PEPCK Activity Research Articles

Differential regulation of glyceroneogenesis by glucocorticoids in epididymal and retroperitoneal white adipose tissue from rats.

Investigate the glycerol-3-phosphate generation pathways in epididymal (EPI) and retroperitoneal (RETRO) adipose tissues from dexamethasone-treated rats. Rats were treated with dexamethasone for 7 days. Glycerol-3-phosphate generation pathways via glycolysis, glyceroneogenesis and direct phosphorylation of glycerol were evaluated, respectively, by 2-deoxyglucose uptake, phosphoenolpyruvate carboxykinase (PEPCK-C) activity and pyruvate incorporation into triacylglycerol (TAG)-glycerol, and glycerokinase activity and glycerol incorporation into TAG-glycerol. Dexamethasone treatment markedly decreased the body weight, but increased the weight and lipid content of EPI and RETRO and plasma insulin, glucose, non-esterified fatty acid and TAG levels. EPI and RETRO from dexamethasone-treated rats showed increased rates of de novo fatty acid synthesis (80 and 100%) and basal lipolysis (20%). In EPI, dexamethasone decreased the 2-deoxyglucose uptake (50%), as well as glyceroneogenesis, evidenced by a decrease of PEPCK-C activity (39%) and TAG-glycerol synthesis from pyruvate (66%), but increased the glycerokinase activity (50%) and TAG-glycerol synthesis from glycerol (72%) in this tissue. In spite of a similar reduction in 2-deoxyglucose uptake in RETRO, dexamethasone treatment increased glyceroneogenesis, evidenced by PEPCK activity (96%), and TAG-glycerol synthesis from pyruvate (110%), accompanied by a decrease in glycerokinase activity (50%) and TAG-glycerol synthesis from glycerol (50%). Dexamethasone effects on RETRO were accompanied by a decrease in p-Akt content and by lower insulin effects on the rates of glycerol release in the presence of isoproterenol and on the rates of glucose uptake in isolated adipocytes. Our data demonstrated differential regulation of glyceroneogenesis and direct phosphorylation of glycerol by glucocorticoids in EPI and RETRO from rats.

Effect of Ocimum sanctum and Crataegus pubescens aqueous extracts on obesity, inflammation, and glucose metabolism

This study aimed to evaluate the effect of Ocimum sanctum and Crataegus pubescens extracts on obesity, inflammation, and glucose metabolism in obese rats. Both extracts exerted anti-obesogenic effects, which was associated to increased fecal triacylglycerol excretion and decreased their accumulation in adipose tissue, decreasing adipocyte hypertrophy. O. sanctum showed the greatest hypoglycemic effect, which was associated to its anti-inflammatory effect, as well as an increased hepatic glycogen content and decreased PEPCK and G6Pase hepatic activities. Conversely, C. pubescens (0.5 and 1.0mg/mL) increased Glut4, Irs1 and Pi3k gene expression in 3T3 L1 adipocyte cells. The beneficial effect of O. sanctum was associated to its high content of ellagic acid, epicatechin, epigallocatehin gallate and rutin, whereas the hypoglycemic effect of C. pubescens was mainly associated with its content of gallic acid. These results suggest that these herbs may be used for the elaboration of functional beverages with hypoglycemic properties.

Transfer of dysbiotic gut microbiota has beneficial effects on host liver metabolism

Gut microbiota dysbiosis has been implicated in a variety of systemic disorders, notably metabolic diseases including obesity and impaired liver function, but the underlying mechanisms are uncertain. To investigate this question, we transferred caecal microbiota from either obese or lean mice to antibiotic‐free, conventional wild‐type mice. We found that transferring obese‐mouse gut microbiota to mice on normal chow (NC) acutely reduces markers of hepatic gluconeogenesis with decreased hepatic PEPCK activity, compared to non‐inoculated mice, a phenotypic trait blunted in conventional NOD2 KO mice. Furthermore, transferring of obese‐mouse microbiota changes both the gut microbiota and the microbiome of recipient mice. We also found that transferring obese gut microbiota to NC‐fed mice then fed with a high‐fat diet (HFD) acutely impacts hepatic metabolism and prevents HFD‐increased hepatic gluconeogenesis compared to non‐inoculated mice. Moreover, the recipient mice exhibit reduced hepatic PEPCK and G6Pase activity, fed glycaemia and adiposity. Conversely, transfer of lean‐mouse microbiota does not affect markers of hepatic gluconeogenesis. Our findings provide a new perspective on gut microbiota dysbiosis, potentially useful to better understand the aetiology of metabolic diseases.

Insulin Promotes the Expression of the Gluconeogenic Rate-Limiting Enzymes Phosphoenolpyruvate Carboxykinase (Pepck) and Glucose 6-Phosphatase (G6pase) through PI3k/Akt/mTOR Signaling Pathway in Goose Hepatocytes

To identify what makes insulin have an activating or inhibiting role in gluconeogenesis in goose hepatocytes and whether insulin regulates PEPCK and G6Pase through the PI3k/Akt/mTOR pathway or not, goose primary hepatocytes were isolated and cultured in vitro. After 12h cultured in serum-free medium, hepatocytes were incubated for 24 h in the medium with no addition (control) or with the addition of50, 100, and 150 nM of insulin, 1000 nM NVP-BEZ235, or co-addition of 150nM insulin and 1000nM NVP-BEZ235. Glucose concentration and PEPCK and G6Pase expression were determined. The results showed that PEPCK and G6Pase mRNA levels and activities were up regulated in the 50, 100, and 150nM insulin treatments, while glucose concentration was not significantly altered (p> 0.05). Compared with the activation role of 150nM insulin alone, the co-treatment with1000nM NVP-BEZ235 and 150nM insulin significantly down regulated PEPCK mRNA level and G6Pase protein activity (p< 0.05). However, there is a different result on mRNA level of G6Pase. In conclusion, G6Pase and PEPCK are up regulated by insulin through PI3k/Akt/mTOR pathway in goose hepatocytes. However, G6Pase mRNA and protein levels may be regulated by insulin through different signaling pathways.

Effects of exposure to hypoxia on metabolic pathways in northern shrimp (Pandalus borealis) and Greenland halibut (Reinhardtius hippoglossoides)

In the Estuary and Gulf of St. Lawrence, northern shrimp (Pandalus borealis) and Greenland halibut (Reinhardtius hippoglossoides) are usually found at depths >150m and thus frequently inhabit hypoxic areas (18–50% saturation). The impact of a one-week exposure to different levels of dissolved oxygen (100, 40, 30, and 20% saturation) at 5°C was evaluated in adult shrimp and juvenile Greenland halibut; the effect of acute exposure to severe hypoxia was also assessed in Greenland halibut. The activities of key enzymes involved in aerobic (citrate synthase [CS], cytochrome c oxidase [COX]) and anaerobic (pyruvate kinase [PK], phosphoenolpyruvate carboxykinase [PEPCK], lactate dehydrogenase [LDH]) pathways, and of enzymes involved in antioxidant defence (superoxide dismutase, glutathione peroxidase [GPx], and catalase [CAT]) were measured. Quantitative real-time PCR analysis was also performed in Greenland halibut. In northern shrimp exposed to chronic hypoxia, muscle CS activity decreased by ~40%. Muscle LDH activity was significantly reduced, with a more intense reduction in males. At the same time, hepatopancreas GPx activity increased under hypoxia, and this response was stronger in males. Overall, the results suggest the presence of a threshold above 40% saturation and higher hypoxia tolerance in males. In juvenile Greenland halibut, exposure to chronic hypoxia elicited a more wide-ranging enzymatic response than did acute exposure to severe hypoxia. Under chronic hypoxia, CS activity decreased and PK and LDH activity were respectively 46% and 57% lower than in normoxia. There were no major changes in the activity of antioxidant enzymes, but activity in normoxia was high compared to other fish species. Interestingly, the relative expression of genes coding for muscle COX (severe hypoxia), liver PEPCK (chronic), and CAT (chronic) activities were triggered in hypoxia. The absence of a corresponding change in enzyme activity makes the interpretation of these results difficult, but clearly there was a response at the transcription level. Overall, the results indicate that these two species are particularly well adapted to withstand severe hypoxia.

Identification and phylogeny of putative PEPC genes in three toxin‐producing Karenia (Dinophyta) species

Dense blooms of toxin-producing Karenia brevis increase local surface ocean pH through CO2 uptake. To identify genes that may contribute to bloom-related environmental pH and pCO2 changes, transcriptomes with RNA from K.brevis Wilson cultures that had been acclimated to low CO2 (250ppm) or recent CO2 (350ppm) pCO2 levels were assembled. Among the annotated transcripts were PEPC, PPDK, and PEPCK enzymes found in the model C4 carbon fixation pathway. Previous studies have demonstrated that the enzymatic activity of PEPC, PPDK, and/or PEPCK in some algae species, including marine diatoms, is influenced by variations in dissolved inorganic carbon. We found significantly similar PEPC, PPDK, and PEPCK enzymes in the transcriptomes of K.brevis and two sister species Karenia papilionacea, and Karenia mikimotoi. One or more isoforms of PEPC were also identified in the transcriptomes of thirty additional photosynthetic phytoplankton species from nine phyla. Phylogenetic trees were constructed with neighbor joining and maximum likelihood techniques to characterize the evolutionary relationship among phytoplankton, terrestrial plant C4, and terrestrial plant C3 PEPC sequences. Based on the nucleotide trees constructed during this study, the Karenia PEPC transcripts were more closely related to the terrestrial C4 genes than the terrestrial C3 genes. Furthermore, PEPC phylogeny among phytoplankton closely resembles phylogenetic trees constructed with ribosomal RNA. This study confirmed that the toxin-producing dinoflagellates K.brevis, K.mikimotoi, and K. papilionacea express putative PEPC, PEPCK, and PPDK transcripts.

Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase

This work reports the first functional characterization of leishmanial PEPCK. The recombinant Leishmania major enzyme (Lmj_PEPCK) exhibits equivalent kcat values for the phosphoenolpyruvate (PEP) and oxaloacetate (OAA) forming reactions. The apparent Km towards OAA is 10-fold lower than that for PEP, while the Km values for ADP and ATP are equivalent. Mutagenesis studies showed that D241, D242 and H205 of Lmj_PEPCK like the homologous residues of all known PEPCKs are implicated in metal ions binding. In contrast, the replacement of R43 for Q nearly abolishes Lmj_PEPCK activity. Moreover, the Y180F variant exhibits unchanged Km values for PEP, Mn2+, and HCO3−, being the kcat for PEP- but not that for OAA-forming reaction more notably decreased. Instead, the Y180A mutant displays an increase in the Km value towards Mn2+. Therefore in Lmj_PEPCK, Y180 seems to exert different functions to those of the analogous residue in ATP- and GTP-dependant enzymes. Besides, the guanidinium group of R43 appears to play an essential but yet unknown role. These findings promote the need for further structural studies to disclose whether Y180 and R43 participate in the catalytic mechanism or/and in the transitions between the open and the catalytically competent (closed) forms of Lmj_PEPCK

The pepper phosphoenolpyruvate carboxykinase CaPEPCK1 is involved in plant immunity against bacterial and oomycete pathogens.

Phosphoenolpyruvate carboxykinase, a member of the lyase family, is involved in the metabolic pathway of gluconeogenesis in organisms. Although the major function of PEPCK in gluconeogenesis is well established, it is unclear whether this enzyme is involved in plant immunity. Here, we isolated and identified the pepper (Capsicum annuum) PEPCK (CaPEPCK1) gene from pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). CaPEPCK1 was strongly expressed in pepper leaves during the incompatible interaction with avirulent Xcv and in response to environmental stresses, especially salicylic acid (SA) treatment. PEPCK activity was low in healthy leaves but dramatically increased in avirulent Xcv-infected leaves. Knock-down expression of CaPEPCK1 by virus-induced gene silencing resulted in high levels of susceptibility to both virulent and avirulent Xcv infection. CaPEPCK1 silencing in pepper compromised induction of the basal defense-marker genes CaPR1 (pathogenesis-related 1 protein), CaPR10 (pathogenesis-related 10 protein) and CaDEF1 (defensin) during Xcv infection. SA accumulation was also significantly suppressed in the CaPEPCK1-silenced pepper leaves infected with Xcv. CaPEPCK1 in an Arabidopsis overexpression (OX) line inhibited the proliferation of Pseudomonas syringae pv. tomato (Pst) and Hyaloperonospora arabidopsidis (Hpa). CaPEPCK1-OX plants developed more rapidly, with enlarged leaves, compared to wild-type plants. The T-DNA insertion Arabidopsis orthologous mutants pck1-3 and pck1-4 were more susceptible to the bacterial Pst and oomycete Hpa pathogens than the wild type. Taken together, these results suggest that CaPEPCK positively contributes to plant innate immunity against hemibiotrophic bacterial and obligate biotrophic oomycete pathogens.

Stanniocalcin 1 effects on the renal gluconeogenesis pathway in rat and fish

The mammalian kidney contributes significantly to glucose homeostasis through gluconeogenesis. Considering that stanniocalcin 1 (STC1) regulates ATP production, is synthesized and acts in different cell types of the nephron, the present study hypothesized that STC1 may be implicated in the regulation of gluconeogenesis in the vertebrate kidney. Human STC1 strongly reduced gluconeogenesis from 14C-glutamine in rat renal medulla (MD) slices but not in renal cortex (CX), nor from 14C-lactic acid. Total PEPCK activity was markedly reduced by hSTC1 in MD but not in CX. Pck2 (mitochondrial PEPCK isoform) was down-regulated by hSTC1 in MD but not in CX. In fish (Dicentrarchus labrax) kidney slices, both STC1-A and -B isoforms decreased gluconeogenesis from 14C-acid lactic, while STC1-A increased gluconeogenesis from 14C-glutamine. Overall, our results demonstrate a role for STC1 in the control of glucose synthesis via renal gluconeogenesis in mammals and suggest that it may have a similar role in teleost fishes.

Polybrominated Diphenylether (PBDE) Suppression of Phosphoenolpyruvate Carboxykinase (PEPCK) Activity Alters Liver Lipid Metabolism

PBDE, a class of flame retardant chemicals that leach into the environment and enter the human body, have been shown to suppress activity of PEPCK (Nash et al., 2013), a key enzyme in fatty acid esterification via hepatic glyceroneogenesis. The objective of this research was to evaluate the metabolic fate of fatty acids in liver of PBDE-treated rats. Two separate studies were conducted with identical experimental design. Male, weanling Wistar rats were gavaged daily for 28 days with 14 mg/kg body weight commercial PBDE mixture (TRT) or corn oil (CON). After a 48 hour fast, rats were euthanized, blood sampled, and livers removed. In study 1 (n=8 per group), serum ketone bodies were elevated in TRT rats compared to CON (27.7 vs 21.8 mg/dL, respectively, p<0.05) In study 2 (n=6 per group), hepatic glyceroneogenesis, as measured by 14C-pyruvate incorporation into triglycerides, was 41% lower in explants from TRT rats compared to CON (8.21 vs 13.97 pmol/hr/mg tissue, respectively, p<0.05). Liver lipid content was 29% lower in TRT compared to CON (2.69% vs 3.80%, respectively, p<0.05). Taken together, these findings suggest that a PBDE-induced suppression in hepatic PEPCK activity alters lipid metabolism by redirecting fatty acids away from esterification and storage, and toward ketone synthesis. Supported by NH Agricultural Experiment Station Grant NH00541

The antigluconeogenic activity of cucurbitacins from Momordica charantia.

Five new cucurbitacins, kuguacins II-VI (1-5), along with five known analogues (6-10), were obtained from the fruit of Momordica charantia. Structures of the new compounds were elucidated as 5β,19-epoxycucurbit-23-en-7-on-3β,25-diol (1), 5β,19-epoxycucurbit-7,23-dion-3β,25-diol (2), 5β,19-epoxycucurbit-6-en-19,23-dion-3β,25-diol (3), 5β,19-epoxy-23,24,25,26,27-pentanorcucurbit-6-en-7,19-dion-3β,22-diol (4), and cucurbit-5-en-7,23-dion-3β,19,25-triol (5) by extensive spectroscopic and single-crystal X-ray diffraction analyses. Some cucurbitane compounds from this species were screened for their potential antidiabetic properties in terms of antigluconeogenic activity. As a result, compounds 1, 10, 11, and 12 (at 25-100 µM) showed concentration-dependent inhibition on glucose production from liver cells. In addition, compounds 11 and 12 (at 100 µM) showed around 20-30 % inhibition on PEPCK activity.

Potent effects of the total saponins from Dioscorea nipponica Makino against streptozotocin-induced type 2 diabetes mellitus in rats.

The aim of the present paper was to investigate the effects and possible mechanisms of the total saponins from Dioscorea nipponica Makino (TSDN) against type 2 diabetes mellitus. Streptozotocin (STZ) with high-fat diet induced type 2 diabetes mellitus (T2DM) rats were treated with TSDN. Some biochemical parameters, target proteins and genes were investigated. The results showed that TSDN decreased the levels of food/water intake, fasting blood glucose and serum lipid parameters, ameliorated oral glucose and insulin tolerance test levels, markedly increased body weight and serum insulin, reduced excess free radicals and affected ossification and renal protection. Histopathological examination indicated that TSDN increased liver glycogen, decreased the production of lipid vacuoles and lightened liver damage. Further investigation showed that TSDN down-regulated the protein expressions of NF-κB, GRP78, ATF6, eIF2 and the levels of MAPK phosphorylation and up-regulated the protein expressions of IRS-1, GLUT-4, p-Akt and p-AMPK. In addition, TSDN obviously decreased the gene expressions of TNF-a, IL-6, PEPCK, G6Pase, GSK-3β and GSK-3β activity, and increased the gene expressions of PFK, PK and GK activity. These findings show the anti-diabetic activity of total saponins from D. nipponica Makino, which should be developed as a new potent drug for treatment of diabetes mellitus in future.

High fat diet induces an increase in the direct phosphorylation of glycerol which can contribute for the increase in white adipose tissue mass (575.2)

Triacylglycerol (TAG) synthesis in mammalian tissues requires a continuous glycerol‐3‐phosphate (G3P) generation for fatty acids esterification and TAG synthesis. The pathways of G3P generation were examined in retroperitoneal (RWAT) and epididymal (EWAT) adipose tissues from mice fed a high‐fat (HF) diet for 8 wk. After this period we evaluate the in vitro rates of glucose uptake and incorporation of 1‐[14C]pyruvate or U‐[14C]glycerol into TAG‐glycerol, and also the glycerol kinase (GyK) and P‐enolpyruvate carboxykinase (PEPCK‐C) activities in both tissues. Protein content was evaluated by western blot. The plasma glucose and insulin were measured by commercial kits. HF diet induced marked increase in body fat mass and in the plasma levels of glucose and insulin. HF diet feeding induced a marked increase in activity (3,4X and 2,5X) and content (42% and 87%) of GyK in RWAT and EWAT, accompanied by a higher TAG‐glycerol synthesis from glycerol in EWAT (47%), but not in RWAT. In contrast, there was a decrease in G3P generation via glycolysis in RWAT (71%) and EWAT (39%) and also a reduction in TAG‐glycerol synthesis from pyruvate (72 and 75%, respectively), without changes in the PEPCK activity and content in both tissues. HF diet induces loss of reciprocal changes in glyceroneogenesis and glycolysis for G3P generation, and seems to favour the direct phosphorylation of glycerol for TAG synthesis and increase in fat mass.Grant Funding Source: Supported by CAPES, FAPESP and CNPQ

Effects of Glycyrrhetic Acid (GE) on Some Gluconeogenic Enzymes, Lipoprotein Lipase and Peroxisome Proliferator-Activated Receptors Alpha and Gamma

The aim of this study was to examine the role of glycyrrhetic acid (GE) as a potential compound in the amelioration of metabolic syndrome. Rats given intraperitoneal injection of GE were sacrificed after 24 hours. Blood was collected for the determination of glucose, insulin and lipid profiles; while tissues were used for 11β-HSD1, gluconeogenic enzymes activities, PPAR-α/-γ and LPL expression by RT-PCR. Intraperitoneal injection of 50mg/kg GE to normal rats significantly lowered blood glucose while insulin level and HOMA-IR showed no significant changes. H6PDH activities increased in the liver, kidney, subcutaneous and visceral adipose tissues and quadriceps femoris but decreased in the abdominal muscle. PEPCK activities were significantly reduced in the kidney and decreased in the liver but showed an increase in the subcutaneous and visceral adipose tissues. G6Pase activities were found to be reduced in both the liver and kidney. 11β-HSD1 activities increased in the liver but decreased in all other tissues. There were improvements in lipid profiles in GE-treated rats. Up-regulation of LPL activity was seen in all tissues except quadriceps femoris. PPAR-α expression was up-regulated in the liver, heart and abdominal muscle while down-regulated in the kidney and quadriceps femoris but were undetectable in the subcutaneous and visceral adipose tissues. PPAR-γ expression was up-regulated in all tissues except the kidney. GE prevented hyperglycaemia and improved lipid profiles possibly through 11β-HSD1 inhibition instead of via PPAR agonism.

Suppression of mitochondrial dehydrogenases accompanying post-glyoxylate cycle activation of gluconeogenesis and reduced lipid peroxidation events during dormancy breakage of walnut kernels by moist chilling

Cold stratification supposedly allows dormant seeds to germinate by removing their metabolic blocks. Nothing is known about the role of mitochondrial enzymes in metabolic regulation of seeds during dormancy release. As cold stratification removes walnut (Juglans regia L.) kernel dormancy, the activities of three mitochondrial enzymes i.e. pyruvate dehydrogenase complex (PDH), NAD+-isocitrate dehydrogenase (NAD+-IDH; EC 1.1.1.41), succinate dehydrogenase (SDH; EC 1.3.99.1) and the extra-mitochondrial glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) were compared in both cold-stratified (5°C; 60days) and warm-incubated (27°C; 20days) kernels. The kernel gluconeogenic competence was also assessed by measuring the activity of phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.49). These were then correlated to kernel lipid hydroperoxide contents and lipoxygenase (EC 1.13.11.12) activity. Lipid hydroperoxides and lipoxygenase activity were significantly greater in warm-incubated kernels. The mitochondrial NAD+-IDH activity was indifferent between cold-stratified and warm-incubated kernels. The activities of PDH and SDH though undetectable in cold-stratified kernels, were relatively high under warm conditions. Succinate oxidation in cold stratified kernels however, was achieved by a glyoxysomal succinate oxidase activity formerly reported in monocotyledons. Furthermore, the kernel succinate oxidase, PEPCK and G6PDH activities were significantly greater at cold conditions. It was concluded that cold induced dormancy release of walnut kernels is associated with suppression of kernel mitochondrial respiration and oxidative stress and meanwhile it allows activation of oxidative pentose phosphate (OPP) and post-glyoxylate cycle gluconeogenesis pathways. The build-up of oxidative stress and toxic lipid hydroperoxide derivatives however, probably result in the compromised germination of warm-incubated walnut kernels.

Significant involvement of PEP-CK in carbon assimilation of C4 eudicots.

C4 eudicot species are classified into biochemical sub-types of C4 photosynthesis based on the principal decarboxylating enzyme. Two sub-types are known, NADP-malic enzyme (ME) and NAD-ME; however, evidence for the occurrence or involvement of the third sub-type (phosphoenolpyruvate carboxykinase; PEP-CK) is emerging. In this study, the presence and activity of PEP-CK in C4 eudicot species of Trianthema and Zaleya (Sesuvioideae, Aizoaceae) is clarified through analysis of key anatomical features and C4 photosynthetic enzymes. Three C4 species (T. portulacastrum, T. sheilae and Z. pentandra) were examined with light and transmission electron microscopy for leaf structural properties. Activities and immunolocalizations of C4 enzymes were measured for biochemical characteristics. Leaves of each species possess atriplicoid-type Kranz anatomy, but differ in ultrastructural features. Bundle sheath organelles are centripetal in T. portulacastrum and Z. pentandra, and centrifugal in T. sheilae. Bundle sheath chloroplasts in T. portulacastrum are almost agranal, whereas mesophyll counterparts have grana. Both T. sheilae and Z. pentandra are similar, where bundle sheath chloroplasts contain well-developed grana while mesophyll chloroplasts are grana deficient. Cell wall thickness is significantly greater in T. sheilae than in the other species. Biochemically, T. portulacastrum is NADP-ME, while T. sheilae and Z. pentandra are NAD-ME. Both T. portulacastrum and Z. pentandra exhibit considerable PEP-CK activity, and immunolocalization studies show dense and specific compartmentation of PEP-CK in these species, consistent with high PEP-CK enzyme activity. Involvement of PEP-CK in C4 NADP-ME T. portulacastrum and NAD-ME Z. petandra occurs irrespective of biochemical sub-type, or the position of bundle sheath chloroplasts. Ultrastructural traits, including numbers of bundle sheath peroxisomes and mesophyll chloroplasts, and degree of grana development in bundle sheath chloroplasts, coincide more directly with PEP-CK recruitment. Discovery of high PEP-CK activity in C4 Sesuvioideae species offers a unique opportunity for evaluating PEP-CK expression and suggests the possibility that PEP-CK recruitment may exist elsewhere in C4 eudicots.

Nesfatin-1 Action in the Brain Increases Insulin Sensitivity Through Akt/AMPK/TORC2 Pathway in Diet-Induced Insulin Resistance

Nesfatin-1, derived from nucleobindin 2, was recently identified as an anorexigenic signal peptide. However, its neural role in glucose homeostasis and insulin sensitivity is unknown. To evaluate the metabolic impact and underlying mechanisms of central nesfatin-1 signaling, we infused nesfatin-1 in the third cerebral ventricle of high-fat diet (HFD)–fed rats. The effects of central nesfatin-1 on glucose metabolism and changes in transcription factors and signaling pathways were assessed during euglycemic-hyperinsulinemic clamping. The infusion of nesfatin-1 into the third cerebral ventricle markedly inhibited hepatic glucose production (HGP), promoted muscle glucose uptake, and was accompanied by decreases in hepatic mRNA and protein expression and enzymatic activity of PEPCK in both standard diet- and HFD-fed rats. In addition, central nesfatin-1 increased insulin receptor (InsR)/insulin receptor substrate-1 (IRS-1)/AMP-dependent protein kinase (AMPK)/Akt kinase (Akt)/target of rapamycin complex (TORC) 2 phosphorylation and resulted in an increase in Fos immunoreactivity in the hypothalamic nuclei that mediate glucose homeostasis. Taken together, these results reveal what we believe to be a novel site of action of nesfatin-1 on HGP and the PEPCK/InsR/IRS-1/AMPK/Akt/TORC2 pathway and suggest that hypothalamic nesfatin-1 action through a neural-mediated pathway can contribute to increased peripheral and hepatic insulin sensitivity by decreasing gluconeogenesis and promoting peripheral glucose uptake in vivo.

On the influence of overexpression of phosphoenolpyruvate carboxykinase in Streptomyces lividans on growth and production of human tumour necrosis factor-alpha

Streptomyces lividans has shown potential as an expression system for heterologous proteins. Overexpression of proteic factors important for heterologous protein production is a valuable approach to improve yields of such proteins. Comparative transcriptomic analysis revealed that several genes were differentially expressed in strains involved in heterologous protein production. For instance, the gene-encoding phosphoenolpyruvate carboxykinase (pepck) showed a significant twofold change in recombinant S. lividans producing human tumour necrosis factor-alpha (hTNF-α). The effect of pepck overexpression on S. lividans TK24 and its hTNF-α producing recombinant was thus investigated in bench-top fermenters. Results obtained revealed that pepck overexpression resulted into a twofold increase in specific PEPCK activity during growth. This overexpression is correlated with slower growth rate, reduced excretion of pyruvate and less alkalinisation of the growth medium when compared with the control strain. After 26h of fermentation, hTNF-α yields were enhanced (up to 1.7-fold) in the pepck-overexpressing S. lividans TK24, demonstrating that this metabolic engineering approach is indeed promising for heterologous protein production.

Non-protein nitrogen supplementation increases gluconeogenic capacity in sheep

The effect of non-protein nitrogen (NPN) supplementation on the gluconeogenic capacity of sheep was investigated. Three groups of six sheep each, control, moderate (MN=0.55gN/kg0.75/d) and high (HN=1.28gN/kg0.75/d) NPN were used. A propionate loading test (PLT) was performed in each animal after 7 or 15 days of NPN supplementation by infusing a bolus of sodium propionate, and plasma glucose, urea, lactate and insulin concentrations were measured up to 88min after infusion. Liver glycogen concentration and the activity of gluconeogenic enzymes and complementary ureagenic enzymes were determined at 9 or 17 days of the supplementation period. Glycemia and lactacidemia increased during the PLT in all groups, being highest in HN sheep. Liver activity of PEPCK was higher in HN than in the control sheep. NPN supplementation in sheep increased plasma urea and lactate concentrations and liver PEPCK activity associated to an increased gluconeogenic capacity from propionate or lower peripheral glucose utilization.

Nur77 inhibits TR4-induced PEPCK expression in 3T3-L1 adipocytes

Nur77 is a member of the nuclear receptor 4A (NR4A) subgroup, which has been implicated in energy metabolism. Although Nur77 is found in adipose tissue, where TR4 plays a key role in lipid homeostasis, the role of Nur77 in adipogenesis is still controversial. Although the Nur77 responsive element (AAAGGTCA) is partially overlapped with TR4-binding sites (AGGTCA n AGGTCA: n=0–6), the regulatory role of Nur77 in TR4 function associated with adipocyte biology remains unclear. Here, we found that Nur77 inhibits adipogenesis and TR4 transcriptional activity. Treatment with a Nur77 agonist, 1,1-bis(3′-indolyl)-1-(p-anisyl)-methane, during 3T3-L1 adipocyte differentiation reduced adipogenesis. In reporter gene analysis, Nur77 specifically suppressed TR4 transcription activity but had little effect on PPARγ transcription activity. Consistently, Nur77 also suppressed TR4-induced promoter activity of the TR4 target gene PEPCK, which is known to be important for glyceroneogenesis in adipose tissue. Furthermore, Nur77 suppressed TR4 binding to TR4 response elements without direct interaction with TR4, suggesting that Nur77 may inhibit TR4 transcription activity via binding competition for TR4-binding sites. Furthermore, DIM-C-pPhOCH3 substantially suppressed TR4-induced PEPCK expression in 3T3-L1 adipocytes. Together, our data demonstrate that Nur77 plays an inhibitory role in TR4-induced PEPCK expression in 3T3-L1 adipocytes.