Glycerol Kinase Expression Research Articles

Glycerol Kinase Drives Hepatic de novo Lipogenesis and Triglyceride Synthesis in Nonalcoholic Fatty Liver by Activating SREBP-1c Transcription, Upregulating DGAT1/2 Expression, and Promoting Glycerol Metabolism.

Glycerol kinase (GK) participates in triglyceride (TG) synthesis by catalyzing glycerol metabolism. Whether GK contributes to nonalcoholic fatty liver (NAFL) is unclear. The expression of hepatic Gk is found to be increased in diet-induced and genetic mouse models of NAFL and is positively associated with hepatic SREBP-1c expression and TG levels. Cholesterol and fatty acids stimulate GK expression in hepatocytes. In HFD-induced NAFL mice, knockdown of hepatic Gk decreases expression of SREBP-1c and its target lipogenic genes as well as DGAT1/2, increases serum glycerol levels, decreases serum TG levels, and attenuates hepatic TG accumulation. Overexpression of GK in hepatocytes in mice or in culture produces opposite results. Mechanistic studies reveal that GK stimulates SREBP-1c transcription directly by binding to its gene promoter and indirectly by binding to SREBP-1c protein, thereby increasing lipogenic gene expression and de novo lipogenesis. Studies with truncated GK and mutant GKs indicate that GK induces SREBP-1c transcription independently of its enzyme activity. GK contributes to lipid homeostasis under physiological conditions by catalyzing glycerol metabolism rather than by regulating SREBP-1c transcription. Collectively, these results demonstrate that increased hepatic GK promotes de novo lipogenesis and TG synthesis in NAFL by stimulating SREBP-1c transcription and DGAT1/2 expression and catalyzing glycerol metabolism.

Glycerol kinase enzyme is a prognostic predictor in esophageal carcinoma and is associated with immune cell infiltration

The influence of lipid metabolism on tumorigenesis and progression has garnered significant attention. However, the role of Glycerol Kinase (GK), a key enzyme in glycerol metabolism, in Esophageal Carcinoma (ESCA) remains unclear. To further elucidate the relationship between GK and ESCA, we investigated GK expression levels using database information. Controlled studies employing immunohistochemistry were conducted on clinical ESCA tumor samples and normal specimens, confirming GK’s elevated expression in ESCA. Analysis of The Cancer Genome Atlas (TCGA) data via Kaplan–Meier (KM) survival plots revealed that increased GK expression correlates with poorer ESCA patient outcomes, particularly in overall survival (OS) and disease-specific survival (DSS). Multiple regression analysis indicated that elevated GK expression is an independent risk factor affecting ESCA prognosis. Statistical analysis of prognostic data from clinical samples further corroborated this finding. Moreover, there appears to be a significant correlation between GK expression and immune infiltration, specifically involving certain T and B lymphocytes. In conclusion, elevated GK expression in ESCA is strongly linked to poor prognosis and increased immune cell infiltration, highlighting its potential as an independent prognostic biomarker and a viable therapeutic target.

Glycerol biosynthetic pathway plays an essential role in proliferation and antioxidative defense in the human enteric protozoan parasite Entamoeba histolytica

Amebiasis is caused by the protozoan parasite Entamoeba histolytica. Treatment options other than metronidazole and its derivatives are few, and their low efficacy against asymptomatic cyst carriers, and experimental evidence of resistance in vitro justify the discovery/repurposing campaign for new drugs against amebiasis. Global metabolic responses to oxidative stress and cysteine deprivation by E. histolytica revealed glycerol metabolism may represent a rational target for drug development. In this study using 14C-labelled glucose, only 11% of the total glucose taken up by E. histolytica trophozoites is incorporated to lipids. To better understand the role of glycerol metabolism in this parasite, we focused on characterizing two important enzymes, glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (G3PDH). Recombinant GK was biochemically characterized in detail, while G3PDH was not due to failure of protein expression and purification. GK revealed novel characteristics and unprecedented kinetic properties in reverse reaction. Gene silencing revealed that GK is essential for optimum growth, whereas G3PDH is not. Gene silencing of G3PDH caused upregulated GK expression, while that of GK resulted in upregulation of antioxidant enzymes as shown by RNA-seq analysis. Although the precise molecular link between GK and the upregulation of antioxidant enzymes was not demonstrated, the observed increase in antioxidant enzyme expression upon GK gene silencing suggests a potential connection between GK and the cellular response to oxidative stress. Together, these results provide the first direct evidence of the biological importance and coordinated regulation of the glycerol metabolic pathways for proliferation and antioxidative defense in E. histolytica, justifying the exploitation of these enzymes as future drug targets.

Measuring the Rate of Lipolysis in Ex vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro.

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are sequentially cleaved from the glycerol backbone, resulting in the release of free fatty acids and glycerol. Due to the low expression of glycerol kinase in white adipocytes, glycerol re-uptake rates are negligible, while fatty acid re-uptake is dictated by the fatty acid binding capacity of media components such as albumin. Both glycerol and fatty acid release into media can be quantified by colorimetric assays to determine the lipolytic rate. By measuring these factors at multiple time points, one can determine the linear rate of lipolysis with high confidence. Here, we provide a detailed protocol for the measurement of lipolysis in in vitro differentiated adipocytes and ex vivo adipose tissue from mice. This protocol may also be optimized for other preadipocyte cell lines or adipose tissue from other organisms; considerations and optimization parameters are discussed. This protocol is designed to be useful in determining and comparing the rate of adipocyte lipolysis between mouse models and treatments.

Pioglitazone Reverses Markers of Islet Beta-Cell De-Differentiation in db/db Mice While Modulating Expression of Genes Controlling Inflammation and Browning in White Adipose Tissue from Insulin-Resistant Mice and Humans.

Obesity, insulin resistance, and type 2 diabetes contribute to increased morbidity and mortality in humans. The db/db mouse is an important mouse model that displays many key features of the human disease. Herein, we used the drug pioglitazone, a thiazolidinedione with insulin-sensitizing properties, to investigate blood glucose levels, indicators of islet β-cell health and maturity, and gene expression in adipose tissue. Oral administration of pioglitazone lowered blood glucose levels in db/db mice with a corresponding increase in respiratory quotient, which indicates improved whole-body carbohydrate utilization. In addition, white adipose tissue from db/db mice and from humans treated with pioglitazone showed increased expression of glycerol kinase. Both db/db mice and humans given pioglitazone displayed increased expression of UCP-1, a marker typically associated with brown adipose tissue. Moreover, pancreatic β-cells from db/db mice treated with pioglitazone had greater expression of insulin and Nkx6.1 as well as reduced abundance of the de-differentiation marker Aldh1a3. Collectively, these findings indicate that four weeks of pioglitazone therapy improved overall metabolic health in db/db mice. Our data are consistent with published reports of human subjects administered pioglitazone and with analysis of human adipose tissue taken from subjects treated with pioglitazone. In conclusion, the current study provides evidence that pioglitazone restores key markers of metabolic health and also showcases the utility of the db/db mouse to understand mechanisms associated with human metabolic disease and interventions that provide therapeutic benefit.

Hepatic transcriptome of the freeze-tolerant Cope\u2019s gray treefrog, Dryophytes chrysoscelis: responses to cold acclimation and freezing

BackgroundCope’s gray treefrog, Dryophytes chrysoscelis, withstands the physiological challenges of corporeal freezing, partly by accumulating cryoprotective compounds of hepatic origin, including glycerol, urea, and glucose. We hypothesized that expression of genes related to cryoprotectant mobilization and stress tolerance would be differentially regulated in response to cold. Using high-throughput RNA sequencing (RNA-Seq), a hepatic transcriptome was generated for D. chrysoscelis, and gene expression was compared among frogs that were warm-acclimated, cold-acclimated, and frozen.ResultsA total of 159,556 transcripts were generated; 39% showed homology with known transcripts, and 34% of all transcripts were annotated. Gene-level analyses identified 34,936 genes, 85% of which were annotated. Cold acclimation induced differential expression both of genes and non-coding transcripts; freezing induced few additional changes. Transcript-level analysis followed by gene-level aggregation revealed 3582 differentially expressed genes, whereas analysis at the gene level revealed 1324 differentially regulated genes. Approximately 3.6% of differentially expressed sequences were non-coding and of no identifiable homology. Expression of several genes associated with cryoprotectant accumulation was altered during cold acclimation. Of note, glycerol kinase expression decreased with cold exposure, possibly promoting accumulation of glycerol, whereas glucose export was transcriptionally promoted by upregulation of glucose-6-phosphatase and downregulation of genes of various glycolytic enzymes. Several genes related to heat shock protein response, DNA repair, and the ubiquitin proteasome pathway were upregulated in cold and frozen frogs, whereas genes involved in responses to oxidative stress and anoxia, both potential sources of cellular damage during freezing, were downregulated or unchanged.ConclusionOur study is the first to report transcriptomic responses to low temperature exposure in a freeze-tolerant vertebrate. The hepatic transcriptome of Dryophytes chrysoscelis is responsive to cold and freezing. Transcriptomic regulation of genes related to particular pathways, such as glycerol biosynthesis, were not all regulated in parallel. The physiological demands associated with cold and freezing, as well as the transcriptomic responses observed in this study, are shared with several organisms that face similar ecophysiological challenges, suggesting common regulatory mechanisms. The role of transcriptional regulation relative to other cellular processes, and of non-coding transcripts as elements of those responses, deserve further study.

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.

Acute Iron Dextran Injection Increases Liver Weight and Reduces Glycerol Kinase Expression in Liver

Iron is essential and needed in a very small amount. When iron exceeds normal need, metabolic alteration occurs, causing hepatosteatosis. The mechanism of iron inducing hepatosteatosis remains unclear. Glycerol kinase, the enzyme responsible in triglyceride synthesis initiation, is assumed to have a role in the pathomechanism of hepatosteatosis. This study aimed to investigate the gene expression of glycerol kinase in an acute iron overload condition. This study was conducted in Animal Laboratory Faculty of Medicine and Central Laboratory Universitas Padjadjaran from May to June 2017. Three groups of mice were divided by the dose of iron dextran injection (0, 0.1, 0.3 mg/day/mice). After 19 days, mice were terminated, liver weight was measured and glycerol kinase gene expression in the liver was determined by semi-qualitative PCR. Quantification of PCR result was calculated by ImageJ software. There was a significant change in liver weight of the mice in a dose-dependent manner of iron injection. The expression of glycerol kinase tended to decrease, but statistically insignificant. Acute iron dextran injection increases liver weight and tends to reduce glycerol kinase gene expression in mice liver.Keywords: Glycerol kinase, hepatosteatosis, iron overload Efek Zat Besi Dosis Tinggi Akut dalam Meningkatkan Berat Organ dan Menurunkan Ekspresi Gliserol Kinase HeparAbstrakZat besi merupakan nutrien esensial dan diperlukan dalam jumlah yang sangat kecil. Ketika kadar zat besi melebihi kadar normal dalam tubuh, terjadi perubahan metabolisme yang menyebabkan hepatosteatosis. Mekanisme zat besi dalam menyebabkan hepatosteatosis masih belum diketahui secara pasti. Gliserol kinase, enzim yang menginisiasi sintesis trigliserida, diduga berperan dalam patomekanisme hepatosteatosis. Penelitian ini bertujuan untuk meneliti ekspresi gen gliserol kinase pada hepar pada kondisi tinggi zat besi akut. Penelitian ini dilakukan di Laboratorium Hewan Fakultas Kedokteran dan Laboratorium Sentral Universitas Padjadjaran dari bulan Mei sampai dengan Juni 2017. Tiga kelompok mencit dibagi berdasarkan dosis injeksi iron dextran intraperitoneal (0, 0,1, 0,3 mg/hari/ekor). Setelah 19 hari, mencit diterminasi, berat hepar ditimbang dan ekspresi gen gliserol kinase diukur dengan metode semi-kualitatif PCR. Kuantifikasi hasil PCR dilakukan dengan menggunakan aplikasi ImageJ. Terdapat peningkatan berat hepar secara signifikan yang sejalan dengan dosis ijeksi zat besi. Ekspresi gen gliserol kinase cenderung menurun, meskipun secara statistik tidak signifikan. Keadaan tinggi kadar zat besi yang akut meningkatkan berat hepar dan cenderung menurunkan ekspresi gen gliserol kinase pada hepar mencit.Kata kunci: Gliserol kinase, hepatosteatosis, zat besi berlebih

To inhibit growth and migration of glioma cells through down regulation glucose metabolism related to aquaporin (AQP)-1

Background: Glioma is one of highly aggressive and fastest growing human tumors. aquaporin (AQP)-1 is a critical factor in cell function, especially in glioma cells. However, its mechanism is unknown. Here, we explored growth and migration mechanism of glioma cells associated with glucose metabolism regulated by AQP-1. Methods: The growth and migration of glioma cells were analyzed after up or down regulation of AQP-1 expression. The expression of glucose metabolism-related proteins G6PC (glucose-6-phosphatase, catalytic subunit), GK (glycerol kinase), and GOT1 (glutamic-oxaloacetic transaminase 1) were examined. Glucose production of glioma cells was determined with colorimetric glucose oxidase method. Results: Our work indicated that AQP-1 facilitated growth and migration of glioma cells by regulating glucose metabolism, which was related to down-expression of gluconeogenesis gene G6PC, GK and GOT1. Interference on AQP-1 expression suppressed these processes of glioma cells. Conclusions: Therefore, our data demonstrated that down regulation of AQP-1 can induce the inhibition of glioma cell growth and migration through the pathway related to gluconeogenesis gene G6PC, GK and GOT1, which may promote the glucose metabolism. AQP-1 is the potential novel target for treatment of glioma.

Glycerol supports growth of the Trypanosoma brucei bloodstream forms in the absence of glucose: Analysis of metabolic adaptations on glycerol-rich conditions.

The bloodstream forms of Trypanosoma brucei (BSF), the parasite protist causing sleeping sickness, primarily proliferate in the blood of their mammalian hosts. The skin and adipose tissues were recently identified as additional major sites for parasite development. Glucose was the only carbon source known to be used by bloodstream trypanosomes to feed their central carbon metabolism, however, the metabolic behaviour of extravascular tissue-adapted parasites has not been addressed yet. Since the production of glycerol is an important primary function of adipocytes, we have adapted BSF trypanosomes to a glucose-depleted but glycerol-rich culture medium (CMM_Glyc/GlcNAc) and compared their metabolism and proteome to those of parasites grown in standard glucose-rich conditions (CMM_Glc). BSF were shown to consume 2-folds more oxygen per consumed carbon unit in CMM_Glyc/GlcNAc and were 11.5-times more sensitive to SHAM, a specific inhibitor of the plant-like alternative oxidase (TAO), which is the only mitochondrial terminal oxidase expressed in BSF. This is consistent with (i) the absolute requirement of the mitochondrial respiratory activity to convert glycerol into dihydroxyacetone phosphate, as deduced from the updated metabolic scheme and (ii) with the 1.8-fold increase of the TAO expression level compared to the presence of glucose. Proton NMR analysis of excreted end products from glycerol and glucose metabolism showed that these two carbon sources are metabolised through the same pathways, although the contributions of the acetate and succinate branches are more important in the presence of glycerol than glucose (10.2% versus 3.4% of the excreted end products, respectively). In addition, metabolomic analyses by mass spectrometry showed that, in the absence of glucose, 13C-labelled glycerol was incorporated into hexose phosphates through gluconeogenesis. As expected, RNAi-mediated down-regulation of glycerol kinase expression abolished glycerol metabolism and was lethal for BSF grown in CMM_Glyc/GlcNAc. Interestingly, BSF have adapted their metabolism to grow in CMM_Glyc/GlcNAc by concomitantly increasing their rate of glycerol consumption and decreasing that of glucose. However, the glycerol kinase activity was 7.8-fold lower in CMM_Glyc/GlcNAc, as confirmed by both western blotting and proteomic analyses. This suggests that the huge excess in glycerol kinase that is not absolutely required for glycerol metabolism, might be used for another yet undetermined non-essential function in glucose rich-conditions. Altogether, these data demonstrate that BSF trypanosomes are well-adapted to glycerol-rich conditions that could be encountered by the parasite in extravascular niches, such as the skin and adipose tissues.

Aberrant expression of miR-451a contributes to 1,2-dichloroethane-induced hepatic glycerol gluconeogenesis disorder by inhibiting glycerol kinase expression in NIH Swiss mice.

The identification of aberrant microRNA (miRNA) expression during chemical-induced hepatic dysfunction will lead to a better understanding of the substantial role of miRNAs in liver diseases. 1,2-Dichloroethane (1,2-DCE), a chlorinated organic toxicant, can lead to hepatic abnormalities in occupationally exposed populations. To explore whether aberrant miRNA expression is involved in liver abnormalities mediated by 1,2-DCE exposure, we examined alterations in miRNA expression patterns in the livers of NIH Swiss mice after dynamic inhalation exposure to 350 or 700 mg m-3 1,2-DCE for 28 days. Using a microarray chip, we discovered that only mmumiR-451a was significantly upregulated in the liver tissue of mice exposed to 700 mg m-3 1,2-DCE; this finding was validated by quantitative real-time polymerase chain reaction. In vitro study revealed that it was metabolite 2-chloroacetic acid, not 1,2-DCE that resulted in the upregulation of mmu-miR-451a in the mouse AML12 cell line. Furthermore, our data showed that the upregulation of mmu-miR-451a induced by 2-chloroacetic acid could suppress the expression of glycerol kinase and lead to the inhibition of glycerol gluconeogenesis in mouse liver tissue and AML12 cells. These observations provide evidence that hepatic mmu-miR-451a responds to 1,2-DCE exposure and might induce glucose metabolism disorders by suppressing the glycerol gluconeogenesis process.

Tissue-specific effects of estrogen on glycerol channel aquaporin 7 expression in an ovariectomized mouse model of menopause

Objective: Elevated fat mass and redistribution of body fat are commonly observed in postmenopausal women. Aquaporin 7 (AQP7), a unique glycerol permeable integral membrane protein, has been associated with the onset of obesity. We hypothesized that estrogen supplementation could counteract this fat accumulation and redistribution through tissue-specific modulation of AQP7.Methods: We measured fat depot weight, adipocyte size, and the expression of AQP7 and glycerol kinase (GK) in visceral and subcutaneous fat tissues of ovariectomized mice supplemented with or without 17β-estradiol.Results: Removal of the ovaries resulted in a significant decrease in AQP7 expression and an increase in GK expression in visceral adipocyte tissue; expression of AQP7 and GK in subcutaneous adipose tissue remained unaltered. Supplementation with estrogen significantly restored the visceral, but not subcutaneous, fat depot mass and adipocyte size to those of sham-operated mice. A marked increase in the expression of AQP7 and a reduction of GK were observed selectively in the visceral fat depots in estrogen-treated mice.Conclusions: Our results suggest that estrogen has tissue-specific effects on AQP7 expression, and modulation of AQP7 by estrogen alters the balance of adipocyte metabolism between adipose tissue depots.

Stimulation of glycerol kinase in grass carp preadipocytes by EPA.

This study was conducted to assess the effect of eicosapentaenoic acid (EPA) on grass carp preadipocyte glycerol kinase (GyK) expression, as well as to explore the mechanism. Here, we cloned partial sequence of grass carp GyK gene and analyzed its tissue distribution. The result showed that GyK gene expressed most in the liver, followed by adipose tissue and the kidney. Besides, 400μM oleic acid (18:1n-9, OA) was used to establish a hypertrophic preadipocyte model. GyK gene expression and enzyme activity were significantly enhanced after model cells were treated with 100μM eicosapentaenoic acid (20:5n-3, EPA) for 6, 12, and 24h. Meanwhile, peroxisome proliferative-activated receptor (PPAR)γ, adipose triglyceride lipase (ATGL), and the two isoforms of grass carp HSL gene were first identified by Sun et al (2016), and they defined the two isoforms as HSLa and HSLb. Therefore, maybe HSLa and HSLb are appropriate.. The content of triglyceride was dramatically increased by EPA treatment for 24h. Further, a competitive ATGL antagonist, HY-15859, attenuated the increase in GyK induced by EPA at 12h. Surprisingly, the enhanced lipolysis and PPARγ gene expression induced by serum deprivation were paralleled by an increase in GyK gene expression, whereas a stabilization in GyK enzyme activity. Other fatty acids, including docosahexaenoic acid, alpha-linolenic acid, linoleic acid, and OA also promoted GyK gene expression. Moreover, an irreversible PPARγ antagonist, GW9662, was used to investigate the role of PPARγ in GyK induction. Data showed that GW9662 abolished the induction of GyK by EPA at 12h. Together, these data suggested that EPA elevated grass carp preadipocytes GyK expression. ATGL and PPARγ contributed to the induction of GyK. PPARγ may be a key regulator in response to GyK expression induced by EPA.

Feeding glycerol-enriched yeast culture improves lactation performance, energy status, and hepatic gluconeogenic enzyme expression of dairy cows during the transition period1

This study aimed to evaluate the effects of feeding glycerol-enriched yeast culture (GY) on feed intake, lactation performance, blood metabolites, and expression of some key hepatic gluconeogenic enzymes in dairy cows during the transition period. Forty-four multiparous transition Holstein cows were blocked by parity, previous 305-d mature equivalent milk yield, and expected calving date and randomly allocated to 4 dietary treatments: Control (no additive), 2 L/d of GY (75.8 g/L glycerol and 15.3 g/L yeast), 150 g/d of glycerol (G; 0.998 g/g glycerol), and 1 L/d of yeast culture (Y; 31.1 g/L yeast). All additives were top-dressed and hand mixed into the upper one-third of the total mixed ration in the morning from -14 to +28 d relative to calving. Results indicated that the DMI, NE intake, change of BCS, and milk yields were not affected by the treatments ( > 0.05). Supplementation of GY or Y increased milk fat percentages, milk protein percentages, and milk protein yields relative to the Control or G group ( < 0.05). Cows fed GY or G had higher glucose levels and lower β-hydroxybutyric acid (BHBA) and NEFA levels in plasma than cows fed the Control ( < 0.05) and had lower NEFA levels than cows fed Y ( < 0.05). On 14 d postpartum, cows fed GY or G had higher enzyme activities, mRNA, and protein expression of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; < 0.05); higher enzyme activities ( < 0.05) and a tendency toward higher mRNA expression ( < 0.10) of glycerol kinase (GK); and a tendency toward higher enzyme activities of pyruvate carboxylase (PC) in the liver ( < 0.10) when compared with cows fed Control or Y. The enzyme activities, mRNA, and protein expression of PEPCK-C, PC, and GK did not differ between cows fed GY and G ( > 0.10). In conclusion, dietary GY or Y supplementation increased the milk fat and protein content of the cows in early lactation and GY or G supplementation improved the energy status as indicated by greater plasma glucose and lower plasma BHBA and NEFA concentrations and upregulated the hepatic gluconeogenic enzymes of dairy cows during the transition period. Feeding cows with a GY mixture in the peripartum period combined the effects of yeast on lactation performance and the effects of glycerol on energy status in dairy cows.

Contrasting the incorporation of glycerol into lipids caused by the presence of two isoforms of diacylglycerol kinase (605.20)

The mammalian isoforms of diacylglycerol kinase (DGK) include the epsilon isoform (DGKε) and the delta isoform (DGKδ). DGKε has specificity for substrates with an arachidonoyl group, while DGKδ shows no acyl chain specificity for the substrate, but it promotes fatty acid synthesis resulting in an increased fraction of lipids with shorter and less unsaturated acyl chains. We have compared the effects of knocking out each of these isoforms individually using SV40 transformed mouse embryo fibroblasts (MEFs). We compared wild‐type (WT) MEFs with those from knock‐out (KO) mice. We find that DGKδ‐KO MEFs have lower incorporation of 3H‐glycerol into lipids compared with their WT counterparts. This is consistent with our recent demonstration (Biochemistry (2013) 52, 7766) that the depletion of this DGK isoform results in less fatty acid synthesis. This change is not caused by a decreased expression of glycerol kinase in the KO cells. In contrast, the DGKε‐KO MEFs show greater incorporation of 3H‐glycerol into lipids compared with their WT counterparts, with no change in the relative amounts of various lipids between the DGKε‐KO and WT MEFs. This result is explained by our observation that glycerol kinase is more highly expressed in the DGKε‐KO cells than in their WT counterparts.

Gender specific regulation of hepatic aquaporin‐9 and glycerol kinase expression in starved rats

Glycerol is known as a product of lipolysis and an important precursor for hepatic gluconeogenesis. Nevertheless, the regulation of hepatic glycerol metabolism is far from fully elucidated. Aquaporin-9 (AQP9) has been linked to the hepatic uptake of glycerol, and inside the cell glyceriol is converted to glycerol-3-phosphate by glycerol kinase (GK). We investigated the effect of starvation on the initial hepatic glycerol metabolism and found that the hepatic AQP9 and GK are regulated in a gender specific manner. Starved male rats increased the expression of both AQP9 and GK by ~ 2-fold, whereas no changes were observed in females. The glycerol permeability of basolateral membranes from hepatocytes of starved rats was higher in males than in females. A corresponding increase in plasma glycerol levels was observed specifically in female rats. Starvation of ovariectomized rats resulted in increased expression of AQP9 as in starved males, whereas GK abundance remained unchanged. By contrast, orchiectomy decreased the abundance of GK and slightly increased AQP9 expression. Taken together, the results demonstrate that sexual dimorphism exists in the initial hepatic glycerol handling. Moreover, the results suggest that ovary derived factors are crucial for the regulation of hepatic AQP9 expression whereas testicular factors govern the expression of hepatic GK. Supported by the Danish National Research Foundation.

Aquaporin 7 expression in postimplantation mouse uteri: a potential role for glycerol transport in uterine decidualization

The aquaglyceroporin aquaporin 7 (AQP7) is dynamically expressed in mouse uteri undergoing decidualization after implantation. The expansion of AQP7 during uterine decidualization is associated with elevated uterine glycerol accumulation and glycerol kinase expression, suggesting that glycerol might be a potential energy substrate involved in the process of decidualization.

Involvement of Inducible 6-Phosphofructo-2-kinase in the Anti-diabetic Effect of Peroxisome Proliferator-activated Receptor γ Activation in Mice

PFKFB3 is the gene that codes for the inducible isoform of 6-phosphofructo-2-kinase (iPFK2), a key regulatory enzyme of glycolysis. As one of the targets of peroxisome proliferator-activated receptor gamma (PPARgamma), PFKFB3/iPFK2 is up-regulated by thiazolidinediones. In the present study, using PFKFB3/iPFK2-disrupted mice, the role of PFKFB3/iPFK2 in the anti-diabetic effect of PPARgamma activation was determined. In wild-type littermate mice, PPARgamma activation (i.e. treatment with rosiglitazone) restored euglycemia and reversed high fat diet-induced insulin resistance and glucose intolerance. In contrast, PPARgamma activation did not reduce high fat diet-induced hyperglycemia and failed to reverse insulin resistance and glucose intolerance in PFKFB3(+/-) mice. The lack of anti-diabetic effect in PFKFB3(+/-) mice was associated with the inability of PPARgamma activation to suppress adipose tissue lipolysis and proinflammatory cytokine production, stimulate visceral fat accumulation, enhance adipose tissue insulin signaling, and appropriately regulate adipokine expression. Similarly, in cultured 3T3-L1 adipocytes, knockdown of PFKFB3/iPFK2 lessened the effect of PPARgamma activation on stimulating lipid accumulation. Furthermore, PPARgamma activation did not suppress inflammatory signaling in PFKFB3/iPFK2-knockdown adipocytes as it did in control adipocytes. Upon inhibition of excessive fatty acid oxidation in PFKFB3/iPFK2-knockdown adipocytes, PPARgamma activation was able to significantly reverse inflammatory signaling and proinflammatory cytokine expression and restore insulin signaling. Together, these data demonstrate that PFKFB3/iPFK2 is critically involved in the anti-diabetic effect of PPARgamma activation.

Gender specific regulation of hepatic aquaglyceroporin 9 and glycerol kinase expression

Increased hepatic glucose production plays a pivotal role in the pathophysiology of diabetes. AQP9 has been suggested to facilitate the transport of glycerol into hepatocytes where glycerol kinase (GK) catalyses the initial step for its conversion into either glucose or glycerolipids. Hence, the regulation of AQP9 and thereby glycerol uptake could be a potential modulator of hepatic glucose production. This study aims to investigate the role of gender in regulation of hepatic AQP9 and GK protein. Immunoblotting showed that starvation of male rats resulted in increased hepatic AQP9 and GK abundance, whereas, starved female rats displayed no increase in AQP9 or GK. In contrast, plasma glycerol levels were doubled in female rats in response to starvation, whereas no statistically significant changes were found in male rats. Interestingly, immunoblotting revealed that castration of male rats resulted in a 1.5 fold increase in hepatic AQP9 expression, but a decrease in GK abundance. This was accompanied by a marked reduction in plasma glycerol together with a small but significant increase in plasma glucose when compared with controls. We conclude that sexual dimorphism exists in hepatic glycerol handling in response to starvation and that castration of male rats has a diverse effect upon liver abundance of AQP9 and GK.Supported by the Danish National Research Foundation

Bovine Oocytes and Early Embryos Express mRNA Encoding Glycerol Kinase but Addition of Glycerol to the Culture Media Interferes with Oocyte Maturation

Glycerol plays multi-functional roles in cellular physiology. Other than forming the backbone molecule for glycerophospholipid and triglyceride (TG), glycerol acts as an energy substrate for glycolysis. Spermatozoa are known to utilize glycerol for energy production, but there are no reports of this in oocytes. In this study, the value of glycerol as an energy substrate for bovine oocyte maturation (Exp. 1) and the gene expression of glycerol kinase (GK), an enzyme crucial for cellular glycerol utilization, in bovine oocytes and early embryos (Exp. 2) were examined. In Exp. 1, in vitro maturation (IVM) was conducted using synthetic oviduct fluid supplemented with/without glucose (1.5 mM) and/or glycerol (1.0 mM), and maturation rate, degree of cumulus expansion, glucose consumption and lactate production by cumulus-oocyte complexes (COC) were examined. In Exp. 2, to examine the developmental expression of GK mRNA, cumulus cells, oocytes and embryos at the 2-, 8- and 16-cell, morula, expanded blastocyst and hatched blastocyst stages were obtained in separate experiments, and the expression of GK mRNA was quantified using a real-time PCR. Glycerol did not support oocyte maturation or cumulus expansion. Addition of glycerol to glucose-supplemented media significantly decreased the maturation rate. Expression of GK mRNA was very low in cumulus cells, whereas an appreciable level of the transcript was observed in the oocytes. GK mRNA was detected in embryos at all the stages examined, and its expression significantly increased at the morula stage. These results indicate that glycerol, at least at the present concentration, is not beneficial as a constituent of the medium for bovine oocyte maturation. However, the appreciable levels of GK mRNA found in the oocyte and embryo imply a physiological role for glycerol in bovine oocyte maturation and embryo development.