Cytosolic phosphoenolpyruvate carboxykinase as a cataplerotic pathway in the small intestine

Abstract

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK) is a gluconeogenic enzyme that is highly expressed in the liver and kidney but is also expressed at lower levels in a variety of other tissues where it may play adjunct roles in fatty acid esterification, amino acid metabolism, and/or TCA cycle function. PEPCK is expressed in the enterocytes of the small intestine, but it is unclear whether it supports a gluconeogenic rate sufficient to affect glucose homeostasis. To examine potential roles of intestinal PEPCK, we generated an intestinal PEPCK knockout mouse. Deletion of intestinal PEPCK ablated ex vivo gluconeogenesis but did not significantly affect glycemia in chow, high-fat diet, or streptozotocin-treated mice. In contrast, postprandial triglyceride secretion from the intestine was attenuated in vivo, consistent with a role in fatty acid esterification. Intestinal amino acid profiles and ¹³C tracer appearance into these pools were significantly altered, indicating abnormal amino acid trafficking through the enterocyte. The data suggest that the predominant role of PEPCK in the small intestine of mice is not gluconeogenesis but rather to support nutrient processing, particularly with regard to lipids and amino acids. NEW & NOTEWORTHY The small intestine expresses gluconeogenic enzymes for unknown reasons. In addition to glucose synthesis, the nascent steps of this pathway can be used to support amino acid and lipid metabolisms. When phosphoenolpyruvate carboxykinase, an essential gluconeogenic enzyme, is knocked out of the small intestine of mice, glycemia is unaffected, but mice inefficiently absorb dietary lipid, have abnormal amino acid profiles, and inefficiently catabolize glutamine. Therefore, the initial steps of intestinal gluconeogenesis are used for processing dietary triglycerides and metabolizing amino acids but are not essential for maintaining blood glucose levels.

Keywords: amino acids; cataplerosis; gluconeogenesis; glyceroneogenesis; small intestine.

Fig. 1.

Intestinal cytosolic phosphoenolpyruvate carboxykinase (Pck1) is responsive to feeding and fasting and is lost in small intestine PEPCK knockout (SIPKO) mice. A: quantitative real-time PCR of Pck1 mRNA in the duodenum (D), jejunum (J), and ileum (I) from fed and 24-h fasted control and SIPKO mice. B: Western blot analysis of PEPCK protein content in response to feeding and fasting. *P < 0.05 by t-test, n = 3 mice. PEPCK; phosphoenolpyruvate carboxykinase.

Fig. 2.

Effect of small intestine PEPCK knockout (SIPKO) on glycemia. A: body weight on chow diet; n = 8. B: fed/fasted plasma glucose concentration on chow diet; n = 8. C: oral glucose tolerance test; n = 10. D: mass-enriched glucose production by everted small intestine perfused with [U-¹³C₅]glutamine; n = 3. E: glycogen content of small intestine; n = 6. F: endogenous glucose production determined by dilution of infused [U-¹³C₆]glucose; n = 7. G: fasting plasma glucose concentration after 16 wk of a 60% high-fat diet (HFD); n = 6. H: oral glucose tolerance test after 16 wk of a 60% HFD; n = 3. I: plasma glucose concentration after streptozotocin (STZ) treatment; n = 7 mice. *P < 0.05 by two-tailed t-test. n = no. of mice. OGTT, Oral Glucose Tolerance Test.

Fig. 3.

Cystolic phosphoenolpyruvate carboxykinase (Pck1) deletion impairs small intestine triglyceride metabolism. A: plasma triglyceride concentration; n = 10. B: plasma triglyceride concentration and area under the curve (AUC) following an oral dose of 200 µl of olive oil; n = 10. C: plasma triglyceride concentration and AUC after 500 mg/kg Tyloxapol; n = 14. D: total triglyceride secretion following an oral dose of olive oil; n = 14. E: body weights throughout 32 wk of a 60% high-fat diet (HFD); n = 6. F: total dietary lipid absorption; n = 4. *P < 0.05 by two-way ANOVA or two-tailed t-test. TG, triglycerides. n = no. of mice.

Fig. 4.

Cystolic phosphoenolpyruvate carboxykinase (Pck1) deletion impairs small intestine amino acid metabolism. Nonessential (A) and essential (B) amino acids from jejuna of fed mice; n = 8. Jejunum glutamine (C) enrichment and (D) content, and alanine (E) enrichment, (F) content, and (G) correlation between aspartate and alanine percent enrichment 10 min after a labeled [U-¹³C₅]glutamine oral challenge; n = 4. *P < 0.05 by two-tailed t-test or Pearson correlation. n = no. of mice.

Fig. 5.

Cystolic phosphoenolpyruvate carboxykinase (Pck1) deletion causes a buildup of jejunum TCA cycle metabolites and an oxidized redox state. Jejunum organic acids in fed (A) and 24-h fasted (B) mice; n = 8. Mitochondrial redox state represented by the αKG x NH4/Glu ratio; n = 7 (C). Cytosolic redox state represented by the lactate/pyruvate ratio; n = 8 (D). Oxygen consumption by isolated villus cells incubated in modified DMEM/F-12 containing 5 mM glutamine and 5 mM glucose; n = 5 (E). Total intestine length; n = 15 (F). Hematoxylin-eosin (H&E) staining of jejunum slices (G). *P < 0.05 by two-tailed t-test. n = no. of mice. αKG, α-ketoglutarate; Cit, citrate; Fum, fumarate; Lac, lactate; OAA, oxaloacetate; Pyr, pyruvate; Succ, succinate.

Fig. 6.

Metabolic pathways impacted by PEPCK in the small intestine of mice. αKG, α-ketoglutarate; DHAP, dihydroxyacetone phosphate; FFA, free fatty acid; GA3P, glyceraldehyde 3-phosphate; Lac, lactate; MAG, monoacylglyceride; Mal, malate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; Succ, succinate.