In summary, the mouse model presented herein carries a homozygous Dguok point mutation (F180S) leading to an aberrant metabolic phenotype, characterized by reduced body weight and subcutaneous fat pads, while no other phenotypic abnormalities, such as hepatic or neurological involvement are present that typically characterize MDDS. Thereby, Dguok F180S/F180S mice extend the spectrum of MDDS with the possibility to specifically characterize metabolic consequences of the disease.
MDDS due to DGUOK mutations is a rare, progressive, and in most cases fatal monogenetic disease, affecting individuals. However, the phenotypical abnormalities, the time of onset and the course of the disease present with a large variety among genetically affected patients. Several frameshift or non-sense and missense mutations (e.g., M1I, M1V, S52F, E44K, K51Q, R105*, S107P, E165V, Q170R, W178X, Y191C, H226R, L248P, L250S, F256*) have been reported in patients with DGUOK deficiency (7, 11, 17–20), addressing the need for various in vitro and in vivo models to improve understanding of the varying clinical characteristics of this disease. So far, two Dguok animal models have been described: a complete Dguok knock-out (KO) rat model which did not display a pathological phenotype (21) and a complete murine Dguok KO model, indicative of a hepatic phenotype (22). Both human and mouse Dguok genes contain 277 amino acids and present homology with 75% identities and 85% positive residues (23). The position F180 is well conserved among species (among others: Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus, Macaca fascicularis, Supplemental Fig. 3). In this position, phenylalanine is the amino acid connecting two subsequent helices of the DGUOK protein. In the case of F180S substitution, the non-polar, hydrophobic phenylalanine is replaced by a polar and hydrophilic serine, affecting helix propensity, and causing structural changes in the predicted three-dimensional structure of the protein (Supplemental Fig. 4, source: https://zhanggroup.org/I-TASSER/). The previously described W178X mutation in close proximity to the F180S mutation, is associated with a severe and lethal hepato-cerebral form of MDDS in the affected individual (11). In contrast, as the F180S mutation originates from an ENU mutagenesis screen, this setting might have favored a milder phenotype.
In variance to currently described DGUOK animal models, the Dguok F180S/F180S mutant mouse line provides a metabolic phenotype. Specifically, mutant animals of both sexes had significantly lower body and inguinal fat pad weights, but no other gross phenotypical abnormalities. Furthermore, Dguok F180S/F180S mice presented hypoglycemia, which is a typical initial sign of MDDS due to Dguok deficiency in humans. However, Pronicka et al. describe an islet cell hyperplasia and a hyperinsulinemia in two patients with Dguok deficiency (17), and, similarly, a further case report presents a hyperinsulinemic hypoglycemia due to a homozygous DGUOK Phe256* mutation (24). Unlike these cases, the Dguok F180S/F180S animals presented with significantly lower insulin levels than their unaffected littermates, suggestive of a negative feedback mechanism to compensate for the hypoglycemia. In parallel, these mutant mice displayed low corticosterone values, originating from the adrenal glands, that also presented a very low mitochondrial DNA content. As adrenal steroidogenesis is partly dependent on mitochondrial function, Dguok deficiency could contribute to lack of adrenal counter-regulation thereby aggravating hypoglycemia. The Dguok F180S/F180S mouse line also displayed significantly lower cholesterol, HDL and triglyceride levels. The lipid profile of affected individuals with MDDS due to DGUOK deficiency has not been described in detail so far, but the Dguok KO mouse line, previously described, presents an opposed biochemical profile, with significantly increased cholesterol levels in the KO animals (22).
Dguok F180S/F180S mutant animals had normal or even increased intestinal absorption, excluding a malabsorption of nutrients as causative for their body weight phenotype, but presented with increased energy expenditure. The latter may be due to browning of white adipose tissue as reflected by increased Ucp1 expression in inguinal WAT as well as the appearance of multilocular fat cells on histological examination. These mice did not display any neurological abnormalities, possibly due to the lower but maintained at > 50% mitochondrial DNA content in the brain. The fact that they did not present a hepatic pathology, despite the almost nonexistent mitochondrial DNA in their liver can potentially be explained by the increased hepatic expression of deoxycytidine kinase, the key enzyme for the rescue pathway for dNTP synthesis. Surprisingly, these animals present increased mitochondrial DNA in the BAT. Nonetheless, the expression of various enzymes involved in the Krebs cycle and electron chain transport presented alterations, suggestive of a reduced flow of substrates in the Krebs cycle and a compensatory increased catalyzing of intermediate products. The current mouse model has some phenotypic overlap with the previously described Dguok KO model, that is characterized by low body weight and decreased subcutaneous fat layer but also with liver damage (22). Furthermore, this mouse line also presents an altered expression of the enzymes of the Krebs cycle (25).
DguokF180S/F180S mice have not yet been systematically observed for the assessment of their life span. According to our preliminary observation, these animals survived up to 30 weeks without further apparent phenotypical or behavioral abnormalities, in line with the preliminary survival estimates of the murine Dguok KO mouse line (22, 25). The differential mitochondrial DNA content in various tissues might play a role in the lean phenotype presented herein, and it seems, that the DguokF180S/F180S mouse line also possesses sufficient compensatory pathways ensuring sufficient mitochondrial DNA levels, that do not further influence their phenotypical appearance and survival.
Taken together, we herein describe the generation and metabolic characterization of a Dguok F180S/F180S mutant mouse line, that displays a lean phenotype, with reduced subcutaneous fat pads, characteristics of WAT browning, and increased energy expenditure. Furthermore, mutant animals are characterized by hypoglycemia, hypoinsulinemia and low lipid levels. This mouse line presents differential mitochondrial DNA quantities in various tissues and altered metabolic function in the mitochondria, but no further phenotypical abnormalities observed in MDDS forms. This mouse model could serve in the future not only in the study of MDDS forms but also in the understanding of mechanisms resulting in a lean phenotype.