Direct anabolic metabolism of three-carbon propionate to a six-carbon metabolite occurs in vivo across tissues and species

Abstract

Anabolic metabolism of carbon in mammals is mediated via the one- and two-carbon carriers S-adenosyl methionine and acetyl-coenzyme A. In contrast, anabolic metabolism of three-carbon units via propionate has not been shown to extensively occur. Mammals are primarily thought to oxidize the three-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. Here, we found that this may not be absolute as, in mammals, one nonoxidative fate of propionyl-CoA is to condense to two three-carbon units into a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this reaction pathway using purified protein extracts provided limited substrates and verified the product via LC-MS using a synthetic standard. In whole-body in vivo stable isotope tracing following infusion of ¹³C-labeled valine at steady state, 2M2PE-CoA was found to form via propionyl-CoA in multiple murine tissues, including heart, kidney, and to a lesser degree, in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA also formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three- to six-carbon reaction conserved in humans and mice that utilizes propionate.

Keywords: 2M2PE-CoA; LC-MS/HRMS; Metabolism; TCA cycle; acetyl-CoA; anabolism; condensation reaction; propionate; stable isotope tracing; valine.

Fig. 1

Isotope incorporation into propionyl-CoA and 2M2PE-CoA from U-¹³C-valine maintains integers of three contiguous carbon atoms. Carbon-13 from U-¹³C-valine or U-¹³C-propionate is indicated by red, blue, and blue open circles. Catabolism of valine or activation of propionate produces [¹³C₃]-propionyl-CoA which retains three labeled atoms (blue). Condensation of two molecules of propionate results in the formation of trans-2-methyl-pentenoyl-CoA with [¹³C₃] labeling of all six-carbon atoms: three deriving from one propionyl-CoA (blue) and three deriving from a second propionyl-CoA (blue open). 2M2PE-CoA, trans-2-methyl-2-pentenoyl-CoA.

Fig. 2

Comparison of synthetic and biologically generated 2M2PE-CoA. Coelution of (A) synthetic standard, (B) 2M2PE-CoA produced in HepG2 cells treated with 100 μM propionate, and (C) 2M2PE-CoA produced in an active fraction of HepG2 protein extract incubated with 100 μM propionyl-CoA, 1 mM NADH, and 1 mM NADPH. D: Chemical structure of 2M2PE-CoA. (E) LC-MS/MS of the synthetic standard. 2M2PE-CoA, trans-2-methyl-2-pentenoyl-CoA; HepG2, human hepatocellular carcinoma cells.

Fig. 3

Acyl-CoA abundance and generation from valine varies by tissue type. Relative amounts (pmol/mg tissue wet weight) of (A) acetyl-CoA, (B) succinyl-CoA, and (C) propionyl-CoA, and isotopolog enrichment (%) for (D) acetyl-CoA, (E) succinyl-CoA, and (F) propionyl-CoA in BAT, heart, kidney, liver, and TA after 120-min infusion of ¹³C-valine at a physiologically relevant steady state concentration in conscious mice. acetyl-CoA, acetyl-coenzyme A; BAT, brown adipose tissue; TA, tibialis anterior muscle.

Fig. 4

2M2PE-CoA varies in amount and enrichment from valine catabolism across tissues and displays a similar trend to propionyl-CoA in normal and diabetic mice. A: Area under the curve and (E) isotopolog enrichment of 2M2PE-CoA labeling incorporating the ¹³C-label from infused ¹³C-valine in murine tissues. Spearman correlation (r) was calculated for the indicated relationships in (B and F) C57/blk6 (n=13), (C and G) db/db (n=22) and (D and H) both mouse groups combined (n=35). Strong significant correlations were found in all variables, with the expectation of (F) 2M2PE-CoA M3 + M6 and propionyl-CoA M3 enrichment % in normal mice (r= 0.4542, P=0.120). 2M2PE-CoA, trans-2-methyl-2-pentenoyl-CoA.

Fig. 5

Increased valine labeling correlates with 3C metabolism. Data shows labeling (%) of (A) succinyl-CoA M3, (B) propionyl-CoA M3, and (C) 2M2PE-CoA M3+M6 from plasma valine after constant infusion of [U-¹³C]-valine for 120 min in conscious mice. 2M2PE-CoA, trans-2-methyl-2-pentenoyl-CoA.

Fig. 6

2M2PE-CoA metabolism by propionyl-CoA is not dependent on entrance carbon to the TCA cycle. Time course of generation of (A) acetyl-CoA, (B) succinyl-CoA, (C) propionyl-CoA, and (D) 2M2PE-CoA by LC-MS/HRMS analysis following treatment of heart tissues with 100 μM [¹³C₃]-sodium propionate in Tyrode’s solution containing 5 mM glucose. 2M2PE-CoA, trans-2-methyl-2-pentenoyl-CoA; acetyl-CoA, acetyl-coenzyme A.