The mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) and glucose homeostasis: Has it been overlooked?

Background

Plasma glucose levels are tightly regulated within a narrow physiologic range. Insulin-mediated glucose uptake by tissues must be balanced by the appearance of glucose from nutritional sources, glycogen stores, or gluconeogenesis. In this regard, a common pathway regulating both glucose clearance and appearance has not been described. The metabolism of glucose to produce ATP is generally considered to be the primary stimulus for insulin release from beta-cells. Similarly, gluconeogenesis from phosphoenolpyruvate (PEP) is believed to be the primarily pathway via the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C). These models cannot adequately explain the regulation of insulin secretion or gluconeogenesis.

Scope of review

A metabolic sensing pathway involving mitochondrial GTP (mtGTP) and PEP synthesis by the mitochondrial isoform of PEPCK (PEPCK-M) is associated with glucose-stimulated insulin secretion from pancreatic beta-cells. Here we examine whether there is evidence for a similar mtGTP-dependent pathway involved in gluconeogenesis. In both islets and the liver, mtGTP is produced at the substrate level by the enzyme succinyl CoA synthetase (SCS-GTP) with a rate proportional to the TCA cycle. In the beta-cell PEPCK-M then hydrolyzes mtGTP in the production of PEP that, unlike mtGTP, can escape the mitochondria to generate a signal for insulin release. Similarly, PEPCK-M and mtGTP might also provide a significant source of PEP in gluconeogenic tissues for the production of glucose. This review will focus on the possibility that PEPCK-M, as a sensor for TCA cycle flux, is a key mechanism to regulate both insulin secretion and gluconeogenesis suggesting conservation of this biochemical mechanism in regulating multiple aspects of glucose homeostasis. Moreover, we propose that this mechanism may be important for regulating insulin secretion and gluconeogenesis compared to canonical nutrient sensing pathways.

Major conclusions

PEPCK-M, initially believed to be absent in islets, carries a substantial metabolic flux in beta-cells. This flux is intimately involved with the coupling of glucose-stimulated insulin secretion. PEPCK-M activity may have been similarly underestimated in glucose producing tissues and could potentially be an unappreciated but important source of gluconeogenesis.

General significance

The generation of PEP via PEPCK-M may occur via a metabolic sensing pathway important for regulating both insulin secretion and gluconeogenesis. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Anatomical and enzymic studies of leaves of a C3 × C4Flaveria F1 hybrid exhibiting reduced photorespiration

Flaveria pringlei exhibits C₃ CO₂ compensation concentration (Г) values averaging 53 μl CO₂/l at 21% (v/v) O₂ and 25 ± 2°C. When this species is hybridized with the C₄ species, F. brownii (male) ($\text{Г = 6 μ}\text{l CO}{}_2\text{/}\text{l}$), the F₁ hybrid plants exhibit an average Г value of 31 μl CO₂/l at 21% O₂.Although light micrographs of leaf cross-sections show that the leaves of the hybrid plants possess the mesophyll arrangement characteristic of F. pringlei leaves, the hybrid plants have some bundle-sheath chloroplasts. However, the numbers of these organelles do not appear to be intermediate with respect to the numbers in the parents and are closest to the small number present in the bundle-sheath cells of F. pringlei leaves. The activities of key C₄ enzymes (in μmol · mg Chl^?1 · h^?1) are: phosphoenolpyruvate (PEP) carboxylase, 121; pyruvate, orthophosphate (P_i) dikinase, 26; NADP-malate dehydrogenase, 2529; and NADP-malic enzyme, 82. All of these activities are substantially higher than in F. pringlei, but are only 7–10% of those in F. brownii (with the exception of the NADP-malate dehydrogenase activity). These data suggest that a C₄ cycle might be operating to a limited extent in the hybrid plants resulting in reduced photorespiration.Whether or not C₄ photosynthesis occurs in these hybrid plants, they represent the first reported C₃ × C₄ F₁ hybrids to exhibit reduced Γ-values. This cross and its reciprocal should be useful models for studying the anatomical and biochemical factors determining the development of limited C₄ photosynthesis in C₃ species.     

TR4 promotes fatty acid synthesis in 3T3-L1 adipocytes by activation of pyruvate carboxylase expression

We show that testicular orphan nuclear receptor 4 (TR4) increases the expression of pyruvate carboxylase (PC) gene in 3T3-L1 adipocytes by direct binding to a TR4 responsive element in the murine PC promoter. While TR4 overexpression increased PC activity, oxaloacetate (OAA) and glycerol levels with enhanced incorporation of ¹⁴C from ¹⁴C-pyruvate into fatty acids in 3T3-L1 adipocytes, PC knockdown by short interfering RNA (siRNA) or inhibition of PC activity by phenylacetic acid (PAA) abolished TR4-enhanced fatty acid synthesis. Moreover, TR4 microRNA reduced PC expression with decreased fatty acid synthesis in 3T3-L1 adipocytes, suggesting that TR4-mediated enhancement of fatty acid synthesis in adipocytes requires increased expression of PC gene.     

The impact of PEPC phosphorylation on growth and development of Arabidopsis thaliana: Molecular and physiological characterization of PEPC kinase mutants

Two phosphoenolpyruvate carboxylase (PEPC) kinase genes (PPCk1 and PPCk2) are present in the Arabidopsis genome; only PPCk1 is expressed in rosette leaves. Homozygous lines of two independent PPCk1 T-DNA-insertional mutants showed very little (dln1), or no (csi8) light-induced PEPC phosphorylation and a clear retard in growth under our greenhouse conditions. A mass-spectrometry-based analysis revealed significant changes in metabolite profiles. However, the anaplerotic pathway initiated by PEPC was only moderately altered. These data establish the PPCk1 gene product as responsible for leaf PEPC phosphorylation in planta and show that the absence of PEPC phosphorylation has pleiotropic consequences on plant metabolism.     

Chloroembryos: A unique photosynthesis system

The embryos of some angiosperm taxa contain chlorophyll and this chlorophyllous stage is persisting until the embryo matures (further referred as chloroembryos). Besides being chlorophyllous, these embryos seem to have the ability to photosynthesize. This suggests that the chlorophyllous state of the embryo has an important role in seed development. The photosynthesis of chloroembryos is highly shade adaptive in nature as it is embedded within the supporting tissues (several layers of pod wall, seed coat and endosperm). Moreover, these chloroembryos are developing in a highly osmotic environment, and contain various components of the photosynthetic machinery. Detailed studies were performed in these chloroembryos in order to elucidate the structure of the chloroplasts, pigment composition, the photochemical activities, the rate of carbon assimilation and also the shade adaptive features. It has been shown that the respired CO₂ within these chloroembryos is recycled by the efficient photosynthetic components of the chloroembryos and thus potentially influences the seed's carbon economy. Thus, the major role of embryonic photosynthesis is to produce both energy-rich molecules and oxygen, of which the former can be directly used for biosynthesis. During embryogenesis oxygen production is especially important, in a situation wherein the oxygen is limited within the enclosed seed. As these chloroembryos grow in an environment of a sugar rich endosperm, it requires some adaptive mechanisms in this high osmotic environment. The additional polypeptides found in the thylakoids of chloroembryo chloroplasts in comparison to the thylakoids of leaf chloroplast have been suggested to have a role in protecting the photosynthetic components in the chloroembryos in an environment of high osmotic strength. An attempt to understand osmotic stress tolerance existing in these chloroembryos may lead to a better understanding of tolerance of photosynthesis to osmotic stress.     

Isolation and characterization of an apple cytosolic malate dehydrogenase gene reveal its function in malate synthesis

Cytosolic NAD-dependent malate dehydrogenase (cyMDH) is an enzyme crucial for malate synthesis in the cytosol. The apple MdcyMDH gene (GenBank Accession No. DQ221207) encoding the cyMDH enzyme in apple was cloned and functionally characterized. The protein was subcellularly localized to the cytoplasm and plasma membrane. Based on kinetic parameters, it mainly catalyzes the reaction from oxalacetic acid (OAA) to malate in vitro. The expression level of MdcyMDH was positively correlated with malate dehydrogenase (MDH) activity throughout fruit development, but not with malate content, especially in the ripening apple fruit. MdcyMDH overexpression contributed to malate accumulation in the apple callus and tomato. Taken together, our results support the involvement of MdcyMDH directly in malate synthesis and indirectly in malate accumulation through the regulation of genes/enzymes associated with malate degradation and transportation, gluconeogenesis and the tricarboxylic acid cycle.     

Mapping photoautotrophic metabolism with isotopically nonstationary (13)C flux analysis

Understanding in vivo regulation of photoautotrophic metabolism is important for identifying strategies to improve photosynthetic efficiency or re-route carbon fluxes to desirable end products. We have developed an approach to reconstruct comprehensive flux maps of photoautotrophic metabolism by computational analysis of dynamic isotope labeling measurements and have applied it to determine metabolic pathway fluxes in the cyanobacterium Synechocystis sp. PCC6803. Comparison to a theoretically predicted flux map revealed inefficiencies in photosynthesis due to oxidative pentose phosphate pathway and malic enzyme activity, despite negligible photorespiration. This approach has potential to fill important gaps in our understanding of how carbon and energy flows are systemically regulated in cyanobacteria, plants, and algae.