Loss of glycerol catabolism confers carbon-source-dependent artemisinin resistance in Mycobacterium tuberculosis.

In view of the urgent need for new antibiotics to treat human infections caused by multidrug-resistant pathogens, drug repurposing is gaining strength due to the relatively low research costs and shorter clinical trials. Such is the case of artemisinin, an antimalarial drug that has recently been shown to display activity against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To gain insight into how Mtb is affected by artemisinin, we used RNAseq to assess the impact of artemisinin on gene expression profiles, revealing the induction of several efflux pumps and the KstR2 regulon. To anticipate the artemisinin resistance-conferring mutations that could arise in clinical Mtb strains, we performed an in vitro evolution experiment in the presence of lethal concentrations of artemisinin. We obtained artemisinin-resistant isolates displaying different growth kinetics and drug phenotypes, suggesting that resistance evolved through different pathways. Whole-genome sequencing of nine isolates revealed alterations in the glpK and glpQ1 genes, both involved in glycerol metabolism, in seven and one strains, respectively. We then constructed a glpK mutant and found that loss of glpK increases artemisinin resistance only when glycerol is present as a major carbon source. Our results suggest that mutations in glycerol catabolism genes could be selected during the evolution of resistance to artemisinin when glycerol is available as a carbon source. These results add to recent findings of mutations and phase variants that reduce drug efficacy in carbon-source-dependent ways.

Deletion of pleiotrophin impairs glucose tolerance and liver metabolism in pregnant mice: Moonlighting role of glycerol kinase.

Pleiotrophin is a pleiotropic cytokine that has been demonstrated to have a critical role in regulating energy metabolism, lipid turnover and plasticity of adipose tissue. Here, we hypothesize that this cytokine can be involved in regulatory processes of glucose and lipid homeostasis in the liver during pregnancy. Using 18-days pregnant Ptn-deficient mice, we evaluated the biochemical profile (circulating variables), tissue mRNA expression (qPCR) and protein levels of key enzymes and transcription factors involved in main metabolic pathways. Ptn deletion was associated with a reduction in body weight gain, hyperglycemia and glucose intolerance. Moreover, we observed an impairment in glucose synthesis and degradation during late pregnancy in Ptn-/- mice. Hepatic lipid content was significantly lower (73.6%) in Ptn-/- mice and was associated with a clear reduction in fatty acid, triacylglycerides and cholesterol synthesis. Ptn deletion was accompanying with a diabetogenic state in the mother and a decreased expression of key proteins involved in glucose and lipid uptake and metabolism. Moreover, Ptn-/- pregnant mice have a decreased expression of transcription factors, such as PPAR-α, regulating lipid uptake and glucose and lipid utilization. Furthermore, the augmented expression and nuclear translocation of glycerol kinase, and the decrease in NUR77 protein levels in the knock-out animals can further explain the alterations observed in hepatic glucose metabolism. Our results point out for the first time that pleiotrophin is an important player in maintaining hepatic metabolic homeostasis during late gestation, and further highlighted the moonlighting role of glycerol kinase in the regulation of maternal glucose homeostasis during pregnancy.

Glycerol biosynthesis plays an essential role in mediating cold tolerance the red imported fire ant, Solenopsis invicta.

The red imported fire ant (RIFA), Solenopsis invicta Buren is native to South America and known as a global problematic invasive species. At low temperatures, several investigations have demonstrated an increase in glycerol as a primary rapid cold hardening (RCH) component and an increase in the supercooling point. Two genes, glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK), have been identified as being involved in the glycerol production process. In this study, one GPDH and two GK sequences were extracted from RIFA transcriptome analysis (Si-GPDH, Si-GK1, and Si-GK2). All three genes were expressed in different body parts and different tissues of S. invicta that Si-GK2 showed a higher expression level than the others. According to gene expression levels by qRT-PCR analysis, the highest expression levels of three genes were observed in fat body tissues. After 1 h of exposure to low temperatures (5°C or lower), the mRNA levels of these genes significantly increased, according to expression analyses. RNA interference (RNAi) of Si-GPDH or Si-GK1 and Si-GK2 exhibited a significant downregulation at the mRNA level. The mortality rate of treated RIFA by double-stranded RNA (dsRNA) specific to GPDH and GK2 significantly increased at low temperatures. This study indicates that GPDH and GK2 as glycerol biosynthesis genes in RIFA have a high expression level to synthesize a high level of glycerol as an RCH factor and they play crucial roles in survival during the cold period.

Delayed diagnosis of complex glycerol kinase deficiency in a Chinese male infant: a case report.

BACKGROUND: Xp21 contiguous gene deletion syndrome is a rare genetic metabolic disorder with poor prognosis in infants, involving deletions of one or more genes in Xp21. When deletions of adrenal hypoplasia (AHC), Duchenne muscular dystrophy (DMD), and chronic granulomatosis (CGD) loci are included, complex glycerol kinase deficiency (CGKD) can be diagnosed. We present a case of CGKD that was initially misdiagnosed and died during treatment in our hospital in terms of improving our understanding of the clinical features and diagnosis of this disease, as well as highlighting the need for more precise dosing of corticosteroid replacement therapy. CASE PRESENTATION: A 48-day-old full-term male infant was transferred to our medical center with global growth delay and persistent vomiting. Routine laboratory tests revealed hyperkalemia, hyponatremia, and a high level of creatine kinase. The initial diagnosis was adrenal cortical hyperplasia (ACH), then revised to adrenocortical insufficiency with a normal level of ACTH detected. After supplementing the routine lipid test and urinary glycerol test, CGKD was diagnosed clinically due to positive triglyceridemia and urinary glycerol, and the follow-up gene screening further confirmed the diagnosis. The boy kept thriving after corticosteroid replacement and salt supplementation. While levels of serum ACTH and cortisol decreased and remained low after corticosteroid replacement was administered. The patient died of acute type 2 respiratory failure and hypoglycemia after an acute upper respiratory tract infection, which may be the result of adrenal crisis after infection. Infants with CGKD have a poor prognosis, so physicians should administer regular follow-ups, and parents counseling during treatment to improve the survival of patients. CONCLUSIONS: Overall, CGKD, although rare, cannot be easily excluded in children with persistent vomiting. Extensive blood tests can help to detect abnormal indicators. Adrenal crisis needs to be avoided as much as possible during corticosteroid replacement therapy.

Improvement of the catalytic performance of glycerol kinase from Bacillus subtilis by chromosomal site-directed mutagenesis.

Glycerol kinase is the key enzyme in glycerol metabolism, and its catalytic efficiency has an important effect on glycerol utilization. Based on an analysis of the glycerol utilization pathway and regulation mechanism in B. subtilis, we conducted site-directed mutagenesis of the key glycerol kinase gene (glpK) on the chromosome to improve the glycerol utilization efficiency of Bacillus subtilis. Recombinant wild-type Bacillus subtilis glycerol kinase (BsuGlpKWT) and two mutants (BsuGlpKM270I and BsuGlpKS71V) were successfully overexpressed in Escherichia coli BL21(DE3) and purified by Ni-IDA metal chelate chromatography. The specific activity of the BsuGlpKM270I mutant (62.6 U/mg) was significantly higher (296.2%) than that of wild-type BsuGlpKWT (15.8 U/mg). By contrast, the mutant BsuGlpKS71V (4.89 U/mg) exhibited lower (69.1%) activity than BsuGlpKWT, which suggested that variant S71V exhibited reduced catalytic efficiency for the substrate. Furthermore, the mutant strain B. subtilis M270I was constructed using a markerless delivery system, and exhibited a higher specific growth rate (improved by 11.3%, from 0.453 ± 0.012 to 0.511 ± 0.017 h-1) and higher maximal biomass (cell dry weight increased by 16%, from 0.577 ± 0.033 to 0.721 ± 0.015 g/L) than the parental strain with a shortened lag phase (2 ~ 4 h shorter) in M9 minimal medium with glycerol. These results indicate that the mutated glpK resulted in improved glycerol utilization, which has broad application prospects.

GlcA-mediated glycerol-3-phosphate synthesis contributes to the oxidation resistance of Aspergillus fumigatus via decreasing the cellular ROS.

Fungi activate corresponding metabolic pathways in response to different carbon sources to adapt to different environments. Previous studies have shown that the glycerol kinase GlcA that phosphorylates glycerol to the intermediate glycerol-3-phosphate (G3P) is required for the growth of Aspergillus fumigatus when glycerol is used as the sole carbon source. The present study identified there were two putative glycerol kinases, GlcA and GlcB, in A. fumigatus but glycerol activated only glcA promoter but not glcB promoter, although both glcA and glcB could encode glycerol kinase. Under normal culture conditions, the absence of glcA caused no detectable colony phenotypes on glucose and other tested carbon sources except glycerol, indicating dissimilation of glucose and these tested carbon sources bypassed requirement of glcA. Notably, the oxidative stress agent H2O2 on the background of glucose medium clearly induced GlcA expression and promoted G3P synthesis. Deletion and overexpression of glcA elicited sensitivity and resistance to oxidative stress agent H2O2, respectively, accompanied by decrease and increase of G3P production. In addition, the sensitivity to oxidative stress in the glcA mutant was probably associated with dysfunction of mitochondria with a decreased mitochondrial membrane potential and an abnormal accumulation of the cellular reactive oxygen species (ROS). Furthermore, overexpressing the glycerol-3-phosphate dehydrogenase GfdA thatcatalyzes the reduction of dihydroxyacetone phosphate (DHAP) to G3P rescued phenotypes of the glcA null mutant to H2O2. Therefore, the present study suggests that GlcA-involved G3P synthesis participates in oxidative stress tolerance of A. fumigatus via regulating the cellular ROS level.

A rare co-occurrence of duchenne muscular dystrophy, congenital adrenal hypoplasia and glycerol kinase deficiency due to Xp21 contiguous gene deletion syndrome: case report.

BACKGROUND: Contiguous gene deletion syndromes are rare genomic disorders caused by deletion of large segments of DNA resulting in co-occurrence of apparently unrelated multiple clinical phenotypes. We report a boy with contiguous gene deletion involving Xp21 genomic location. CASE PRESENTATION: A Sri Lankan boy with developmental delay and failure to thrive first presented at three years of age with hypovolaemia, hyperpigmentation and drowsiness. Investigations done at that time revealed hypoglycaemia, hyponatraemia, hyperkalaemia, low cortisol, low aldosterone, high ACTH and low 17-hydroxyprogesterone. He was diagnosed to have primary adrenal insufficiency. During follow-up at five years, he was noted to have progressive difficulty in walking, waddling gait, hypotonia, calf hypertrophy and positive Gower's sign. His creatine kinase was very high, and the electromyogram showed myopathy. Genetic analysis revealed hemizygous deletion involving the final 35 exons of the dystrophin gene confirming the diagnosis of Duchenne muscular dystrophy. Further investigations revealed pseudohypertriglyceridemia, large glycerol peak on urine organic acid analysis and hemizygous deletion of the glycerol kinase gene confirming glycerol kinase deficiency. Based on the presence of Duchenne muscular dystrophy, glycerol kinase deficiency and probable congenital adrenal hypoplasia along with genetic confirmation of deletions involving dystrophin and glycerol kinase genes, the diagnosis of Xp21 contiguous gene deletion syndrome was made. CONCLUSIONS: We report a child with contiguous gene deletion syndrome who was initially diagnosed as having isolated primary adrenal insufficiency probably due to congenital adrenal hypoplasia. Later he was confirmed to have Duchenne muscular dystrophy and glycerol kinase deficiency, as well. This case report highlights the importance of pre-emptive evaluation and identification of genetic defects when patients present with seemingly unrelated diseases that could aid in accurate diagnoses of contiguous gene deletion syndromes.

Exposure to particulate matter 2.5 and cigarette smoke induces the synthesis of lipid droplets by glycerol kinase 5.

Particulate matter (PM2.5) and cigarette smoke exposure are leading factors contributing to various diseases, especially respiratory diseases. The purpose of this research was to study the effects of PM2.5 and cigarette smoke on glycerol kinase 5 (GK5) expression and the possible mechanisms by which GK5 participates in lipid droplet (LD) synthesis in alveolar epithelial A549 cells. Real-time polymerase chain reaction (RT-PCR) and western blotting have been used for the detection of messenger RNA (mRNA) and protein expression respectively. GK5 overexpressing cells were established by lentivirus transfection, whereby lentiviral vectors deliver the gene into chromosomes, allowing stable expression. Affymetrix microarray analysis, a widely used tool for measuring genome-wide gene expression, has been used to explore differential gene expression profiles. A549 cells stimulated with PM2.5 and cigarette smoke extract (CSE) showed elevated GK5 expression in a dose-dependent manner. Transmission electron microscopy and oil red O staining were used to observe LDs in cells. Further, GK5 overexpressing cells showed increased LDs and upregulation of genes and proteins related to lipogenesis and lipid transportation. Affymetrix microarray analysis revealed that GK5 overexpression resulted in the differential expression of more than 109 genes, which were mainly involved in the regulation of cell death, cell survival, cellular movement and migration, and those involved in cellular growth and proliferation pathways. Overall, this study demonstrates that GK5 is upregulated during PM2.5 and cigarette smoke exposure and induces LD synthesis.

Plasma glycerol levels in men with hypertriglyceridemia.

When plasma triglyceride is assessed in standard laboratories, it is a measurement of plasma glycerol after hydrolysis of triglycerides into fatty acids and glycerol. In most patients, the plasma level of free glycerol will only marginally influence the measurement of plasma triglyceride. However, in rare cases elevated free glycerol concentrations causes pseudohypertriglyceridemia and blanking for free glycerol becomes important. In this study, we investigated the plasma free glycerol level in 100 adult men with mild to moderate hypertriglyceridemia to assess the need for providing a free glycerol measurement in our clinical biochemistry department. The plasma samples were obtained in our blood sampling facility that receives both in- and outpatients. The highest plasma level of free glycerol observed was 300 µmol/L and in 99% of the investigated men the inclusion of plasma free glycerol in the measurement of plasma triglyceride cause a less than 10% false increase in plasma triglyceride. A weak positive correlation between the plasma levels of free glycerol and triglyceride was observed. When subdividing the cohort into mild and moderate hypertriglyceridemia, the positive correlation was only maintained in the moderate hypertriglyceridemia group that also demonstrated a 23% higher plasma glycerol level than men with mild hypertriglyceridemia. We conclude that even though glycerol blanking is relevant in rare occasions, then this study does not support providing such a measurement in our department. The positive correlation between free glycerol and triglyceride in this cohort likely reflects a shared association with metabolic dysregulation.

Glycerol kinase enhances hepatic lipid metabolism by repressing nuclear receptor subfamily 4 group A1 in the nucleus.

Glycerol kinase (GYK) plays a critical role in hepatic metabolism by converting glycerol to glycerol 3-phosphate in an ATP-dependent reaction. GYK isoform b is the only glycerol kinase present in whole cells, and has a non-enzymatic moonlighting function in the nucleus. GYK isoform b acts as a co-regulator of nuclear receptor subfamily 4 group A1 (NR4A1) and participates in the regulation of hepatic glucose metabolism by protein-protein interaction with NR4A1. Herein, GYK expression was found to upregulate the expression of NR4A1-mediated lipid metabolism-related genes (SREBP1C, FASN, ACACA, and GPAM) in HEK293T and L02 cells, and in mouse in vivo studies. GYK expression increased blood levels of cholesterol, triglyceride, and high-density lipoprotein cholesterol, but not low-density lipoprotein cholesterol levels. It enhanced the transcriptional activity of Nr4a1 target genes by negatively cooperating with NR4A1 and its enzymatic activity or by other undefined moonlighting functions. This enhancement was observed in both normal and diabetic mice. We also found a feed-forward regulation loop between GYK and NR4A1, serving as part of a GYK-NR4A1 regulatory mechanism in hepatic metabolism. Thus, GYK regulates the effect of NR4A1 on hepatic lipid metabolism in normal and diabetic mice, partially through the cooperation of GYK and NR4A1.