Absence of the SRC-2 Coactivator Results in a Glycogenopathy Resembling Von Gierke's Disease

In mammals, when exogenous glucose is not available, between meals and during fasting, the liver produces glucose from substrates such as glycogen, lactate, pyruvate, glycerol, and amino acids (1). During fasting, the liver contributes about 85 to 90% of the glucose load, and the remainder is contributed by the kidney (1, 2). This function of the liver is vital for basal brain function, and thus for the survival of the fasting organism (1, 3).

Glucose-6-phosphatase (G6Pase) is an essential, rate-limiting enzyme that catalyzes the terminal reaction that produces glucose during fasting (2, 4, 5). This reaction is common to glycogenolysis and gluconeogenesis, the two processes that provide the body with glucose when the organism is subjected to a fast. This metabolic location, at the crossroad between glycogenolysis and gluconeogenesis, allows G6Pase to control both the short-term and longer-term responses to fasting (2, 4, 5). G6Pase is present at the membrane of the endoplasmic reticulum in the liver and kidney, where it dephosphorylates glucose-6-phosphate to produce free glucose (2, 4, 5). Without dephosphorylation, glucose remains trapped in the liver, because the glucose transporters that shuttle it into the plasma cannot transport the phosphorylated form (2, 4, 5). Loss-of-function mutations in G6Pase result in Von Gierke's disease (glycogen storage disorder–1a), an autosomal recessive disorder with an incidence of 1 in 100,000 live births (6). Von Gierke's disease is clinically characterized by impaired growth, fasting hypoglycemia, and an increase in concentrations of triglycerides, cholesterol, free fatty acids, ketone bodies, uric acid, and lactic acid in the plasma of fasting animals. Increased liver glycogen stores and hepatic steatosis also occur (6).

Steroid receptor coactivator 2 (SRC-2), a member of the p160 family of transcriptional co-activators, has been implicated in a number of physiological processes, from reproduction, mammary morphogenesis, and uterine function to energy metabolism in adipose tissue via regulation of adaptive thermogenesis (7-10). Its role in liver-mediated glucose homeostasis, however, is unknown. Because fasting is a powerful inducer of the glycogenolytic and gluconeogenic response through increased transcription of the G6Pase gene, we tested the role of SRC-2 in fasting modulation of G6Pase expression. In the absence of SRC-2 in mice, there was a deficit in fasting G6Pase expression in both the liver and the kidney (Fig. 1A). This deficit in expression was accompanied by a deficiency in the catalytic activity of hepatic G6Pase.

Because G6Pase expression is enhanced by fasting and suppressed by feeding, we tested the effect of a lack of SRC-2 in those settings. SRC-2 was critical for both basal as well as induced expression of G6Pase (Fig. 1B). The fasting expression of other genes involved in the control of gluconeogenic activity such as phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase 1 (FBP1), peroxi-some proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1a), and Glucose-6-phosphate translocase (G6P translocase) was essentially unaffected by the absence of SRC-2 (Fig. 1C). Of 21 other genes that are important for fasting glucose homeostasis, in the glycogenolysis, glycogenesis, β-oxidation, ketogenesis, and hexose monophosphate shunt pathways, expression of none of these genes was significantly decreased in mice lacking SRC-2 (table S1). To determine whether other p160 coactivator family members (SRC-1 and SRC-3) function in G6Pase regulation during fasting, we determined G6Pase expression in the livers of SRC-1 and SRC-3 null animals that were fasted for 24 hours. The absence of SRC-1 and SRC-3 did not affect the expression of G6Pase, suggesting an SRC-2–specific effect on the regulation of fasting hepatic G6Pase expression (fig. S10).

It was possible that the expression deficit of G6Pase that we observed in SRC-2 null animals was due to an indirect systemic effect. To rule out this possibility we isolated primary hepatocytes from wild-type (WT) mice and depleted SRC-2 using RNA interference (RNAi). RNAi-mediated knockdown of SRC-2 resulted in a down-regulation of G6Pase expression, suggesting a cell-autonomous effect (Fig. 1D). G6Pase is positively regulated, at the transcriptional level, by fasting-associated hormones such as glucagon, catecholamines, and glucocorticoids. We exposed primary hepatocytes from WT and SRC-2 null animals to dexamethasone to stimulate the glucocorticoid receptor or forskolin to simulate hormonal stimulation of adenylyl cyclase. Ablation of SRC-2 decreased both control and hormone-mediated transcription of G6Pase (fig. S6, A and B).

The primary manifestations of Von Gierke's disease, which occur as a result of mutations in the G6Pase gene, are diminished growth; fasting hypoglycemia; increased concentrations of triglycerides, cholesterol, free fatty acids, ketone bodies, uric acid, and lactic acid in the plasma; increased liver glycogen stores; and hepatic steatosis (6). SRC-2 null mice showed fasting hypoglycemia, although the animals maintained normoglycemia in the fed state (Fig. 2A). Considered along with the observation of fasting hypoinsulinemia (Fig. 2B), this suggests a defect in liver function. Further plasma analysis demonstrated increased concentrations of triglycerides, cholesterol, free fatty acids (FFAs), and ketone bodies in SRC-2^−/− animals that were fasted for 24 hours (Fig. 2C). Histologically, liver morphology was distorted with a characteristic mosaic pattern like that of patients with Von Gierke's disease(Fig.2D)(11). Periodic acid–Schiff staining, Oil Red O staining, and electron microscopy (EM) revealed heavy accumulation of glycogen and triglycerides in the liver of fasted SRC-2 null animals (Fig. 2, E through G). Livers of 24-hour fasted SRC-2 null animals contained about twice as much glycogen and about three times as much triglycerides as did livers from WT animals (Fig. 2H). This was accompanied by increased concentrations of circulating glucagon and catecholamine in SRC-2 null animals, suggesting a compensatory attempt to mobilize liver glycogen (fig. S1). The growth deficit that accompanies the lack of SRC-2 has been described (12).

To unequivocally assess the effect of SRC-2 ablation on fasting (basal) liver glucose production, we measured hepatic glucose production using radiolabeled glucose in awake SRC-2 null animals and WT littermates that had been fasted overnight. Basal hepatic glucose production in SRC-2 null animals was depressed compared with that of WT littermates (fig. S3A). In vitro glucose production from gluconeogenic precursors lactate and pyruvate, by primary hepatocytes subjected to RNAi-mediated knockdown of SRC-2, was one-half that by control cells (fig. S3B).

Given that glucose homeostasis in fasting animals is primarily a function of the liver, we tested whether SRC-2 ablation, specifically in the liver, would reproduce the features of Von Gierke's disease. We infused adenovirus carrying the CRE recombinase gene into the tail veins of homozygous SRC-2 floxed (F/F) mice and exposed control SRC-2 F/F mice to empty adenovirus. This approach produces liver-selective transgenesis, because most of the adenoviral load is localized in the liver (13-15). Liver-selective depletion of SRC-2 resulted in decreased abundance of G6Pase mRNA in the liver of fasting animals (Fig. 3A). G6Pase expression in the kidney was increased, possibly as a compensatory response (Fig. 3A). By contrast, the results in fasted SRC-2 (whole-body) knockout mice showed decreased renal G6Pase expression, and thus exemplifies the critical nature of SRC-2's influence over G6Pase expression (Fig. 1A). Liver-selective depletion of SRC-2 in fasted animals resulted in hypoglycemia (Fig. 3B), along with increased concentrations of triglycerides, cholesterol, ketone bodies, and uric acid in the plasma (Fig. 3C). This plasma profile was accompanied by an accumulation of hepatic glycogen and triglycerides in fasted liver-selective SRC-2 null animals (Fig. 3D), thereby reproducing the phenotypic characteristics of Von Gierke's disease.

Because SRC-2 is a “coactivator” and lacks specific DNA binding activity, it is presumably recruited to a gene promoter by a transcription factor in order to function. Although transcriptional coactivators often function at multiple genes, our results indicated that SRC-2 might regulate the expression of just one gene in the gluconeogenesis and glycogenolysis pathways—G6Pase (Fig.1,A and B, and table S1).We therefore searched the G6Pase promoter sequence for transcription factor response elements that might explain such selectivity. We found an evolution-arily conserved retinoid-related orphan receptor α (RORα) response element (RORE) close to the transcriptional start site (fig. S11). SRC-2 and RORα have been reported to bind and synergize in reporter assays (16). Consequently, we tested the human WT G6Pase promoter (−1227, +57) by coexpressing it with expression plasmids carrying SRC-2 and RORα. SRC-2 and RORα, when expressed together, synergized strongly to produce G6Pase promoter activation (Fig. 4A). This synergism (coactivation) was completely abolished upon specific mutation of the RORE (Fig. 4A). We also tested other transcription factors that bind SRC-2 and are important for regulation of fasting glucose homeostasis such as the glucocorticoid receptor and orphan nuclear receptor Nurr77, and found that SRC-2 did not synergize with them in this context (fig. S7C).

To demonstrate the effect of RORα on trans-activation of G6Pase in the correct chromatin context, we transduced primary hepatocytes with adenoviral RORα. This resulted in increased expression of the endogenous G6Pase gene, without affecting the expression of other key genes involved in the gluconeogenic pathway such as PEPCK, FBP1, PGC-1a and G6P translocase (Fig. 4B). The RORα-mediated increase in G6Pase transactivation was abolished in a SRC-2 null background (fig. S4B). Furthermore, RNAi-mediated depletion of RORα in primary hepatocytes resulted in decreased expression of G6Pase (fig. S4). To assess whether RORα affects G6Pase regulation mediated by fasting hormones, we treated primary hepatocytes that had been exposed to RNAi-mediated depletion of RORα with dexamethasone or forskolin. Depletion of RORα diminished the effect of dexamethasone and forskolin stimulation of G6Pase gene expression (fig. S6C), although forskolin retained a strong activating effect. We also tested the effects of the promoter-proximal RORE on mediation of the signal from dexamethasone and forskolin (fig.S6D). Basal activity of the G6Pase promoter and that induced by a combination of dexamethasone and forskolin was inhibited upon mutation of the RORE, suggesting that this response element functions in the signaling from fasting hormones that results in G6Pase promoter activation. In vivo chromatin immunoprecipitation (ChIP) experiments showed that both SRC-2 and RORα proteins bound to the G6Pase promoter, selectively in the region of the RORE (Fig. 4C). To further substantiate that SRC-2 was recruited to the G6Pase promoter by RORα, we overexpressed RORα in primary hepatocytes. This resulted in increased promoter occupancy of RORα, which in turn increased the promoter occupancy of SRC-2, suggesting that RORα could indeed recruit SRC-2 to the G6Pase promoter (Fig. S5A). Conversely, SRC-2 promoter occupancy was abolished in cells depleted of RORα (Fig. S5B). These results suggest that RORα is necessary and sufficient to recruit SRC-2 to the G6Pase promoter.

The phenotype and the gene-expression profile of SRC-2 null animals suggests that the perturbation of glucose homeostasis is due to a deficiency in G6Pase expression. However, the possibility that SRC-2 might regulate another gene that might contribute to the phenotype cannot be ruled out. Therefore, we reintroduced G6Pase into SRC-2 null mice by infusion of a recombinant adenovirus containing G6Pase cDNA, or an adenovirus containing no cDNA insert. We measured blood glucose concentration in three independent feeding states—fed, fasted for 4 hours, and fasted for 18 hours. Fasting hypoglycemia caused by lack of SRC-2 was diminished in animals that received the G6Pase adenovirus compared to that in animals that were exposed to an empty adenovirus (Fig. 5). This result is consistent with the possibility that some of the glucose homeostasis deficit observed in fasted SRC-2 null animals results from a deficiency in G6Pase expression.

Glycogenolysis is the primary process of endogenous glucose production in the first 4 hours after a meal and is followed by the transition to gluconeogenesis upon prolonged fasting (2, 4, 5). During glycogenolysis, there is no requirement for the activation of PEPCK and FBP1, enzymes that are limited to the process of gluconeogenesis (1). We report the identification of two transcriptional entities, SRC-2 and RORα, that regulate G6Pase without regulating PEPCK and FBP1. Unlike systems in which transcriptional coactivators exert control over multiple downstream target genes to modulate physiology (17-19), SRC-2 appears to control glucose availability through the regulation of one key mediator (Fig. 1, A and B, and table S1). This study unmasks a biological entity, other than mutations in the G6Pase gene itself, that might contribute to cases of glycogen storage disorders and unexplained hypoglycemia. Accordingly, SRC-2 and RORα may represent new therapeutic targets against these diseases.

Supplementary Material