The Source of Glycolytic Intermediates in Mammalian Tissues

Abstract

Glycolysis plays a central role in organismal metabolism, but its quantitative inputs across mammalian tissues remain unclear. Here we use ¹³C-tracing in mice to quantify glycolytic intermediate sources: circulating glucose, intra-tissue glycogen, and circulating gluconeogenic precursors. Circulating glucose is the main source of circulating lactate, the primary end product of tissue glycolysis. Yet circulating glucose highly labels glycolytic intermediates in only a few tissues: blood, spleen, diaphragm, and soleus muscle. Most glycolytic intermediates in the bulk of body tissue, including liver and quadriceps muscle, come instead from glycogen. Gluconeogenesis contributes less but also broadly to glycolytic intermediates, and its flux persists with physiologic feeding (but not hyperinsulinemic clamp). Instead of suppressing gluconeogenesis, feeding activates oxidation of circulating glucose and lactate to maintain glucose homeostasis. Thus, the bulk of the body slowly breaks down internally stored glycogen while select tissues rapidly catabolize circulating glucose to lactate for oxidation throughout the body.

Keywords: compartmentalized metabolism; glucose homeostasis; glycogen; glycolysis; glycolytic intermediates; glycolytic specialist; isotope tracing; metabolic heterogeneity; red muscle.

Figure 1.. Circulating glucose supplies only a fraction of glycolytic intermediates.

(A) Schematic of experimental procedures. [U-¹³C]glucose is infused into jugular vein catheterized mice. At the end of the infusion, serum and tissue are collected for LC-MS analysis. (B) Schematic of glycolysis showing hexose phosphate (hp), fructose-1,6-bisphosphate (fbp), and 3-phosphoglycerate (3pg). (C) Serum glucose and lactate labeling from [U-¹³C]glucose infusion (n=5 mice). Data is fit with a single exponential. (D) Normalized labeling of circulating lactate when sampled from the tail (n=10) versus from a carotid artery catheter (n=16). P-value calculated by unpaired t-test; **p=0.0021. (E) Labeling in arterial serum glucose, arterial serum lactate (n=16 from 1D), and tissue glycolytic intermediates at the end of 2.5 h fasted [U-¹³C]glucose infusions. n=4 for most tissues except gastroc (n=2) and diaphragm (n=9). Tissue abbreviations are quadriceps fermoris muscle (quad), gastrocnemius muscle (gastroc), diaphragm (diaph), small intestine (small int), gonadal white adipose tissue (gwat), inguinal white adipose tissue (iwat), and brown adipose tissue (bat). Mean ± SEM. Replicates indicate number of mice in which measurements were made.

Figure 2.. Hyperinsulinemia increases glucose use in glycolysis more than physiologic feeding.

(A) Illustration of 2.5 h fasted and refed [U-¹³C]glucose infusion. (B) Normalized labeling in arterial serum lactate after 2.5 h infusion in fasted (n=16 from Figure 1B) and refed (n=6) states. P-value calculated by unpaired t-test; *p=0.015. (C) Normalized labeling from 2.5 h [U-¹³C]glucose infusion of glycolytic intermediates hexose-phosphate (hp), fructose-1,6-bisphosphate (fbp), and 3-phosphoglycerate (3pg) in the quadriceps muscles, the soleus, and the liver for fasted (n = 4 from Figure 1E) or refed (n=6 for liver and quad, n=5 for soleus) mice. P-values calculated by two-way ANOVA; *p<0.05 (D) Illustration of hyperinsulinemic-euglycemic clamp with additional [U-¹³C]glucose tracer. Mice infused with either 2.4 or 0.8 mU min⁻¹ kg⁻¹ insulin, variable glucose to maintain euglycemia (required only in 2.5 mU min⁻¹ kg⁻¹ insulin group), and tracer amounts of stable isotope labeled glucose. Red blood cells were also infused to maintain hematocrit to allow for repeated blood sampling. (E) Insulin levels measured by ELISA in different states: fasted (n=10), refed (n=7), 0.8 mU min⁻¹ kg⁻¹ insulin infusion (n=5), or 2.5 mU min⁻¹ kg⁻¹ insulin infusion (n=6). P-value calculated with one-way ANOVA; ****p<0.0001 (F) Normalized label in arterial serum lactate after infusion of either 0.8 (n=2) or 2.5 (n=3) mU min⁻¹ kg⁻¹ of insulin with [U-¹³C]glucose. (G) Normalized labeling of glycolytic intermediates hexose-phosphate (hp), fructose-1,6-bisphosphate (fbp), and 3-phosphoglycerate (3pg) in the quadriceps muscles, the soleus, and the liver after infusion of either 0.8 (n=2) or 2.5 (n=3) mU min⁻¹ kg⁻¹ of insulin with [U-¹³C]glucose. P-value calculated with two-way ANOVA; ***p=0.0005, ****p<0.0001. Mean ± SEM. Replicates indicate number of mice in which measurements were made.

Figure 3.. Glycogen supplies glycolytic intermediates in all tissues.

(A) Schematic of pulse-chase experiment. Mice were infused with [U-¹³C]glucose for 16 h with the goal of labeling glycogen. Next an 8 h chase was performed in which the infusion was stopped during 8 h of fasting to allow circulating metabolite labeling to decrease. (B) Arterial serum glucose and lactate labeling over the course of the pulse-chase experiments. Combined data during the infusion is fit with a single exponential. Labeling in liver glycogen collected at the end of the pulse-chase is also displayed. Mean ± SEM; n=4 mice for most time points except serum at 20 and 22 h (n=2), serum at 24 h (n=9), liver glycogen at 20 h (n=5) and liver glycogen at 24 h (n=8). Error bars for some data points are too small to be visible. (C-D) Labeling in serum glucose and lactate and tissue glycolytic intermediates normalized to glycogen labeling in the liver (C) or the quadriceps (D) at the end of the pulse-chase experiment. Expected labeling from circulating glucose is calculated based on the 2.5 h glucose infusion data from Figure 1 as described by the displayed equation; n=6 mice. (E) Labeling in glycolytic intermediates at the end of the pulse chase experiment normalized to glycogen labeling measured in each tissue. n vary by tissue; n=4 mice for most tissues; n=6 for liver, quad, heart, and small intestine; n=5 for soleus and diaphragm, n=3 for iwat, bat and gastroc, and n=2 for brain. Mean ± SEM. Replicates indicate number of mice in which measurements were made.

Figure 4.. Gluconeogenic substrates broadly contribute to glycolytic intermediates in mammalian tissues.

(A) Schematic of the entry points of measured substrates into glycolysis. (B-E) Labeling in circulating metabolites (lactate, glucose, alanine, glycerol, and glutamine) and tissue glycolytic intermediates after 2.5 h fasted infusion (normalized to enrichment of the tracer in serum). Each panel represents data from a different tracer: [U-¹³C]lactate (B), [U-¹³C]alanine (C), [U-¹³C]glycerol (D), and [U-¹³C]glutamine (E). Mean ± SEM; n=4 mice in all cases except f in [U-¹³C]lactate infusion where n=10 mice for serum measurements and n=8 for liver and kidney measurements.

Figure 5.. Gluconeogenesis persists in the physiologic fed state.

(A) Illustration of substrate interconversions used to calculate the direct contributions to circulating glucose (B) Direct substrate contributions to circulating glucose production flux. The total height of each bar is reflective of total glucose turnover, calculated by the $F_{circ}^{atom}$. n ≥ 3 mice for each nutrient and condition. (C) Illustration of hyperinsulinemic-euglycemic clamps performed in combination with U-¹³C-lactate infusion. Mice infused with either 2.4 or 0.8 mU min⁻¹ kg⁻¹ insulin, variable glucose to maintain euglycemia (required only in 2.5 mU min⁻¹ kg⁻¹ insulin group), and tracer amounts of stable isotope labeled lactate. Red blood cells were also infused to maintain hematocrit allowing for repeated blood sampling. (D) Normalized labeling in arterial serum glucose after the indicated ¹³C-lactate infusions; n vary by condition; fasted (n=10 mice from Figure 4B); fed (n=7 mice); insulin infusions (n=3 mice). (E) Normalized labeling of liver glycolytic intermediates hexose-phosphate (hp, n=3 mice), fructose-1,6-bisphosphate (fbp, n=2), and 3-phosphoglycerate (3pg, n=2) during U-¹³C-lactate infusion in combination with either 0.8 or 2.5 mU min⁻¹ kg⁻¹ of insulin with variable glucose. Mean ± SEM.

Figure 6.. Carbohydrate is primarily cleared through oxidation.

(A) Schematic indicating the measured fates of glucose. (B) Glucose turnover in the fasted (n=9 mice) and fed states (n=5 mice) from Figure 5B. (C) The rate of carbohydrate oxidation measured by indirect calorimetry (n=6 mice per condition) calculated based on data from (Hui et al., 2020). (D) Carbohydrate consumption fluxes. (E) Fluxes of carbohydrate oxidative measured independently by stable isotope tracing (as consumption flux not flowing into other metabolites) or by indirect calorimetry. (F) Model summarizing carbohydrate production and consumption fluxes in fasting and feeding. The model is calculated from metabolite interconverting fluxes determined by isotope labeled glucose, lactate, glycerol, alanine, and glutamine infusions in each of these conditions. Fluxes <50 nmol C min⁻¹ g body weight⁻¹ are not displayed. Mean ± SEM.

Figure 7.. Select tissues convert circulating glucose to circulating lactate, bypassing the bulk of whole-body glycolytic intermediates.

(A) Direct nutrient contributions to production of glycolytic intermediates in each tissue. (B) Direct nutrient contributions to production of circulating lactate. The total height of each bar represents total lactate turnover. (C) Illustration showing the amount of whole-body glycolytic intermediates that come from circulating glucose or glycogen, compared to the amount of circulating lactate that comes from each of these sources. Mean ± SEM; n ≥ 3 mice for each nutrient and condition.