Hepatocytes contribute to residual glucose production in a mouse model for glycogen storage disease type Ia.

It is a long-standing enigma how glycogen storage disease (GSD) type I patients retain a limited capacity for endogenous glucose production despite the loss of glucose-6-phosphatase activity. Insight into the source of residual endogenous glucose production is of clinical importance given the risk of sudden death in these patients, but so far contradictory mechanisms have been proposed. We investigated glucose-6-phosphatase-independent endogenous glucose production in hepatocytes isolated from a liver-specific GSD Ia mouse model (L-G6pc-/- mice) and performed real-time analysis of hepatic glucose fluxes and glycogen metabolism in L-G6pc-/- mice using state-of-the-art stable isotope methodologies. Here we show that G6pc-deficient hepatocytes are capable of producing glucose. In vivo analysis of hepatic glucose metabolism revealed that the hepatic glucokinase flux was decreased by 95% in L-G6pc-/- mice. It also showed increased glycogen phosphorylase flux in L-G6pc-/- mice, which is coupled to the release of free glucose through glycogen debranching. Although the ex vivo activities of debranching enzyme and lysosomal acid maltase, two major hepatic α-glucosidases, were unaltered in L-G6pc-/- mice, pharmacological inhibition of α-glucosidase activity almost completely abolished residual glucose production by G6pc-deficient hepatocytes. CONCLUSION: Our data indicate that hepatocytes contribute to residual glucose production in GSD Ia. We show that α-glucosidase activity, i.e. glycogen debranching and/or lysosomal glycogen breakdown, contributes to residual glucose production by GSD Ia hepatocytes. A strong reduction in hepatic GCK flux in L-G6pc-/- mice furthermore limits the phosphorylation of free glucose synthesized by G6pc-deficient hepatocytes, allowing the release of glucose into the circulation. The almost complete abrogation of GCK flux in G6pc-deficient liver also explains the contradictory reports on residual glucose production in GSD Ia patients. (Hepatology 2017;66:2042-2054).

Hepatic lentiviral gene transfer prevents the long-term onset of hepatic tumours of glycogen storage disease type 1a in mice.

Glycogen storage disease type 1a (GSD1a) is a rare disease due to the deficiency in the glucose-6-phosphatase (G6Pase) catalytic subunit (encoded by G6pc), which is essential for endogenous glucose production. Despite strict diet control to maintain blood glucose, patients with GSD1a develop hepatomegaly, steatosis and then hepatocellular adenomas (HCA), which can undergo malignant transformation. Recently, gene therapy has attracted attention as a potential treatment for GSD1a. In order to maintain long-term transgene expression, we developed an HIV-based vector, which allowed us to specifically express the human G6PC cDNA in the liver. We analysed the efficiency of this lentiviral vector in the prevention of the development of the hepatic disease in an original GSD1a mouse model, which exhibits G6Pase deficiency exclusively in the liver (L-G6pc(-/-) mice). Recombinant lentivirus were injected in B6.G6pc(ex3lox/ex3lox). SA(creERT2/w) neonates and G6pc deletion was induced by tamoxifen treatment at weaning. Magnetic resonance imaging was then performed to follow up the development of hepatic tumours. Lentiviral gene therapy restored glucose-6 phosphatase activity sufficient to correct fasting hypoglycaemia during 9 months. Moreover, lentivirus-treated L-G6pc(-/-) mice presented normal hepatic triglyceride levels, whereas untreated mice developed steatosis. Glycogen stores were also decreased although liver weight remained high. Interestingly, lentivirus-treated L-G6pc(-/-) mice were protected against the development of hepatic tumours after 9 months of gene therapy while most of untreated L-G6pc(-/-) mice developed millimetric HCA. Thus the treatment of newborns by recombinant lentivirus appears as an attractive approach to protect the liver from the development of steatosis and hepatic tumours associated to GSD1a pathology.

In vivo hepatic lipid quantification using MRS at 7 Tesla in a mouse model of glycogen storage disease type 1a.

The assessment of liver lipid content and composition is needed in preclinical research to investigate steatosis and steatosis-related disorders. The purpose of this study was to quantify in vivo hepatic fatty acid content and composition using a method based on short echo time proton magnetic resonance spectroscopy (MRS) at 7 Tesla. A mouse model of glycogen storage disease type 1a with inducible liver-specific deletion of the glucose-6-phosphatase gene (L-G6pc(-/-)) mice and control mice were fed a standard diet or a high-fat/high-sucrose (HF/HS) diet for 9 months. In control mice, hepatic lipid content was found significantly higher with the HF/HS diet than with the standard diet. As expected, hepatic lipid content was already elevated in L-G6pc(-/-) mice fed a standard diet compared with control mice. L-G6pc(-/-) mice rapidly developed steatosis which was not modified by the HF/HS diet. On the standard diet, estimated amplitudes from olefinic protons were found significantly higher in L-G6pc(-/-) mice compared with that in control mice. L-G6pc(-/-) mice showed no noticeable polyunsaturation from diallylic protons. Total unsaturated fatty acid indexes measured by gas chromatography were in agreement with MRS measurements. These results showed the great potential of high magnetic field MRS to follow the diet impact and lipid alterations in mouse liver.

Control of blood glucose in the absence of hepatic glucose production during prolonged fasting in mice: induction of renal and intestinal gluconeogenesis by glucagon.

OBJECTIVE: Since the pioneering work of Claude Bernard, the scientific community has considered the liver to be the major source of endogenous glucose production in all postabsorptive situations. Nevertheless, the kidneys and intestine can also produce glucose in blood, particularly during fasting and under protein feeding. The aim of this study was to better define the importance of the three gluconeogenic organs in glucose homeostasis. RESEARCH DESIGN AND METHODS: We investigated blood glucose regulation during fasting in a mouse model of inducible liver-specific deletion of the glucose-6-phosphatase gene (L-G6pc(-/-) mice), encoding a mandatory enzyme for glucose production. Furthermore, we characterized molecular mechanisms underlying expression changes of gluconeogenic genes (G6pc, Pck1, and glutaminase) in both the kidneys and intestine. RESULTS: We show that the absence of hepatic glucose release had no major effect on the control of fasting plasma glucose concentration. Instead, compensatory induction of gluconeogenesis occurred in the kidneys and intestine, driven by glucagon, glucocorticoids, and acidosis. Moreover, the extrahepatic action of glucagon took place in wild-type mice. CONCLUSIONS: Our study provides a definitive quantitative estimate of the capacity of extrahepatic gluconeogenesis to sustain fasting endogenous glucose production under the control of glucagon, regardless of the contribution of the liver. Thus, the current dogma relating to the respective role of the liver and of extrahepatic gluconeogenic organs in glucose homeostasis requires re-examination.

Protein-induced satiety is abolished in the absence of intestinal gluconeogenesis.

Protein-enriched diets are well known to initiate satiety effects in animals and humans. It has been recently suggested that this might be dependent on the induction of gluconeogenesis in the intestine. The resulting intestinal glucose release, detected by a "so-called" glucose sensor located within the walls of the portal vein and connected to peripheral afferents, activates hypothalamic nuclei involved in the regulation of food intake, in turn initiating a decrease in hunger. To definitively demonstrate the role of intestinal gluconeogenesis in this mechanism, we tested the food intake response to a protein-enriched diet in mice with an intestine-specific deletion (using an inducible Cre/loxP strategy) of the glucose-6 phosphatase gene (I-G6pc(-/-) mice) encoding the mandatory enzyme for glucose production. There was no effect on food intake in I-G6pc(-/-) mice fed on a standard rodent diet compared to their wild-type counterparts. After switching to a protein-enriched diet, the food intake of wild-type mice decreased significantly (by about 20% of daily calorie intake), subsequently leading to a decrease of 12 ± 2% of initial body weight after 8 days. On the contrary, I-G6pc(-/-) mice were insensitive to the satiety effect induced by a protein-enriched diet and preserved their body weight. These results provide molecular evidence of the causal role of intestinal gluconeogenesis in the satiety phenomenon initiated by protein-enriched diets.

Targeted deletion of liver glucose-6 phosphatase mimics glycogen storage disease type 1a including development of multiple adenomas.

BACKGROUND AND AIMS: Glycogen storage disease type 1a (GSD1a) is an inherited disease caused by a deficiency in the catalytic subunit of the glucose-6 phosphatase enzyme (G6Pase). GSD1a is characterized by hypoglycaemia, hyperlipidemia, and lactic acidosis with associated hepatic (including hepatocellular adenomas), renal, and intestinal disorders. A total G6pc (catalytic subunit of G6Pase) knock-out mouse model has been generated that mimics the human pathology. However, these mice rarely live longer than 3 months and long-term liver pathogenesis cannot be evaluated. Herein, we report the long-term characterization of a liver-specific G6pc knock-out mouse model (L-G6pc(-/-)). METHODS: We generated L-G6pc(-/-) mice using an inducible CRE-lox strategy and followed up the development of hepatic tumours using magnetic resonance imaging. RESULTS: L-G6pc(-/-) mice are viable and exhibit normoglycemia in the fed state. They develop hyperlipidemia, lactic acidosis, and uricemia during the first month after gene deletion. However, these plasmatic parameters improved after 6 months. L-G6pc(-/-) mice develop hepatomegaly with glycogen accumulation and hepatic steatosis. Using an MRI approach, we could detect hepatic nodules with diameters of less than 1 mm, 9 months after induction of deficiency. Hepatic nodules (1 mm) were detected in 30-40% of L-G6pc(-/-) mice at 12 months. After 18 months, all L-G6pc(-/-) mice developed multiple hepatocellular adenomas of 1-10 mm diameter. CONCLUSIONS: This is the first report of a viable animal model of the hepatic pathology of GSD1a, including the late development of hepatocellular adenomas.

Metabolic and melanocortin gene expression alterations in male offspring of obese mice.

To study the consequences of maternal obesity during gestation and suckling periods on metabolic features and expression of genes belonging to the melanocortinergic system, we developed Diet-Induced-Obesity (DIO) in mice fed high-fat-diet (HFD). After weaning, F1-descendants were fed the same diet than dams up to 16 weeks or received a 2-week standard chow at several time points. From birth, F1-DIO displayed higher body weight than F1-control. Hyperinsulinemia, hypertriglyceridemia, hyperleptinemia were detected from P10 and fasting hyperglycaemia from 2 week-post-weaning. From late gestation to 16-week-post-weaning the expression of MC4-R gene and/or the POMC/AgRP ratio was increased, suggesting an activation of this pathway to compensate the deleterious effects of HFD. Standard chow replacement at weaning normalized metabolic status but a partial recovery was obtained for later changes. Concomitant variations in the expression of the melanocortinergic genes were observed. Therefore, early nutritional intervention could override the impact of maternal and postnatal over-nutrition.