Increased atherosclerosis in a mouse model of glycogen storage disease type 1a

Glycogen storage disease type 1a (GSD Ia) is an inborn error of carbohydrate metabolism. Despite severe hyperlipidemia, GSD Ia patients show limited atherogenesis compared to age-and-gender matched controls. Employing a GSD Ia mouse model that resembles the severe hyperlipidemia in patients, we here found increased atherogenesis in GSD Ia. These data provide a rationale for investigating atherogenesis in GSD Ia in a larger patient cohort.


Introduction
Glycogen storage disease type 1a (GSD Ia) is an inborn error of carbohydrate metabolism, caused by mutations in the gene encoding the catalytic subunit of glucose-6-phosphatase enzyme (G6PC1) [1]. G6PC1 is expressed by liver, kidney, and intestine, and is essential for conversion of glucose-6-phosphate (G6P) into glucose [2]. GSD Ia patients show severe hypoglycemia upon fasting and show 10-fold higher plasma very-low-density lipoprotein (VLDL)-triglycerides (TG) and 2-fold higher VLDL-and LDL-cholesterol compared to age-and-gender matched controls [3,4].
Despite severe hyperlipidemia, GSD Ia patients show a ~ 10% reduction in carotid intima media thickness compared to age-andgender matched controls [5], indicative of decreased atherosclerotic lesion size in the carotid arteries. This decrease has been attributed to LDL particles being less susceptible to oxidation [4], due to increased saturated fatty acids (SFA) in plasma VLDL/LDL [4]. However, diets rich in SFA are associated with increased atherosclerosis in humans [6], and replacement of SFA with polyunsaturated fatty acids decreases atherosclerosis in mice [7]. Therefore, the decrease in atherosclerosis in GSD Ia cannot be explained by increased plasma SFA levels. Further research into the mechanisms driving the decreased atherogenesis in GSD Ia patients is thus needed. We previously employed hepatocyte-specific G6pc deficient mice as a model of GSD Ia. Although these mice are slightly hyperlipidemic during fasting, their plasma cholesterol mainly circulates in high-density lipoproteins (HDL) and not in VLDL or LDL [8]. This is similar to wild-type mice. For this reason wild-type mice do not develop atherosclerosis [9]. For atherosclerosis studies, mice deficient in the LDL receptor (Ldlr − /− mice) have been generated [10]. When fed a cholesterol-rich Western-type diet (WTD), Ldlr − /− mice show high levels of VLDL/LDL-cholesterol, and develop advanced atherogenesis with plaques comparable to humans [9]. Therefore, to study atherogenesis under hyperlipidemic conditions similar to those in GSD Ia patients, we generated hepatocyte-specific G6pc deficient mice on the Ldlr − /− background and fed these mice WTD.

Animals
B6.G6pc lox/lox and B6.G6pc lox/lox .SA CreERT2 mice were intercrossed with Ldlr − /− (stock 002207; Jackson Laboratories, Bar Harbor, ME, USA) mice to generate B6.G6pc lox/lox Ldlr − /− and B6.G6pc lox/lox .SA CreERT2 Ldlr − /− mice. At 8-12 weeks of age, mice received intraperitoneal tamoxifen injections (T5648; Sigma-Aldrich, St. Louis, MO, USA) (1 mg/ day in 95% sunflower oil/5% ethanol) for five consecutive days to induce hepatocyte-specific G6pc deficiency, as described previously [11]. We refer to B6.G6pc lox/lox .SA CreERT2 Ldlr − /− and littermate B6. G6pc lox/lox Ldlr − /− mice as L-G6pc − /− Ldlr − /− and Ldlr − /− mice. Mice were housed in a light (lights on at 7:00 AM, lights off at 7:00 PM) and temperature (21 • C)-controlled facility and had ad libitum access to water and food. After tamoxifen injections, male and female mice were fed a chow diet (RMH-B, AB diets, Woerden, The Netherlands) for 4 weeks (recovery period), followed by WTD (40% fat, 0.15% cholesterol; D12079B, Research Diets, New Brunswick, NJ, USA) for 8 or 15 weeks. Mice were randomly assigned to experimental groups. The number of mice used for each experiment and the period of WTD feeding are indicated in the figure legends. No inclusion or exclusion criteria were used. Experiments were performed at 8:00 AM in fed condition or at 2:00 PM after a 6 h fasting period during the inactive period. Mice were sacrificed after 8 (~20 weeks old) or 15 (~27 weeks old) weeks of WTD feeding. All animal studies were approved by the Institutional Animal Care and Use Committee from the University of Groningen under permit number AVD105002015244 and adhered to guidelines set out in the 2010/63/EU directive.

Plasma lipoprotein analysis
Blood samples were collected by tail bleeding into EDTA-coated tubes. Plasma was separated by centrifugation and plasma cholesterol and TG levels were measured using enzymatic kits (113,009,910,026 and 157,109,910,917, respectively; Diasys Diagnostic Systems, Holzheim, Germany) with Cholesterol FS or Precimat Glycerol standard (113,009,910,030; Diasys Diagnostic Systems and 10,166,588; Roche, Mannheim, Germany, respectively) for the calibration curve. Lipoprotein cholesterol and triglyceride distribution were measured by fast performance liquid chromatography (FPLC) using a system containing a PU-4180 pump with a linear degasser and UV-4075 UV/VIS detectors (Jasco, Tokyo, Japan). Pooled plasma samples were injected onto a Superose 6 Increase 10/300 GL column (GE Healthcare, Hoevelaken, The Netherlands) and eluted at a constant flow rate of 0.31 mL/min in PBS (pH 7.4). Cholesterol or triglycerides were measured in line by addition of cholesterol or triglyceride reagent at a constant flow rate of 0.1 mL/min using an additional PU-4080i infusion pump (Jasco, Tokyo, Japan). Data acquisition and analysis were performed using ChromNav software (version 1.0; Jasco, Tokyo, Japan).

Atherosclerotic lesion analysis
Female Ldlr − /− and L-G6pc − /− Ldlr − /− mice were fed a WTD for 8 weeks and males for 15 weeks. Mice were sacrificed, hearts were isolated and fixed in 4% phosphate buffered paraformaldehyde, embedded in paraffin, and 4 μm sections of the aortic root area were made and stained with hematoxylin-eosin (H&E). Atherosclerotic lesion area was quantified using Image J software (NIH) and the average of 5 sections with 40 μm distance between the sections was calculated for each mouse.

Plasma uric acid measurement
Blood samples were collected by tail bleeding into EDTA-coated tubes. Plasma was separated by centrifugation and uric acid levels were measured using a uric acid kit (KA1651, Abnova, Tapei, Taiwan) according to the manufacturer's instructions.

Statistical analysis
All data are presented as mean ± SEM. The unpaired t-test was used to compare two datasets. Group size and statistical test are reported in the figure legends. The criterion for significance was set at P < 0.05. Statistical analysis was performed using GraphPad Prism 5.

Results
To investigate the role of hepatocyte-specific G6pc in atherogenesis, we generated L-G6pc − /− Ldlr − /− mice and Ldlr − /− littermate controls. On the Ldlr − /− background, hepatocyte-specific G6pc deficiency increased plasma cholesterol levels during WTD feeding by ~2.3-fold in female and by ~1.7-fold in male Ldlr − /− mice (Fig. 1A, B). Plasma cholesterol levels increased further over the course of WTD feeding, while the difference between the genotypes remained similar (Fig. 1A, B). Hepatocyte-specific G6pc deficiency increased plasma TG by ~2.3-fold in females and by ~1.9-fold in males at 2 weeks of WTD (Fig. 1C, D). These increases gradually declined during the study and were no longer different for male mice at 9 weeks of WTD (Fig. 1C, D). Sharing similarities with findings in GSD Ia patients, the increases in plasma lipids were reflected by an increase in VLDL-and LDL-cholesterol, as well as VLDL-TG ( Fig. 1E-H). The decreased susceptibility of LDL to oxidation in GSD Ia patients has been attributed to elevated plasma concentrations of uric acid [12]. Consistently, L-G6pc − /− Ldlr − /− mice showed increased plasma uric acid levels compared to Ldlr − /− female and male mice fed WTD (Fig. 1I, J). We then measured blood glucose levels in mice fed chow or WTD. Similar to previous data [11,13], hepatocyte-specific G6pc1 deficiency induced hypoglycemia (blood glucose ≤4.0 mM) upon a 6 h fasting period (Fig. 1K-N). However, after 2 weeks of WTD, L-G6pc − /− Ldlr − /− mice showed blood glucose levels of ~5.5 mM in both fed and fasted conditions (Fig. 1K-N). This is likely due to the WTD being rich in sucrose (35% sucrose). Sucrose rapidly increases blood glucose levels to promote insulin release [14]. When fed chronically, sucrose lowers blood glucose levels in L-G6pc − /− mice as also shown upon high fat/high sucrose (HF/HS) diet feeding in a previous study [15].
We subsequently assessed atherosclerotic lesion area in these mice. In line with guidelines for atherosclerosis studies [9], we evaluated lesion area in mice of both genders. Given that atherosclerosis develops faster in female Ldlr − /− mice than in males [9], we fed female L-G6pc − / − Ldlr − /− and Ldlr − /− mice WTD for 8 weeks, and used 15 weeks of WTD for males. In contrast to findings on carotid intima media thickness in humans [5], but in line with the increases in plasma lipid levels, hepatocyte-specific G6pc deficiency increased atherosclerotic lesion area by ~1.5-fold in female Ldlr − /− mice after 8 weeks of WTD (Fig. 1O). Male Ldlr − /− mice fed WTD for 15 weeks showed larger atherosclerotic plaques than expected based on earlier studies [16]. Hepatocyte-specific G6pc deficiency tended to increase atherosclerotic lesion area in Ldlr − /− males fed WTD (Fig. 1P). As a consequence of the longer WTD feeding in males and the lesions in Ldlr − /− mice being larger than usual, we may have missed detecting a significant increase of hepatocyte-specific G6pc deficiency on atherosclerotic plaques, though we observed a tendency that did not reach statistical significance (p = 0.10). Together, hepatocyte-specific G6pc deficiency increases plasma lipid levels, which increases atherogenesis, at least in female WTD-fed Ldlr − /− mice, and shows a tendency to an increase in lesion size in males.

Discussion
GSD Ia patients show a ~ 10% decrease in carotid intima media thickness compared to age-and-gender matched controls [5], despite hyperlipidemia reflected by 10-fold higher plasma TG and 2-fold higher cholesterol levels [4,17]. Even though the patient cohort was small (n = 9 per group), these findings suggest that GSD Ia patients are less susceptible to atherogenesis [5]. To investigate mechanisms underlying this observation, we bred L-G6pc − /− mice on the Ldlr − /− background and fed them a cholesterol-rich WTD to induce hyperlipidemia and atherosclerosis. Similar to GSD Ia patients, hepatocyte-specific G6pc deficiency increased plasma lipids in mice of both genders, and increased plasma uric acid levels. However, unlike in GSD Ia patients, hepatocyte-specific G6pc deficiency increased atherosclerotic lesion size in female WTD-fed Ldlr − /− mice, while showing a tendency towards an increase in males.
Our data suggest, in contrast to findings in GSD Ia patients [5], that hyperlipidemia in a mouse model of GSD Ia does accelerate atherosclerosis. We also found that hepatocyte-specific G6pc deficiency increased plasma uric levels. While this increase is in line with findings in GSD Ia patients, it has been suggested that elevated plasma uric acids in GSD Ia decreases LDL oxidation, and may thus be athero-protective [12]. In contrast, several epidemiological studies and studies in animal models have shown that uric acid levels are associated with an increase in atherosclerosis [18]. These studies [18] thus suggest that the increase in plasma uric acid in mice with hepatocyte-specific G6pc deficiency enhances atherosclerosis.
Further, the discrepancy between our observations and the limited atherogenesis in GSD Ia patients may be due to the model that we used. Feeding Ldlr − /− mice WTD is one of the most frequently used models to induce atherogenesis [9]. WTD contains a high percentage of sucrose (35%), and sucrose rapidly increases blood glucose levels, leading to insulin release and hypoglycemia [14]. GSD Ia patients adhere to a strict diet to prevent fasting hypoglycemia [19], including avoiding sucrose intake [20]. The recommended diet for GSD Ia patients consists of 60-70% carbohydrates with a low glycemic index, 10-15% protein, and 15-30% fat [20][21][22]. We cannot exclude that the continuous low level of blood glucose in our model may have affected cells locally in the vessel wall, and therefore, increased atherogenesis, similar to findings in humans with hypoglycemia [23]. However, elevated plasma TG levels, as observed in GSD Ia patients [3,8,17,24], have, except for one study employing Apolipoprotein C3 (APOC3) overexpression in mice [25], a clear pro-atherogenic role in mice and in humans [26][27][28][29][30][31][32]. Since only 9 patients were included in the study on atherogenesis in GSD Ia [5], and in view of the pro-atherogenic role of triglycerides and plasma uric acid, our findings do provide a rationale to investigate atherogenesis in a larger GSD Ia patient cohort.