Uric acid-dependent inhibition of AMP kinase induces hepatic glucose production in diabetes and starvation: evolutionary implications of the uricase loss in hominids

Abstract

Reduced AMP kinase (AMPK) activity has been shown to play a key deleterious role in increased hepatic gluconeogenesis in diabetes, but the mechanism whereby this occurs remains unclear. In this article, we document that another AMP-dependent enzyme, AMP deaminase (AMPD) is activated in the liver of diabetic mice, which parallels with a significant reduction in AMPK activity and a significant increase in intracellular glucose accumulation in human HepG2 cells. AMPD activation is induced by a reduction in intracellular phosphate levels, which is characteristic of insulin resistance and diabetic states. Increased gluconeogenesis is mediated by reduced TORC2 phosphorylation at Ser171 by AMPK in these cells, as well as by the up-regulation of the rate-limiting enzymes PEPCK and G6Pc. The mechanism whereby AMPD controls AMPK activation depends on the production of a specific AMP downstream metabolite through AMPD, uric acid. In this regard, humans have higher uric acid levels than most mammals due to a mutation in uricase, the enzyme involved in uric acid degradation in most mammals, that developed during a period of famine in Europe 1.5 × 10(7) yr ago. Here, working with resurrected ancestral uricases obtained from early hominids, we show that their expression on HepG2 cells is enough to blunt gluconeogenesis in parallel with an up-regulation of AMPK activity. These studies identify a key role AMPD and uric acid in mediating hepatic gluconeogenesis in the diabetic state, via a mechanism involving AMPK down-regulation and overexpression of PEPCK and G6Pc. The uricase mutation in the Miocene likely provided a survival advantage to help maintain glucose levels under conditions of near starvation, but today likely has a role in the pathogenesis of diabetes.

Keywords: gluconeogenesis; insulin; phosphate; urate.

Figure 1.

Decreased AMPK phosphorylation and increased AMPD activity in the livers of diabetic mice. A) Serum glucose levels in nondiabetic and diabetic mice. B) Blood HbA1c levels in nondiabetic and diabetic mice. C) Representative Western blot demonstrating reduced AMPK phosphorylation (at Thr172) in the livers of diabetic mice compared to nondiabetics (n=5 mice/group) with no difference in the expression of both total AMPK and total AMPD2. Reduced AMPK phosphorylation is associated with higher PEPCK and G6Pc expression. D) AMPD2 Western blot densitometry, demonstrating no significant change in expression. E) AMPD activity is higher in livers from diabetic mice compared to nondiabetics. F) Intrahepatic uric acid levels in control (nondiabetic) and diabetic mice. **P < 0.01 vs. control nondiabetic mice.

Figure 2.

Generation of high AMPD activity in human HepG2 cells demonstrate increased expression of gluconeogenic enzymes and glucose production. A) Total AMPD activity in livers of mice (left side of histogram; white bar denotes nondiabetic control; gray bar denotes diabetic cells) and HepG2 cells (right side of histogram; white bar denotes control cells, while gray bar denotes AMPD2-overexpressing cells); n = 4 clones/group. **P < 0.05 vs. respective control. B) Representative Western blot for AMPD2 and actin loading control in control HepG2 cells and cells stably overexpressing AMPD2. C) Representative Western blot for AMPD2, PEPCK, G6Pc, and actin loading control in control HepG2 cells (n=4) and cells stably overexpressing AMPD2 (n=4). D) Western blot densitometry for PEPCK and G6Pc in control HepG2 cells (n=4) and cells stably overexpressing AMPD2 (n=4). **P < 0.01 vs. control. E) Glucose production in HepG2 cells control (left) and stably overexpressing AMPD2 (right) in control conditions (white bars), in serum-free medium supplemented with lactate, pyruvate, and Bt₂-cAMP (gray bars) and serum-free medium without lactate, pyruvate, and Bt₂-cAMP (black bars). **P < 0.01 vs. control and no L/P; ^##P < 0.01.

Figure 3.

Inhibition of AMPD activity results in blockade of gluconeogenesis. A) Total AMPD activity in HepG2 cells stably overexpressing AMPD2 control (white bar) or in the presence of the AMPD inhibitor pentostatin (100 μM, black bar). **P < 0.01 vs. control. B) Glucose production in HepG2 cells stably overexpressing AMPD2 (right) in control conditions (white bar), in serum-free medium supplemented with lactate, pyruvate, and Bt₂-cAMP alone (gray bar), or in the presence of pentostatin (100 μM, black bar). **P < 0.01 vs. control and serum-free medium plus pentostatin.

Figure 4.

AMPD2 controls AMPK phosphorylation in HepG2 cells acutely exposed to serum-free medium. A) Representative Western blot for AMPD2, AMPK, pAMPK, PEPCK, and actin loading control in control HepG2 cells in serum-free medium for different time points (0, 1, and 3 h; n=2/time point) and cells stably overexpressing AMPD2 under the same conditions (n=2 for each time point). B) Western blot densitometry for pAMPK in control HepG2 cells under serum-free medium conditions 3 h and cells stably overexpressing AMPD2 under the same conditions (n=4). ***P < 0.001 vs. control. C) Representative Western blot for AMPD2, AMPK, pAMPK, PEPCK, and actin loading control in control HepG2 cells under serum-free conditions for 3 h in the presence or absence of the AMPK agonist AICAR (1 μM) and cells stably overexpressing AMPD2 under the same conditions (n=2/condition). D) Western blot densitometry for pAMPK and PEPCK in HepG2 cells stably overexpressing AMPD2 under serum-free conditions for 3 h in the presence or absence of the AMPK agonist AICAR (1 μM, n=4/condition). ***P < 0.001 vs. control. E) Glucose production in HepG2 cells control (left) and stably overexpressing AMPD2 (right) in control conditions (white bars), in serum-free conditions supplemented with lactate, pyruvate, and Bt₂-cAMP (gray bars) alone or in the presence of AICAR (1 μM). **P < 0.01 vs. respective control and serum-free plus lactate and pyruvate.

Figure 5.

Effects of inosine in hepatic glucose production are mediated by the generation of uric acid. A) Intracellular inosine levels in HepG2 cells control (left) and stably overexpressing AMPD2 (right). ***P < 0.001 vs. control. B) Intracellular uric acid levels in HepG2 control cells (left) and cells stably overexpressing AMPD2 (right). ***P < 0.001 vs. control. C) Glucose production in HepG2 cells under control conditions (white bar), serum-free conditions supplemented with lactate, pyruvate and Bt₂-cAMP (light gray bar), and the same serum-free conditions plus inosine (dark gray bar) or uric acid (black bar) **P < 0.01, ***P < 0.001 vs. control; ^#P < 0.05. D) Intracellular inosine levels in HepG2 stably overexpressing AMPD2 (left) or stably silenced for purine nucleoside phosphorylase (PNP, right). E) Intracellular uric acid levels in HepG2 stably overexpressing AMPD2 (left) or stably silenced for purine nucleoside phosphorylase (PNP, right). ***P < 0.001 vs. control AMPD2-overexpressing cells. F) Glucose production in HepG2 cells stably overexpressing AMPD2 (left) or silenced for PNP (right) under control conditions (white bars) and serum-free conditions supplemented with lactate, pyruvate, and Bt₂-cAMP (gray bars). **P < 0.01 vs. control.

Figure 6.

Expression of resurrected ancestral uricases from early hominids in HepG2 cells stimulate AMPK phosphorylation and block TORC2 nuclear translocation. A) Representative Western blot for AMPD2, AMPK, pAMPK, PEPCK, G6Pc, uricase, and actin loading control in HepG2 control cells or cells stably expressing ancestral uricase 19 (Anc19) or uricase 27 (Anc27) under normal conditions or serum-free conditions for 3 h in the presence of lactate, pyruvate, and Bt₂-cAMP. B) Western blot densitometry for PEPCK (left) and G6Pc (right) under serum-free conditions in control cells (white bars) and cells expressing Anc19 (gray bars) or Anc27 (black bars) (n=4). ***P < 0.001 vs. control. C) Representative confocal image of TORC2 (green) location in HepG2 cells control (top row) or expressing Anc19 (bottom row) under serum-free condition supplemented with lactate, pyruvate, and Bt₂-cAMP. The nuclear marker DAPI is shown in red; merged pseudocolor of TORC2 and DAPI is shown in yellow. View: ×63 in left 3 panels; ×100 magnification in right 2 panels. D) Representative Western blot for TORC2 and pTORC2 in HepG2 cells control or stably expressing ancestral uricase 19 (Anc19) or uricase 27 (Anc27) under normal conditions or serum-free conditions for 3 h in the presence of lactate, pyruvate, and Bt₂-cAMP. E) Western blot densitometry for pTORC2 (normalized to total TORC2) under serum-free conditions in control cells (white bar) and cells expressing Anc19 (gray bar) or Anc27 (black bar) (n=4). ***P < 0.001 vs. control.

Figure 7.

Expression of resurrected ancestral uricases from early hominids in HepG2 cells page glucose production by reducing intracellular uric acid levels. Glucose production in HepG2 cells control (left), stably overexpressing Anc19 (middle), or Anc27 (right) in control conditions (white bars) or serum-free conditions supplemented with lactate, pyruvate, and Bt₂-cAMP (gray bars). **P < 0.01 vs. respective control. B) Glucose production in control cells (left) and stably overexpressing Anc19 (right) in control conditions or serum-free condition supplemented with lactate, pyruvate and Bt₂-cAMP and increasing concentrations of uric acid (from 0 to 12 mg/dl). **P < 0.01 vs. respective control. C) Representative Western blot for pAMPK, AMPK, actin, and uricase in the same conditions as in B.

Figure 8.

Intracellular phosphate regulates AMPD activity and glucose production in HepG2 cells. A) AMPD activity in cell lysates incubated with increasing levels of phosphate. **P < 0.01 and ***P < 0.001 vs. addition of 0 mM phosphate. B) Intrahepatic phosphate levels in nondiabetic and diabetic mice (n=5). *P < 0.05 vs. nondiabetic control mice. C) Glucose production in HepG2 cells under normal (control) conditions (white bar), serum-free conditions supplemented with lactate, pyruvate, and Bt₂-cAMP with 1 mM phosphate (gray bar), or 2.5 mM phosphate (black bar). **P < 0.01 vs. control and serum-free plus 2.5 mM phosphate. D) Percentage of change in intracellular phosphate levels in HepG2 cells under serum-free conditions supplemented with lactate, pyruvate, and Bt₂-cAMP with 1 mM phosphate (white bar) or 2.5 mM phosphate (black bar) compared to control conditions. *P < 0.05 vs. control and starvation. E) AMPD activity in cells exposed to 1 and 2.5 mM phosphate for 3 d. **P < 0.001 vs. serum-free condition.

Figure 9.

Proposed role of AMPD2 and uric acid in hepatic glucose production in diabetic states. Under normal conditions (left side of panel), insulin secretion favors both glucose and phosphate uptake by the liver. When ATP depletion occurs and the AMP/ATP ratio rises, AMPK is activated by its phosphorylation at threonine-172 to block anabolic routes, including gluconeogenesis, while AMPD activity is reduced by intracellular phosphate. The mechanism whereby AMPK blocks gluconeogensis is mediated by the phosphorylation of TORC2 and PEPCK and G6Pc-reduced transcriptional activity. In contrast, in insulin-deficient or insulin-resistant states (right side of panel), intrahepatic phosphate levels decrease, and AMPD2 is thus activated and uric acid generated. Increased AMPD activity and uric acid levels block the activation of AMPK, leading to the translocation of TORC2 to the nucleus and the transcription of PEPCK and G6Pc that along with increased availability of gluconeogenic substrates in diabetic states (lactate and pyruvate) stimulate de novo production of glucose.