Hepatic anaplerotic outflow fluxes are redirected from gluconeogenesis to lactate synthesis in patients with Type 1a glycogen storage disease

Abstract

Hepatic glucose production and relative Krebs cycle fluxes (indexed to a citrate synthase flux of 1.0) were evaluated with [U-¹³C]glycerol tracer in 5 fed healthy controls and 5 Type 1a glycogen storage disease (GSD1a) patients. Plasma glucose, hepatic glucose-6-phosphate (G6P) and glutamine ¹³C-isotopomers were analyzed by ¹³C NMR via blood sampling and chemical biopsy. In healthy subjects, 35±14% of plasma glucose originated from hepatic G6P while GSD1a patients had no detectable G6P contribution. Compared to controls, GSD1a patients had an increased fraction of acetyl-CoA from pyruvate (0.5±0.2 vs. 0.3±0.1, p<0.01), and increased pyruvate recycling fluxes (14.4±3.8 vs. 8.7±2.8, p<0.05). Despite negligible gluconeogenic flux, net anaplerotic outflow was not significantly different from controls (2.2±0.8 vs. 1.3±0.5). The enrichment of lactate with ¹³C-isotopomers derived from the Krebs cycle suggests that lactate was the main anaplerotic product in GSD1a patients.

Introduction

In Type 1a glycogen storage disease (GSD1a), hepatic glucose-6-phosphatase (G6P-ase) is inactive resulting in major changes of systemic glucose metabolism both upstream and downstream of hepatic glucose-6-phosphate (G6P) (Bandsma et al., 2002a, Bandsma et al., 2002b). The capacity to maintain plasma glucose levels during fasting is impaired because endogenous glucose cannot be synthesized from either glycogen or gluconeogenic precursors since G6P is a common intermediate for both pathways. Normally, when glucose release via the absorption of dietary carbohydrate is insufficient to meet systemic demand, the balance is supplied by the hydrolysis of hepatic G6P to glucose by glucose-6-phosphatase. In GSD1a patients, the lack of G6P-ase activity means that hepatic carbon fluxes that would normally be destined for glucose production are necessarily redirected to other pathways. Although G6P can be disposed as glycogen, the hydrolysis of glycogen to glucose—which is largely dependent on G6P-ase—is also blocked. This results in the characteristic overabundance of hepatic glycogen, which cannot be relieved by fasting glycogenolysis. Under these conditions, the capacity for converting G6P to glycogen on a daily basis is likely very small and can only account for a very minor portion of G6P flux that is normally destined for endogenous glucose production.¹

Under both fasting and fed conditions, a significant fraction of hepatic G6P and plasma glucose is derived from gluconeogenic carbons originating from the anaplerotic pathways of the Krebs cycle, with pyruvate being the principal precursor. Gluconeogenesis is considered to account for the majority of anaplerotic outflow from the Krebs cycle (Rognstad and Katz, 1977; Magnusson et al., 1991; Katz et al., 1993; Beylot et al., 1995). Gluconeogenic flux is largely sustained by the conversion of pyruvate to phosphoenolpyruvate (PEP) via pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEP-ck). Pyruvate may also be oxidized to acetyl-CoA via pyruvate dehydrogenase (PDH) hence substrate competition between PC and PDH can potentially regulate the rate of pyruvate conversion to G6P (Beylot et al., 1995; Large and Beylot, 1999). Another potential regulatory mechanism is the recycling of PEP to pyruvate via pyruvate kinase that completes a futile cycle (pyruvate→oxaloacetate→PEP→pyruvate) (Rognstad and Katz, 1977; Magnusson et al., 1991; Jones et al., 1997; Diraison et al., 1998; She et al., 2003; Jin et al., 2005). Operation of this cycle may attenuate gluconeogenic flux in two ways: first by redirecting anaplerotic flux from all sources (including anaplerotic substrates that enter the Krebs cycle as 4 and 5-carbon skeletons, such as aspartate, glutamate and glutamine) to pyruvate, and second, by consuming ATP that could otherwise fuel gluconeogenesis. In GSD1a patients, gluconeogenesis is negligible; hence the carbon outflow that would normally be destined for glucose synthesis must either be attenuated by the mechanisms described or redirected into alternative disposal pathways.

To study hepatic G6P-ase and Krebs cycle fluxes, we designed a stable-isotope tracer study that is summarized in Fig. 1. The procedure is non-invasive and can be accommodated into the cornstarch feeding regime of GSD1a patients. [U-¹³C]Glycerol was used as means of delivering ¹³C into the hepatic triose-P pool. This substrate has some key advantages in that it is palatable and can be mixed with the cornstarch meal and is also efficiently extracted and metabolized by the liver. [U-¹³C]Glycerol was accompanied by two xenobiotic agents, acetaminophen and phenylbutyric acid, designed to safely sample key hepatic metabolites as conjugates that are cleared into urine. Acetaminophen is metabolized to acetaminophen glucuronide with the glucuronide moiety derived from UDP-glucose (Hellerstein et al., 1986). Phenylbutyric acid can undergo β-oxidation to phenylacetyl-CoA, which then combines with hepatic glutamine to form phenylacetylglutamine (PAGN) (Comte et al., 2002). Hepatic glutamine is derived in part from the Krebs cycle intermediate α-ketoglutarate, hence the ¹³C-enrichment distribution of acetyl-CoA and the Krebs cycle can be inferred from PAGN (Yang et al., 1993; Jones et al., 1998, Jones et al., 2001; Diraison et al., 1999). The ¹³C-enrichment and isotopomer distributions of PAGN and acetaminophen glucuronide, as well as plasma glucose and lactate, can be quantified by ¹³C NMR following simple extraction and purification procedures (Jones et al., 1998, Jones et al., 2001). The relative activities of anaplerotic and oxidative Krebs cycle fluxes and the pyruvate futile cycle can then be derived from the ¹³C-enrichment or isotopomer distribution of PAGN (Diraison et al., 1998; Jones et al., 1998, Jones et al., 2001).