Glycerophosphate Dehydrogenase Activity Research Articles

Deficient Activity of FAD-Linked Glycerophosphate Dehydrogenase in the Mitochondria of Insulin-Producing Pancreatic Islets B-Cells: A Possible Cause of the Metabolic Syndrome

A deficient activity of mitochondrial FAD-linked glycerophosphate dehydrogenase in insulin-producing pancreatic islet B-cells was recently identified in selected animal models of non-insulin-dependent diabetes mellitus. The incidence of this enzymatic defect remains however to be unambiguously documented in type-2 diabetic patients. The present review mainly aims at speculating that the altered recognition of nutrients, especially D-glucose, by pancreatic B-cells, as resulting from the abovementioned enzymatic perturbation could also eventually lead to the development of the metabolic syndrome (MetS).

Deficient Activity of Mitochondrial Flavine Adenine Dinucleotide-Linked Glycerophosphate Dehydrogenase in Pancreatic Islet Beta Cells as a Determinant of Non-Insulin-Dependent Diabetes Mellitus

Extensive experimental investigations in animal models of diabetes mellitus have led to the proposal that a deficient activity of mitochondrial flavine adenine dinucleotide (FAD)-linked glycerophosphate dehydrogenase represents a far-from-uncommon, albeit not invariable, determinant of pancreatic islet B-cell insulin secretory dysfunction. The major aim of the present review is to summarize the results of these investigations and, hence, to encourage further work on the prevalence of this enzymatic defect in the islet B cell of human subjects, especially non-insulin-dependent diabetic patients. J Endocrinol Metab. 2018;8(2-3):16-26 doi: https://doi.org/10.14740/jem496w

Deficient Activity of Mitochondrial Flavine Adenine Dinucleotide-Linked Glycerophosphate Dehydrogenase in Pancreatic Islet Beta Cells as a Determinant of Non-Insulin-Dependent Diabetes Mellitus

Deficient Activity of Mitochondrial Flavine Adenine Dinucleotide-Linked Glycerophosphate Dehydrogenase in Pancreatic Islet Beta Cells as a Determinant of Non-Insulin-Dependent Diabetes Mellitus

ｎ‐３系多価不飽和脂肪酸の摂取が糖代謝調節に及ぼす影響

It is believed that fish oil (FO), contained a large amount of n-3 polyunsaturated fatty acids, reduces the risk of developing type 2 diabetes mellitus. However, mechanism of FO-induced reduction of diabetes risk is unclear. The effects of FO on regulation of plasma glucose level were studied in male Crlj:CD1 (ICR) mice. Weekly changes in plasma glucose and lipid levels were measured for 16 weeks. Plasma phospholipid, total cholesterol and glucose levels were significantly lower in mice fed FO diet than in mice fed lard (LD) diet at week 2, 6 and 8, respectively. Those results suggest that the decreases in plasma glucose in response to intakes of FO in mice take place over a longer period of time than similar decreases in the plasma lipid. There were no marked differences in plasma glucose and insulin levels changes on glucose tolerance test between LD and FO groups. Hepatic glycerophosphate dehydrogenase activity was 53-fold higher in the FO group than in the LD group. It is possible that glycerol synthesis is related to reduction in plasma glucose in mice fed the FO diet. Plasma adiponectin levels were significantly higher in mice fed the FO diet than in mice fed the LD diet. However, no phosphorylation of AMP-activated protein kinase (AMPK) in both liver and skeletal muscle was increased by the FO intake. Those results suggest that FO-induced increases in plasma adiponectin dose not induce the AMPK activation.

Effects of dietary fish oil on thyroid hormone signaling in the liver

n−3 polyunsaturated fatty acids (PUFAs) present in fish oil (FO) potently decrease serum lipids, which is also an effect of thyroid hormones. Both PUFAs and thyroid hormones affect hepatic lipid metabolism, and here we hypothesized that a long-term diet rich in n−3 PUFAs would enhance thyroid hormone action in the liver. Female rats received isocaloric and normolipid diets containing either soybean oil (SO) or FO during lactation. Male offspring received the same diet as their dams since weaning until sacrifice when they were 11 weeks old. FO group, as compared to SO group, exhibited lower body weight since 5 weeks of age until sacrifice, with no alterations in food ingestion, lower retroperitoneal white fat mass and elevated inguinal fat mass relative to body weight, with unchanged water and lipid but reduced protein percentage in their carcasses. FO diet resulted in lower serum triglycerides and cholesterol. Serum total triiodothyronine, total thyroxine and thyrotropin were similar between groups. However, liver thyroid hormone receptor (TR) β1 protein expression was higher in the FO group and correlated negatively with serum lipids. Liver 5′-deiodinase activity, which converts thyroxine into triiodothyronine, was similar between groups. However, the activity of hepatic mitochondrial glycerophosphate dehydrogenase, the enzyme involved in thermogenesis and a well-characterized target stimulated by T3 via TRβ1, was higher in the FO group, suggesting enhancement of thyroid hormone action. These findings suggest that the increase in thyroid hormone signaling pathways in the liver may be one of the mechanisms by which n−3 PUFAs exert part of their effects on lipid metabolism.

Automated microscopic quantification of adipogenic differentiation of human gland stem cells

Detection of differentiation in general and adipogenesis specifically is conventionally practised by taking only the few cells into account which are visible in the field of view provided by optical microscopy using high-resolution objectives. Other methods of quantification of adipogenic differentiation such as real time PCR, measurement of glycerophosphate dehydrogenase activity or adipogenesis assays only provide integral information lacking spatial resolution and information on the fraction of differentiated cells. Here we used high-resolution scanning and automated image processing to automatically analyze and quantify cell numbers in the range of 20,000. For optimisation of the approach, human gland stem cells (GSC) were differentiated to the adipogenic phenotype comprising inclusion of lipid vesicles. Oil red O and 4',6'-diamidino-2-phenylindole (DAPI) staining made it possible to derive the number of differentiated cells in relation to the total number of cells. For evaluation of the image processing software we verified our results using adipogenesis assay and phase contrast based cell counting. We developed a method of determining differentiation efficiencies covering the range from 10% down to 100ppm with the same image processing and an identical set of parameters, matching the results of the adipogenesis assay. Our approach is based on a statistically significant number of cells and shows high sensitivity taking into account the heterogeneous differentiation pattern of adipogenesis in GSC and other stem cells.

Levels of plasma insulin, leptin and adiponectin, and activities of key enzymes in carbohydrate metabolism in skeletal muscle and liver in fasted ICR mice fed dietary n-3 polyunsaturated fatty acids

The aim of this study was to clarify the mechanisms related to plasma glucose concentration in mice fed a diet rich in n-3 polyunsaturated fatty acids (n-3 PUFAs). Male Crlj:CD-1 (ICR) mice were fed experimental diets containing 6% lard (LD), 6% fish oil (FO) or 4.1% lard plus 1.5% docosahexaenoic acid ethyl ester and 0.4% eicosapentaenoic acid ethyl ester (DE) for 12 weeks. There were no marked differences in plasma glucose and insulin concentration changes on glucose tolerance test between the three dietary groups. At the end of the feeding trial, plasma glucose concentration was significantly lower in fasted mice in the FO group than in those in the LD group ( P<.005). Plasma adiponectin concentration was significantly higher in the FO group than in the LD group ( P<.05). Hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and glycerophosphate dehydrogenase activities in skeletal muscle tended to be lower in the FO group than in the LD group, while there were no differences in glucokinase and phosphofructokinase activities in liver between the three dietary groups. However, hepatic glycerophosphate dehydrogenase activity was 53-fold and 4.2-fold higher in the FO group than in the LD and DE groups, respectively ( P<.0005 and P<.05, respectively). These results suggest that the reduction in plasma glucose concentration in mice fed n-3 PUFAs is mainly caused by acceleration of glucose uptake and glycerol synthesis in the liver rather than in the skeletal muscle.

In vitro effects of chorionic gonadotropin hormone on human adipose development

It is well known that pregnancy is associated with fat weight gain. However, the mechanisms whereby fat mass accumulation is controlled during this period are poorly understood. Therefore, we attempted to determine whether human chorionic gonadotropin (HCG), in vitro, influences human adipose tissue development and/or metabolism. For the first time, HCG/LH receptor was characterized in human adipose cells. We also demonstrated that physiological concentrations of HCG, while unaltering both lipolysis and expression of two markers of lipogenesis (FAS and ADD1) in human mature adipocytes, stimulate human preadipocyte growth via the activation of a protein kinase A-independent mitogen-activated protein kinase/c-fos signaling pathway. HCG also moderately increases the preadipocyte differentiation capacity as reflected by enhanced glycerophosphate dehydrogenase activity and expression of key adipogenic transcriptional factors (C/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma 2). Finally, HCG significantly stimulates the secretion of the pro-adipogenic factor, leptin, from human adipose tissue. Taken altogether, these data suggest that the pro-adipogenic effect of HCG in human preadipocytes contributes to explain why increased fat storage occurs during the first trimester of pregnancy.

Diet and Dysfunctional Metabolism in BB Rats

This review deals with the age- and diet-related metabolic and functional anomalies found in BB rats, currently considered as an animal model of type 1 diabetes mellitus. The metabolism of nutrients, the secretion of insulin, the production of NO, the effects of IL-1β and the activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase in pancreatic islets are first considered. The metabolism and mitotic activity in Peyers patch cells and both mesenteric and pancreatic lymph node cells are then evoked. The occurrence of intestinal dysfunction is mentioned, with emphasis on recent findings made in BB rats before weaning. The participation of zonulins and mucins in the perturbation of gut permeability are also discussed. Last, attention is drawn to the perturbation of the anomeric specificity of glucose-induced insulin release, considered as a manifestation of pancreatic islet beta-cell glucotoxicity. Keywords: BioBreeding rats, type 1 diabetes, diet, pancreatic islets, lymph node cells, gut, mucins, anomeric specificity of glucose-induced insulin release

Chromium picolinate depressed proliferation and differentiation of 3T3-L1 preadipocytes

This in vitro study investigated the effects of trivalent chromium picolinate (CrPic) on the proliferation and differentiation of preadipocytes. The mouse preadipocyte cell line (3T3-L1) used in this study included control and the 50 ppb CrPic groups. Preadipocyte differentiation results showed that the number of differentiated adipocytes and their triacylglycerol content in the CrPic group is significantly less (P < .01) than that in the control group. Transcription factor expression of peroxisome proliferator–activated receptors γ in the CrPic group was substantially depressed (P < .0001) compared with that in the control group. Glycerophosphate dehydrogenase activity in the CrPic group was markedly depressed (P < .0001) compared with that in the control group; fatty acid synthase activity in the CrPic group was also significantly repressed (P < 0.0001). For the proliferation of preadipocytes, the number of cells in the CrPic group was lower than that in the control group (P < .01). Results of this study indicated that 50 ppb CrPic significantly depressed preadipocyte proliferation and differentiation.

High activity of mitochondrial glycerophosphate dehydrogenase and glycerophosphate-dependent ROS production in prostate cancer cell lines

Most malignant cells are highly glycolytic and produce high levels of reactive oxygen species (ROS) compared to normal cells. Mitochondrial glycerophosphate dehydrogenase (mGPDH) participates in the reoxidation of cytosolic NADH by delivering reducing equivalents from this molecule into the electron transport chain, thus sustaining glycolysis. Here, we investigate the role of mGPDH in maintaining an increased rate of glycolysis and evaluate glycerophosphate-dependent ROS production in prostate cancer cell lines (LNCaP, DU145, PC3, and CL1). Immunoblot, polarographic, and spectrophotometric analyses revealed that mGPDH abundance and activity was significantly elevated in prostate cancer cell lines when compared to the normal prostate epithelial cell line PNT1A. Furthermore, both the glycolytic capacity and glycerophosphate-dependent ROS production was increased 1.68- to 4.44-fold and 5- to 7-fold, respectively, in prostate cancer cell lines when compared to PNT1A cells. Overall, these data demonstrate that mGPDH is involved in maintaining a high rate of glycolysis and is an important site of electron leakage leading to ROS production in prostate cancer cells.

Optimization of the differentiation of human preadipocytes in vitro

This study aimed at developing an optimal protocol for proliferation and differentiation of preadipocytes that is a prerequisite for constructing an ideal biohybrid composed of viable adipose precursor cells in a three-dimensional matrix. Such an implant could represent an adequate solution for correcting soft tissue defects, e.g., extensive deep burns or tumor resections. Preadipocytes were isolated from human subcutaneous adipose tissue samples and cultured in Dulbecco's modified eagle medium (DMEM)/Ham's F12 medium (F12) or OPTIMEM medium with or without the addition of human serum (hS) or fetal calf serum (FCS). The advantages of fibronectin-coated culture dishes for preadipocyte yield after isolation and differentiation were evaluated. After culture expansion, differentiation was induced by insulin, isobutylmethylxanthine, pioglitazone, dexamethasone, and transferrin in the absence of serum. The extent of differentiation was assayed by measuring the activity of glycerophosphate dehydrogenase as well as counting of differentiated versus undifferentiated cells. Our results show that fibronectin coating does not only strongly increase the yield of preadipocytes after isolation from adipose tissue but also significantly enhances differentiation of precursor cells to mature adipocytes. For optimal cell expansion, DMEM/F12 is more promoting than OPTIMEM and culturing with FCS shows a slightly better proliferation compared with hS supplementation. Differentiation, in contrast, is significantly improved when hS is used instead of FCS during proliferation. Our results smooth the way for autologous preadipocyte culturing and show that hS for preadipocyte culturing opens new and promising perspectives for adipose tissue engineering by optimizing in vitro expansion in cell culture and inducing substantial differentiation.

Glycerophosphate-dependent hydrogen peroxide production by rat liver mitochondria.

We studied the extent to which hormonally-induced mitochondrial glycerophosphate dehydrogenase (mGPDH) activity contributes to the supply of reducing equivalents to the mitochondrial respiratory chain in the rat liver. The activity of glycerophosphate oxidase was compared with those of NADH oxidase and/or succinate oxidase. It was found that triiodothyronine-activated mGPDH represents almost the same capacity for the saturation of the respiratory chain as Complex II. Furthermore, the increase of mGPDH activity induced by triiodothyronine correlated with an increase of capacity for glycerophosphate-dependent hydrogen peroxide production. As a result of hormonal treatment, a 3-fold increase in glycerophosphate-dependent hydrogen peroxide production by liver mitochondria was detected by polarographic and luminometric measurements.

A simple and sensitive assay for GH activity based on 3T3-F442A cell differentiation

We describe a fast, sensitive, specific, and simple in vitro assay for GH biological activity, based on the differentiation of 3T3-F442A cells into adipocytes. The 3T3-F442A cells were directly plated at 1.5 × 10 4 cells/cm 2 in medium with or without various concentrations of human growth hormone (hGH). After 7 days, cells were lysed with buffer containing 0.5 % (v/v) Triton X-100, and adipose conversion was quantitated by the activity of the adipogenic enzyme glycerophosphate dehydrogenase. The assay is highly sensitive and specific for GH from different species. These culture conditions have shortened the time for the cells to undergo adipose differentiation, and they might also be useful to design and test drugs or agents that modify adipocyte differentiation or lipid metabolism, or for evaluation of cytotoxic and pharmacologic effects of drugs and other compounds.

Salacia reticulata and Its Polyphenolic Constituents with Lipase Inhibitory and Lipolytic Activities Have Mild Antiobesity Effects in Rats

Salacia (S.) reticulata, a Hippocrateaceae plant distributed in Sri Lankan and Indian forests, has been used as a supplementary food in Japan to prevent obesity and diabetes. We examined the antiobesity effects of the hot water-soluble extract (SRHW) from the roots of S. reticulata using obese rat models and an in vitro study. Body weight (P = 0.07) and periuterine fat storage (P = 0.10) in female Zucker fatty rats (8-9 wk old) tended to be suppressed by oral administration of SRHW (125 mg/kg) for 27 d. Male rats fed a high fat diet were not affected by SRHW. Furthermore, SRHW inhibited porcine pancreatic lipase (PL), rat adipose tissue-derived lipoprotein lipase (LPL) and glycerophosphate dehydrogenase (GPDH) activities with 50% inhibitory concentrations (IC(50)) of 264 (95% confidence limits: 203-393) mg/L, 15 (12-18) mg/L and 54 (35-85) mg/L, respectively, but did not inhibit hormone-sensitive lipase activity in rat adipose tissue. Next, we examined the effects of polyphenols, di- and triterpenes and salacinol isolated from the roots of S. reticulata on lipid metabolizing enzymes and lipolysis. (-)-Epigallocatechin and (-)-epicatechin-(4beta-->8)-(-)-4'-O-methylepigallocatechin inhibited PL activity with IC(50) of 88 (not calculated) and 68 (26-122) mg/L, respectively. (-)-Epicatechin, 3beta, 22beta-dihydroxyolean-12-en-29-oic acid and the tannin fraction inhibited LPL activity with IC(50) of 81 (54-214), 89 (62-214) and 35 (24-62) mg/L. Only the tannin fraction inhibited GPDH activity with an IC(50) of 6.8 (3.4-10.9) mg/L. These constituents may be involved in the lipase and GPDH inhibitory activities of SRHW. On the other hand, SRHW at 100 mg/L tended to enhance lipolysis in rat adipocytes (P = 0.06). Significant lipolytic effects were exerted by mangiferin, (-)-4'-O-methylepigallocatechin and maytenfolic acid at 100 mg/L (P < 0.01). In conclusion, polyphenolic compounds may be involved in the antiobesity effects of SRHW in rats through inhibition of fat metabolizing enzymes (PL, LPL and GPDH) and enhanced lipolysis.

Pancreatic islet insulin secretion and metabolism in adult rats malnourished during neonatal life.

Pancreatic islets were isolated from rats that had been nursed by dams fed with a control or an 8.7% protein diet during the first 12 d of the lactation period. Glucose-induced insulin secretion from islets in the 8.7% protein group was reduced 50%. The islet insulin and DNA content were similar, whereas the pancreatic insulin content was reduced by 30 % in the rats fed 8.7 % protein. In order to elucidate the mechanism responsible for the attenuation of insulin secretion, measurements were performed of the activity of several islet enzymes that had previously been supposed to be involved in the coupling of glucose stimulation to insulin secretion. Islet glucose oxidation was unaffected, but glucose-stimulated hydrolysis of phosphatidylinositol was reduced by one-third in the islets of rats fed 8.7% protein. The activity of mitochondrial glycerophosphate dehydrogenase was similar in islets of rats fed the 8.7% protein diet and those fed the control diet. The activity of Ca-independent phospholipase A2 was increased fourfold in the islets of rats fed 8.7% protein. It is concluded that impairment of glucose-induced insulin secretion in rats fed a low-protein diet may be caused by attenuation of islet phosphatidylinositol hydrolysis, and it is tentatively suggested that the increased activity of Ca-independent phospholipase A2 in islets of rats fed a low-protein diet may participate in the stimulation of apoptosis.

Vitamin C is one of the lipolytic substances in green tea

We have studied the influence of vitamin C contained in green tea on the lipolysis of well-differentiated 3T3-L1 cells. When mature adipocytes were exposed to vitamin C the triglyceride concentration was decreased (p < 0.05) and the activity of glycerophosphate dehydrogenase, a marker of adipose conversion, was significantly inhibited (p < 0.01). These data suggest that green tea may have a lipolytic activity due to the mechanism by which the vitamin C contained in it inhibits triglyceride accumulation.

Distinct effect of diazoxide on insulin secretion stimulated by protein kinase A and protein kinase C in rat pancreatic islets

Protein kinase activation is known to stimulate glucose-induced insulin secretion in the presence of diazoxide. Diazoxide opens the ATP-sensitive K + channel and inhibits FAD-linked glycerophosphate dehydrogenase activity in a concentration-dependent manner. In the present study, we examined the effect of lower (100 μM) and higher (250 μM) concentrations of diazoxide on insulin release by protein kinase A (PKA) and protein kinase C (PKC) activation. Forced depolarization by a high potassium concentration, augmented the intracellular Ca 2+ concentration ([Ca 2+] i ) similarly in the presence of both concentrations of diazoxide. Under this condition, 250 μM diazoxide inhibited insulin release enhanced by PKA activation but not that by PKC. Under a basal concentration of [Ca 2+] i , PKC activation elicited glucose-induced insulin secretion at 100 and 250 μM diazoxide, while PKA activation did so only at 100 μM. These augmentations were completely inhibited by mannoheptulose, a glucokinase inhibitor. Glyceraldehyde, in place of glucose, enhanced insulin secretion by PKC activation under both concentrations of diazoxide. On the other hand, it did not affect PKA-stimulated insulin release under either conditions, but in the case of 100 μM, glucose augmented the insulin secretion in the presence of glyceraldehyde and db-cAMP concentration-dependently. These data suggest that insulin release stimulated by PKA and PKC activation under diazoxide is dependent on glucose metabolism, and that a signal derived from proximal steps in glycolysis may be necessary for the secretion by PKA activation.

The two tumor necrosis factor receptors mediate opposite effects on differentiation and glucose metabolism in human adipocytes in primary culture.

Tumor necrosis factor-alpha (TNF) inhibits fat cell differentiation and may also mediate insulin resistance in adipocytes. Both TNF receptors are expressed in adipose tissue, but it is unknown how both receptors are involved in these biological functions. We therefore studied the effect of receptor-specific TNF muteins on adipose differentiation and insulin-stimulated glucose transport of in vitro differentiated human adipocytes in primary culture. Adipocyte precursor cells exposed to the 60-kDa TNF receptor (p60-TNFR)-specific TNF(R32W-S86T) showed a marked decrease in the percentage of differentiating cells in response to adipogenic factors as well as a reduction in peroxisome proliferator-activated receptor-gamma2 (PPARgamma2) messenger RNA (mRNA) and glycerophosphate dehydrogenase (GPDH) activity, but increased endogenous TNF mRNA expression. When cells were incubated with the p80-TNFR-specific TNF(D143N-A145R), adipogenesis and PPARgamma2 mRNA expression were stimulated, GPDH activity was unchanged, and TNF mRNA was completely suppressed. Insulin-stimulated 2-deoxy-D-glucose transport was inhibited by both muteins. The p60-TNFR-mediated inhibition increased continuously during 6 h of treatment and was associated with a down-regulation of glucose transporter-4 (GLUT4) mRNA and GLUT4 protein, whereas the p80-TNFR-specific mutein caused a transient increase in GLUT4 mRNA, but did not alter GLUT4 protein expression after a 24-h incubation. We conclude that p60-TNFR mediates the antiadipogenic effect as well as the down-regulation of GLUT4 by TNF, thereby leading to long-term inhibition of insulin-stimulated glucose transport. In contrast, activation of the p80-TNFR induces an adipogenic effect and transiently up-regulates GLUT4 expression. Here, the acute inhibition of insulin-stimulated glucose transport may be induced by interference with the insulin signaling pathway.

Enzymes of Glucose and Glycerol Catabolism in in vitro -Propagated Theileria parva Schizonts

Theileria parva schizonts propagated in vitro in peripheral blood lymphocytes were purified and assayed for key enzymes of glucose and glycerol catabolism and the citric acid cycle. The activities of glycolytic enzymes were in the range of 21–100 nmol/min/mg protein. Glycerol kinase and α -glycerophosphate dehydrogenase activities were more than 16 times lower than the activities of other enzymes catalysing the oxidation of the triose phosphates to lactate. It was suggested that the catabolism of glycerol is negligible and that glucose is catabolized to lactate via the Embden–Meyerhof pathway. The activities of the enzymes catalysing the section of the citric acid cycle that involves the formation of citrate to succinyl-CoA were consistently very low (less than 2.0 nmol/min/mg protein), indicating that this part of the cycle plays a minor role in this parasite. Enzyme activities of the cycle catalysing the formation of succinate from oxaloacetate were relatively higher than those catalysing other sections of the citric acid cycle, suggesting that this section of the cycle could be important to the parasite. Pyruvate carboxylase activity was more than 10 times that of phosphoenolpyruvate carboxykinase. It was suggested that pyruvate could be carboxylated to oxaloacetate. Taken together, these results suggest that the catabolism of glucose in Theileria parva schizonts is mainly via the Embden–Meyerhof pathway and that the citric acid cycle plays a minor role in energy production.