The operation of PEPCK increases light harvesting plasticity in C4 NAD-ME and NADP-ME photosynthetic subtypes: A theoretical study.

The repeated emergence of NADP-malic enzyme (ME), NAD-ME and phosphoenolpyruvate carboxykinase (PEPCK) subtypes of C4 photosynthesis are iconic examples of convergent evolution, which suggests that these biochemistries do not randomly assemble, but are instead specific adaptations resulting from unknown evolutionary drivers. Theoretical studies that are based on the classic biochemical understanding have repeatedly proposed light-use efficiency as a possible benefit of the PEPCK subtype. However, quantum yield measurements do not support this idea. We explore this inconsistency here via an analytical model that features explicit descriptions across a seamless gradient between C4 biochemistries to analyse light harvesting and dark photosynthetic metabolism. Our simulations show that the NADP-ME subtype, operated by the most productive crops, is the most efficient. The NAD-ME subtype has lower efficiency, but has greater light harvesting plasticity (the capacity to assimilate CO2 in the broadest combination of light intensity and spectral qualities). In both NADP-ME and NAD-ME backgrounds, increasing PEPCK activity corresponds to greater light harvesting plasticity but likely imposed a reduction in photosynthetic efficiency. We draw the first mechanistic links between light harvesting and C4 subtypes, providing the theoretical basis for future investigation.

Inhibition of Sodium-Glucose Cotransporter-2 during Serum Deprivation Increases Hepatic Gluconeogenesis via the AMPK/AKT/FOXO Signaling Pathway.

BACKGRUOUND: Sodium-dependent glucose cotransporter 2 (SGLT2) mediates glucose reabsorption in the renal proximal tubules, and SGLT2 inhibitors are used as therapeutic agents for treating type 2 diabetes mellitus. This study aimed to elucidate the effects and mechanisms of SGLT2 inhibition on hepatic glucose metabolism in both serum deprivation and serum supplementation states. METHODS: Huh7 cells were treated with the SGLT2 inhibitors empagliflozin and dapagliflozin to examine the effect of SGLT2 on hepatic glucose uptake. To examine the modulation of glucose metabolism by SGLT2 inhibition under serum deprivation and serum supplementation conditions, HepG2 cells were transfected with SGLT2 small interfering RNA (siRNA), cultured in serum-free Dulbecco's modified Eagle's medium for 16 hours, and then cultured in media supplemented with or without 10% fetal bovine serum for 8 hours. RESULTS: SGLT2 inhibitors dose-dependently decreased hepatic glucose uptake. Serum deprivation increased the expression levels of the gluconeogenesis genes peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), glucose 6-phosphatase (G6pase), and phosphoenolpyruvate carboxykinase (PEPCK), and their expression levels during serum deprivation were further increased in cells transfected with SGLT2 siRNA. SGLT2 inhibition by siRNA during serum deprivation induces nuclear localization of the transcription factor forkhead box class O 1 (FOXO1), decreases nuclear phosphorylated-AKT (p-AKT), and p-FOXO1 protein expression, and increases phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK) protein expression. However, treatment with the AMPK inhibitor, compound C, reversed the reduction in the protein expression levels of nuclear p- AKT and p-FOXO1 and decreased the protein expression levels of p-AMPK and PEPCK in cells transfected with SGLT2 siRNA during serum deprivation. CONCLUSION: These data show that SGLT2 mediates glucose uptake in hepatocytes and that SGLT2 inhibition during serum deprivation increases gluconeogenesis via the AMPK/AKT/FOXO1 signaling pathway.

Sodium butyrate alleviates deoxynivalenol-induced porcine intestinal barrier disruption by promoting mitochondrial homeostasis via PCK2 signaling.

Deoxynivalenol (DON) is one of the most plentiful trichothecenes occurring in food and feed, which brings severe health hazards to both animals and humans. This study aims to investigate whether sodium butyrate (NaB) can protect the porcine intestinal barrier from DON exposure through promoting mitochondrial homeostasis. In a 4-week feeding experiment, 28 male piglets were allocated according to a 2 by 2 factorial arrangement of treatments with the main factors including supplementation of DON (< 0.8 vs. 4.0 mg/kg) and NaB (0.0 vs. 2 g/kg) in a corn/soybean-based diet. Dietary NaB supplementation mitigated the damaged mitochondrial morphology within the jejunal mucosa and the disrupted gut epithelial tight junctions irritated by DON. In IPEC-J2 cells, we found efficient recovery of the intestinal epithelial barrier occurred following NaB administration. This intestinal barrier reparation was facilitated by NaB-induced PCK2-mediated glyceroneogenesis and restoration of mitochondrial structure and function. In conclusion, we elucidated a mechanism of PCK2-mediated improvement of mitochondrial function by NaB to repair porcine intestinal barrier disruption during chronic DON exposure. Our findings highlight the promise of NaB for use in protecting against DON-induced gut epithelial tight junction disruption in piglets.

PEPCK and glucose metabolism homeostasis in arthropods.

The fat body is responsible for a variety of functions related to energy metabolism in arthropods, by controlling the processes of de novo glucose production (gluconeogenesis) and glycogen metabolism. The rate-limiting factor of gluconeogenesis is the enzyme phosphoenolpyruvate carboxykinase (PEPCK), generally considered to be the first committed step in this pathway. Although the study of PEPCK and gluconeogenesis has been for decades restricted to mammalian models, especially focusing on muscle and liver tissue, current research has demonstrated particularities about the regulation of this enzyme in arthropods, and described new functions. This review will focus on arthropod PEPCK, discuss different aspects to PEPCK regulation and function, its general role in the regulation of gluconeogenesis and other pathways. The text also presents our views on potentially important new directions for research involving this enzyme in a variety of metabolic adaptations (e.g. diapause), discussing enzyme isoforms, roles during arthropod embryogenesis, as well as involvement in vector-pathogen interactions, contributing to a better understanding of insect vectors of diseases and their control.

Biochemical, structural, and kinetic characterization of PPi -dependent phosphoenolpyruvate carboxykinase from Propionibacterium freudenreichii.

Phosphoenolpyruvate carboxykinases (PEPCK) are a well-studied family of enzymes responsible for the regulation of TCA cycle flux, where they catalyze the interconversion of oxaloacetic acid (OAA) and phosphoenolpyruvate (PEP) using a phosphoryl donor/acceptor. These enzymes have typically been divided into two nucleotide-dependent classes, those that use ATP and those that use GTP. In the 1960's and early 1970's, a group of papers detailed biochemical properties of an enzyme named phosphoenolpyruvate carboxytransphosphorylase (later identified as a third PEPCK) from Propionibacterium freudenreichii (PPi -PfPEPCK), which instead of using a nucleotide, utilized PPi to catalyze the same interconversion of OAA and PEP. The presented work expands upon the initial biochemical experiments for PPi -PfPEPCK and interprets these data considering both the current understanding of nucleotide-dependent PEPCKs and is supplemented with a new crystal structure of PPi -PfPEPCK in complex with malate at a putative allosteric site. Most interesting, the data are consistent with PPi -PfPEPCK being a Fe2+ activated enzyme in contrast with the Mn2+ activated nucleotide-dependent enzymes which in part results in some unique kinetic properties for the enzyme when compared to the more widely distributed GTP- and ATP-dependent enzymes.

Mitochondrial phosphoenolpyruvate carboxykinase promotes tumor growth in estrogen receptor-positive breast cancer via regulation of the mTOR pathway.

BACKGROUND: Tumor cells may aberrantly express metabolic enzymes to adapt to their environment for survival and growth. Targeting cancer-specific metabolic enzymes is a potential therapeutic strategy. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and links the tricarboxylic acid cycle and glycolysis/gluconeogenesis. Mitochondrial PEPCK (PEPCK-M), encoded by PCK2, is an isozyme of PEPCK and is distributed in mitochondria. Overexpression of PCK2 has been identified in many human cancers and demonstrated to be important for the survival program initiated upon metabolic stress in cancer cells. We evaluated the expression status of PEPCK-M and investigated the function of PEPCK-M in breast cancer. METHODS: We checked the expression status of PEPCK-M in breast cancer samples by immunohistochemical staining. We knocked down or overexpressed PCK2 in breast cancer cell lines to investigate the function of PEPCK-M in breast cancer. RESULTS: PEPCK-M was highly expressed in estrogen receptor-positive (ER+ ) breast cancers. Decreased cell proliferation and G0 /G1 arrest were induced in ER+ breast cancer cell lines by knockdown of PCK2. PEPCK-M promoted the activation of mTORC1 downstream signaling molecules and the E2F1 pathways in ER+ breast cancer. In addition, glucose uptake, intracellular glutamine levels, and mTORC1 pathways activation by glucose and glutamine in ER+ breast cancer were attenuated by PCK2 knockdown. CONCLUSION: PEPCK-M promotes proliferation and cell cycle progression in ER+ breast cancer via upregulation of the mTORC1 and E2F1 pathways. PCK2 also regulates nutrient status-dependent mTORC1 pathway activation in ER+ breast cancer. Further studies are warranted to understand whether PEPCK-M is a potential therapeutic target for ER+ breast cancer.

Metformin can mitigate skeletal dysplasia caused by Pck2 deficiency.

As an important enzyme for gluconeogenesis, mitochondrial phosphoenolpyruvate carboxykinase (PCK2) has further complex functions beyond regulation of glucose metabolism. Here, we report that conditional knockout of Pck2 in osteoblasts results in a pathological phenotype manifested as craniofacial malformation, long bone loss, and marrow adipocyte accumulation. Ablation of Pck2 alters the metabolic pathways of developing bone, particularly fatty acid metabolism. However, metformin treatment can mitigate skeletal dysplasia of embryonic and postnatal heterozygous knockout mice, at least partly via the AMPK signaling pathway. Collectively, these data illustrate that PCK2 is pivotal for bone development and metabolic homeostasis, and suggest that regulation of metformin-mediated signaling could provide a novel and practical strategy for treating metabolic skeletal dysfunction.

MiR-34a-5p promotes hepatic gluconeogenesis by suppressing SIRT1 expression.

Elevated hepatic gluconeogenesis is a major contributor of fasting hyperglycemia in diabetes. MicroRNAs (miRNAs) are tightly linked to glucose metabolism, but their role in hepatic gluconeogenesis remains largely unkown. In this current study, miR-34a-5p expression was significantly increased in liver tissues of db/db mice. Overexpression of miR-34a-5p promoted hepatic glucose production in mouse primary hepatocytes with increased expressions of gluconeogenic genes while miR-34a-5p inhibition displayed a contrary action. MiR-34a-5p overexpression in mouse primary hepatocytes repressed SIRT1 expression. SIRT1 inhibition by EX527 blocked phosphoenolpyruvate carboxykinase (PEPCK) protein degradation and enhanced hepatic gluconeogenesis. Treatment of A485 (a CBP/p300 inhibitor) decreased miR-34a-5p and PEPCK expressions in the livers of db/db mice, but elevated SIRT1 protein expression. In mouse primary hepatocytes, A485 exhibited a similar result. Overexpression of miR-34a-5p attenuated A485-inhibited gluconeogenic gene expressions and A485-induced SIRT1 protein expression. Finally, after miR-34a-5p was inhibited in the livers of db/db mice, hepatic glucose production and gluconeogenic gene expressions were markedly lowered. Our findings highlight a critical role of miR-34a-5p in the regulation of hepatic gluconeogenesis and miR-34a-5p may be a potential target in the treatment of type 2 diabetes.

FOXO1 is downregulated in obese mice subjected to short-term strength training.

Obesity is a worldwide health problem and is directly associated with insulin resistance and type 2 diabetes. The liver is an important organ for the control of healthy glycemic levels, since insulin resistance in this organ reduces phosphorylation of forkhead box protein 1 (FOXO1) protein, leading to higher hepatic glucose production (HGP) and fasting hyperglycemia. Aerobic physical training is known as an important strategy in increasing the insulin action in the liver by increasing FOXO1 phosphorylation and reducing gluconeogenesis. However, little is known about the effects of strength training in this context. This study aimed to investigate the effects of short-term strength training on hepatic insulin sensitivity and glycogen synthase kinase-3ß (GSK3ß) and FOXO1 phosphorylation in obese (OB) mice. To achieve this goal, OB Swiss mice performed the strength training protocol (one daily session for 15 days). Short-term strength training increased the phosphorylation of protein kinase B and GSK3ß in the liver after insulin stimulus and improved the control of HGP during the pyruvate tolerance test. On the other hand, sedentary OB animals reduced FOXO1 phosphorylation and increased the levels of nuclear FOXO1 in the liver, increasing the phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) content. The bioinformatics analysis also showed positive correlations between hepatic FOXO1 levels and gluconeogenic genes, reinforcing our findings. However, strength-trained animals reverted to this scenario, regardless of body adiposity changes. In conclusion, short-term strength training is an efficient strategy to enhance the insulin action in the liver of OB mice, contributing to glycemic control by reducing the activity of hepatic FOXO1 and lowering PEPCK and G6Pase contents.

Glycosylation defects, offset by PEPCK-M, drive entosis in breast carcinoma cells.

On glucose restriction, epithelial cells can undergo entosis, a cell-in-cell cannibalistic process, to allow considerable withstanding to this metabolic stress. Thus, we hypothesized that reduced protein glycosylation might participate in the activation of this cell survival pathway. Glucose deprivation promoted entosis in an MCF7 breast carcinoma model, as evaluated by direct inspection under the microscope, or revealed by a shift to apoptosis + necrosis in cells undergoing entosis treated with a Rho-GTPase kinase inhibitor (ROCKi). In this context, curbing protein glycosylation defects with N-acetyl-glucosamine partially rescued entosis, whereas limiting glycosylation in the presence of glucose with tunicamycin or NGI-1, but not with other unrelated ER-stress inducers such as thapsigargin or amino-acid limitation, stimulated entosis. Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is upregulated by glucose deprivation, thereby enhancing cell survival. Therefore, we presumed that PEPCK-M could play a role in this process by offsetting key metabolites into glycosyl moieties using alternative substrates. PEPCK-M inhibition using iPEPCK-2 promoted entosis in the absence of glucose, whereas its overexpression inhibited entosis. PEPCK-M inhibition had a direct role on total protein glycosylation as determined by Concanavalin A binding, and the specific ratio of fully glycosylated LAMP1 or E-cadherin. The content of metabolites, and the fluxes from 13C-glutamine label into glycolytic intermediates up to glucose-6-phosphate, and ribose- and ribulose-5-phosphate, was dependent on PEPCK-M content as measured by GC/MS. All in all, we demonstrate for the first time that protein glycosylation defects precede and initiate the entosis process and implicates PEPCK-M in this survival program to dampen the consequences of glucose deprivation. These results have broad implications to our understanding of tumor metabolism and treatment strategies.

Role of PCK2 in the proliferation of vascular smooth muscle cells in neointimal hyperplasia.

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets (GSE28829, GSE43292, GSE100927, and GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.

Reduced insulin signaling and high glucagon in early insulin resistance impaired fast-fed regulation of renal gluconeogenesis via insulin receptor substrate.

Gluconeogenesis is one of the key processes through which the kidney contributes to glucose homeostasis. Urinary exosomes (uE) have been used to study renal gene regulation noninvasively in humans and rodents. Recently, we demonstrated fast-fed regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme for gluconeogenesis, in human uE. The regulation was impaired in subjects with early insulin resistance. Here, we studied primary human proximal tubule cells (hPT) and human uE to elucidate a potential link between insulin resistance and fast-fed regulation of renal PEPCK. We demonstrate that fasted hPTs had higher PEPCK and insulin receptor substrate-2 (IRS2) mRNA and protein levels, relative to fed cells. The fast-fed regulation was, however, attenuated in insulin receptor knockdown (IRKO) hPTs. The IRKO was confirmed by the blunted insulin-induced response on PEPCK, PGC1α, p-IR, and p-AKT expression in IRKO cells. Exosomes secreted by the wild-type or IRKO hPT showed similar regulation to the respective hPT. Similarly, in human uE, the relative abundance of IRS-2 mRNA (to IRS1) was higher in the fasted state relative to the fed condition. However, the fast-fed difference was absent in subjects with early insulin resistance. These subjects had higher circulating glucagon levels relative to subjects with optimal insulin sensitivity. Furthermore, in hPT cells, glucagon significantly induced PEPCK and IRS2 gene, and gluconeogenesis. IR knockdown in hPT cells further increased the gene expression levels. Together the data suggest that reduced insulin sensitivity and high glucagon in early insulin resistance may impair renal gluconeogenesis via IRS2 regulation.

Low expression of PCK2 in breast tumors contributes to better prognosis by inducing senescence of cancer cells.

Cell cycle arrest, one of the main characteristics of cellular senescence, has been described as a crucial barrier that needs to be bypassed for cancer progression. Typically, cellular senescence can be induced by multiple stresses including telomere shortening, oncogenic activation as well as therapy treatment, and contributes to the inhibition of epithelial-mesenchymal transition (EMT), tumor suppression or progression depending on the senescence-associated secretory phenotype (SASP) components. However, the mechanisms underlying cancer cell senescence remain partially understood. Here, according to METABRIC database, we identified that patients with senescent-like breast tumors show better short-term survival, lower tendency of neoplasm histological grades, lower tumor stages, and negative status of estrogen receptor (ER) and progesterone receptor (PR) compared with non-senescent ones. Interestingly, Kyoto encyclopedia of genes and genomes (KEGG) analysis identified insulin signaling was significantly repressed in senescent breast tumors. Further verification in cultured breast cancer cells indicated that phosphoenolpyruvate carboxykinase 2 (PCK2) was significantly inhibited after therapy treatment. In addition, knockdown of PCK2 induced a senescent phenotype of breast cancer cells. Moreover, comparing with the non-senescent group, the senescent breast cancers displayed lower EMT capacity both in patients and breast cancer cell lines after knocking down PCK2. In conclusion, we described for the first time that low expression level of PCK2 may contribute to better prognosis via triggering senescent phenotype and thereby inhibiting EMT capacity in breast cancers.

Adenylate Kinase Isozyme 3 Regulates Mitochondrial Energy Metabolism and Knockout Alters HeLa Cell Metabolism.

The balance between oxidative phosphorylation and glycolysis is important for cancer cell growth and survival, and changes in energy metabolism are an emerging therapeutic target. Adenylate kinase (AK) regulates adenine nucleotide metabolism, maintaining intracellular nucleotide metabolic homeostasis. In this study, we focused on AK3, the isozyme localized in the mitochondrial matrix that reversibly mediates the following reaction: Mg2+ GTP + AMP ⇌ Mg2+ GDP + ADP. Additionally, we analyzed AK3-knockout (KO) HeLa cells, which showed reduced proliferation and were detected at an increased number in the G1 phase. A metabolomic analysis showed decreased ATP; increased glycolytic metabolites such as glucose 6 phosphate (G6P), fructose 6 phosphate (F6P), and phosphoenolpyruvate (PEP); and decreased levels of tricarboxylic acid (TCA) cycle metabolites in AK3KO cells. An intracellular ATP evaluation of AK3KO HeLa cells transfected with ATeam plasmid, an ATP sensor, showed decreased whole cell levels. Levels of mitochondrial DNA (mtDNA), a complementary response to mitochondrial failure, were increased in AK3KO HeLa cells. Oxidative stress levels increased with changes in gene expression, evidenced as an increase in related enzymes such as superoxide dismutase 2 (SOD2) and SOD3. Phosphoenolpyruvate carboxykinase 2 (PCK2) expression and PEP levels increased, whereas PCK2 inhibition affected AK3KO HeLa cells more than wild-type (WT) cells. Therefore, we concluded that increased PCK2 expression may be complementary to increased GDP, which was found to be deficient through AK3KO. This study demonstrated the importance of AK3 in mitochondrial matrix energy metabolism.

Posttranscriptional regulation of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase in Komagataella phaffii.

The yeast Komagataella phaffii (a.k.a. Pichia pastoris) harbours a unique glutamate utilization pathway in which the cytosolic enzymes glutamate dehydrogenase 2 (GDH2), aspartate aminotransferase 2 (AAT2) and phosphoenolpyruvate carboxykinase (PEPCK) catalyze the sequential conversion of glutamate to α-ketoglutarate, oxaloacetate and phosphoenolpyruvate respectively. GDH2 and PEPCK are essential for glutamate catabolism. Their synthesis is induced by autophagy during carbon starvation and are essential for cell survival. Here, we demonstrate that GDH2 and PEPCK reciprocally regulate each other's protein levels during glutamate catabolism such that GDH2 is downregulated in Δpepck and PEPCK is downregulated in Δgdh2. We further demonstrate that sequential conversion of glutamate to α-ketoglutarate and oxaloacetate by GDH2 and AAT2, respectively, is essential for PEPCK synthesis in cells metabolizing glutamate. Our studies indicate that translation of GDH2 mRNA is induced by glutamate while oxaloacetate derived from glutamate is likely to be the inducer of PEPCK mRNA translation during glutamate catabolism. Thus, GDH2- and PEPCK-catalyzed reactions are essential for ATP generation and gluconeogenesis respectively during carbon starvation and glutamate catabolism in K. phaffii. We conclude that K. phaffii harbours a unique translational regulatory circuit in which substrates of GDH2 and PEPCK act as inducers of their synthesis, a phenomenon not reported in any yeast species.

Acid-base effects of combined renal deletion of NBCe1-A and NBCe1-B.

The molecular mechanisms regulating ammonia metabolism are fundamental to acid-base homeostasis. Deletion of the A splice variant of Na+-bicarbonate cotransporter, electrogenic, isoform 1 (NBCe1-A) partially blocks the effect of acidosis to increase urinary ammonia excretion, and this appears to involve the dysregulated expression of ammoniagenic enzymes in the proximal tubule (PT) in the cortex but not in the outer medulla (OM). A second NBCe1 splice variant, NBCe1-B, is present throughout the PT, including the OM, where NBCe1-A is not present. The purpose of the present study was to determine the effect of combined renal deletion of NBCe1-A and NBCe1-B on systemic and PT ammonia metabolism. We generated NBCe1-A/B deletion using Cre-loxP techniques and used Cre-negative mice as controls. As renal NBCe1-A and NBCe1-B expression is limited to the PT, Cre-positive mice had PT NBCe1-A/B deletion [PT-NBCe1-A/B knockout (KO)]. Although on a basal diet, PT-NBCe1-A/B KO mice had severe metabolic acidosis, yet urinary ammonia excretion was not changed significantly. PT-NBCe1-A/B KO decreased the expression of phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase and increased the expression of glutamine synthetase, an ammonia-recycling enzyme, in PTs in both the cortex and OM. Exogenous acid loading increased ammonia excretion in control mice, but PT-NBCe1-A/B KO prevented any increase. PT-NBCe1-A/B KO significantly blunted acid loading-induced changes in phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamine synthetase expression in PTs in both the cortex and OM. We conclude that NBCe1-B, at least in the presence of NBCe1-A deletion, contributes to PT ammonia metabolism in the OM and thereby to systemic acid-base regulation.NEW & NOTEWORTHY The results of the present study show that combined deletion of both A and B splice variants of electrogenic Na+-bicarbonate cotransporter 1 from the proximal tubule impairs acid-base homeostasis and completely blocks changes in ammonia excretion in response to acidosis, indicating that both proteins are critical to acid-base homeostasis.

Propofol ameliorates acute postoperative fatigue and promotes glucagon-regulated hepatic gluconeogenesis by activating CREB/PGC-1α and accelerating fatty acids beta-oxidation.

Postoperative fatigue (POF) is the most common and long-lasting complication after surgery, which brings heavy burden to individuals and society. Recently, hastening postoperative recovery receives increasing attention, but unfortunately, the mechanisms underlying POF remain unclear. Propofol is a wildly used general anesthetic in clinic, and inspired by the rapid antidepressant effects induced by ketamine at non-anesthetic dose, the present study was undertaken to investigate the anti-fatigue effects and underlying mechanisms of propofol at a non-anesthetic dose in 70% hepatectomy induced POF model in rats. We first showed here that single administration of propofol at 0.1 mg/kg ameliorated acute POF in hepatectomy induced POF rats. Based on metabonomics analysis, we hypothesized that propofol exerted anti-fatigue activity in POF rats by facilitating free fatty acid (FFA) oxidation and gluconeogenesis. We further confirmed that propofol restored the deficit in FFA oxidation and gluconeogenesis in POF rats, as evidenced by the elevated FFA utilization, acetyl coenzyme A content, pyruvic acid content, phosphoenolpyruvic acid content, hepatic glucose output and glycogen storage. Moreover, propofol stimulated glucagon secretion and up-regulated expression of cAMP-response element binding protein (CREB), phosphorylated CREB, peroxlsome prolifeator-activated receptor-γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinade1 and carnitine palmitoltransferase 1A. In summary, our study suggests for the first time that propofol ameliorates acute POF by promoting glucagon-regulated gluconeogenesis via CREB/PGC-1α signaling and accelerating FFA beta-oxidation.

Optimized 3D Culture of Hepatic Cells for Liver Organoid Metabolic Assays.

The liver is among the principal organs for glucose homeostasis and metabolism. Studies of liver metabolism are limited by the inability to expand primary hepatocytes in vitro while maintaining their metabolic functions. Human hepatic three-dimensional (3D) organoids have been established using defined factors, yet hepatic organoids from adult donors showed impaired expansion. We examined conditions to facilitate the expansion of adult donor-derived hepatic organoids (HepAOs) and HepG2 cells in organoid cultures (HepGOs) using combinations of growth factors and small molecules. The expansion dynamics, gluconeogenic and HNF4α expression, and albumin secretion are assessed. The conditions tested allow the generation of HepAOs and HepGOs in 3D cultures. Nevertheless, gluconeogenic gene expression varies greatly between conditions. The organoid expansion rates are limited when including the TGFß inhibitor A8301, while are relatively higher with Forskolin (FSK) and Oncostatin M (OSM). Notably, expanded HepGOs grown in the optimized condition maintain detectable gluconeogenic expression in a spatiotemporal distribution at 8 weeks. We present optimized conditions by limiting A8301 and incorporating FSK and OSM to allow the expansion of HepAOs from adult donors and HepGOs with gluconeogenic competence. These models increase the repertoire of human hepatic cellular tools available for use in liver metabolic assays.

Simultaneous Tests of Theaflavin-3,3'-digallate as an Anti-Diabetic Drug in Human Hepatoma G2 Cells and Zebrafish (Danio rerio).

Theaflavin-3,3'-digallate (TF3) is the most important theaflavin monomer in black tea. TF3 was proved to reduce blood glucose level in mice and rats. However, the elaborate anti-diabetic mechanism was not well elucidated. In this work, human hepatoma G2 (HepG2) cells and zebrafish (Danio rerio) were used simultaneously to reveal anti-diabetic effect of TF3. The results showed that TF3 could effectively rise glucose absorption capacity in insulin-resistant HepG2 cells and regulate glucose level in diabetic zebrafish. The hypoglycemic effect was mediated through down-regulating phosphoenolpyruvate carboxykinase and up-regulating glucokinase. More importantly, TF3 could significantly improve ß cells regeneration in diabetic zebrafish at low concentrations (5 µg/mL and 10 µg/mL), which meant TF3 had a strong anti-diabetic effect. Obviously, this work provided the potential benefit of TF3 on hypoglycemic effect, regulating glucose metabolism enzymes, and protecting ß cells. TF3 might be a promising agent for combating diabetes.

Role of the renal androgen receptor in sex differences in ammonia metabolism.

There are sex differences in renal ammonia metabolism and structure, many of which are mediated by testosterone. The goal of the present study was to determine the role of renal expression of testosterone's canonical receptor, androgen receptor (AR), in these sexual dimorphisms. We studied mice with kidney-specific AR deletion [KS-AR-knockout (KO)] generated using Cre/loxP techniques; control mice were Cre-negative littermates (wild type). In male but not female mice, KS-AR-KO increased ammonia excretion, which eliminated sex differences. Although renal structural size typically parallel ammonia excretion, KS-AR-KO decreased kidney size, cortical proximal tubule volume density, and cortical proximal tubule cell height in males-neither were altered in females and collecting duct volume density was unaltered in both sexes. Analysis of key protein involved in ammonia handling showed in male mice that KS-AR-KO increased both phosphoenolpyruvate carboxykinase (PEPCK) and Na+-K+-2Cl- cotransporter (NKCC2) expression and decreased Na+/H+ exchanger isoform 3 (NHE3) and electrogenic Na+-bicarbonate cotransporter 1 (NBCe1)-A expression. In female mice, KS-AR-KO did not alter these parameters. These effects occurred even though KS-AR-KO did not alter plasma testosterone, food intake, or serum Na+, K+, or [Formula: see text] significantly in either sex. In conclusion, AR-dependent signaling pathways in male, but not female, kidneys regulate PEPCK and NKCC2 expression and lead to the sexual differences in ammonia excretion. Opposing effects on NHE3 and NBCe1-A expression likely limit the magnitude of ammonia excretion changes. As AR is not present in the thick ascending limb, the effect of KS-AR-KO on NKCC2 expression is indirect. Finally, AR mediates the greater kidney size and proximal tubule volume density in male compared with female mice.NEW & NOTEWORTHY Sexual dimorphisms in ammonia metabolism involve androgen receptor (AR)-dependent signaling pathways in male, but not female, kidneys that lead to altered proximal tubule (PT), phosphoenolpyruvate carboxykinase, and thick ascending limb Na+-K+-2Cl- cotransporter expression. Adaptive responses in Na+/H+ exchanger 3 and electrogenic Na+-bicarbonate cotransporter 1-A expression limit the magnitude of the effect on ammonia excretion. Finally, the greater kidney size and PT volume density in male mice is the result of PT androgen signaling through AR.