Structural basis for the recruitment of glycogen synthase by glycogenin

Glycogen is a primary form of energy storage in eukaryotes that is essential for glucose homeostasis. The glycogen polymer is synthesized from glucose through the cooperative action of glycogen synthase (GS), glycogenin (GN), and glycogen branching enzyme and forms particles that range in size from 10 to 290 nm. GS is regulated by allosteric activation upon glucose-6-phosphate binding and inactivation by phosphorylation on its N- and C-terminal regulatory tails. GS alone is incapable of starting synthesis of a glycogen particle de novo, but instead it extends preexisting chains initiated by glycogenin. The molecular determinants by which GS recognizes self-glucosylated GN, the first step in glycogenesis, are unknown. We describe the crystal structure of Caenorhabditis elegans GS in complex with a minimal GS targeting sequence in GN and show that a 34-residue region of GN binds to a conserved surface on GS that is distinct from previously characterized allosteric and binding surfaces on the enzyme. The interaction identified in the GS-GN costructure is required for GS–GN interaction and for glycogen synthesis in a cell-free system and in intact cells. The interaction of full-length GS-GN proteins is enhanced by an avidity effect imparted by a dimeric state of GN and a tetrameric state of GS. Finally, the structure of the N- and C-terminal regulatory tails of GS provide a basis for understanding phosphoregulation of glycogen synthesis. These results uncover a central molecular mechanism that governs glycogen metabolism.Glycogen forms the major rapidly accessible energy reserve in eukaryotes and, as such, is essential for cellular and whole-body energy supply and glucose homeostasis. In mammals, glucose is stored as glycogen mainly in muscle and liver cells (and to a lesser extent in astrocytes, adipocytes, and kidney and pancreatic cells) when blood glucose levels are high, and then released for utilization within the cell, or systemically when glucose and energy levels are low. The dysregulation of glycogen metabolism contributes to glycogen storage diseases (1), cardiac myopathies (2), neurodegeneration (3), insulin resistance (4), and cancer (5). Notably, the up-regulation of glycogen synthesis provides an alternate source of energy under hypoxic conditions and contributes to cancer cell survival in preangiogenic states (5).Glycogen is a branched polymer of glucose formed primarily through α1,4 glycosidic linkages, with periodic intersecting α1,6 linkages serving as branch points. In eukaryotes, glycogen is synthesized through the cooperative action of three enzymes, namely glycogen synthase (GS), glycogenin (GN), and glycogen branching enzyme (GBE) (1), which use UDP-glucose (UDP-G) as a glucose donor. A GN dimer initiates the glycogen polymer by autoglucosylation of a conserved tyrosine residue (Tyr195 in human GN1, Tyr230 in yeast GN1, and Tyr194 in Caenorhabditis elegans GN), leading to an α1,4-linked chain of 8–12 glucose units (6). This oligosaccharide remains attached to glycogenin and forms a primer that is converted into a full-size glycogen particle by the combined actions of GS and GBE (1, 7). Once fully elaborated, a glycogen particle can comprise up to ∼55,000 glucose residues with a size distribution in muscle tissue of 10–44 nm in diameter, termed β particles (8, 9). In liver, glycogen particles of 110–290 nm in diameter, termed α particles, are formed by the assembly of several β particles, possibly through covalent linkage (9, 10). Glycogen particle size varies greatly between tissues and species (11), but the basis and significance of these size differences are poorly understood.Glycogenin is a member of the GT8 family of glycosyltransferases with a GT-A architecture containing an N-terminal catalytic domain with a single Rossmann fold that operates as an obligate dimer (12–14). The core catalytic domain is followed by a C-terminal extension of variable length and undefined structure. The last 35 amino acids of this tail in human and yeast contain a conserved motif that is sufficient for binding to GS in cell lysates (15). The region that separates the core catalytic domain of GN and the GS binding motif is highly variable both in sequence and in length (Fig. 1A). Most eukaryotes possess two versions of GN that differ in the length of this linker, which ranges from 50 to 257 residues in yeast, 7–142 residues in worm and 34–170 residues in human. In addition, the linker region is a site of alternative splicing in humans that imparts further length variation (16). The functional significance of the variability in linker length has yet to be explored.Open in a separate windowFig. 1.Structure of CeGS in complex with CeGN³⁴. (A) Domain architecture of CeGS (GT-B fold) and CeGN (GT-A fold). (B) Interaction of CeGN³⁴ to CeGS determined by fluorescence polarization. K_d value ± SEM is the average of three independent experiments carried out in duplicate. (C) Structure of the CeGS tetramer bound to the CeGN³⁴ peptide. (D) Comparison of CeGS protomers (blue) and the budding yeast ScGS (PDB ID code 3NAZ, gray). The N-terminal extension of CeGS (green) and C-terminal extensions of CeGS (red) and ScGS (orange) are highlighted. Phosphoregulatory sites 2 (S12), 2a (T19), 3a (S654), 3b (S658), and 3c (S662) and the allosteric regulatory site (Arg cluster) on the CeGS protomer are shown as ball and stick models with violet-colored carbon atoms. (E) Peel away surface representations of the CeGS–CeGN complex with contact residues (CeGS, Upper; CeGN, Lower). (F) Detailed stereoview of the CeGS–CeGN³⁴ complex. CeGS is shown in blue and CeGN in green. Side chain residues that make direct contacts are shown as sticks with colored heteroatoms (oxygen, red; nitrogen, dark blue; sulfur, yellow). Glycine residues are depicted as blue spheres, and carbonyl oxygens are shown as red spheres. Hydrogen bonds are depicted as dotted lines.GS synthesizes α1,4-linked glucose polymers. The mammalian and yeast GS enzymes belong to the GT3 family of glycosyltransferases and are regulated by covalent phosphorylation and allosteric ligand interactions. In contrast, the bacterial and plant GS enzymes belong to the GT5 family and appear to lack these regulatory features, although both the GT3 and GT5 families have a GT-B architecture comprised of two tightly associated Rossmann fold domains (13, 17). GS, unlike GN, lacks the ability to initiate glucose chain formation and instead only catalyzes the extension of primed chains generated by GN. Because GN is a constitutively active enzyme, glycogen synthesis is regulated at the level of GS catalytic function. GS activity is tightly repressed by phosphorylation (1, 18) on regulatory sites within N-terminal (sites 2 and 2a) and C-terminal (sites 3a, 3b, and 3c) extensions to the core Rossmann fold domains that differ significantly in sequence between metazoan and fungal species. GS activity is potently activated by the combination of dephosphorylation and the binding of the allosteric activator glucose-6-phosphate (G6P) (1, 19, 20). The structures of the yeast GS enzyme have yielded insights into allosteric regulation by G6P and provide a template for understanding phosphoregulation in fungal species, features of which are likely conserved in metazoan orthologs (19). The structural basis by which GS and GN interact and how this interaction contributes to glycogen synthesis remains uncharacterized.Here, we report the X-ray crystal structure of GS in complex with a minimal targeting region of GN from C. elegans. The structure reveals that CeGN binds a conserved surface on CeGS that is remote from previously characterized sites for G6P, UDP, sugar, and tetramer interactions. This interaction surface is required for glycogen synthesis in vitro and in vivo, and the CeGS–CeGN interaction is enhanced by an avidity effect between the CeGN dimer and the CeGS tetramer. Finally, the structure also reveals conserved features of the phosphoregulatory elements of CeGS. Collectively, these results explain how CeGN initiates glycogen chain synthesis by CeGS.     

C-S bond cleavage by a polyketide synthase domain

Leinamycin (LNM) is a sulfur-containing antitumor antibiotic featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. The 1,3-dioxo-1,2-dithiolane moiety is essential for LNM’s antitumor activity, by virtue of its ability to generate an episulfonium ion intermediate capable of alkylating DNA. We have previously cloned and sequenced the lnm gene cluster from Streptomyces atroolivaceus S-140. In vivo and in vitro characterizations of the LNM biosynthetic machinery have since established that: (i) the 18-membered macrolactam backbone is synthesized by LnmP, LnmQ, LnmJ, LnmI, and LnmG, (ii) the alkyl branch at C-3 of LNM is installed by LnmK, LnmL, LnmM, and LnmF, and (iii) leinamycin E1 (LNM E1), bearing a thiol moiety at C-3, is the nascent product of the LNM hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS). Sulfur incorporation at C-3 of LNM E1, however, has not been addressed. Here we report that: (i) the bioinformatics analysis reveals a pyridoxal phosphate (PLP)-dependent domain, we termed cysteine lyase (SH) domain (LnmJ-SH), within PKS module-8 of LnmJ; (ii) the LnmJ-SH domain catalyzes C-S bond cleavage by using l-cysteine and l-cysteine S-modified analogs as substrates through a PLP-dependent β-elimination reaction, establishing l-cysteine as the origin of sulfur at C-3 of LNM; and (iii) the LnmJ-SH domain, sharing no sequence homology with any other enzymes catalyzing C-S bond cleavage, represents a new family of PKS domains that expands the chemistry and enzymology of PKSs and might be exploited to incorporate sulfur into polyketide natural products by PKS engineering.Sulfur is found in many primary metabolites, such as thiamin, biotin, molybdenum cofactors, lipoic acid, iron-sulfur clusters, and nucleosides including 2-thiocytidine, 4-thiouridine, and 2-methylthio-N⁶-isopentenyl adenosine (1). There is a wealth of information on how sulfur is incorporated into these primary metabolites, the key step of which is catalyzed by a desulfurase to produce a protein-bound cysteine persulfide by using cysteine as a substrate (2–5). Sulfur is also found in many secondary metabolites (interchangeably referred to as natural products here). The major groups of sulfur-containing natural products are peptides produced by ribosome or nonribosomal peptide synthetases (NRPSs). Sulfur incorporation into these natural products from intact cysteine or methionine is well characterized. However, sulfur incorporation into other natural products remains poorly understood. Only a few cases featured by C-S bond formation or C-S bond cleavage steps were reported to date, and most of the mechanisms require further investigation (6–15).Leinamycin (LNM) is a sulfur-containing antitumor antibiotic produced by Streptomyces atroolivaceus S-140 (16). The structure of LNM is characterized by an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring (Fig. 1). The 1,3-dioxo-1,2-dithiolane moiety is essential for LNM’s antitumor activity by virtue of its ability to alkylate the N7 position of the deoxyguanosine of DNA through an episulfonium ion intermediate (17–19). We have previously cloned and sequenced the lnm gene cluster from S. atroolivaceus S-140 (20–22). In vivo and in vitro characterizations of the LNM biosynthetic machinery have since established that: (i) the 18-membered hybrid peptide-polyketide macrolactam backbone is synthesized by a hybrid NRPS-acyltransferase (AT)-less type I polyketide synthase (PKS), consisting of LnmQ (adenylation protein), LnmP (peptide carrier protein), LnmI (a hybrid NRPS-AT-less type I PKS), LnmJ (PKS), and LnmG (AT) (21, 23, 24); (ii) the alkyl branch at C-3 of LNM is installed by a novel pathway for β-alkylation in polyketide biosynthesis, featuring LnmK (AT/decarboxylase), LnmL (ACP), LnmM (hydroxymethylglutaryl-CoA synthase), and LnmF (enoyl-CoA hydratase) that act on the growing polyketide intermediate tethered to the ACP domain of PKS module-8 of LnmJ (25–27); and (iii) leinamycin E1 (LNM E1), bearing a thiol moiety at C-3, is the nascent product of the LNM hybrid NRPS-AT-less type I PKS (28) (Fig. 1). Although sulfur incorporation into the 1,3-dioxo-1,2-dithiolane moiety of LNM has not been addressed, the structures of biosynthetic intermediates and shunt metabolites accumulated in the SB3029 (ΔlnmK), SB3030 (ΔlnmL), and SB3031 (ΔlnmM) mutant strains suggest that the β-alkyl branch at C-3 is installed before the sulfur incorporation, within the PKS module-8 of LnmJ (26), and the structure of LNM E1 further suggests that one sulfur at C-3 is introduced after the β-alkyl branch formation, but before the linear polyketide is released from the PKS module-8 of LnmJ by the thioesterase (TE)-catalyzed cyclization (28). Therefore, the region of approximately 850 aa between the ACP and TE domains in PKS module-8 of LnmJ represent potential candidates responsible for the sulfur incorporation at C-3 of LNM E1 and LNM (Fig. 1).Open in a separate windowFig. 1.Proposed biosynthetic pathway for LNM featuring (i) the LnmQPIJ hybrid NRPS-AT–less type I PKS with the discrete LnmG AT loading the malonyl CoA extender units to all six PKS modules (21, 23, 24), (ii) the LnmKLMF enzymes catalyzing introduction of the β-alkyl branch at C-3 (25-27), (iii) the DUF and SH domains of PKS module-8 of LnmJ catalyzing sulfur incorporation into C-3 of LNM from l-cysteine characterized in this study, and (iv) LNM E1 as the nascent product of the LNM hybrid NRPS-AT-lee type I PKS (28), setting the stage to investigate the tailoring steps that convert LNM E1 to LNM. Color coding indicates the moieties installed by NRPS (blue), PKS (red), β-alkyl branch (green), and other tailing enzyme (black), and the green ovals denote AT docking domains. A, adenylation domain; AT, acyltransferase; Cy, condensation/cyclization; DH, dehydratase; DUF, domain of unknown function; KR, ketoreductase; KS, ketosynthase; MT, methyltransferase; Ox, oxidation; PCP, peptidyl carrier protein; SAM, S-adenosylmethionine; SH, PLP-dependent cysteine lyase domain; TE, thioesterase.We here report that: (i) the bioinformatics analysis reveals a PLP-dependent domain, we termed cysteine lyase (SH) domain (LnmJ-SH), together with a domain of unknown function (DUF), located between the ACP and TE domains in PKS module-8 of LnmJ; (ii) the LnmJ-SH domain catalyzes C-S bond cleavage by using l-cysteine and l-cysteine S-modified analogs as substrates through a PLP-dependent β-elimination reaction, establishing l-cysteine as the origin of sulfur at C-3 of LNM and LNM E1; and (iii) the LnmJ-SH domain, sharing no sequence homology with any other enzymes catalyzing C-S bond cleavage in natural product biosynthesis, represents a new family of PKS domains that expands the chemistry and enzymology of PKSs.     

Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independent of AMPK

The multifunctional AMPK-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth, and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because although on one hand active AMPK inhibits mammalian target of rapamycin (mTOR) and lipogenesis—two crucial arms of cancer growth—AMPK also ensures viability by metabolic reprogramming in cancer cells. AMPK activation by two indirect AMPK agonists AICAR and metformin (now in over 50 clinical trials on cancer) has been correlated with reduced cancer cell proliferation and viability. Surprisingly, we found that compared with normal tissue, AMPK is constitutively activated in both human and mouse gliomas. Therefore, we questioned whether the antiproliferative actions of AICAR and metformin are AMPK independent. Both AMPK agonists inhibited proliferation, but through unique AMPK-independent mechanisms and both reduced tumor growth in vivo independent of AMPK. Importantly, A769662, a direct AMPK activator, had no effect on proliferation, uncoupling high AMPK activity from inhibition of proliferation. Metformin directly inhibited mTOR by enhancing PRAS40’s association with RAPTOR, whereas AICAR blocked the cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. Together, our results suggest that although AICAR and metformin are potent AMPK-independent antiproliferative agents, physiological AMPK activation in glioma may be a response mechanism to metabolic stress and anticancer agents.AMP-activated protein kinase (AMPK) is a molecular hub for cellular metabolic control (1–4). It is a heterotrimer of catalytic α, regulatory β, and γ subunits. The rising AMP:ATP ratio during energy stress leads to AMP-dependent phosphorylation of the catalytic α subunits. This activates AMPK which then phosphorylates numerous substrates to restore energy homeostasis. It phosphorylates acetyl CoA carboxylase (ACCα) to inhibit fatty acid (FA) synthesis (5) and TSC2 and RAPTOR (6, 7) to inhibit mammalian target of rapamycin (mTOR)C1. Because fatty acid synthesis and mTORC1 activity are essential for cell proliferation and growth (8), AMPK activation with two indirect AMPK agonists AICAR and metformin have been correlated with suppression of cell proliferation and growth (9–11).AICAR is metabolized to an AMP mimetic, ZMP that activates AMPK (12). Although AICAR does inhibit proliferation (11–15), it also causes AMPK-independent cellular and metabolic effects (12, 16) including inhibition of glucokinase, glycogen phosphorylase, and nucleotide biosynthesis (17, 18). Whether AICAR requires AMPK to suppress proliferation is questionable because although both AICAR and 2-deoxyglucose activated AMPK, only AICAR inhibited proliferation of trisomic mouse fibroblasts (11). Moreover, although AICAR strongly increases glucose uptake through AMPK activation in muscle cells, it reduced fluorodeoxyglucose-PET signals and inhibited glioma growth in vivo (9), suggesting that reduced PET signals could be due to its AMPK-independent antiglioma action.The antiproliferative mechanisms of metformin also remain unclear. It is argued that because metformin inhibits mitochondrial respiration (19), it induces an energy crisis (metabolic stress), leading to AMPK activation, mTOR inhibition, and suppression of proliferation (20). However, Dykens et al. (21) showed that net cellular ATP is not affected by metformin. Other suggested mechanisms include disruption of cross-talk between GPCRs and insulin receptors (22), inhibition of the ErbB2/IGF1 receptor (23), and mTOR inhibition by blocking RAG function (24). In vivo, metformin and the direct AMPK agonist A769662 delayed onset but not progression of lymphoma in Pten^+/−;LKB1^+/− mice (25) (LKB1 is the upstream kinase that activates AMPK). Moreover, these experiments were not conducted on AMPK-deficient animals, making it unclear whether the drug effects were AMPK dependent. Contrary to these results, metformin prevented tumorigenesis without activating AMPK in lung tumors (26), and in fact, LKB1-deficient lung tumors were actually more responsive to the metformin analog phenformin (27). The latter results suggest that the LKB1–AMPK pathway protects cancer cells from antiproliferative agents and may support tumorigenesis.In line with the above idea, genetic studies showed a procancer role of AMPK in the in vivo growth of H-RAS–transformed fibroblasts and astrocytic tumors, in pancreatic cancer, and in a subtype of renal cell carcinoma (28–31). Additional genetic studies also underscore the requirement of AMPK in cancer cell metabolic programming (32, 33); cell division (34–37); migration (38), protection against stress; and anticancer therapy (39–41). However, in Myc-driven mouse lymphoma, AMPK was shown to function as a tumor suppressor (42), suggesting a context-dependent role of AMPK in cancer.To definitively determine whether AMPK is necessary for the antiproliferative actions of AICAR and metformin, we conducted a comprehensive pharmacogenetic study in glioma. First, we found that gliomas express constitutively active AMPK, and that AICAR and metformin inhibit proliferation by distinct AMPK-independent and unique mechanisms. Second, A769662, a direct AMPK activator (43) showed no antiproliferative effects. Therefore, many agents that inhibit proliferation with concomitant AMPK activation may not require AMPK for their action. Instead, AMPK activation could be a response mechanism to counter stress induced by anticancer agents.     

Regulation of PTEN inhibition by the pleckstrin homology domain of P-REX2 during insulin signaling and glucose homeostasis

Insulin activation of phosphoinositide 3-kinase (PI3K) signaling regulates glucose homeostasis through the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3). The dual-specificity phosphatase and tensin homolog deleted on chromosome 10 (PTEN) blocks PI3K signaling by dephosphorylating PIP3, and is inhibited through its interaction with phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 2 (P-REX2). The mechanism of inhibition and its physiological significance are not known. Here, we report that P-REX2 interacts with PTEN via two interfaces. The pleckstrin homology (PH) domain of P-REX2 inhibits PTEN by interacting with the catalytic region of PTEN, and the inositol polyphosphate 4-phosphatase domain of P-REX2 provides high-affinity binding to the postsynaptic density-95/Discs large/zona occludens-1-binding domain of PTEN. P-REX2 inhibition of PTEN requires C-terminal phosphorylation of PTEN to release the P-REX2 PH domain from its neighboring diffuse B-cell lymphoma homology domain. Consistent with its function as a PTEN inhibitor, deletion of Prex2 in fibroblasts and mice results in increased Pten activity and decreased insulin signaling in liver and adipose tissue. Prex2 deletion also leads to reduced glucose uptake and insulin resistance. In human adipose tissue, P-REX2 protein expression is decreased and PTEN activity is increased in insulin-resistant human subjects. Taken together, these results indicate a functional role for P-REX2 PH-domain–mediated inhibition of PTEN in regulating insulin sensitivity and glucose homeostasis and suggest that loss of P-REX2 expression may cause insulin resistance.Phosphatases are essential for the regulation of many signal transduction pathways, and altered phosphatase activity disrupts various cellular processes. Phosphatases are divided into two families, the serine (Ser)/threonine (Thr) phosphatases and the tyrosine (Tyr) phosphatases, which include the subfamily of dual-specificity phosphatases (1). Serine/threonine phosphatases are predominantly regulated by the formation of inhibitor complexes (2). Direct phosphorylation of both phosphatases and their inhibitors has also been implicated in serine/threonine phosphatase regulation (2). Protein tyrosine phosphatases (PTPs) are mainly regulated by reversible oxidation of the catalytic pocket (3). However, phosphorylation has also been implicated in their regulation (4).The dual-specificity phosphatase and tensin homolog deleted from chromosome 10 (PTEN) was discovered through the mapping of homozygous deletions in cancer (5, 6). PTEN has the conserved PTP catalytic motif within its phosphatase domain (PD) and a C2 domain, both of which are required to dephosphorylate its primary substrate, phosphatidylinositol 3,4,5-trisphosphate (PIP3). This generates phosphatidylinositol 4,5-bisphosphate. thereby inhibiting PIP3-mediated recruitment and activation of the serine/threonine kinase AKT (7–9). Beyond these domains, the C-terminal tail of PTEN is phosphorylated at Ser-366, Ser-370, Ser-380, Thr-382, Thr-383, and Ser-385. High stoichiometry phosphorylation has been reported at Ser-370, -380. and -385 in vivo (10, 11). Furthermore, incorporation of ³²P into PTEN during orthophosphate labeling in vivo is substantially reduced when the cluster of Ser-380, Thr-382, and Thr-383 are mutated to alanines. Phosphorylation at this cluster of three residues has been implicated in the regulation of PTEN stability and phosphatase activity (12). C-terminal tail phosphorylation is also required for the formation of an intramolecular interaction that occurs between the tail and the catalytic region of PTEN, which inhibits PTEN membrane recruitment and PIP3 access (13). In addition, the C terminus of PTEN contains a postsynaptic density-95/Discs large/zona occludens-1 (PDZ)-binding domain (PDZ-BD), providing a binding site for several PDZ-domain–containing proteins (14).P-REX2A and P-REX2B were identified by two different groups through database searches for proteins homologous to P-REX1 (15, 16). P-REX2A is a guanine nucleotide exchange factor (GEF) for the small GTPase RAC and responds to both PIP3 and the beta-gamma subunits of G proteins (15). The structural domains of P-REX2A include the catalytic DHPH (Diffuse B-cell lymphoma homology and pleckstrin homology) domain tandem, two DEP (Disheveled, EGL-10, and pleckstrin homology) domains, two PDZ domains, and a C-terminal inositol polyphosphate-4 phosphatase (IP4P) domain. P-REX2B, a splice variant of P-REX2, lacks the C-terminal phosphatase domain. P-REX2 plays an important role in endothelial cell RAC1 activation and migration, as well as Purkinje cell dendrite morphology in the cerebellum (17, 18). Recently, we reported that P-REX2 interacts with PTEN to inhibit its phosphatase activity in a noncompetitive manner, thereby activating the phosphoinositide-3 kinase (PI3K) signaling pathway in cells (19). Here, we examine the mechanism by which P-REX2 inhibits PTEN and uncover that the PH domain of P-REX2 is responsible for inhibiting PTEN phosphatase activity. PH-domain–mediated inhibition is highly regulated by both the DH domain of P-REX2 and PTEN C-terminal tail phosphorylation. Furthermore, P-REX2 inhibition of PTEN plays a physiological role in the regulation of insulin-stimulated PI3K signaling and glucose metabolism.     

以癫痫为主要表现的儿童遗传代谢性疾病

目的提高对遗传代谢性疾病所致儿童癫痫的认识。方法采用回顾性方法,对我院以癫痫发作为主诉就诊,经血氨、乳酸、血同型半胱氨酸、尿有机酸气相色谱质谱联用分析、骨髓涂片、脑脊液检查、溶酶体酶活性分析、线粒体基因检测、线粒体酶活性分析确诊的先天代谢性缺陷病26例患儿的临床表现、生化特点以及诊疗情况进行回顾和分析。结果所有患儿均有癫痫发作,以部分性发作、痉挛发作为最常见发作形式,少数患者可有肌阵挛发作。患者多有不同程度的体格或智力运动发育迟滞,年长儿发病的线粒体脑肌病患者发病前智力正常,发病后出现智力运动倒退。脑电图表现以多灶独立性棘波、高度失律、背景慢波为主要表现。患者癫痫发作多难以控制,部分患者对因治疗后癫痫发作明显好转。结论先天代谢性疾病是儿童癫痫的病因之一;临床上应根据患儿发病急缓、生长发育、智力行为等,选择适宜的检查,以早期诊断。除了抗癫痫治疗以外,应根据不同的病因给予病因治疗。     

Androgen metabolism in relation to growth stimulation by a uterine cell line

The metabolism of radioactive testosterone, 5alpha-dihydrotestosterone, 4-androstene-3beta,17beta-diol or 4-androstene-3alpha,17beta-diol by the human cell line NHIK 3025, derived from a carcinoma of the uterine cervix, was studied. The cells were grown in Eagle's minimal essential medium (MEM) with a steriod concentration of 10-(7) M for 4 days. Androgen metabolism by this cell line is essentially the same as for other androgen-responsive cells. The most interesting testosterone metabolite in this system is 4-androstene-3beta,17beta-diol, and the separation of this compound from 4-androstene-3alpha,17beta-diol and the two corresponding 5alpha-reduced diols is described. Since 4-androsterone-3beta,17beta-diol is a more potent growth factor for these cells than testosterone, the small conversion of testosterone to 4-androstene-3beta, 17beta-diol observed could be responsible for the growth stimulation by testosterone.     

胰高血糖素样肽1的胰腺外作用研究进展

胰高血糖素样肽1(GLP-1)是体内重要的肠肽激素,在调节体内葡萄糖稳态中起重要作用.它通过促进胰岛素分泌、抑制胰高血糖素产生以及减慢餐后胃排空降低血糖.在胰腺外组织它也可通过调节葡萄糖代谢来参与全身血糖的调节.一方面通过激活磷脂酰肌醇3激酶、蛋白激酶B、蛋白激酶C、1型蛋白磷酸酶和丝裂原活化蛋白酶等增加糖原合酶a活性,促进糖原合成和糖利用.另一方面,在脂肪组织直接促进葡萄糖利用或增强胰岛素对葡萄糖的利用.此外,GLP-1还具有其它生物学作用包括舒张血管、保护血管内皮功能并激活垂体前叶激素分泌等.exendin-4是GLP-1的长效类似物,具有比GLP-1更持久的生物学活性和更强的降血糖作用,是一种治疗2型糖尿病的新型药物.     

Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo

Δ⁹-Tetrahydrocannabinol (THC), the psychoactive component of marijuana, and other direct cannabinoid receptor (CB1) agonists produce a number of neurobehavioral effects in mammals that range from the beneficial (analgesia) to the untoward (abuse potential). Why, however, this full spectrum of activities is not observed upon pharmacological inhibition or genetic deletion of either fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), enzymes that regulate the two major endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), respectively, has remained unclear. Here, we describe a selective and efficacious dual FAAH/MAGL inhibitor, JZL195, and show that this agent exhibits broad activity in the tetrad test for CB1 agonism, causing analgesia, hypomotilty, and catalepsy. Comparison of JZL195 to specific FAAH and MAGL inhibitors identified behavioral processes that were regulated by a single endocannabinoid pathway (e.g., hypomotility by the 2-AG/MAGL pathway) and, interestingly, those where disruption of both FAAH and MAGL produced additive effects that were reversed by a CB1 antagonist. Falling into this latter category was drug discrimination behavior, where dual FAAH/MAGL blockade, but not disruption of either FAAH or MAGL alone, produced THC-like responses that were reversed by a CB1 antagonist. These data indicate that AEA and 2-AG signaling pathways interact to regulate specific behavioral processes in vivo, including those relevant to drug abuse, thus providing a potential mechanistic basis for the distinct pharmacological profiles of direct CB1 agonists and inhibitors of individual endocannabinoid degradative enzymes.     

Baseline tumor growth and immune control in laboratory mice are significantly influenced by subthermoneutral housing temperature

We show here that fundamental aspects of antitumor immunity in mice are significantly influenced by ambient housing temperature. Standard housing temperature for laboratory mice in research facilities is mandated to be between 20–26 °C; however, these subthermoneutral temperatures cause mild chronic cold stress, activating thermogenesis to maintain normal body temperature. When stress is alleviated by housing at thermoneutral ambient temperature (30–31 °C), we observe a striking reduction in tumor formation, growth rate and metastasis. This improved control of tumor growth is dependent upon the adaptive immune system. We observe significantly increased numbers of antigen-specific CD8⁺ T lymphocytes and CD8⁺ T cells with an activated phenotype in the tumor microenvironment at thermoneutrality. At the same time there is a significant reduction in numbers of immunosuppressive MDSCs and regulatory T lymphocytes. Notably, in temperature preference studies, tumor-bearing mice select a higher ambient temperature than non-tumor-bearing mice, suggesting that tumor-bearing mice experience a greater degree of cold-stress. Overall, our data raise the hypothesis that suppression of antitumor immunity is an outcome of cold stress-induced thermogenesis. Therefore, the common approach of studying immunity against tumors in mice housed only at standard room temperature may be limiting our understanding of the full potential of the antitumor immune response.Mouse models are widely used in cancer research to investigate the antitumor immune response and its role in disease progression, as well as to test new therapies. Unfortunately, there is growing appreciation that these models may not accurately predict which new therapies will be effective in the clinic (1, 2). Therefore, identification of factors that impact experimental outcomes could improve our ability to identify the most promising therapies.One variable that has received little attention in cancer research is the relatively cool ambient housing temperature in research facilities. This factor is important because mice have a high surface area to body mass ratio and lose heat rapidly. In nature, mice seek warm environments and build nests to minimize metabolic demands for heat production (3), and thermal preference studies have clearly shown that healthy mice will select an ambient temperature of 30–31 °C (termed “thermoneutrality”) at which their basal metabolism is sufficient to maintain body temperature (3–7). However, at subthermoneutral temperatures, mice experience cold stress, which induces a systemic sympathetic response involving adaptive metabolic changes and secretion of catecholamines, particularly norepinephrine (8). These changes drive a highly energetically demanding process known as “adaptive thermogenesis” to maintain normal body temperature (8).For research facilities, the room temperature that the National Research Council Guide for the Care and Use of Laboratory Animals (9) requires is considerably cooler than thermoneutrality to facilitate some aspects of husbandry, to reduce frequency of cage cleaning, and to ensure thermal comfort of animal care technicians (4, 7). Institutes must select and maintain a constant room temperature between 20 °C and 26 °C; until 2011, an even cooler range between 18 °C and 24 °C was permitted. Despite the significant impact of ambient temperature on the metabolism of laboratory mice, the room temperature of mouse colonies has not concerned researchers because mice are able to maintain a normal body temperature. However, cool housing temperature is not always a benign variable and there is a disconcerting possibility that it may affect the outcome of a broad range of experimental endpoints (4, 5, 7). Although researchers interested in measuring fever in LPS-treated rodents have long recognized the importance of ambient temperature (4, 10), more recent studies demonstrate that an expected obesity phenotype in uncoupling protein 1 (UCP1)-deficient mice could only be observed when mice were housed at thermoneutrality (11). In another study, it was shown that adaptation to standard housing temperatures is associated with an increased polarization of macrophages to the alternatively activated state (12). Because there is little or no information on the effect of housing temperature on tumor growth or whether tumor growth affects thermal preference, we began to study the effects of cold stress in mouse tumor models. Here, using several different widely studied tumor models, we compared tumor formation, growth, and metastasis at either subthermoneutral or thermoneutral housing temperatures, and found significant differences that we were able to directly relate to differences in the status of the antitumor immune response.     

First pass metabolism of ethanol is strikingly influenced by the speed of gastric emptying

Background—Ethanol undergoes a first passmetabolism (FPM) in the stomach and liver. Gastric FPM of ethanolprimarily depends on the activity of gastric alcohol dehydrogenase(ADH). In addition, the speed of gastric emptying (GE) may modulateboth gastric and hepatic FPM of ethanol.
Aims—To study the effect of modulation of GE onFPM of ethanol in the stomach and liver.
Methods—Sixteen volunteers (eight men andeight women) received ethanol (0.225 g/kg body weight) orally andintravenously, and the areas under the ethanol concentration timecurves were determined to calculate FPM of ethanol. In seven of thesesubjects, FPM of ethanol was measured after the intravenousadministration of 10 mg metoclopramide (MCP) and 20 mgN-butylscopolamine (NBS) in separate experiments to eitheraccelerate or delay GE. GE was monitored sonographically by integrationof the antral area of the stomach every five minutes for 90 minutesafter oral ethanol intake. In addition, gastric biopsy specimens weretaken to determine ADH activity and phenotype, as well as to evaluategastric histology. Blood was also drawn for ADH genotyping.
Results—GE time was significantly delayed by theadministration of NBS as compared with controls (p<0.0001) and ascompared with the administration of MCP (p<0.0001). This wasassociated with a significantly enhanced FPM of ethanol with NBScompared with MCP (p = 0.0004). A significant correlation was notedbetween GE time and FPM of ethanol (r = 0.43, p = 0.0407).Gastric ADH activity did not significantly correlate with FPM of ethanol.
Conclusion—FPM of ethanol is strikingly modulatedby the speed of GE. Delayed GE increases the time of exposure ofethanol to gastric ADH and may therefore increase gastric FPM ofethanol. In addition, hepatic FPM of ethanol may also be enhanced asthe result of slower absorption of ethanol from the small intestine. Thus a knowledge of GE time is a major prerequisite for studying FPM ofethanol in humans.

Keywords:first pass metabolism of ethanol; gastric emptying; alcohol dehydrogenase; ethanol metabolism; stomach

     

Specificity of insulin signalling in human skeletal muscle as revealed by small interfering RNA

Insulin action on metabolically active tissues is a complex process involving positive and negative feedback regulation to control whole body glucose homeostasis. At the cellular level, glucose and lipid metabolism, as well as protein synthesis, are controlled through canonical insulin signalling cascades. The discovery of small interfering RNA (siRNA) allows for the molecular dissection of critical components of the regulation of metabolic and gene regulatory events in insulin-sensitive tissues. The application of siRNA to tissues of human origin allows for the molecular dissection of the mechanism(s) regulating glucose and lipid metabolism. Penetration of the pathways controlling insulin action in human tissue may aid in discovery efforts to develop diabetes prevention and treatment strategies. This review will focus on the use of siRNA to validate critical regulators controlling insulin action in human skeletal muscle, a key organ important for the control of whole body insulin-mediated glucose uptake and metabolism.     

Rescue of Notch signaling in cells incapable of GDP-l-fucose synthesis by gap junction transfer of GDP-l-fucose in Drosophila

Notch (N) is a transmembrane receptor that mediates cell–cell interactions to determine many cell-fate decisions. N contains EGF-like repeats, many of which have an O-fucose glycan modification that regulates N-ligand binding. This modification requires GDP-l-fucose as a donor of fucose. The GDP-l-fucose biosynthetic pathways are well understood, including the de novo pathway, which depends on GDP-mannose 4,6 dehydratase (Gmd) and GDP-4-keto-6-deoxy-d-mannose 3,5-epimerase/4-reductase (Gmer). However, the potential for intercellularly supplied GDP-l-fucose and the molecular basis of such transportation have not been explored in depth. To address these points, we studied the genetic effects of mutating Gmd and Gmer on fucose modifications in Drosophila. We found that these mutants functioned cell-nonautonomously, and that GDP-l-fucose was supplied intercellularly through gap junctions composed of Innexin-2. GDP-l-fucose was not supplied through body fluids from different isolated organs, indicating that the intercellular distribution of GDP-l-fucose is restricted within a given organ. Moreover, the gap junction-mediated supply of GDP-l-fucose was sufficient to support the fucosylation of N-glycans and the O-fucosylation of the N EGF-like repeats. Our results indicate that intercellular delivery is a metabolic pathway for nucleotide sugars in live animals under certain circumstances.     

An enzyme regulating triacylglycerol composition is encoded by the ROD1 gene of Arabidopsis

The polyunsaturated fatty acids (PUFAs) linoleic acid (18:2) and α-linolenic acid (18:3) in triacylglycerols (TAG) are major factors affecting the quality of plant oils for human health, as well as for biofuels and other renewable applications. These PUFAs are essential fatty acids for animals and plants, but also are the source of unhealthy trans fats during the processing of many foodstuffs. PUFAs 18:2 and 18:3 are synthesized in developing seeds by the desaturation of oleic acid (18:1) esterified on the membrane lipid phosphatidylcholine (PC) on the endoplasmic reticulum. The reactions and fluxes involved in this metabolism are incompletely understood, however. Here we show that a previously unrecognized enzyme, phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), encoded by the Arabidopsis ROD1 gene, is a major reaction for the transfer of 18:1 into PC for desaturation and also for the reverse transfer of 18:2 and 18:3 into the TAG synthesis pathway. The PDCT enzyme catalyzes transfer of the phosphocholine headgroup from PC to diacylglycerol, and mutation of rod1 reduces 18:2 and 18:3 accumulation in seed TAG by 40%. Our discovery of PDCT is important for understanding glycerolipid metabolism in plants and other organisms, and provides tools to modify the fatty acid compositions of plant oils for improved nutrition, biofuel, and other purposes.     

自由基在实验性胃癌及癌前病变发生中的作用

目的探讨自由基在胃癌及其癌前病变发生中的作用．方法将１００只Ｗｉｓｔａｒ大鼠分为２组，实验组（７０只），给予１００ｍｇ／Ｌ甲基硝基亚硝基胍（ＭＮＮＧ）水溶液自由饮用３０ｗｋ，对照组（３０只）饮用自来水．选５个时相点，动态观察ＭＮＮＧ诱发实验性胃癌及其癌前病变过程中大鼠体内丙二醛（ＭＤＡ）、脂质过氧化物（ＬＰＯ）、谷胱甘肽过氧化物酶（ＧＳＨＰＸ）及超氧化物歧化酶（ＳＯＤ）等的变化情况．结果在实验组，ＭＤＡ平均含量在５２ｗｋ非常显著地大于０ｗｋ（Ｐ＜００１），并显著地大于１６ｗｋ以前（Ｐ＜００５）．胃癌组织ＭＤＡ含量显著高于胃癌癌前病变组织（Ｐ＜００５）．癌组织ＬＰＯ的含量显著高于癌前病变组织（Ｐ＜００５）．实验组，总ＳＯＤ和ＣｕＺｎＳＯＤ活性在５２ｗｋ明显低于１６ｗｋ之前（分别为Ｐ＜００５和Ｐ＜００１）．癌组织ＣｕＺｎＳＯＤ含量非常显著地小于正常胃粘膜（Ｐ＜００１），亦明显低于胃粘膜异型增生和肠上皮化生（Ｐ＜００５）．在３０ｗｋ和５２ｗｋＧＳＨＰＸ活性显著低于１６ｗｋ以前．结论自由基在实验性胃癌及其癌前病变发生中具有一定作用，自由基清除剂可能对胃癌的综合防治具有积极意义     

Induction of glucokinase in chicken liver by dietary carbohydrates

We recently provided evidence of the presence of glucokinase (GCK) in the chicken liver [Berradi, H., Taouis, M., Cassy, S., Rideau, N., 2005. Glucokinase in chicken (Gallus gallus). Partial cDNA cloning, immunodetection and activity determination. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 141, 129-139]. In the present study we addressed the question of whether nutritional regulation of GCK occurs. Several nutritional conditions were compared in chickens (5 weeks old) previously trained to meal-feeding. One group was left in the fasted state (F: 24 h) and one was tested at the end of the 2 h meal (refed: RF). Two other 2 h meal-refed groups received an acute oral saccharose load (6 ml/kg BW) just before the 2 h meal and were sacrificed either at the end of the meal (Saccharose refed, SRF) or 3 h later (SRF+3). Liver GCK mRNA and protein levels did not differ between F, RF and SRF chickens but were significantly increased in SRF+3 chickens (2-fold, p < 0.05). GCK activity did not differ between F and RF chickens but increased significantly in SRF and SRF+3 chickens (1.7-fold, p < 0.05). Chicken liver GCK expression (mRNA and protein) and activity were therefore inducible in these chickens by feeding a meal with acute oral administration of carbohydrate. These and recent findings demonstrating insulin dependency of the liver GCK mRNA and protein strongly suggest that GCK may have an important role in carbohydrate metabolism, including that of the chicken. However, even in these highly stimulatory conditions, liver GCK activity remained relatively low in comparison with other species. The latter result may partly explain the high plasma glucose level in the chicken.     

Differential induction of inositol phosphate metabolism by three adipokinetic hormones

Many (in)vertebrates simultaneously release several structurally and functionally related hormones; however, the relevance of this phenomenon is poorly understood. In the locust e.g. each of three adipokinetic hormones (AKHs) is capable of controlling mobilization of carbohydrate and lipid from fat body stores, but it is unclear why three AKHs coexist. We now demonstrate disparities in the signal transduction of these hormones. Massive doses of the AKHs stimulated total inositol phosphate (InsP_n) production in the fat body biphasicly, but time courses were different. Inhibition of phospholipase C (PLC) resulted in attenuation of both InsP_n synthesis and glycogen phosphorylase activation. The AKHs evoked differential formation of individual [³H]InsP_n isomers (InsP_1–6), the effect being most pronounced for InsP₃. 40 nM of AKH-I and -III induced a substantial rise in total InsP_n and [³H]InsP₃ at short incubations, whereas the AKH-II effect was negligible. At a more physiological dose of 4 nM, the AKHs equally enhanced Ins(1,4,5)P₃ levels. The InsP₃ effect was most prolonged for AKH-III. These subtle differences in InsP_n metabolism, together with earlier findings on differences between the AKHs, support the hypothesis that each AKH exerts specific biological functions in the overall syndrome of energy mobilization during flight.     

Cardiac Metabolism and Mechanics are Altered by Genetic Loss of Lipoprotein Triglyceride Lipolysis

Introduction Most circulating fatty acids are contained in lipoprotein triglycerides. For the heart to acquire these lipids, they must be broken down into free fatty acids via the enzyme lipoprotein lipase (LpL). Although it has long been known that hearts primarily use esterified fatty acids as fuel, different sources of fatty acids were thought to be interchangeable. Materials and methods By creating mice with neonatal and acute LpL deletion we showed that lipoprotein-derived fatty acids could not be replaced by albumin-associated free fatty acids. Loss of cardiac LpL forces the heart to increase its uptake of glucose, reduce fatty acid oxidation, and eventually leads to cardiac dysfunction. In contrast, cardiomyocyte specific overexpression of an anchored form of LpL leads to excess lipid uptake, induction of fatty acid oxidation genes, and dilated cardiomyopathy. Conclusion Increasing lipid secretion from the heart or redirecting lipids to adipose tissue can alleviate this lipotoxic situation.