Glucose-free/high-protein diet improves hepatomegaly and exercise intolerance in glycogen storage disease type III mice

Abstract

Glycogen disease type III (GSDIII), a rare incurable autosomal recessive disorder due to glycogen debranching enzyme deficiency, presents with liver, heart and skeletal muscle impairment, hepatomegaly and ketotic hypoglycemia. Muscle weakness usually worsens to fixed myopathy and cardiac involvement may present in about half of the patients during disease. Management relies on careful follow-up of symptoms and diet. No common agreement was reached on sugar restriction and treatment in adulthood. We administered two dietary regimens differing in their protein and carbohydrate content, high-protein (HPD) and high-protein/glucose-free (GFD), to our mouse model of GSDIII, starting at one month of age. Mice were monitored, either by histological, biochemical and molecular analysis and motor functional tests, until 10 months of age. GFD ameliorated muscle performance up to 10 months of age, while HPD showed little improvement only in young mice. In GFD mice, a decreased muscle glycogen content and fiber vacuolization was observed, even in aged animals indicating a protective role of proteins against skeletal muscle degeneration, at least in some districts. Hepatomegaly was reduced by about 20%. Moreover, the long-term administration of GFD did not worsen serum parameters even after eight months of high-protein diet. A decreased phosphofructokinase and pyruvate kinase activities and an increased expression of Krebs cycle and gluconeogenesis genes were seen in the liver of GFD fed mice. Our data show that the concurrent use of proteins and a strictly controlled glucose supply could reduce muscle wasting, and indicate a better metabolic control in mice with a glucose-free/high-protein diet.

Keywords: Glucose-free diet; Glycogen debranching enzyme; Glycogen storage disease type III; High-protein diet.

Fig. 1

Short- and long-term effects of HPD and GFD on liver and heart in GSDIII mice. A) Liver glycogen content in HPD- and GFD-KO mice did not show any reduction respect to untreated mice. B) Liver weight expressed as percentage of whole body weight. HPD did not reduce hepatomegaly in treated mice. Instead, hepatomegaly was significantly reduced in mice fed with GFD at any time point showing long-term effects of GFD. C) Glycogen content assessed in heart was not reduced in HPD-treated mice at any time point. GFD-KO mice showed a significant reduction of glycogen storage at 2 months of age respect to untreated mice. D) Heart weight expressed as percentage of whole body weight. In HPD-KO mice of 2 and 3 months of age heart appeared enlarged respect to SD-KO mice, whereas heart size was not different between GFD-KO and SD-KO mice. Data are shown as mean ± standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001 vs. SD-KO mice.

Fig. 2

Short- and long-term effects of HPD and GFD on skeletal muscle in GSDIII mice. A, B) Glycogen content in gastrocnemius showed a significant reduction in GFD-KO mice at the last time point respect to untreated mice. When vastus was tested, GFD-KO mice showed a statistically significant decrease of glycogen at each time point. HPD did not show differences in glycogen content neither in gastrocnemius nor in vastus except a statically significant increase at 3 months of age. C) Run test. After 10 min of warm-up, mice were run on a treadmill with a 5° incline and a speed of 30 cm/s for a maximum of 5 min (SD-WT n = 5 (3 M, 2F); SD-KO n = 10 (6 M, 4F), except 5-month-old SD-KO n = 5 (3 M, 2F); HPD-KO n = 10 (7 M, 3F); GFD-KO n = 9 (3 M, 6F), except 9 and 10 month-old GFD-KO n = 5 (5F)). HPD-KO mice were able to run for longer time respect to mice fed with SD until 4 months of age. At 5 months of age, both the treated and the untreated groups were able to run just for few seconds. GFD-KO mice showed a substantial and significant improvement of their motor abilities. They were able to run for longer time respect to mice fed with SD and, even better, the muscle performance was maintained for a long period respect to SD-KO mice. At 10 months of age, the muscle performance of the treated group declined but was still higher than muscle performance of untreated 5 month-old mice. Data are shown as mean ± standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001 vs. SD-KO mice.

Fig. 3

PAS staining. Liver showed a considerable accumulation of glycogen in all the mice tested, independently of the diet (A-D). PAS staining showed an important reduction in glycogen storage in muscular and cardiac tissues from GFD-KO mice compared to HPD- and SD-KO mice (E-H; J-L). Glycogen content in kidney from SD-KO mice did not show any changes respect to WT mice (M-P). Analyzed tissues were from 3-month old mice. Liver (A-D) and heart (J-L): bar 25 μm. Skeletal muscle (vastus) (E-H) and kidney (M-P): bar 40 μm.

Fig. 4

Histological findings in HPD-KO and GFD-KO mice compared to untreated KO mice. Representative light microscopy images showing muscle tissues from WT, SD-KO, HPD-KO and GFD-KO mice at 2, 3, and 9 months of age. No differences were observed in HPD-KO mice in anterior tibialis and diaphragm, that remained very compromised despite the diet. A slight improvement in the morphological features of vastus and gastrocnemius was noted in HPD-KO mice at all ages examined. In GFD-KO mice both vastus and gastrocnemius showed a marked reduction of vacuoles that was maintained even in older mice. Diaphragm is one of the most compromised muscles in GSDIII mice since young age. In mice treated with GFD, diaphragm showed both a reduction of vacuolization and a better preservation of muscle architecture at any time point. Conversely, the anterior tibialis remained a highly compromised muscle. Bar: 40 μm.

Fig. 5

Metabolic outcomes of GFD in fasted and unfasted mice. A, B) Cholesterol and triglyceride evaluation showed a decrease of cholesterol content in fasted GFD-KO mice, whereas triglycerides were the same in both groups and in both conditions. C-E) ALT, AST and ALP showed a high increase in fasted SD-KO mice, while fasted GFD-KO mice showed only a slight increase in these values respect to ad libitum fed mice. F) CK levels dramatically increased in fasted SD-KO mice. In fasted GFD-KO mice CK level showed only a slight increase not significant. G) As expected, in fasted mice blood glucose was lower than in fed mice. H) Because of the high protein intake, BUN was higher in ad libitum-fed GFD-KO mice than in SD-KO. In fasted GFD-KO mice urea in the bloodstream decreased respect to ad libitum fed GFD-KO mice, on the contrary in fasted SD-KO mice BUN increased. These data highlight that GFD exerts a protective role against muscle proteolysis during fasting. The BUN/creatinine ratio decreased in fasted GFD-KO mice respect to ad libitum GFD-KO mice due to the stop of protein intake. Except for G (n = 6, 3 M, 3F), fasted GFD-KO mice: n = 5 (3 M, 2F) for each group; fasted SD-KO mice: n = 4 (2 M, 2F) for each group; ad libitum fed mice: n = 3 (2 M, 1F) for each group. Data are shown as mean ± standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6

Gene expression analysis in the liver and the skeletal muscle of Agl-KO and GFD-KO mice compared to WT mice. A, D) Liver and vastus of 2-month old mice (n = 4, (2 M, 2F)) were examined and the expression profiling of genes involved in glucose and glycogen metabolism was analyzed. B, E) Heat maps of liver (B) and skeletal muscle (E) showed gene expression profile in untreated Agl-KO mice compared to WT mice. C, F) Heat maps of liver (C) and skeletal muscle (F) showed gene expression profile in GFD-KO mice compared to WT mice. G) Color code for the different pathways analyzed.