rats under different nutritional conditions. Rat were diet manipulated as described, saline or epinephrine (0.02 mg/100 g rat) was injected subcutaneously, and epitrochlearis muscles were removed 3 h later and preincubated for 45 min and incubated 30 min in buffer without and with different concentrations of insulin. A–C: GS %I-form1.5 in epitrochlearis muscles from 24-h-fasted rats (A), rats on normal diet (B), and fasted-refed rats (C) after injection of saline (open bars) or epinephrine (filled bars) and incubated with different concentrations of insulin (0, 100, 10,000 ␮U/ml). D–F: GS FV1.5 in epitrochlearis muscles from 24-h-fasted rats (D), rats on normal diet (E), and fasted-refed rats (F) after injection of saline (open bars) or epinephrine (filled bars) and incubated with different concentrations of insulin (0, 100, 10,000 ␮U/ml). G–I: GS %I-form0.03 in epitrochlearis muscles from 24-h-fasted rats (G), rats on normal diet (H), and fasted-refed rats (I) after injection of saline (open bars) or epinephrine (filled bars) and incubated with different concentrations of insulin (0, 100, 10,000 ␮U/ml). J–L: GS FV0.03 in epitrochlearis muscles from 24-h-fasted rats (J), rats on normal diet (K), and fasted-refed rats (L) after injection of saline (open bars) or epinephrine (filled bars) and incubated with different concentrations of insulin (0, 100, 10,000 ␮U/ml). Data are means ⫾ SE; n ⫽ 7– 8 in all groups. aP ⬍ 0.05 vs. muscles from saline-treated rats; bP ⬍ 0.05 vs. muscles from fasted rats treated similarly; cP ⬍ 0.05 vs. normal diet with similar treatment.

and the kinase phosphorylates GS at sites 2a, 1a, and 1b (5, 18, 36, 38). PKA also phosphorylates the PP1 binding protein RGL, which releases PP1 from the glycogen particle and increases GS phosphorylation (40). The present study was designed to stimulate glycogen breakdown in conditions where GS was highly phosphorylated and inactivated, and our data confirmed that GS activity was reduced in the muscles immediately frozen 1 h after epinephrine injection, as expected (9, 18, 38). The main finding in the present study was that GS phosphorylation at sites 2⫹2a and 3a⫹3b was reduced after removal of epinephrine in muscles where epinephrine injection

reduced glycogen content. GSK-3␤ phosphorylates GS sites 3a, 3b, 3c, and 4 (36), but GSK-3␤ phosphorylation was not regulated by epinephrine injection or by glycogen content. We (19) have previously shown that when glycogen content is manipulated by fasting-refeding GS phosphorylation is regulated independent of GSK-3␤. Indeed, reduction in glycogen content by fasting requires epinephrine (41), and our data show that both fasting and epinephrine injection decreased GS phosphorylation independently of GSK-3. Insulin, on the other hand, requires GSK-3 phosphorylation for GS activation (2). In the present study, insulin increased GSK-3␤ Ser9 phosphory-


E237


100

50

a

0

200

Fasted/Refed-Saline Fasted/Refed-Adrenaline 250


150

100

a

a

(a)

50

p-GS Site 2+2a

p-GS Site 2+2a

150

C

250

( % of Basal Normal diet)

200



B

250


p-GS Site 2+2a

A

200

(a)

150

a (a)

100

50

0

Insulin (µU/ml) 0

100

10,000

Insulin (µU/ml) 0

100

0

10,000

Insulin (µU/ml) 0

100

a

50

a

b

(b)

0

H

100

50

10,000

150

100

50

b

0.6 0.4 0.2

10,000

L

10,000

a a

a

50

200

100

10,000


150

100

50

b,(c) b,(c) 0.8

b

b 0.6 0.4

Insulin (µU/ml) 0

100

10,000

1.2 1.0


0.8

b

b,c

b,c

b

0.6 0.4 0.2

0.0

100

100

Insulin (µU/ml) 0


0.2

0.0

Insulin (µU/ml) 0

(Arbitrary units)

(b)

100

1.2 1.0

p-GSK-3β Ser9

0.8

b,(c) b,(c)

150

0

Insulin (µU/ml) 0

K Fasted-Saline Fasted-Adrenaline


250


( Arbitrary units)

10,000

200

Insulin (µU/ml) 0

I

250

200

10,000

0

100

p-GSK-3β Ser9

100

1.2

( Abitrary tnits)

a,(b)

0

Insulin (µU/ml) 0

p-GSK-3β Ser9

a

p-GS Site 1b

p-GS Site 1b


150

1.0

b

a 50

Insulin (µU/ml) 0

0

J

100


10,000



p-GS Site 1b

100

250

200

150

0

Insulin (µU/ml) 0

G


p-GS Site 3a+3b

150

200

100

250



F

250


200

p-GS Site 3a+3b

E

250


p-GS Site 3a+3b

D

0.0

100

10,000

Insulin (µU/ml) 0

100

10,000

Fig. 5. Effects of epinephrine injection on insulin-stimulated phosphorylation of GS and GSK-3␤ in epitrochlearis muscles from rats under different nutritional conditions. Rats were diet manipulated as described, saline or epinephrine (0.02 mg/100 g rat) was injected subcutaneously, and epitrochlearis muscles were removed 3 h later and preincubated for 45 min and incubated 30 min in buffer without and with different concentrations of insulin. A–C: GS site 2⫹2a phosphorylation in epitrochlearis muscles from 24-h-fasted rats (A), rats on normal diet (B), and fasted-refed rats (C) after injection of saline (open bars) or epinephrine (filled bars) and incubated with different concentrations of insulin (0, 100, 10,000 ␮U/ml). D–F: GS site 3a⫹3b phosphorylation in epitrochlearis muscles from 24-h-fasted rats (D), rats on normal diet (E), and fasted-refed rats (F) after injection of saline (open bars) or epinephrine (filled bars) and incubated with different concentrations of insulin (0, 100, 10,000 ␮U/ml). G–I: GS site 1b phosphorylation in epitrochlearis muscles from 24-h-fasted rats (G), rats on normal diet (H), and fasted-refed rats (I) after injection of saline (open bars) or epinephrine (filled bars) and incubated with different concentrations of insulin (0, 100, 10,000 ␮U/ml). J–L: GSK-3␤ Ser9 phosphorylation in epitrochlearis muscles from 24-h-fasted rats (J), rats on normal diet (K), and fasted-refed rats (L) after injection of saline (open bars) or epinephrine (filled bars) and incubated with different concentrations of insulin (0, 100, 10,000 ␮U/ml). Data are means ⫾ SE; n ⫽ 7– 8 in all groups. aP ⬍ 0.05; (a)P ⬍ 0.1 vs. muscles from saline-treated rats; bP ⬍ 0.05; (b)P ⬍ 0.1 vs. muscles from fasted rats treated similarly; cP ⬍ 0.05; (c)P ⬍ 0.1 vs. normal diet with similar treatment.

lation and decreased GS site 3a⫹3b phosphorylation, but not phosphorylation of GS site 2⫹2a, agreeing with previous studies in rat skeletal muscles (13, 16, 19, 29 –31). The insulinmediated increase in GS activity was also less than the increase in muscles where glycogen content was reduced by epinephrine, as expected (9, 19, 29, 33). AMPK phosphorylates GS site 2 (25, 36). However, AMPK phosphorylation was similar in muscles from saline- and epinephrine-injected rats, and the lower GS site 2⫹2a phosphorylation in muscles from epinephrine-injected rats occurred

without regulation of AMPK phosphorylation. Muscle contraction decreases GS phosphorylation at sites 2⫹2a and 3a⫹3b (29, 30, 35), as we observed in muscles where epinephrine injection decreased glycogen content. Muscle contraction requires RGL for activation of GS, suggesting that contraction regulates GS phosphorylation via PP1 activation (1). This agrees with the more general dephosphorylation of GS during muscle contraction compared with insulin stimulation (30). Muscles with high glycogen have reduced protein phosphatase activity, whereas overexpression of RGL increases glycogen


E238


B

250

50

10

5

50

(mmol/kg dw/30 min

a

a

100

1.5

1.0

10,000

Insulin (µU/ml) 0

F a

20

15

a 10

100

10,000

2.5 Normal diet-Saline Normal diet-Adrenaline 2.0

b,c

b,c

1.5

b

(b)

1.0

0.5

5

0.0

0

0

10,000

Ins (µU/ml) 0

H

Fasted/Refed-Saline Fasted/Refed-Adrenaline 300

100

10,000

I

25

200

a a

150

a

100

Glucose uptake


250

20

a

15

a 10

5

50

0

0

Ins (µU/ml)

0

100

10,000

Ins (µU/ml)

Insulin (µU/ml) 0

100

10,000

2.5 Fasted/Refed-Saline Fasted/Refed-Adrenaline 2.0

( Arbitrary units)

100

p-TBC1D4 Thr642

Ins (µU/ml) 0

(mmol/kg dw)

100

25

(mmol/kg dw/30 min

(mmol/kg dw)

a

0

Normal-Saline Normal-Adrenaline

150

b

0.0

Ins (µU/ml)

E

Normal-Saline Normal-Adrenaline

b,c b

b

0.5

(Arbitrary units)

10,000


2.0

( Abitrary tnits)

15

p-TBC1D4 Thr 642

100

Glucose uptake

0

200

Glycogen content

a

0

250

G

p-TBC1D4 Thr 642

a 100

(mmol/kg dw/30 min

Glucose uptake

150

20

0

D

2.5


200

Ins (µU/ml)

Glycogen content

C

25


(mmol/kg dw)

Glycogen content

A

b,c b,c

b 1.5

b

1.0

0.5

0.0

0

100

10,000

Insulin (µU/ml) 0

100

10,000

Fig. 6. Effects of epinephrine injection on glycogen content, insulin-stimulated glucose uptake, and TBC1D4 phosphorylation in epitrochlearis muscles from saline- and epinephrine-treated rats under different nutritional conditions. Epinephrine/saline was injected 3 h before muscles were dissected out for incubation. A: glycogen content in epitrochlearis muscles from 24-h-fasted rats after injection of saline (open bars) or epinephrine (filled bars). Glycogen content was measured after 30-min incubation with 0, 100, 10,000 ␮U/ml of insulin. B: basal and insulin-stimulated glucose uptake in epitrochlearis muscles from 24-h-fasted rats after injection of saline (open bars) or epinephrine (filled bars). Glucose uptake was measured during 30-min incubation with 0, 100, 10,000 ␮U/ml insulin. C: basal and insulin-stimulated TBC1D4 Thr642 phosphorylation in epitrochlearis muscles from 24-h-fasted rats after injection of saline (open bars) or epinephrine (filled bars). TBC1D4 Thr642 phosphorylation was measured after 30-min incubation with 0, 100, 10,000 ␮U/ml insulin. D: glycogen content in epitrochlearis muscles from rats on normal diet after injection of saline (open bars) or epinephrine (filled bars). E: basal and insulin-stimulated glucose uptake in epitrochlearis muscles from rats on normal diet after injection of saline (open bars) or epinephrine (filled bars). F: basal and insulin-stimulated TBC1D4 Thr642 phosphorylation in epitrochlearis muscles from rats on normal diet after injection of saline (open bars) or epinephrine (filled bars). G: glycogen content in epitrochlearis muscles from fasted-refed rats after injection of saline (open bars) or epinephrine (filled bars). H: basal and insulin-stimulated glucose uptake in epitrochlearis muscles from fasted-refed rats after injection of saline (open bars) or epinephrine (filled bars). I: basal and insulin-stimulated TBC1D4 Thr642 phosphorylation in epitrochlearis muscles from fasted-refed rats after injection of saline (open bars) or epinephrine (filled bars). Data are means ⫾ SE; n ⫽ 7– 8 in all groups. aP ⬍ 0.05 vs. muscles from saline-treated rats; bP ⬍ 0.05; (b)P ⬍ 0.1 vs. muscles from fasted rats treated similarly; cP ⬍ 0.05; vs. normal diet with similar treatment.

content (1), supporting the concept that glycogen content regulates GS phosphorylation via PP1. Indeed, epinephrinestimulated glycogen breakdown did not decrease GS site 1b phosphorylation, but overall, our data support that epinephrinestimulated glycogen content decreases GS phosphorylation via activation of PP1. We suggest that RGL becomes dephosphorylated after removal of epinephrine and recruits PP1 back to the glycogen particle and dephosphorylates GS in relation to the glycogen content. This is the first documentation that epinephrine-mediated glycogen breakdown is directly linked to regulation of GS phosphorylation and activation. Indeed, Danforth (9) used epinephrine in one of his experiments to modulate glycogen content to study the relationship between glycogen content and GS activation, but it is not possible to separate effects independent of reduced glycogen content in that study. The data in the present study enable us to separate the effect of glycogen content from that of epinephrine stimulation per se, as epineph-

rine did not significantly reduce glycogen content in muscles from fasted rats. In muscles from fasted rats, epinephrine did not influence GS phosphorylation and GS activation. Therefore, the present study for the first time documents a direct “autoregulation” of GS phosphorylation by glycogen content. The modulation of glycogen content in epitrochlearis muscles prior to epinephrine injection also showed that the glycogen content regulates epinephrine-stimulated glycogenolysis. In agreement with a previous study (34), epinephrine injection decreased muscle glycogen content by 45% in muscle from rats on normal diet. Epinephrine did not significantly decrease glycogen content in epitrochlearis from fasted rats, suggesting that muscles with low glycogen content are protected from complete depletion by the stress hormone. Previously, glycogen phosphorylase activation was reported to be lower in glycogen-depleted muscles and increases as glycogen stores are repleted (3, 12). Glycogen phosphorylase activation and glycogenolytic rate are also elevated during muscle contraction



in muscles with high glycogen (14). Although we did not find a significant reduction in glycogen content in epitrochlearis from fasted rats (⬃10% lower), blood glucose increased after epinephrine injection, and liver glycogen increased, as reported by Cori and Cori (6), showing that epinephrine relocates carbohydrates between organs. Insulin-stimulated glucose uptake was increased in muscles where epinephrine injection reduced glycogen content, agreeing with previous studies in which glycogen content was modulated by fasting-refeeding or exercise and diet (10, 17, 19). An increase in insulin-stimulated glucose uptake after epinephrine injection has been reported previously (22, 34), but this is the first comprehensive investigation where epinephrine was injected in rats with different glycogen content. In epitrochlearis from rats on normal diet and from fasted-refed rats, where epinephrine reduced glycogen content, glucose uptake was increased at both physiological and high insulin concentration, indicating that insulin sensitivity and responsiveness were both increased. Of note, glucose uptake at a physiological insulin concentration was increased in muscles from fasted rats, but these muscles also had a small reduction in glycogen content, and insulin-stimulated glucose uptake was increased in muscles only where epinephrine stimulated glycogen breakdown. Insulin increased TBC1D4 Thr642 phosphorylation as expected (4, 39). Importantly, insulin-stimulated TBC1D4 Thr642 phosphorylation was similar in muscles from salineand epinephrine-injected rats despite the fact that insulinstimulated glucose was elevated in muscles where epinephrine decreased glycogen content. Therefore, epinephrine-stimulated glycogen content did not influence TBC1D4 Thr642 and GSK-3␤ Ser9 phosphorylation, indicating that insulin signaling was not amplified at key molecules regulating glucose uptake and GS activity, respectively. The present data therefore support a mechanistic link between glycogen content and stimulation of glucose uptake, although such a mechanism remains to be documented. In conclusion, epinephrine stimulated glycogen breakdown in epitrochlearis muscles with normal and high glycogen content but not in muscles with low glycogen content. GS phosphorylation decreased and GS activation increased in muscles where epinephrine injection decreased glycogen content. Importantly, epinephrine did not decrease glycogen content in muscles from fasted rats, and GS was neither dephosphorylated nor activated in these muscles. Therefore, our data document for the first time a direct autoregulation of GS phosphorylation by glycogen content after removal of epinephrine. Our data also show that epinephrine-stimulated glycogen breakdown increases insulin-stimulated glucose uptake and therefore that the Cori cycle contributes to increased insulin sensitivity in skeletal muscles. ACKNOWLEDGMENT We thank Betina Bolmgren, Astrid Bolling, Ada Ingvaldsen, Fang-Chin Lin, David Håkensen, and Kristoffer Cumming for technical assistance. Prof. Erik A. Richter is thanked for most useful comments to the manuscript. This work was carried out as a part of the program of the UNIK: Food, Fitness & Pharma for Health and Disease (see www.foodfitnesspharma.ku.dk). GRANTS The UNIK project is supported by the Danish Ministry of Science, Technology and Innovation. The study was funded by the Danish Council for Independent Research Medical Sciences (FSS), the Novo Nordisk Foundation.

E239

The Danish Diabetes Academy supported the study via a Visiting Professorship to JJ at Department of Nutrition, Exercise and Sports, Copenhagen University. DISCLOSURES No conflicts of interest. AUTHOR CONTRIBUTIONS Author contributions: A.J.K., E.E., J.F.W., and J.J. conception and design of research; A.J.K., J.B.B., E.E., J.T.S., and J.J. performed experiments; A.J.K., J.B.B., J.T.S., J.F.W., and J.J. analyzed data; A.J.K., J.B.B., J.F.W., and J.J. interpreted results of experiments; A.J.K., J.B.B., and J.J. prepared figures; A.J.K. drafted manuscript; A.J.K., J.F.W., and J.J. edited and revised manuscript; A.J.K., J.B.B., E.E., J.T.S., J.F.W., and J.J. approved final version of manuscript. REFERENCES 1. Aschenbach WG, Suzuki Y, Breeden K, Prats C, Hirshman MF, Dufresne SD, Sakamoto K, Vilardo PG, Steele M, Kim JH, Jing SL, Goodyear LJ, Paoli-Roach AA. The muscle-specific protein phosphatase PP1G/R(GL)(G(M))is essential for activation of glycogen synthase by exercise. J Biol Chem 276: 39959 –39967, 2001. 2. Bouskila M, Hirshman MF, Jensen J, Goodyear LJ, Sakamoto K. Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle. Am J Physiol Endocrinol Metab 294: E28 –E35, 2008. 3. Bräu L, Ferreira LDMCB, Nikolovski S, Raje G, Palmer TN, Fournier PA. Regulation of glycogen synthase and phosphorylase during recovery from high-intensity exercise in the rat. Biochem J 322: 303–308, 1997. 4. Cartee GD. Roles of TBC1D1 and TBC1D4 in insulin- and exercisestimulated glucose transport of skeletal muscle. Diabetologia 58: 19 –30, 2014. 5. Cohen P. Dissection of the protein phosphorylation cascades involved in insulin and growth factor action. Biochem Soc Trans 214: 555–567, 1993. 6. Cori CF, Cori GT. The mechanism of epinephrine action. I. The influence of epinephrine on the carbohydrate metabolism of fasting rats, with a note on new formation of carbohydrates. J Biol Chem 79: 309 –319, 1928. 7. Cori CF, Cori GT. The mechanism of epinephrine action. III. The influence of epinephrine on the utilization of absorbed glucose. J Biol Chem 79: 343–355, 1928. 8. Cori CF, Cori GT. The mechanism of epineprine action. II. The influence of epinephrine and insulin on the carbohydrate metabolism of rats in the postabsorptive state. J Biol Chem 79: 321–341, 1928. 9. Danforth WH. Glycogen synthase activity in skeletal muscle. J Biol Chem 240: 588 –593, 1965. 10. Derave W, Hansen BF, Lund S, Kristiansen S, Richter EA. Muscle glycogen content affects insulin-stimulated glucose transport and protein kinase B activity. Am J Physiol Endocrinol Metab 279: E947–E955, 2000. 11. Derave W, Lund S, Holman GD, Wojtaszewski J, Pedersen O, Richter EA. Contraction-stimulated muscle glucose transport and GLUT-4 surface content are dependent on glycogen concentration. Am J Physiol Endocrinol Metab 277: E1103–E1110, 1999. 12. Franch J, Aslesen R, Jensen J. Regulation of glycogen synthesis in rat skeletal muscle after glycogen depleting contractile activity: effects of adrenaline on glycogen synthesis and activation of glycogen synthase and glycogen phosphorylase. Biochem J 344: 231–235, 1999. 13. Friedrichsen M, Birk JB, Richter EA, Ribel-Madsen R, Pehmoller C, Hansen BF, Beck-Nielsen H, Hirshman MF, Goodyear LJ, Vaag A, Poulsen P, Wojtaszewski JF. Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NH2-terminal (sites 2 ⫹ 2a) phosphorylation. Am J Physiol Endocrinol Metab 304: E631–E639, 2013. 14. Hespel P, Richter EA. Mechanism linking glycogen concentration and glycogenolytic rate in perfused contracting rat skeletal muscle. Biochem J 284: 777–780, 1992. 15. Højlund K, Birk JB, Klein DK, Levin K, Rose AJ, Hansen BF, Nielsen JN, Beck-Nielsen H, Wojtaszewski JF. Dysregulation of glycogen synthase. J Clin Endocrinol Metab 94: 4547–4556, 2009. 16. Hunter RW, Treebak JT, Wojtaszewski JF, Sakamoto K. Molecular mechanism by which AMP-activated protein kinase activation promotes glycogen accumulation in muscle. Diabetes 60: 766 –774, 2011.


E240


17. Jensen J, Aslesen R, Ivy JL, Brørs O. Role of glycogen concentration and epinephrine on glucose uptake in rat epitrochlearis muscle. Am J Physiol Endocrinol Metab 272: E649 –E655, 1997. 18. Jensen J, Grønning-Wang LM, Jebens E, Whitehead JP, Zorec R, Shepherd PR. Adrenaline potentiates insulin-stimulated PKB activation in the rat fast-twitch epitrochlearis muscle without affecting IRS-1 associated PI 3-kinase activity. Pflügers Arch Eur J Physiol 456: 969 –978, 2008. 19. Jensen J, Jebens E, Brennesvik EO, Ruzzin J, Soos MA, Engebretsen EM, O’Rahilly S, Whitehead JP. Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling. Am J Physiol Endocrinol Metab 290: E154 –E162, 2006. 20. Jensen J, Lai YC. Regulation of muscle glycogen synthase phosphorylation and kinetic properties by insulin, exercise, adrenaline and role in insulin resistance. Arch Physiol Biochem 115: 13–21, 2009. 21. Jensen J, Rustad PI, Kolnes AJ, Lai YC. The role of skeletal muscle glycogen breakdown for regulation of insulin sensitivity by exercise. Front Physiol 2: 112, 2011. 22. Jensen J, Ruzzin J, Jebens E, Brennesvik EO, Knardahl S. Improved insulin-stimulated glucose uptake and glycogen synthase activation in rat skeletal muscles after adrenaline infusion: role of glycogen content and PKB phosphorylation. Acta Physiol Scand 184: 121–130, 2005. 23. Jensen J, Tantiwong P, Stuenaes JT, Molina-Carrion M, DeFronzo RA, Sakamoto K, Musi N. Effect of acute exercise on glycogen synthase in muscle from obese and diabetic subjects. Am J Physiol Endocrinol Metab 303: E82–E89, 2012. 24. Jensen TE, Richter EA. Regulation of glucose and glycogen metabolism during and after exercise. J Physiol 590: 1069 –1076, 2012. 25. Jørgensen SB, Nielsen JN, Birk JB, Olsen GS, Viollet B, Andreelli F, Schjerling P, Vaulont S, Hardie DG, Hansen BF, Richter EA, Wojtaszewski JF. The alpha2-5=AMP-activated protein kinase is a site 2 glycogen synthase kinase in skeletal muscle and is responsive to glucose loading. Diabetes 53: 3074 –3081, 2004. 26. Kawanaka K, Han DH, Nolte LA, Hansen PA, Nakatani A, Holloszy JO. Decreased insulin-stimulated GLUT-4 translocation in glycogensupercompensated muscles of exercised rats. Am J Physiol Endocrinol Metab 276: E907–E912, 1999. 27. Kolnes AJ, Ingvaldsen A, Bolling A, Stuenaes JT, Kreft M, Zorec R, Shepherd PR, Jensen J. Caffeine and theophylline block insulin-stimulated glucose uptake and PKB phosphorylation in rat skeletal muscles. Acta Physiol (Oxf) 200: 65–74, 2010. 28. Lai YC, Lin FC, Jensen J. Glycogen content regulates insulin- but not contraction-mediated glycogen synthase activation in the rat slow-twitch soleus muscles. Acta Physiol (Oxf) 197: 139 –150, 2009. 29. Lai YC, Stuenæs JT, Kuo CH, Jensen J. Glycogen content and contraction regulate glycogen synthase phosphorylation and affinity for UDP-

30. 31.

32. 33. 34. 35.

36. 37. 38. 39.

40. 41. 42.

glucose in rat skeletal muscles. Am J Physiol Endocrinol Metab 293: E1622–E1629, 2007. Lai YC, Zarrinpashneh E, Jensen J. Additive effect of contraction and insulin on glucose uptake and glycogen synthase in muscle with different glycogen contents. J Appl Physiol 108: 1106 –1115, 2010. Lin FC, Bolling A, Stuenaes JT, Cumming KT, Ingvaldsen A, Lai YC, Ivy JL, Jensen J. Effect of insulin and contraction on glycogen synthase phosphorylation and kinetic properties in epitrochlearis muscles from lean and obese Zucker rats. Am J Physiol Cell Physiol 302: C1539 –C1547, 2012. Lowry OH, Passonneau JV. A Flexible System of Enzymatic Analysis. New York: Academic, 1972, p. 1–291. Nielsen JN, Derave W, Kristiansen S, Ralston E, Ploug T, Richter EA. Glycogen synthase localization and activity in rat skeletal muscle is strongly dependent on glycogen content. J Physiol 531: 757–769, 2001. Nolte LA, Gulve EA, Holloszy JO. Epinephrine-induced in vivo muscle glycogen depletion enhances insulin sensitivity of glucose transport. J Appl Physiol 76: 2054 –2058, 1994. Prats C, Helge JW, Nordby P, Qvortrup K, Ploug T, Dela F, Wojtaszewski JF. Dual regulation of muscle glycogen synthase during exercise by activation and compartmentalization. J Biol Chem 284: 15692– 15700, 2009. Roach PJ, DePaoli-Roach AA, Hurley TD, Tagliabracci VS. Glycogen and its metabolism: some new developments and old themes. Biochem J 441: 763–787, 2012. Thomas JA, Schlender KK, Larner J. A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. Anal Biochem 25: 486 –499, 1968. Toole BJ, Cohen PT. The skeletal muscle-specific glycogen-targeted protein phosphatase 1 plays a major role in the regulation of glycogen metabolism by adrenaline in vivo. Cell Signal 19: 1044 –1055, 2007. Treebak JT, Pehmoller C, Kristensen JM, Kjobsted R, Birk JB, Schjerling P, Richter EA, Goodyear LJ, Wojtaszewski JF. Acute exercise and physiological insulin induce distinct phosphorylation signatures on TBC1D1 and TBC1D4 proteins in human skeletal muscle. J Physiol 592: 351–375, 2014. Walker KS, Watt PW, Cohen P. Phosphorylation of the skeletal muscle glycogen-targetting subunit of protein phosphatase 1 in response to adrenaline in vivo. FEBS Lett 466: 121–124, 2000. Winder WW, Duan C. Control of fructose 2,6-diphosphate in muscle of exercising fasted rats. Am J Physiol Endocrinol Metab 262: E919 –E924, 1992. Wojtaszewski JF, MacDonald C, Nielsen JN, Hellsten Y, Hardie DG, Kemp BE, Kiens B, Richter EA. Regulation of 5=-AMP-activated protein kinase activity and substrate utilization in exercising human skeletal muscle. Am J Physiol Endocrinol Metab 284: E813–E822, 2003.
