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How do muscle cells synthesize glycogen?

How do muscle cells synthesize glycogen?


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Hexokinase enzyme is present in all cells (including muscle cells) and can be suppressed by excessive G-6-P product. So that's why in the liver, glucokinase can act on glucose without inhibition of it despite excessive G-6-P product; that's why it can generate glycogen and not let excessive glucose flow back in bloodstream. My question is then: why muscle cells can generate glycogen despite having HK instead of GK? Because as i said above, HK activity is dependent on intracellular glucose level, and cannot phosphorylate glucose to hold it inside the cell. So glucose enters back to bloodstream and prevents the synthesis of glycogen?


The answer to the question is that under conditions favouring glycogen synthesis (e.g. the fed state) G6P only accumulates in skeletal muscle cells when the capacity of those cells for storing glycogen has been reached. Until then the G6P is immediately converted to G1P and on to glycogen, in the reactions in sunboyharry's answer.

The question suggests a misunderstanding of the role of glucokinase in liver, which can be better considered if we first make clear the role of liver and skeletal muscle in glucose metabolism. The role of the liver is to deal with excess glucose after a meal by trapping it in the cell as G6P. Although initially the G6P may be converted to G1P and hence to glycogen, this is not the only fate of G6P. Subsequently it may be directed to glycolysis and to the pentose phosphate shunt to provide the acetyl units and reducing power (NADPH) needed for fatty acid synthesis. (The fatty acids are converted to triglycerides and exported to the adipose tissue.) When the blood glucose falls the role of the liver is to release glucose into the blood to supply those tissues that depend on it (brain and erythrocytes in starvation, skeletal muscle if the fall is due to muscle exercise). The skeletal muscle, in contrast, is only concerned with storing glucose as glycogen when it is available, and converting it to G6P for glycolysis when it needs ATP to power muscle contraction.

Glucokinase has a key role in the regulation of blood glucose concentration by the liver. To paraphrase (and edit slightly) Cornish-Bowden and Cardenas: "Mammals have two types of enzymes to catalyse the formation of G6P from glucose. Glucokinase [hexokinase D in their nomenclature] differs significantly from the other type [generally just referred to as hexokinase]. Its abundance varies markedly with hormonal status; it requires much higher glucose concentrations (about 10 mM) for half saturation, and is insensitive to physiological concentrations of G6P. It is thus well adapted to respond to variations in blood-glucose concentrations."

To clarify, 10mM is in the region of the blood glucose concentration (ca. 5mM) so the glucokinase reaction will be affected by the relative concentrations of blood glucose and intracellular G6P in a standard mass action manner, which will determine whether the liver takes up glucose or releases it into the blood. If glucokinase were inhibited allosterically by G6P like hexokinase, this couldn't work.

Now let's turn to G6P and hexokinase in skeletal muscle. Hexokinase has a much higher affinity for glucose than hexokinase and will convert it efficiently to G6P. As long as G6P is then converted to G1P for glycogen synthesis, G6P will not build up. However when glycogen synthesis stops because the capacity of the muscle to store glycogen is reached, the concentration of G6P will increase and turn hexokinase off. This, in turn, will cause a build up of intracellular glucose and prevent glucose transport into the muscle. This makes sense, because with full glycogen stores, and in the absence of need for contraction, glucose will not be metabolized by the muscle; it will be left in the blood for the liver to handle.

Thus, the key point about G6P inhibition of hexokinase, and the apparent source of confusion in the question, is that it only occurs at concentrations that are reached when the G6P is not being metabolized within the cell.

Reference and Footnote

Ref for citation: Cornish-Bowden, A and Cardenas, M… (1991) Trends in Biochemical Sciences 16, pp281-2. (This article also makes the point that the kinetics of glucokinase are sigmoidal and not hyperbolic as stated in most texts, and that the enzyme is not specific for glucose. Neither of these points affect the argument here. The former does indicate that my reference to glucose control of glucokinase operating in "a standard mass action manner" is a simplification. It does respond to changes in glucose concentration in the blood range, but in a more sophisticated manner than an enzyme with simple Michaelis-Menten kinetics.)


Hexokinase catalyze glucose to form G-6-P is only the first step of the glycogen synthesis, and then the G-6-P will continue to processing a series reaction and finally become glycogen. So once the G-6-P forms, it will keep reacting, which will not inhibit the activity of hexokinase.


Glycogen Muscle Level

Muscle glycogen does not vary significantly over the course of normal daily activity as long as usual meal patterns are followed ( 73 ). But with starvation, its concentration gradually falls over a 5- to 6-day period. On calorically adequate but virtually CHO-free diet, glycogen falls in muscle but more slowly than during starvation ( 73 ). Glucose or fructose infusion increases muscle glycogen content only modestly in a 6-hour period of rest ( 73 ). However, this is not the case in the recovery of muscle previously exercised and glycogen depleted. Even without food intake in the immediate recovery period, muscle glycogen is resynthesized, both in rest and during active recovery at a reduced exercise intensity, presumably using blood glucose from accelerated glyconeogenesis ( 4 ), and using enhanced lactate uptake by muscle ( 67 ). There remains, however, a controversy as to the extent lactate disappearance during recovery represents oxidation and glycogen resynthesis ( 17 , 18 , 49 , 67 ). Nevertheless, complete restitution of muscle glycogen requires food intake, since it has been shown that endogenous substrates do not lead to full recovery, at least over 4–5 ( 101 ) to 20 hours ( 73 ). A mixed diet does replenish glycogen by between 24 hours ( 101 ) to 46 hours ( 118 ). Presumably this difference in rate of restitution depends on the intensity and duration of the preceding exercise and the extent the exercise has depleted muscle glycogen. Also, some of the differences in the glycogen restitution rate of the last two studies, which used different exercise protocols, could be related to differences in blood glucose and insulin levels produced by each exercise. For the prolonged hard exercise of Piehl, blood glucose and insulin levels fell ( 2–4 ) and remained low for some time during recovery, whereas in the short intermittent severe exercise of MacDougall, blood glucose and insulin were elevated at the end and following exercise. So presumably, there is accelerated resynthesis of glycogen in the latter case thereby promoting significant glycogen accumulation within 2 hours even without food, and complete recovery occurs in 24 hours with consumption of a 3100-kcal mixed diet.

For muscle glycogen depletion produced by prolonged exercise, it appears the composition of food intake is important in determining both the rate and extent of glycogen repletion. With a calorically adequate diet of fat and protein, glycogen resynthesis is slow and incomplete even after 4 days ( 73 ). In contrast, a day of fasting after exhaustive exercise, followed by CHO ingestion, nearly restores glycogen in the next 24 hours and produces “supercompensation” of glycogen stores in 2 days ( 73 ). Supercompensation means the glycogen stores increase to a level markedly above the predepletion content. It can amount to three to four times control over a 3-day period of CHO feeding ( 16 ). This supercomposition of glycogen repletion produced by exhausting exercise and CHO feeding is specific to the muscles exercised nonexercised muscles increase their glycogen content only slightly ( 16 ).


What Is the Function of Glycogen?

Glycogen is a polysaccharide that is the storage form of glucose in the human body. Glucose is an important biomolecule that provides energy to cells throughout the entire human body. Humans derive glucose from the foods that they eat. When they are running low on glucose, glycogen can be utilized as a glucose source.

In humans, glycogen is stored and produced by the hepatocytes in the liver. The main function of glycogen is as a secondary long-term energy-storage molecule. The primary energy-storage molecules are adipose cells. Glycogen is also stored in muscle cells. Muscle glycogen is converted into glucose by the muscle cells whenever muscles are overworked and tired. Glycogen from the liver is converted into glucose to be used mainly by the central nervous system, which includes the brain and spinal cord.

In the liver, blood glucose from the foods that humans eat reaches the liver via the portal vein. There, insulin stimulates the liver cells, which stimulates glycogen synthase. This enzyme stimulates the synthesis of glycogen in the liver therefore, glycogen in the liver is formed from the food that humans eat. Muscle-cell glycogen is chemically identical to liver glycogen. However, it functions as an immediate source of glucose for muscle cells. When muscles are tired, they may convert glycogen to glucose to continue to function properly. However, liver glycogen does not convert into glucose unless the body is deprived of food.


RESULTS

Effects of glucose withdrawal on glycogen content and GS activity.

Human myoblasts were routinely maintained in Ham’s F-10 medium, which contains physiological levels of glucose (6.1 mmol/l). Under these culture conditions, the cells accumulate significant amounts of glycogen. To deplete these stores of glycogen, cells were deprived of glucose for increasing periods of time in a glucose-deficient medium of similar composition to Ham’s F-10, i.e., DMEM Glu − (Ham’s F-10 lacking glucose is not commercially available) (Fig. 1). A decrease in cellular glycogen content of ∼50% was observed in myoblasts maintained in DMEM Glu − for 6 h, as compared with control cells maintained in the presence of glucose (glycogen content of 0.54 ± 0.07 μmol glucose/mg protein in glucose-deprived myoblasts vs. 1.12 ± 0.06 μmol glucose/mg protein in control myoblasts). This decrease was time-dependent, with a significant effect being observed after 2 h. During this time period, the fractional activity of GS also fell slightly, decreasing after 5 h of glucose starvation from 0.032 ± 0.008 in control cells to 0.014 ± 0.005 (n = 7, from three different subjects). However, if after incubation in the absence of glucose for 6 h Ham’s F-10 media (containing 6.1 mmol/l glucose) was returned to the cells for 15 min, a dramatic increase in the fractional activity of GS to 0.390 ± 0.03 was observed (Fig. 1). To confirm that the stimulatory effect is because of glucose restoration, after incubation in DMEM Glu − for 6 h, cells were exposed to DMEM containing 5 mmol/l glucose. This again caused a dramatic activation of GS to a fractional activity of 0.064 ± 0.01. Supplementation of DMEM with pyruvate (1 mmol/l) and glucose further increased this value to 0.186 ± 0.02 (pyruvate alone had no effect), whereas DMEM, which contains 5.5 mmol/l glucose and 1 mmol/l pyruvate, activated GS to 0.345 ± 0.033. To confirm that alterations in GS activity were a result of previous glucose withdrawal, cells were incubated with DMEM supplemented with 5.5 mmol/l glucose for 5 h before treatment with Ham’s F-10. No increase in the GS activity was observed after Ham’s F-10 readmission (data not shown). These data indicate that the stimulation of GS is predominantly due to the action of glucose and requires previous glycogen depletion because of glucose removal, but other components of the different media may modulate the magnitude of the response. In subsequent experiments, Ham’s F-10 was used for glucose replenishment, except in Fig. 2C, where the effects of varying amounts of glucose were examined, and in Fig. 5, where DMEM Glu − was supplemented by glucose at t = 0, because of the necessity of adding glucose after preincubation with inhibitors.

The increase in GS fractional activity in response to glucose was dependent on the duration of previous glucose deprivation and, hence, inversely related to the glycogen content of the cells. In the absence of glycogen depletion, no significant activation of GS by glucose was observed (Fig. 1). Total GS activity remained essentially unchanged in all conditions, indicating that alteration in the expression of the GS polypeptide was not involved (not shown).

Time and concentration dependence of GS activation by glucose in glycogen-depleted myoblasts.

Myoblasts were incubated in DMEM Glu − for 5 h before treatment with glucose-containing media for increasing periods of time. Glucose treatment (6.1 mmol/l) caused a rapid time-dependent increase in the fractional activity of GS, with stimulation being observed within 2 min and reaching a maximum fractional activity of ∼0.3 after 10–15 min (Fig. 2A). After 30 min of glucose re-administration, the fractional activity of GS started to decrease, reaching ∼0.1 after 4 h, but remaining constant thereafter up to 8 h (Fig. 2B). During the same period, the glycogen content of the cells increased, reaching a value ∼80% of that in control cells. The stimulation of GS was dependent on the concentration of glucose added, with significant stimulatory effects being observed in response to 1.5 mmol/l (Fig. 2C).

Combined effect of insulin and glucose on GS activity and glycogen synthesis in glycogen-depleted myoblasts.

The combined effects of glucose readdition and insulin on cells cultured in the absence of glucose was then examined. Treatment of cells with 100 nmol/l insulin for 15 min after preincubation in glucose-containing media for 5 h led to an ∼1.6-fold increase in the fractional activity of GS from 0.040 ± 0.010 to 0.071 ± 0.020 (Fig. 3A). Similarly, GS activity was increased by insulin ∼1.7-fold from 0.214 ± 0.021 to 0.337 ± 0.037 in myoblasts that had been glucose-depleted for 5 h and that had glucose re-administered during the 15 min of insulin treatment this finding indicates that the effects of glucose and insulin are additive and act by different mechanisms. In the absence of readministration of glucose, stimulatory effects of insulin on GS activity were minimal after glycogen depletion (data not shown).

Treatment of control cultures with 100 nmol/l insulin for 1 h led to an ∼1.9-fold increase in the rate of glycogen synthesis from 134.7 ± 23.45 to 259.8 ± 31.11 pmol · min –1 · mg –1 protein (Fig. 3B). The rate of glycogen synthesis after glucose re-administation for 1 h to cells preincubated in DMEM Glu − for 5 h was increased dramatically to 944.8 ± 63.79 pmol · min –1 · mg –1 . This value further increased to 1,493.8 ± 110.47 pmol · min –1 · mg –1 when insulin was additionally present during the 1 h of glucose refeeding, representing a 1.6-fold stimulation by insulin.

Effect of glucose deprivation on 2-deoxyglucose uptake in human myoblasts.

An increase in the rate of 2-deoxyglucose uptake was observed in myoblasts deprived of glucose for increasing time, reaching 1.7-fold after 5 h (Fig. 4A). The most significant increase in the rate of 2-deoxyglucose uptake was observed after the first 2 h in DMEM Glu − : 35.45 ± 1.99 pmol · min –1 · mg –1 in cells maintained in the presence of glucose and 49.58 ± 3.93 pmol · min –1 · mg –1 in cells deprived of glucose for 2 h.

The effect of insulin treatment on 2-deoxyglucose uptake was examined in control and glucose-starved myoblasts (Fig. 4B). Incubation of control cells with 100 nmol/l insulin for 15 min led to a modest 1.3-fold increase in the rate of 2-deoxyglucose uptake. Insulin (100 nmol/l) treatment failed to further increase 2-deoxyglucose uptake above the increased basal rate observed in glucose-starved myoblasts.

Mechanisms involved in activation of GS by glucose.

To investigate the mechanisms by which glucose activates GS, selective inhibitors of signaling pathways known to be stimulated by insulin were used (Fig. 5). DMEM Glu − supplemented with 5.5 mmol/l glucose was added for 15 min to myoblasts previously depleted of glucose for 5 h, resulting in an ∼4.2-fold increase in GS activity ratio. Preincubation of cells with rapamycin, which selectively inhibits the activation of p70 s6k , failed to inhibit this stimulatory effect of glucose on GS activation. Treatment of glycogen-depleted myoblasts with either the MEK inhibitor, PD098059, or the PI 3-kinase inhibitor wortmannin partly reduced the activity state of GS obtained in response to glucose readministration. However, this is essentially attributable to these inhibitors reducing the basal activity state of GS, as previously observed (13). Therefore, these findings indicate that the mechanisms involved in the activation of GS by glucose are independent of the activity of PI 3-kinase, the classical mitogen-activated protein kinase pathway, and the rapamycin-sensitive pathway leading to activation of p70 s6k . Furthermore, during glucose readministration, there was no observable decrease in the activity of GSK-3 and no detectable phosphorylation of that protein, as detected by phosphospecific anti–GSK-3 antibodies (Fig. 6). No changes in the activity of protein kinase B were detected (not shown). Thus, the mechanism underlying the effects of glucose/glycogen are apparently distinct from those utilized by insulin.

Glucose-6-phosphate (G6P) is a potent allosteric activator of GS (19), whereas a number of small metabolites, such as ATP, ADP, and AMP, are capable of inhibiting GS activity (20). To confirm that the observed alterations in the fractional activity of GS are because of covalent modification and not carryover of allosteric activators or inhibitors, cell extracts were fractionated using Bio-Spin P-6 polyacrylamide gel spin columns to remove molecules with a mass <6 kDa. Use of 14 C-glucose-1-phosphate as a marker indicated that >95% of small metabolites were removed by this process. After refeeding of glycogen-depleted cells, fractionation of extracts failed to significantly alter GS activity ratio (0.255 ± 0.011 before fractionation and 0.283 ± 0.003 after fractionation), indicating that activation of GS was not because of allosteric activation by G6P or effects of other small molecules.

The activity of endogenous PPs against GS was then measured in extracts from control and glycogen-depleted cells. Because it has been demonstrated that GS becomes a better substrate for PPs in the presence of G6P (21), this was carried out over a range of G6P concentrations. No differences were found in the rate of activation of GS by endogenous phosphatases in extracts prepared from control and glycogen-depleted cells. For example, at a physiological concentration of 0.25 mmol/l G6P in the extract, the effect of endogenous phosphatases on GS activity in extracts of cells preincubated in the presence of glucose led to an increase of 3.44 ± 0.29 pmol · min –1 · mg –1 GS activity in 30 min, compared with an increase of 3.85 ± 0.60 pmol · min –1 · mg –1 in 30 min in cells preincubated in the absence of glucose for 5 h. Therefore, glycogen depletion does not appear to stimulate phosphatase activity against GS, at least when measured subsequently in cell extracts.


Carbohydrate metabolism

James C. Blackstock , in Guide to Biochemistry , 1989

11.5 Glycogenesis and glycogenolysis

Glycogen synthesis (called glycogenesis ) commences from glucose 6-phosphate ( Figure 11.7 ) which may be produced from glucose absorbed from the bloodstream as in skeletal muscle or by gluconeogenesis ( Section 11.6 ) from C3 compounds, e.g. lactate, as in liver. An intramolecular transfer of the phosphate from the C-6 position to C-1 position is performed by phosphoglucomutase.

FIGURE 11.7 . Synthesis and degradation of glycogen

The next reaction is unique to the synthetic pathway and involves the formation of uridine diphosphate glucose (UDP-glucose) which serves as the carrier of the glucosyl residue which participates in the elongation of a primer molecule of glycogen. The enzyme, UTP-glucose-1-phosphate uridylyltransferase utilizes both UTP and glucose 1-phosphate in a readily reversible reaction. Synthesis is promoted by the irreversible hydrolysis of pyrophosphate by inorganic pyrophosphatase. The removal of the pyrophosphate commits the uridylyltransferase reaction to the direction of glycogen synthesis ( Section 10.4 ).

Glycogen synthase transfers the glucosyl moiety of UDP-glucose to the non-reducing end ( Section 3.5 ) of a glycogen primer. Glycogen synthase is highly specific it will only produce a new α-(1 → 4) glycosidic bond. The minimum size for an active primer molecule is four glucose units but the enzyme is more effective with longer polymers. Indeed, the usual primer is a glycogen molecule. The released UDP may be phosphorylated to UTP (at the expense of ATP) which may participate in the formation of another UDP-glucose. Some animal tissues may utilize ADP as a glucosyl carrier but the rate of reaction is lower. Glycogen synthase may repeatedly add glucosyl groups to the primer molecule.

Because of the catalytic constraints of glycogen synthase, branching through α-(1 → 6) glycosidic bonds occurs by the action of another enzyme, glycogen branching enzyme, which transfers terminal hexa- or septa-saccharide units from growing chains of at least 11 residues to the hydroxyl group of glucose residues in internal positions ( Figure 11.8 ). Branch points are not created closer than every fourth residue. Since similar chemical linkages are involved, the free-energy change is very small. Branching increases the number of non-reducing ends which may be simultaneously elongated or degraded by glycogen synthase or Phosphorylase respectively. In plant tissues, starch is synthesized by an analogous pathway which employs starch synthase and ADP-glucose ( Section 14.6 ).

FIGURE 11.8 . Branching of glycogen

Glycogen degradation (called glycogenolysis) proceeds by the action of the enzyme, glycogen Phosphorylase ( Figure 11.7 ). The reaction involves the cleavage of the α-(1 → 4) glycosidic linkage between the terminal glucose residue of a branch and its neighbour by phosphorolysis. The products of the reaction are glucose 1-phosphate which retains the α-configuration and a glycogen molecule which is one glucose residue smaller. Glucose 1-phosphate is rearranged into glucose 6-phosphate. Glycogen Phosphorylase may sequentially remove residues from the non-reducing ends of glycogen chains until it approaches a branching point. Like glycogen synthase, glycogen Phosphorylase cannot negotiate α-(1→6) glucosidic linkages which require an enzyme system called the glycogen debranehing system. The debranching system of mammals and yeast contains two enzymic activities: 4-α-glucanotransferase and amylo-1,6-glucosidase ( Figure 11.9 ). The activity of glycogen Phosphorylase ceases at the fourth residue from an α-(1 → 6) linkage. The 4-α-glucanotransferase transfers a trisaccharide unit to the end of another chain. The solitary glucose remaining at the branch is removed by the amylo-l,6-glucosidase activity. Glycogen Phosphorylase resumes its activity.

FIGURE 11.9 . Debranching of glycogen

The fate of glucose 6-phosphate and glucose depends on the nature of the tissue. In skeletal muscle cells, the non-phosphorylated glucose which accounts for about 10% of the cleavage products (branches occur every 8–12 glucose residues) may be phosphorylated into glucose 6-phosphate. Glucose 6-phosphate from either route may be utilized in energy production through glycolysis.

The liver utilizes glucose-6-phosphatase to remove the phosphate from glucose 6-phosphate. Non-phosphorylated glucose from liver glycogen can traverse the plasma membrane and be transported via the blood circulation to other tissues. Glucose-6-phosphatase is absent from skeletal muscle and brain and so glucose is retained by these tissues as glucose 6-phosphate which cannot permeate the plasma membrane.


RESULTS

Wheel running

HR normal mice ran more than three-fold more revolutions per day than C mice on all days, and HR mini ran even more, with values 6–35% above those of HR normal (Tables S1–S5 in supplementary material). HR mice ran significantly more minutes per day than C (37–62% increase Table S5 in supplementary material), and also at higher average speeds (HR mini also ran significantly faster than HR normal Table S5 in supplementary material).

Gastrocnemius GLUT-4

In mice that never had wheel access (Group 4), GLUT-4 abundance did not significantly differ among C, HR normal and HR mini individuals(Table 2). Wheel access for five days caused an increase in GLUT-4 concentration for all mice(Table 2), but the magnitude of the increase was much greater for HR normal (3.15-fold) and HR mini(4.46-fold) than for C mice (1.80-fold), as indicated by a highly significant wheel access × line type interaction in the combined analyses of Groups 1 and 4 (Table 3, P=0.0011). On average, HR mice had approximately 2.4 times higher GLUT-4 abundance than C lines after five days of wheel access, and, within the HR or C groups, GLUT-4 abundance was unrelated to the amount of wheel running performed by individual mice on the day before sacrifice(Fig. 1, Table 2). Results (not shown)were similar when the amount of running on days 1, 2, 3 or 4 was used as a covariate instead of running on day 5.

Plasma glucose, glycogen in gastrocnemius, soleus and liver, and GLUT-in gastrocnemius, of control (C) and high runner (HR) lines of house mice

Trait . Group . Transform . N . Control . HR normal . HR mini . Line type . Mini muscle . Estrous cycle . Age . Time . Revolutions .
Plasma glucose 1 ^1.5 57 10.72 10.40 10.43 F1,6=0.16 F1,41=0.00 F4,41=0.72 F1,41=2.02 F1,41=0.48 F1,41=0.19
P=0.7068 P=0.9663 P=0.5822 P=0.1632 P=0.4926 P=0.6620 [–]
2 ^1.8 65 8.84 9.56 9.37 F1,6=2.40 F1,49=0.38 F4,49=0.81 F1,49=16.23F1,49=4.60F1,49=6.53
P=0.1725 P=0.5424 P=0.5223 P=0.0002P=0.0370P=0.0138 [–]
3 ^1.8 61 8.81 8.91 9.45 F1,6=0.04 F1,45=1.79 F4,45=0.27 F1,45=0.94 F1,45=4.25F1,45=7.60
P=0.8465 P=0.1873 P=0.8987 P=0.3369 P=0.0451P=0.0084 [–]
4 ^0.5 44 9.87 9.51 10.19 F1,6=0.74 F1,29=1.44 F4,29=5.85F1,29=2.03 F1,29=0.01
P=0.4224 P=0.2406 P=0.0014P=0.1651 P=0.9296
Glycogen gastrocnemius 1 ^0.3 65 16.96 12.38 37.52 F1,6=1.70 F1,49=35.03F4,49=1.32 F1,49=0.23 F1,49=0.11 F1,49=2.89
P=0.2396 P<0.0001P=0.2774 P=0.6346 P=0.7450 P=0.0954 [+]
2 ^0.3 67 16.46 16.65 42.48 F1,6=0.00 F1,51=40.74F4,51=2.23 F1,51=0.59 F1,51=4.17F1,51=0.03
P=0.9593 P<0.0001P=0.0785 P=0.4468 P=0.0463P=0.8739 [–]
3 ^0.5 65 19.54 23.28 36.69 F1,6=0.55 F1,47=7.69F4,47=0.26 F1,47=3.42 F1,47=0.10 F1,47=1.45
P=0.4876 P=0.0079P=0.9018 P=0.0708 P=0.7476 P=0.2349 [+]
4 None 45 15.93 10.08 40.98 F1,6=2.05 F1,30=42.99F4,30=0.64 F1,30=2.16 F1,30=0.02
P=0.2025 P<0.0001P=0.6356 P=0.1524 P=0.8973
Glycogen soleus 1 ^0.2 65 21.95 25.83 32.52 F1,6=0.52 F1,49=1.61 F4,49=1.05 F1,49=0.02 F1,49=2.00 F1,49=0.17
P=0.4960 P=0.2101 P=0.3923 P=0.8790 P=0.1631 P=0.6828 [+]
2 ^0.8 67 18.06 20.72 42.02 F1,6=0.30 F1,50=24.00F4,50=3.46F1,50=0.00 F1,50=9.14F1,50=2.47
P=0.6061 P<0.0001P=0.0142P=0.9607 P=0.0039P=0.1226 [–]
3 ^1.3 65 22.57 44.62 50.02 F1,6=10.93F1,47=1.23 F4,47=1.08 F1,47=1.53 F1,47=1.18 F1,47=0.39
P=0.0163P=0.2733 P=0.3766 P=0.2224 P=0.2828 P=0.5335 [–]
4 ^0.2 44 21.83 20.22 32.48 F1,6=0.16 F1,29=4.04 F4,29=2.28 F1,29=1.02 F1,29=0.02
P=0.7013 P=0.0539 P=0.0847 P=0.3216 P=0.8774
Glycogen liver 1 ^0.05 65 76.41 67.58 55.59 F1,6=0.09 F1,49=0.29 F4,49=3.19F1,49=0.01 F1,49=0.07 F1,49=0.36
P=0.7791 P=0.5936 P=0.0208P=0.9389 P=0.7992 P=0.5486 [+]
2 ^0.3 67 135.78 139.01 153.35 F1,6=0.01 F1,51=0.30 F4,51=2.09 F1,51=2.15 F1,51=2.35 F1,51=9.10
P=0.9235 P=0.5844 P=0.0952 P=0.1486 P=0.1317 P=0.0040 [–]
3 ^2.3 65 228.42 292.47 223.70 F1,6=3.82 F1,47=5.46F4,47=0.34 F1,47=0.14 F1,47=0.04 F1,47=0.19
P=0.0983 P=0.0237P=0.8487 P=0.7125 P=0.8520 P=0.6626 [–]
4 Log1043 26.13 29.21 56.55 F1,6=0.25 F1,28=4.89F4,28=3.42F1,28=1.19 F1,28=5.60
P=0.6356 P=0.0353P=0.0213P=0.2841 P=0.0251
GLUT-4 gastrocnemius 1 Log1065 15.82 37.34 39.69 F1,6=89.61F1,49=0.66 F4,49=0.25 F1,49=3.28 F1,49=2.09 F1,49=0.00
P<0.0001P=0.4189 P=0.9063 P=0.0761 P=0.1542 P=0.9573 [+]
4 None 41 8.81 11.85 8.90 F1,6=2.31 F1,26=1.34 F4,26=2.73 F1,26=0.26 F1,26=0.00
P=0.1794 P=0.2584 P=0.0506 P=0.6117 P=0.9765
Glycogen synthase total activity 1 Log1063 1.68 1.73 2.33 F1,6=0.06 F1,48=5.89F4,48=0.55 F1,48=0.42 F1,48=20.32
P=0.8118 P=0.0190P=0.7006 P=0.5179 P<0.0001 a
4 Log1042 1.32 1.24 2.30 F1,6=0.33 F1,27=26.43F4,27=2.78F1,27=1.37 F1,27=10.27
P=0.5850 P<0.0001P=0.0468P=0.2519 P=0.0035 a
Glycogen synthase activity ratio 1 Log1063 19.44 20.65 15.90 F1,6=0.45 F1,48=5.34F4,48=0.55 F1,48=1.90 F1,48=2.64
P=0.5269 P=0.0252P=0.7008 P=0.1749 P=0.1110 a
4 Log1042 19.68 21.03 9.80 F1,6=0.37 F1,27=20.74F4,27=0.98 F1,27=0.64 F1,27=0.49
P=0.5665 P=0.0001P=0.4347 P=0.4300 P=0.4919 a
Glycogen synthase fractional velocity 1 Log1063 85.61 85.90 78.72 F1,6=0.01 F1,48=3.55 F4,48=0.10 F1,48=0.60 F1,48=1.28
P=0.9263 P=0.0655 P=0.9808 P=0.4433 P=0.2631 a
4 Log1042 85.35 84.10 76.70 F1,6=0.13 F1,27=2.10 F4,27=1.45 F1,27=0.04 F1,27=0.72
P=0.7328 P=0.1584 P=0.2458 P=0.8422 P=0.4040 a
Trait . Group . Transform . N . Control . HR normal . HR mini . Line type . Mini muscle . Estrous cycle . Age . Time . Revolutions .
Plasma glucose 1 ^1.5 57 10.72 10.40 10.43 F1,6=0.16 F1,41=0.00 F4,41=0.72 F1,41=2.02 F1,41=0.48 F1,41=0.19
P=0.7068 P=0.9663 P=0.5822 P=0.1632 P=0.4926 P=0.6620 [–]
2 ^1.8 65 8.84 9.56 9.37 F1,6=2.40 F1,49=0.38 F4,49=0.81 F1,49=16.23F1,49=4.60F1,49=6.53
P=0.1725 P=0.5424 P=0.5223 P=0.0002P=0.0370P=0.0138 [–]
3 ^1.8 61 8.81 8.91 9.45 F1,6=0.04 F1,45=1.79 F4,45=0.27 F1,45=0.94 F1,45=4.25F1,45=7.60
P=0.8465 P=0.1873 P=0.8987 P=0.3369 P=0.0451P=0.0084 [–]
4 ^0.5 44 9.87 9.51 10.19 F1,6=0.74 F1,29=1.44 F4,29=5.85F1,29=2.03 F1,29=0.01
P=0.4224 P=0.2406 P=0.0014P=0.1651 P=0.9296
Glycogen gastrocnemius 1 ^0.3 65 16.96 12.38 37.52 F1,6=1.70 F1,49=35.03F4,49=1.32 F1,49=0.23 F1,49=0.11 F1,49=2.89
P=0.2396 P<0.0001P=0.2774 P=0.6346 P=0.7450 P=0.0954 [+]
2 ^0.3 67 16.46 16.65 42.48 F1,6=0.00 F1,51=40.74F4,51=2.23 F1,51=0.59 F1,51=4.17F1,51=0.03
P=0.9593 P<0.0001P=0.0785 P=0.4468 P=0.0463P=0.8739 [–]
3 ^0.5 65 19.54 23.28 36.69 F1,6=0.55 F1,47=7.69F4,47=0.26 F1,47=3.42 F1,47=0.10 F1,47=1.45
P=0.4876 P=0.0079P=0.9018 P=0.0708 P=0.7476 P=0.2349 [+]
4 None 45 15.93 10.08 40.98 F1,6=2.05 F1,30=42.99F4,30=0.64 F1,30=2.16 F1,30=0.02
P=0.2025 P<0.0001P=0.6356 P=0.1524 P=0.8973
Glycogen soleus 1 ^0.2 65 21.95 25.83 32.52 F1,6=0.52 F1,49=1.61 F4,49=1.05 F1,49=0.02 F1,49=2.00 F1,49=0.17
P=0.4960 P=0.2101 P=0.3923 P=0.8790 P=0.1631 P=0.6828 [+]
2 ^0.8 67 18.06 20.72 42.02 F1,6=0.30 F1,50=24.00F4,50=3.46F1,50=0.00 F1,50=9.14F1,50=2.47
P=0.6061 P<0.0001P=0.0142P=0.9607 P=0.0039P=0.1226 [–]
3 ^1.3 65 22.57 44.62 50.02 F1,6=10.93F1,47=1.23 F4,47=1.08 F1,47=1.53 F1,47=1.18 F1,47=0.39
P=0.0163P=0.2733 P=0.3766 P=0.2224 P=0.2828 P=0.5335 [–]
4 ^0.2 44 21.83 20.22 32.48 F1,6=0.16 F1,29=4.04 F4,29=2.28 F1,29=1.02 F1,29=0.02
P=0.7013 P=0.0539 P=0.0847 P=0.3216 P=0.8774
Glycogen liver 1 ^0.05 65 76.41 67.58 55.59 F1,6=0.09 F1,49=0.29 F4,49=3.19F1,49=0.01 F1,49=0.07 F1,49=0.36
P=0.7791 P=0.5936 P=0.0208P=0.9389 P=0.7992 P=0.5486 [+]
2 ^0.3 67 135.78 139.01 153.35 F1,6=0.01 F1,51=0.30 F4,51=2.09 F1,51=2.15 F1,51=2.35 F1,51=9.10
P=0.9235 P=0.5844 P=0.0952 P=0.1486 P=0.1317 P=0.0040 [–]
3 ^2.3 65 228.42 292.47 223.70 F1,6=3.82 F1,47=5.46F4,47=0.34 F1,47=0.14 F1,47=0.04 F1,47=0.19
P=0.0983 P=0.0237P=0.8487 P=0.7125 P=0.8520 P=0.6626 [–]
4 Log1043 26.13 29.21 56.55 F1,6=0.25 F1,28=4.89F4,28=3.42F1,28=1.19 F1,28=5.60
P=0.6356 P=0.0353P=0.0213P=0.2841 P=0.0251
GLUT-4 gastrocnemius 1 Log1065 15.82 37.34 39.69 F1,6=89.61F1,49=0.66 F4,49=0.25 F1,49=3.28 F1,49=2.09 F1,49=0.00
P<0.0001P=0.4189 P=0.9063 P=0.0761 P=0.1542 P=0.9573 [+]
4 None 41 8.81 11.85 8.90 F1,6=2.31 F1,26=1.34 F4,26=2.73 F1,26=0.26 F1,26=0.00
P=0.1794 P=0.2584 P=0.0506 P=0.6117 P=0.9765
Glycogen synthase total activity 1 Log1063 1.68 1.73 2.33 F1,6=0.06 F1,48=5.89F4,48=0.55 F1,48=0.42 F1,48=20.32
P=0.8118 P=0.0190P=0.7006 P=0.5179 P<0.0001 a
4 Log1042 1.32 1.24 2.30 F1,6=0.33 F1,27=26.43F4,27=2.78F1,27=1.37 F1,27=10.27
P=0.5850 P<0.0001P=0.0468P=0.2519 P=0.0035 a
Glycogen synthase activity ratio 1 Log1063 19.44 20.65 15.90 F1,6=0.45 F1,48=5.34F4,48=0.55 F1,48=1.90 F1,48=2.64
P=0.5269 P=0.0252P=0.7008 P=0.1749 P=0.1110 a
4 Log1042 19.68 21.03 9.80 F1,6=0.37 F1,27=20.74F4,27=0.98 F1,27=0.64 F1,27=0.49
P=0.5665 P=0.0001P=0.4347 P=0.4300 P=0.4919 a
Glycogen synthase fractional velocity 1 Log1063 85.61 85.90 78.72 F1,6=0.01 F1,48=3.55 F4,48=0.10 F1,48=0.60 F1,48=1.28
P=0.9263 P=0.0655 P=0.9808 P=0.4433 P=0.2631 a
4 Log1042 85.35 84.10 76.70 F1,6=0.13 F1,27=2.10 F4,27=1.45 F1,27=0.04 F1,27=0.72
P=0.7328 P=0.1584 P=0.2458 P=0.8422 P=0.4040 a

Plasma glucose (mmol/l), glycogen (glucosyl units: μmol g –1 wet mass) in gastrocnemius, soleus and liver, and GLUT-4(% of standard) in gastrocnemius of control (C) and high runner (HR) lines of house mice based on separate analyses of Groups 1–4 (nested ANCOVAs). For the effect of wheel revolutions as a covariate, the sign in brackets indicates the direction of the effect. Data were transformed as indicated to improve normality of residuals. Values are back-transformed least-squares means from SAS Procedure Mixed. Bold font indicates 2-tailed P<0.05, unadjusted for multiple comparisons

Amount of time tissue was stored in freezer used as covariate

Nested ANCOVAs comparing Groups 1 (five days wheel access) and 4 (no wheel access)

. Transform . N . Wheel access . Line type . Mini muscle . Wheel access× line type . Wheel access ×mini muscle . Estrous cycle . Age .
Plasma glucose None 101 F1,6=8.31F1,6=1.91 F1,78=0.55 F1,6=0.10 F1,78=0.00 F4,78=1.02 F1,78=4.11
P=0.0280P=0.2163 P=0.4595 P=0.7591 P=0.9901 P=0.4011 P=0.0459
Glycogen gastrocnemius ^0.2 110 F1,6=0.48 F1,6=2.93 F1,87=82.38F1,6=1.06 F1,87=0.10 F4,87=1.41 F1,87=1.73
P=0.5150 P=0.1376 P<0.0001P=0.3439 P=0.7552 P=0.2371 P=0.1915
Glycogen soleus ^0.3 109 F1,6=0.08 F1,6=0.59 F1,86=0.48 F1,6=0.81 F1,86=0.28 F4,86=0.79 F1,86=0.00
P=0.7863 P=0.4698 P=0.4891 P=0.4037 P=0.5962 P=0.5359 P=0.9974
Glycogen liver ^0.1 109 F1,6=3.39 F1,6=0.00 F1,86=0.71 F1,6=0.01 F1,86=0.59 F4,86=1.60 F1,86=0.21
P=0.1150 P=0.9881 P=0.4007 P=0.9197 P=0.4456 P=0.1823 P=0.6485
GLUT-4 gastrocnemius ^0.5 106 F1,6=33.79F1,6=72.17F1,83=0.22 F1,6=34.48F1,83=3.61 F4,83=0.70 F1,83=4.59
P=0.0011P<0.0001P=0.6390 P=0.0011P=0.0610 P=0.5921 P=0.0351
Glycogen synthase activity ratio Log10105 F1,6=4.01 F1,6=0.29 F1,82=25.51F1,6=0.02 F1,82=3.44 F4,82=0.33 F1,86=0.57
P=0.0921 P=0.6085 P<0.0001P=0.8934 P=0.0672 P=0.8572 P=0.4518
Glycogen synthase fractional velocity Log10105 F1,6=0.08 F1,6=0.05 F1,82=4.49F1,6=0.17 F1,82=0.16 F4,82=0.68 F1,86=0.32
P=0.7809 P=0.8365 P=0.0370P=0.6919 P=0.6923 P=0.6055 P=0.5715
. Transform . N . Wheel access . Line type . Mini muscle . Wheel access× line type . Wheel access ×mini muscle . Estrous cycle . Age .
Plasma glucose None 101 F1,6=8.31F1,6=1.91 F1,78=0.55 F1,6=0.10 F1,78=0.00 F4,78=1.02 F1,78=4.11
P=0.0280P=0.2163 P=0.4595 P=0.7591 P=0.9901 P=0.4011 P=0.0459
Glycogen gastrocnemius ^0.2 110 F1,6=0.48 F1,6=2.93 F1,87=82.38F1,6=1.06 F1,87=0.10 F4,87=1.41 F1,87=1.73
P=0.5150 P=0.1376 P<0.0001P=0.3439 P=0.7552 P=0.2371 P=0.1915
Glycogen soleus ^0.3 109 F1,6=0.08 F1,6=0.59 F1,86=0.48 F1,6=0.81 F1,86=0.28 F4,86=0.79 F1,86=0.00
P=0.7863 P=0.4698 P=0.4891 P=0.4037 P=0.5962 P=0.5359 P=0.9974
Glycogen liver ^0.1 109 F1,6=3.39 F1,6=0.00 F1,86=0.71 F1,6=0.01 F1,86=0.59 F4,86=1.60 F1,86=0.21
P=0.1150 P=0.9881 P=0.4007 P=0.9197 P=0.4456 P=0.1823 P=0.6485
GLUT-4 gastrocnemius ^0.5 106 F1,6=33.79F1,6=72.17F1,83=0.22 F1,6=34.48F1,83=3.61 F4,83=0.70 F1,83=4.59
P=0.0011P<0.0001P=0.6390 P=0.0011P=0.0610 P=0.5921 P=0.0351
Glycogen synthase activity ratio Log10105 F1,6=4.01 F1,6=0.29 F1,82=25.51F1,6=0.02 F1,82=3.44 F4,82=0.33 F1,86=0.57
P=0.0921 P=0.6085 P<0.0001P=0.8934 P=0.0672 P=0.8572 P=0.4518
Glycogen synthase fractional velocity Log10105 F1,6=0.08 F1,6=0.05 F1,82=4.49F1,6=0.17 F1,82=0.16 F4,82=0.68 F1,86=0.32
P=0.7809 P=0.8365 P=0.0370P=0.6919 P=0.6923 P=0.6055 P=0.5715

Data were transformed as indicated to improve normality of residuals. Bold font indicates 2-tailed P<0.05, unadjusted for multiple comparisons

Gastrocnemius glycogen synthase activity

Glycogen synthase total activity was significantly increased, whereas the activity ratio was significantly reduced in mini-muscle gastrocnemius, in comparison with both HR normal and C mice, but was not generally affected by line type or wheel access (Tables 2, 3). Because freezer storage time significantly affected total activity within both groups(Table 2), and time was confounded with group membership, groups 1 and 4 were not compared statistically (Table 3). Inspection of the least-squares means shows that, uniquely, HR mini exhibited a 60% increase in activity ratio following five days of wheel access (from 9.80 to 15.90 Tables 2, 3). Glycogen synthase fractional velocity was also significantly reduced in HR mini(Table 3).

Glycogen concentration

As shown in Table 2, the mini-muscle phenotype was associated with higher glycogen concentration in gastrocnemius, soleus and liver in mice without wheel access (Group 4). When mice had access to wheels for 5–6 days, HR mini also showed significantly elevated gastrocnemius glycogen at all three time points (Tables 2, 3, 4). Although HR mini also showed elevated soleus glycogen at all time points, the effect was statistically significantly only at 02:00 h (Group 2), when values for HR mini were approximately twice those of other mice. HR also had significantly higher soleus glycogen concentration at 07:00 h (Group 3 Table 2). In contrast to results for muscles, liver glycogen concentration was significantly lower in HR mini than HR normal at 07:00 h (Group 3 Table 2). Finally, the number of wheel revolutions was negatively related to glycogen concentration in liver at 02:00 h (Group 2 see Fig. 2), and this was the only significant relationship between[glycogen] and the amount of running (Table 2).

Nested ANCOVAs comparing Groups 1, 2 and 3 sacrificed at different times on the sixth day of wheel access

. Transform . N . Group . Line type . Mini muscle . Group×line type . Group×mini muscle . Estrous cycle . Age .
Plasma glucose None 186 F2,12=7.10F1,6=3.27 F1,154=0.01 F2,12=1.1 F2,154=2.37 F4,154=085 F1,154=6.12
P=0.0092P=0.1203 P=0.9196 P=0.3625 P=0.0969 P=0.4972 P=0.0145
Glycogen gastrocnemius ^0.4 197 F2,12=0.13 F1,6=1.24 F1,165=92.39F2,12=0.98 F2,165=3.04 F4,165=0.88 F1,165=0.01
P=0.8761 P=0.3084 P<0.0001P=0.4045 P=0.0503 P=0.4754 P=0.9140
Glycogen soleus ^0.6 196 F2,12=1.27 F1,6=6.53F1,164=4.06F2,12=4.63F2,164=3.58F4,164=3.20F1,164=0.03
P=0.3170 P=0.0431P=0.0456P=0.0324P=0.0302P=0.0146P=0.8708
Glycogen liver ^0.5 197 F2,12=11.06F1,6=0.68 F1,165=1.25 F2,12=3.28 F2,165=0.12 F4,165=4.00F1,165=3.88
P=0.0019P=0.4422 P=0.2659 P=0.0729 P=0.8854 P=0.0040P=0.0505
. Transform . N . Group . Line type . Mini muscle . Group×line type . Group×mini muscle . Estrous cycle . Age .
Plasma glucose None 186 F2,12=7.10F1,6=3.27 F1,154=0.01 F2,12=1.1 F2,154=2.37 F4,154=085 F1,154=6.12
P=0.0092P=0.1203 P=0.9196 P=0.3625 P=0.0969 P=0.4972 P=0.0145
Glycogen gastrocnemius ^0.4 197 F2,12=0.13 F1,6=1.24 F1,165=92.39F2,12=0.98 F2,165=3.04 F4,165=0.88 F1,165=0.01
P=0.8761 P=0.3084 P<0.0001P=0.4045 P=0.0503 P=0.4754 P=0.9140
Glycogen soleus ^0.6 196 F2,12=1.27 F1,6=6.53F1,164=4.06F2,12=4.63F2,164=3.58F4,164=3.20F1,164=0.03
P=0.3170 P=0.0431P=0.0456P=0.0324P=0.0302P=0.0146P=0.8708
Glycogen liver ^0.5 197 F2,12=11.06F1,6=0.68 F1,165=1.25 F2,12=3.28 F2,165=0.12 F4,165=4.00F1,165=3.88
P=0.0019P=0.4422 P=0.2659 P=0.0729 P=0.8854 P=0.0040P=0.0505

Data were transformed as indicated to improve normality of residuals. Bold font indicates 2-tailed P<0.05, unadjusted for multiple comparisons

Analyses comparing Groups 1 and 4 failed to show an effect of wheel access on [glycogen] in any of the organs measured(Table 3), despite the fact that liver glycogen in normal mice (i.e. not mini) with wheel access was on average higher than in mice without wheels, regardless of selection history(2.92 and 2.31 times higher for C and HR normal, respectively Table 2).

Combined analyses of all mice with wheel access for 5–6 days (Groups 1, 2 and 3) showed that [glycogen] was affected in different ways by time of measurement, depending on the tissue (Table 4). Liver [glycogen] was significantly affected by time of measurement (Table 4), as all mice showed a significant increase in its mean value from 16:00 h to 02:00 h and from 02:00 h to 07:00 h (Table 2). In soleus, while HR mini showed an increase in [glycogen] from both 16:00 h to 02:00 h (29.2%) and 02:00 h to 07:00 h (19.0%), C and HR normal showed a decrease from 16:00 h to 02:00 h (17.7% and 19.8% lower values at 02:00 h for C and HR normal, respectively) followed by a rebound from 02:00 h to 07:00 h (25.0% and 115.4% higher values at 07:00 h for C and HR normal,respectively Table 2). Such differences in pattern and magnitude of variation of soleus [glycogen] with time of measurement were reflected in the significant interaction between time and both line type and mini-muscle factors(Table 4). In comparison with C mice, HR mice in general had higher soleus [glycogen] for Group 3 (Tables 2, 4).

Control, HR normal and HR mini also differed in the way that gastrocnemius[glycogen] varied with measurement time. For example, HR mini showed an increase from 16:00 h to 02:00 h (13.2%), followed by a similar decrease from 02:00 h to 07:00 h (13.6%) (Tables 2, 4). By contrast, C mice showed essentially no change from 16:00 h to 02:00 h (+3.0%), but an increase from 02:00 h to 07:00 h (18.7%), whereas mean values for HR normal increased from both 16:00 h to 02:00 h (34.5%) and 02:00 h to 07:00 h (39.8% Table 2).

GLUT-4 abundance in gastrocnemius muscle was much higher in the selectively bred `high runner' (HR) lines of mice than in non-selected control (C) lines after five days of wheel access (Group 1, as outlined in Table 1). Clearly, this differential was not a simple linear function of the amount of wheel running performed by individual mice on the previous night(Table 2, P=0.9573 for effect of wheel revolutions). As discussed in the text, this seems to represent a case of increased `self-induced adaptive plasticity' in the HR lines of mice (Swallow et al.,2005 Garland and Kelly,2006). A representative western blot for GLUT-4 from six individuals is also shown.

GLUT-4 abundance in gastrocnemius muscle was much higher in the selectively bred `high runner' (HR) lines of mice than in non-selected control (C) lines after five days of wheel access (Group 1, as outlined in Table 1). Clearly, this differential was not a simple linear function of the amount of wheel running performed by individual mice on the previous night(Table 2, P=0.9573 for effect of wheel revolutions). As discussed in the text, this seems to represent a case of increased `self-induced adaptive plasticity' in the HR lines of mice (Swallow et al.,2005 Garland and Kelly,2006). A representative western blot for GLUT-4 from six individuals is also shown.

Liver glycogen concentration showed a significant (P=0.0040)negative relation with amount of wheel revolutions in the 02:00 h group (Group 2, as outlined in Table 1 see Table 2 for statistical analysis), but no differences among control (C), `high runner' (HR) normal(`selected') or HR mini-muscle mice.

Liver glycogen concentration showed a significant (P=0.0040)negative relation with amount of wheel revolutions in the 02:00 h group (Group 2, as outlined in Table 1 see Table 2 for statistical analysis), but no differences among control (C), `high runner' (HR) normal(`selected') or HR mini-muscle mice.

Additionally, [glycogen] was influenced by the estrous cycle in both soleus and liver (Table 4). On average, values in estrous 2 were lowest in liver: values during postestrous,estrous 1, diestrous and proestrous were 13%, 15%, 27% and 46% higher,respectively. In soleus, females during postestrous had the lowest glycogen concentrations, with values for estrous 2, estrous 1, proestrous and diestrous being 2%, 15%, 17% and 37% higher, respectively.

Plasma glucose

Plasma glucose concentration was not affected by line type or mini-muscle phenotype in any group (Table 2). In mice with wheel access for 5 to 6 days, [glucose] was significantly negatively related to the number of revolutions for mice sampled at both 02:00 h and 07:00 h (Groups 2 and 3, respectively).

Comparison of groups 1 and 4 showed a significant increase in plasma[glucose] when mice were housed with access to wheels(Table 3). Mean values for Group 1 were 8.6%, 9.4% and 2.4% higher for C, HR normal and HR mini from Group 4, respectively (Table 2).

Analysis comparing the three groups of mice that had wheel access showed a significant effect of time of measurement on plasma glucose concentration in addition to the positive age influence(Table 4). All mice showed a decrease from 16:00 h to 02:00 h (17.5%, 8.1% and 10.2% for C, HR normal and HR mini, respectively Table 2). Plasma glucose concentration showed little change from 02:00 h to 07:00 h for C and HR mini, but HR normal showed a mean value 6.8% lower at 07:00 h (Table 2).


Metabolic Processes Controlled by Allosteric Enzymes (With Diagram)

An excellent example of allosteric enzyme regulation of metabolic processes is provided by the interrela­tionship in animals between the metabolic pathways that result in:

(1) The synthesis of glycogen from glu­cose and

(2) The oxidation of glucose to CO2 and water.

Nearly all of the energy-consuming processes in the body proceed at the expense of ATP and much of this ATP is derived through the oxidation of glucose. Dur­ing periods of elevated activity (e.g., exercise), glyco­gen is broken down to yield glucose, which then enters the metabolic pathway converting it to CO2 and water, with consequent generation of ATP. In contrast, dur­ing periods of rest or low energy demand, absorbed glucose is converted to glycogen.

Three of the en­zymes involved in glucose metabolism are allosteric these are phosphofructokinase (an enzyme required in the series of reactions that convert glucose-6-phosphate to CO2 and water), glycogen synthetase (involved in the incorporation of glucose-l-phosphate into glyco­gen), and glycogen phosphorylase (which removes glu­cose as glucose-l-phosphate from glycogen during glycogen catabolism).

When ATP levels are high and no major consump­tion of energy is taking place in the body, glucose is diverted into glycogen (i.e., “glycogenesis” predomi­nates). This is achieved because ATP acts as a nega­tive effector of phosphofructokinase and glycogen phosphorylase and as a positive effector, along with glucose-6-phosphate, of glycogen synthetase (Fig. 11-8a).

When the ATP level falls (e.g., during exercise) and there is an increased demand for ATP, glycogen syn­thesis is halted as absorbed glucose is directly con­sumed in the production of ATP and additional glu­cose is made available through the catabolism of glycogen (i.e., “glycogenolysis”). This pathway is acti­vated by the positive effects on phosphofructokinase and glycogen phosphorylase of the ATP precursor, AMP.

The hormone epinephrine, secreted into the blood­stream during periods of great activity, also has an ef­fect on these metabolic pathways in muscle and in liver. When epinephrine in the bloodstream reaches the muscles, it binds to the surface of the muscle cells and promotes the synthesis of cyclic AMP (cAMP) by the enzyme adeny Icy close.

The cAMP then allosterically activates a second enzyme (protein kinase), which ultimately activates glycogen phosphorylase but inactivates glycogen synthetase (Fig. 11-8b). This phe­nomenon is also considered with the functions of hormones and the role of protein phosphorylation as a metabolic regulatory mechanism.

The pathways described above illustrate the mecha­nisms for turning allosteric enzymes on and off. In the absence of such mechanisms, both pathways would simultaneously be active so that their effects cancel one another—a most unproductive state! Allosterism thus provides a basis for regulating the levels of activ­ity of related metabolic pathways.

The Regulation of Amino Acid Synthesis:

Escherichia coli provide a clear example of control of divergent metabolic pathways by feedback inhibition. An outline of the metabolic pathways for the synthesis of three amino acids is shown in Figure 11-9. Lysine, methionine, and threonine are each synthesized from aspartate and each may be utilized in protein synthe­sis.

Without metabolic controls, the consumption or utilization of any one of these amino acids would stim­ulate the pathways and cause unneeded synthesis of the unused amino acids as well as the one utilized. Such an unregulated system would consume vital re­sources and energy both factors could have survival implications to the organism and evolutionary conse­quences to the species.

However, in E. coli, the allo­steric regulatory mechanisms are most effective. The accumulation of each amino acid produces a feedback inhibition of the first enzyme in the specific branch of the pathway leading to the synthesis of that amino acid. In Figure 11-9, this negative effect is shown by dashed lines.

Moreover, an additional level of regula­tion is achieved through effects on the enzyme aspartokinase, which catalyzes and phosphorylation of aspartate. This enzyme exists in three forms (i.e., there are three isozymes), symbolized in Figure 11-9 by using three separate arrows to show the conversion of aspartate to aspartylphosphate.

One of the isozy­mes is specifically and completely inhibited by threonine the second (which is present only in small amounts) is specifically inhibited by homoserine and the third isozyme is specifically inhibited by lysine. In addition, synthesis of the latter isozyme is repressed by lysine. (Repression is a regulatory mechanism that reduces the number of enzyme molecules in the cell.


INTRODUCTION

Muscle glycogen has been recognized as an important fuel during prolonged exercise since the early studies by Bergström et al ( 1– 3). The reliance on muscle glycogen increases with increasing exercise intensity and a direct relation between fatigue and depletion of muscle glycogen stores has been described ( 1– 3). Therefore, the postexercise glycogen synthesis rate is an important factor in determining the time needed to recover. Glycogen synthesis is affected not only by the extent of glycogen depletion but also in a more direct manner by the type, duration, and intensity of the preceding exercise because these will differentially influence the acute enzymatic changes as well as recovery from the acute changes that are induced by strenuous exercise ( 4– 6).

To optimize glycogen synthesis rates, adequate amounts of carbohydrate should be ingested ( 1, 7, 8). Blom et al ( 9) suggested initially that a carbohydrate intake of 0.35 g•kg body wt −1 •h −1 , provided at 2-h intervals, maximized muscle glycogen synthesis. Ivy et al ( 7) observed no differences in glycogen storage rates after subjects ingested 0.75 or 1.5 g carbohydrate•kg −1 •h −1 provided at 2-h intervals. In a follow-up study, Ivy ( 10) reported that an intake of >0.5 g•kg −1 •h −1 is necessary to maximize postexercise glycogen synthesis if supplements are administered at 2-h intervals. Higher glycogen synthesis rates have been reported in studies in which carbohydrates were ingested more frequently and at higher ingestion rates than in Ivy's study ( 4, 11). Other efforts to increase glycogen synthesis rates by changing the form of administration (ie, as a solution, as a solid, or intravenously) have been unsuccessful ( 8, 12).

Zawadzki et al ( 13) reported that addition of an intact protein to a carbohydrate-containing solution resulted in higher glycogen synthesis rates in subjects after exercise than did ingestion of carbohydrate only at a rate of 0.8 g•kg −1 •h −1 . This was explained by the observed additional increase in plasma insulin concentrations after ingestion of the carbohydrate-protein mixture. Elevated insulin concentrations may lead to increased glucose uptake ( 14) and to an increase in glycogen synthase activity ( 15, 16), which forms the major factor in determining the rate of glycogen synthesis when the substrate supply is adequate ( 13, 17). The stimulating effect of the combined ingestion of carbohydrate and protein on plasma insulin concentrations was investigated previously ( 18– 21) and is described in detail in an accompanying article ( 22). We showed that ingestion of a mixture of a wheat hydrolysate, free leucine, and free phenylalanine in combination with a carbohydrate drink leads to a substantial increase in plasma insulin in healthy subjects after an overnight fast ( 22).

The first aim of this study was to investigate whether, and to what extent, the ingestion of this highly insulinotropic protein hydrolysate and amino acid mixture in combination with carbohydrate (0.8 g•kg −1 •h −1 ) can accelerate postexercise muscle glycogen synthesis. The second aim was to determine whether glycogen synthesis rates can also be elevated by increasing carbohydrate intake. The ingestion rates chosen were based on those used by Zawadzki et al ( 13). However, we decided to provide the supplements at 30-min intervals because more frequent carbohydrate ingestion could result in higher glycogen synthesis rates ( 4, 11). To realize these aims, 8 highly trained cyclists performed a glycogen-depletion test on 3 occasions, after which beverages containing carbohydrate (0.8 g•kg −1 •h −1 ), carbohydrate and an amino acid and protein hydrolysate mixture (0.8 and 0.4 g•kg −1 •h −1 , respectively), or an isoenergetic amount of carbohydrate (1.2 g•kg −1 •h −1 ) were ingested during a 5-h period. Plasma insulin and glucose concentrations were measured and muscle biopsies were taken immediately postexercise and 5 h later to determine glycogen synthase activity and muscle glycogen content.


Gluconeogenesis vs glycolysis - key enzymes

Gluconeogenesis steps

The pathway for gluconeogenesis utilizes many, but not all, of the enzymes of glycolysis.

The reactions that are common to glycolysis and gluconeogenesis are the reversible reactions.

Two of these irreversible steps are the two ATP-requiring activation reactions of glycolysis catalyzed by glucokinase and phosphofructokinase-1. They are bypassed by glucose 6-phosphatase and fructose 1,6-bisphosphatase, respectively.

The third irreversible step of glycolysis is the second ATP-generating reaction, which is catalyzed by pyruvate kinase.

The gluconeogenesis pathway utilizes the reactions catalyzed by pyruvate carboxylase and phosphoenolpyruvate carboxykinase to bypass the irreversible pyruvate kinase reaction of glycolysis.

Video - Gluconeogenesis - Biochemistry


Fasted state metabolism

Approximately 2 hours after a meal, the decrease in serum glucose levels will lead to decreased insulin production in the pancreas. At this point in fasted state metabolism, the insulin to glucagon ratio becomes <1 (insulin low glucagon high) with an additional increase of cortisol and epinephrine. Under these conditions tissues will transition to utilizing alternative fuels for energy as a means of maintaining glucose homeostasis. Fasted state metabolism will have limited impact on the oxidation of glucose by the brain and red blood cells but it will lead to an increase in fatty acid oxidation by both the skeletal muscle and the liver (Figure 3.6). The fatty acids oxidized by these tissues are released through the process of epinephrine mediated lipolysis from the adipose. In the fasted state, the liver will primarily release glucose using both gluconeogenesis and glycogenolysis for the maintenance of blood glucose.

TISSUE TYPE FUEL UTILIZED IN THE FASTED STATE PATHWAY PROVIDING THE FUEL
Liver Fatty acids Lipolysis in the adipose
Red blood cells Glucose Hepatic glycogenolysis and gluconeogenesis
Brain Glucose Hepatic glycogenolysis and gluconeogenesis
Skeletal muscle Fatty acids Lipolysis in the adipose
Table 3.3 Summary table of fuels used in the fasted state and the pathways providing the fuel source. (Kindred Gray 2020 via LibreTexts CC BY 4.0)

Liver metabolism

The primary role of the liver in the fasted state is to synthesize and release glucose. To facilitate this task, the liver, will use circulating free fatty acids as the primary fuel source to generate energy (ATP) for these homeostatic processes. (These processes are summarized in Figure 3.2 and Tables 3.3 and 3.4)

  1. Glycogenolysis. Hepatic glycogenolysis provides glucose that is released into the blood stream to maintain blood glucose and provide an oxidizable substrate for the brain and RBCs (Section 4.5).
  2. Gluconeogenesis. This is an anabolic process that synthesizes glucose from lactate, amino acids and/or glycerol. This process is heavily reliant on the ATP generated from (eta)- oxidation. The glucose produced is released into the blood stream to maintain blood glucose and provide an oxidizable substrate for the brain and RBCs (Section 5.1).
  3. Fatty acid (eta)-oxidation. This is the process by which free fatty acids are oxidized to produce acetyl-CoA, NADH and FADH(_2). It is a high energy yielding process and is required to generate ATP in the fasted state (Section 5.2).
  4. Ketogenesis. This process utilizes the acetyl-CoA produced through (eta)-oxidation to produce (eta)-hydroxybutyrate and acetoacetate. These ketone bodies can be oxidized by peripheral tissues the liver can not oxidize ketone bodies (Section 5.2).
  5. Urea cycle. Cortisol initiated protein catabolism provides amino acids needed as a substrate for gluconeogenesis. In order to use the carbon skeletons (keto-acids) the amino acids must be deaminated and the ammonia is disposed of through the synthesis of urea nitrogen will enter the urea cycle as aspartate or free ammonia (Section 5.3).

Red blood cell metabolism

The red blood cell lacks mitochondria, therefore it oxidizes glucose under both fed and fasted conditions. The metabolism of this tissue remains largely unchanged.

Brain metabolism

The brain will oxidize glucose under most conditions with the exception of starvation states. Under normal fasting conditions, although ketones will be synthesized the brain will not transition to utilizing them as a predominant source of fuel until extended fasting has occurred (days).

Skeletal muscle metabolism

The skeletal muscle will increase uptake of fatty acids and ketones.

  1. Fatty acid (eta)-oxidation. This is the process by which free fatty acids are oxidized to produce acetyl-CoA, NADH and FADH(_2) (Section 5.2).
  2. Oxidation of ketones. Ketone bodies taken up by the skeletal muscle can be reconverted to acetyl-CoA and oxidized in the TCA cycle (Section 5.2).
  3. Protein catabolism. Cortisol mediated protein catabolism is also active and supplying amino acids for gluconeogenesis in the liver.

Adipose metabolism

The most important process in the adipose during the fasted state is lipolysis.

  1. Lipolysis. This process will release fatty acids from stored triacylglycerols and provides an oxidizable substrate for the skeletal muscle and liver (Section 5.2).

Fructose 1,6 biphosphatase

Driven by substrate availability

Table 3.4: Summary of metabolism during the fasted state. (Kindred Gray 2020 via LibreTexts CC BY 4.0)

Figure 3.2: Overview of fasted state metabolism. (Kindred Gray 2020 via LibreTexts CC BY 4.0 Adapted from Pelley&rsquos Rapid Reviews)



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