High carbohydrate and high fat fed <i>Rai1</i>^+/− mice have altered body fat and fat distribution.

<p>(<b>A</b>). Wild type and <i>Rai1^+/−</i> mice fed normal chow did not have significantly different total body fat. Both high carbohydrate and high fat fed <i>Rai1^+/−</i> mice had significantly more body fat than high carbohydrate and high fat fed wild type mice. (<b>B</b>). <i>Rai1</i>^+/− mice on a high fat diet had significantly more subcutaneous and abdominal fat relative to high fat fed wild type mice. However high carbohydrate fed <i>Rai1</i>^+/− mice only displayed alterations to abdominal fat portions but not subcutaneous. Normal chow diet regimen did not alter the distribution of fat in either genotype. All data are plotted as means +/− SEM; ** <i>P</i><0.01; *** <i>P</i><0.001; † <i>P</i><0.05; †† <i>P</i><0.01. NC: WT n = 3, <i>Rai1^+/−</i> n = 3. HC: WT n = 2, <i>Rai1^+/−</i> n = 4. HF WT n = 3, <i>Rai1^+/−</i> n = 3.</p

Blood glucose levels are not altered due to diet regimens.

<p>Mice fed either normal chow, high carbohydrate, or high fat do not have altered blood glucose levels after 12 hours of fasting. All data are plotted as mean +/− SEM. NC: WT n = 5, <i>Rai1^+/</i>⁻n = 8. HC: WT n = 3, <i>Rai1^+/−</i> n = 7. HF: WT n = 3, <i>Rai1^+/−</i> n = 5.</p

High carbohydrate and high fat diets alter growth rates in <i>Rai1</i>^+/− mixed background mice.

<p>(<b>A</b>). All mice fed a normal chow diet had similar growth rates during adolescence (weeks 5–9) or adulthood (weeks 10–16). No significant difference was observed in the amount of weight gained during each developmental stage. (<b>B</b>). <i>Rai1^+/−</i> mice fed a high carbohydrate diet had significantly faster growth during adolescence and adulthood relative to wild type mice. <i>Rai1^+/−</i> mice gained significantly more weight during adolescence and adulthood relative to wild type. (<b>C</b>). <i>Rai1^+/−</i> mice grew significantly faster and gained more weight when fed a high fat diet during both adolescent and adult stages of development relative to wild type mice. All data are plotted as means +/− SEM; dashed line represents the separation between developmental stages. Left panel; adolescence (weeks 5–9); n.s. = not significant; *<i>P</i><0.05; **<i>P</i><0.01; adulthood (weeks 10–16); n.s. = not significant; † <i>P</i><0.05; †† <i>P</i><0.01. Right panel; * <i>P</i><0.05. NC: WT n = 4, <i>Rai1^+/−</i> n = 8. HC: WT n = 3, <i>Rai1^+/−</i> n = 7. HF: WT n = 4, <i>Rai1^+/−</i> n = 3.</p

<i>Rai1^+/−</i> mice have improved fecundity in a FVB/NJ genetic background.

<p>(<b>A</b>). <i>Rai1^+/−</i> mice in the C57Bl/6J background when mated to C57Bl/6J produced significantly fewer progeny relative to <i>Rai1^+/−</i> mice mated to a FVB/NJ genetic background. In either background, the proportion of male and female progeny was not significantly different. No significant difference was observed for transmission of the <i>Rai1^+/−</i> allele by either parent (data not shown). (<b>B</b>). The number of <i>Rai1^+/−</i> progeny produced in a C57Bl/6J genetic background is significantly less than the number of wild type progeny produced, indicating altered Mendelian ratios; however, the number of <i>Rai1^+/−</i> progeny produced when mating occurs in the FVB/NJ genetic background is not significantly different than the number of wild type progeny, consistent with Mendelian transmission. Number of litters: C57Bl/6J x C57Bl/6J n = 19; C57Bl/6J x FVB/NJ n = 8, with either parent carrying the <i>Rai1^+/−</i> allele. (<b>C</b>). C57Bl/6J:FVB/NJ mixed background mice that are heterozygous at the <i>Rai1</i> locus have significantly reduced <i>Rai1</i> expression. All data are plotted as mean +/− SEM; n.s. = not significant; **<i>P</i><0.01; ***<i>P</i><0.001; ****<i>P</i><0.0001, WT n = 8, <i>Rai1^+/−</i> n = 8.</p

<i>Rai1</i>^+/− breeding in the C57Bl/6J genetic background.

<p><i>Rai1</i>^+/− breeding in the C57Bl/6J genetic background.</p

Biochemical phenotyping unravels novel metabolic abnormalities and potential biomarkers associated with treatment of GLUT1 deficiency with ketogenic diet

<div><p>Global metabolomic profiling offers novel opportunities for the discovery of biomarkers and for the elucidation of pathogenic mechanisms that might lead to the development of novel therapies. GLUT1 deficiency syndrome (GLUT1-DS) is an inborn error of metabolism due to reduced function of glucose transporter type 1. Clinical presentation of GLUT1-DS is heterogeneous and the disorder mirrors patients with epilepsy, movement disorders, or any paroxysmal events or unexplained neurological manifestation triggered by exercise or fasting. The diagnostic biochemical hallmark of the disease is a reduced cerebrospinal fluid (CSF)/blood glucose ratio and the only available treatment is ketogenic diet. This study aimed at advancing our understanding of the biochemical perturbations in GLUT1-DS pathogenesis through biochemical phenotyping and the treatment of GLUT1-DS with a ketogenic diet. Metabolomic analysis of three CSF samples from GLUT1-DS patients not on ketogenic diet was feasible inasmuch as CSF sampling was used for diagnosis before to start with ketogenic diet. The analysis of plasma and urine samples obtained from GLUT1-DS patients treated with a ketogenic diet showed alterations in lipid and amino acid profiles. While subtle, these were consistent findings across the patients with GLUT1-DS on ketogenic diet, suggesting impacts on mitochondrial physiology. Moreover, low levels of free carnitine were present suggesting its consumption in GLUT1-DS on ketogenic diet. 3-hydroxybutyrate, 3-hydroxybutyrylcarnitine, 3-methyladipate, and N-acetylglycine were identified as potential biomarkers of GLUT1-DS on ketogenic diet. This is the first study to identify CSF, plasma, and urine metabolites associated with GLUT1-DS, as well as biochemical changes impacted by a ketogenic diet. Potential biomarkers and metabolic insights deserve further investigation.</p></div

Significant free carnitine and carnitine derivatives for 6 patients with GLUT1-DS patients.

<p>Significant free carnitine and carnitine derivatives for 6 patients with GLUT1-DS patients.</p

Plasma biochemical profiles of GLUT1-DS patients on KD.

<p>A) Low plasma free carnitine in GLUT1-DS patients on ketogenic diet. Box-plot of free and grouped bound-carnitine compounds z-scores for 6 patients with GLUT1-DS on ketogenic diet. Low levels of free carnitine were detected, while all carnitine conjugates showed higher levels in GLUT1-DS patients on ketogenic diet. B) Carnitine-bound metabolites are elevated in plasma of patients on ketogenic diet. Box-plot of free carnitine and specific carnitine-derived compound for 6 patients with GLUT1-DS on ketogenic diet. Higher levels of 3-hydroxybutyrylcarnitine is detected in face of low levels of other carnitine derived compounds (isovalerylcarnitine. 2-methylbutyrylcarnitine. 2-methylmalonyl carnitine. propionylcarnitine. succinylcarnitine) in GLUT1-DS patients on ketogenic diet.</p

Z-scores for consistently elevated compounds in plasma and urine from GLUT1-DS on KD diet.

<p>Z-scores for consistently elevated compounds in plasma and urine from GLUT1-DS on KD diet.</p

Z-scores of carbohydrate levels in CSF from patients diagnosed with GLUT1-DS not being treated with a KD.

<p>Z-scores of carbohydrate levels in CSF from patients diagnosed with GLUT1-DS not being treated with a KD.</p