Lactate Contributes to Glyceroneogenesis and Glyconeogenesis in Skeletal Muscle by Reversal of Pyruvate Kinase

Abstract

Phosphoenolpyruvate (PEP) generated from pyruvate is required for de novo synthesis of glycerol and glycogen in skeletal muscle. One possible pathway involves synthesis of PEP from the citric acid cycle intermediates via PEP carboxykinase, whereas another could involve reversal of pyruvate kinase (PK). Earlier studies have reported that reverse flux through PK can contribute carbon precursors for glycogen synthesis in muscle, but the physiological importance of this pathway remains uncertain especially in the setting of high plasma glucose. In addition, although PEP is a common intermediate for both glyconeogenesis and glyceroneogenesis, the importance of reverse PK in de novo glycerol synthesis has not been examined. Here we studied the contribution of reverse PK to synthesis of glycogen and the glycerol moiety of acylglycerols in skeletal muscle of animals with high plasma glucose. Rats received a single intraperitoneal bolus of glucose, glycerol, and lactate under a fed or fasted state. Only one of the three substrates was (13)C-labeled in each experiment. After 3 h of normal awake activity, the animals were sacrificed, and the contribution from each substrate to glycogen and the glycerol moiety of acylglycerols was evaluated. The fraction of (13)C labeling in glycogen and the glycerol moiety exceeded the possible contribution from either plasma glucose or muscle oxaloacetate. The reverse PK served as a common route for both glyconeogenesis and glyceroneogenesis in the skeletal muscle of rats with high plasma glucose. The activity of pyruvate carboxylase was low in muscle, and no PEP carboxykinase activity was detected.

Keywords: citric acid cycle; glycerol; glycobiology; glycogen synthase; phosphoenolpyruvate carboxykinase; pyruvate carboxylase (PC); pyruvate dehydrogenase complex (PDC); pyruvate kinase; skeletal muscle metabolism.

FIGURE 1.

Excess [1,2,3-¹³C₃] labeling in muscle glycerol and glycogen compared with plasma glucose. All the ¹³C NMR spectra (of glucose from plasma, the glucosyl units of glycogen from muscle, the glycerol moiety of acylglycerols from muscle, and lactate from muscle) are from a fasted rat given [U-¹³C₃]lactate plus glucose and glycerol. The doublets (¹³C-¹³C) were dominant in C2 and C5 regions of plasma glucose, whereas the quartet (¹³C-¹³C-¹³C) was dominant in C2 of muscle lactate. The labeling pattern in glycogen was intermediate between plasma glucose and muscle lactate, and the quartets were the major signals in C2 and C5 regions of the glucosyl units of glycogen. In the glycerol moiety C2 region of acylglycerols, the signal from triplet (¹³C-¹³C-¹³C) was 2-fold that of the doublet. Insets: D12, doublet from coupling of C1 with C2; D23, doublet from coupling of C2 with C3; D45, doublet from coupling of C4 with C5; D56, doublet from coupling of C5 with C6; D, doublet from coupling of adjacent carbon; Q, doublet of doublets, or quartet, arising from coupling of C2 with both C1 and C3 or from coupling of C5 with both C4 and C6; T, triplet arising from coupling of C2 with C1 and C3; S, singlet; open circle = ¹²C; filled circle = ¹³C.

FIGURE 2.

¹³C labeling in glutamate and oxaloacetate after the entry of [U-¹³C₃]pyruvate to the citric acid cycle. Rats receiving a mixture of [U-¹³C₆]glucose, glycerol, and lactate showed dominant [U-¹³C₃]lactate in skeletal muscle among ¹³C-labeled lactate isotopomers after 3 h of the mixture administration (A). [U-¹³C₃]Pyruvate entry to the citric acid cycle through PDH led to [4,5-¹³C₂]α-ketoglutarate (α-kg) and consequently [4,5-¹³C₂]glutamate. After decarboxylation, the α-ketoglutarate was converted to [1,2-¹³C₂]- and [3,4-¹³C₂]fumarate (fum; a symmetric molecule), which was in exchange with oxaloacetate (OAA) and then aspartate (Asp) producing [1,2-¹³C₂]- and [3,4-¹³C₂]aspartate. The condensation of [1,2-¹³C₂]- and [3,4-¹³C₂]oxaloacetate with [1,2-¹³C₂]acetyl-CoA and subsequent one turn through the citric acid cycle may produce [1,2,3-¹³C₃]- and [2,3,4-¹³C₃]aspartate (B). In contrast, [U-¹³C₃]pyruvate entry through pyruvate carboxylase (PC) produced [1,2,3-¹³C₃]oxaloacetate first and then [2,3,4-¹³C₃]oxaloacetate after back-scrambling with fumarate, which became [2,3-¹³C₂]- and [1,2,3-¹³C₃]glutamate, respectively, through the forward turn of the citric acid cycle (C). Because oxaloacetate C2-C4 are equivalent with α-ketoglutarate C1-C3 in a reverse order, the same ¹³C labeling pattern is expected between aspartate C3 and glutamate C2 (D). In panel D the resonances from glutamate and aspartate were from the skeletal muscle of a fed rat received [U-¹³C₆]glucose, glycerol, and lactate. Signals from glutamate were stronger than those from aspartate; they are not on the same scale. Open circle = ¹²C; filled circle = ¹³C; gray circle = ¹²C or ¹³C.

FIGURE 3.

¹³C enrichment in muscle glycogen and evidence for glyconeogenesis from [U-¹³C₃]lactate through reversal of PK. Fed or fasted rats received a mixture of glucose (2 g/kg), glycerol (0.5 g/kg), and lactate (0.5 g/kg), but only one substrate was enriched with ¹³C in each experiment. 3 h after the mixture administration, ¹³C enrichment in muscle glycogen was higher in rats given [U-¹³C₆]glucose than rats given [U-¹³C₃]glycerol or [U-¹³C₃]lactate (A). Excess ¹³C in glycogen from rats given [U-¹³C₃]lactate plus glucose and glycerol was mainly from glyconeogenesis from ¹³C-labeled lactate through reversal of PK. The contribution from ¹³C-labeled plasma glucose was minor (B). NS, not significant. n = 4–7 in each group.

FIGURE 4.

Evidence for glyconeogenesis from [U-¹³C₃]lactate through reversal of PK. ¹³C NMR spectra of plasma glucose, muscle glycogen, and muscle lactate are from a fed rat given [U-¹³C₃]lactate plus glucose and glycerol. Quartets (¹³C-¹³C-¹³C) are dominant in muscle lactate and glycogen resonances, whereas doublets (¹³C-¹³C) are prominent in plasma glucose. This demonstrates that [1,2,3-¹³C₃] in lactate was preserved in glycogen, which was possible via PK reversal. An alternative pathway through oxaloacetate (pyruvate → oxaloacetate → PEP) cannot keep the intact ¹³C distribution in 3-units due to extensive ¹³C scrambling in the citric acid cycle intermediates including oxaloacetate. DHAP, dihydroxyacetone phosphate; GA3P, d-glyceraldehyde 3-phosphate.

FIGURE 5.

¹³C enrichment in the glycerol moiety of muscle acylglycerols and evidence for glyceroneogenesis from [U-¹³C₃]lactate through reversal of PK. In rats given a mixture of glucose (2 g/kg), glycerol (0.5 g/kg), and lactate (0.5 g/kg), glucose was the major source for the glycerol moiety of acylglycerols in muscle followed by free glycerol and lactate (A). Noticeably, ¹³C enrichment in the glycerol moiety from free [U-¹³C₃]glycerol reached half that from [U-¹³C₆]glucose. In rats given [U-¹³C₃]lactate plus glucose and glycerol, ¹³C in the glycerol moiety originated dominantly through reversal of PK rather than glycolysis (B). NS, not significant. n = 7–8 in each group.

FIGURE 6.

Evidence for glycerol kinase activity in skeletal muscle. In situ hind limb perfusion was performed using perfusate containing 0.2 mm [U-¹³C₃]glycerol. The glycerol moiety of acylglycerols from muscle had 0.14% [U-¹³C₃]glycerol, and lactate released from hind limbs had 0.34% [U-¹³C₃]lactate. The excess enrichments in these metabolites were not possible without [U-¹³C₃]glycerol phosphorylation via glycerol kinase (GK) in skeletal muscle. DHAP, dihydroxyacetone phosphate; GA3P, d-glyceraldehyde 3-phosphate; G3P, glycerol 3-phosphate.

FIGURE 7.

¹³C NMR analysis of muscle glutamate and aspartate: evidence for PDH activity and low pyruvate carboxylase activity. NMR spectra of glutamate and aspartate were derived from skeletal muscle of a fed rat (A) or a fasted rat (B) receiving a mixture of [U-¹³C₆]glucose, glycerol, and lactate. The signal from [4,5-¹³C₂]glutamate indicates active PDH flux, which was dominant in fed animals and strong in fasted animals. The signals from [1,2-¹³C₂]glutamate, [1,2-¹³C₂]aspartate, and [3,4-¹³C₂]aspartate demonstrate forward flux of [4,5-¹³C₂]α-ketoglutarate (in exchange with [4,5-¹³C₂]glutamate) through the citric acid cycle. The equivalent labeling pattern between aspartate C2 and C3 indicates a rapid exchange of aspartate with oxaloacetate that is also in exchange with fumarate, a symmetric molecule of the citric acid cycle. The small peaks from [1,2,3-¹³C₃]- and [2,3,4-¹³C₃]aspartate could be produced through (i) PHD flux followed by forward turns of the citric acid cycle or (ii) [U-¹³C₃]pyruvate carboxylation followed by oxaloacetate exchange with fumarate. Because fluxes through PDH and the citric acid cycle are obvious in skeletal muscle, the small signals from [1,2,3-¹³C₃]- and [2,3,4-¹³C₃]aspartate are most likely due to PDH flux followed by forward turns of the citric acid cycle. Fed animals show an equivalent labeling pattern among glutamate C2, aspartate C2, and aspartate C3. In contrast, the fraction from [2,3-¹³C₂]glutamate is slightly increased than the fraction from [2,3-¹³C₂]aspartate in fasted animals. Thus the ratio of D23/D12 in glutamate was higher than the corresponding ratio of D23/D34 or D23/D12 in aspartate from fasted animals (C). This small increase could be due to [U-¹³C₃]pyruvate carboxylation to [1,2,3-¹³C₃]oxaloacetate and immediate forward flux through the citric acid cycle without exchanging with aspartate ([U-¹³C₃]pyruvate → [1,2,3-¹³C₃]oxaloacetate → [2,3,6-¹³C₃]citrate→ [2,3-¹³C₂]α-ketoglutarate →[2,3-¹³C₂]glutamate). Open circle = ¹²C; filled circle = ¹³C; gray circle = ¹²C or ¹³C; NS, not significant. n = 3–6 spectra in aspartate or glutamate analysis from each group.