Palmitate Induces Insulin Resistance in H4IIEC3 Hepatocytes through Reactive Oxygen Species Produced by Mitochondria

Visceral adiposity in obesity causes excessive free fatty acid (FFA) flux into the liver via the portal vein and may cause fatty liver disease and hepatic insulin resistance. However, because animal models of insulin resistance induced by lipid infusion or a high fat diet are complex and may be accompanied by alterations not restricted to the liver, it is difficult to determine the contribution of FFAs to hepatic insulin resistance. Therefore, we treated H4IIEC3 cells, a rat hepatocyte cell line, with a monounsaturated fatty acid (oleate) and a saturated fatty acid (palmitate) to investigate the direct and initial effects of FFAs on hepatocytes. We show that palmitate, but not oleate, inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate 2 and serine phosphorylation of Akt, through c-Jun NH₂-terminal kinase (JNK) activation. Among the well established stimuli for JNK activation, reactive oxygen species (ROS) played a causal role in palmitate-induced JNK activation. In addition, etomoxir, an inhibitor of carnitine palmitoyltransferase-1, which is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation, as well as inhibitors of the mitochondrial respiratory chain complex (thenoyltrifluoroacetone and carbonyl cyanide m-chlorophenylhydrazone) decreased palmitate-induced ROS production. Together, our findings in hepatocytes indicate that palmitate inhibited insulin signal transduction through JNK activation and that accelerated β-oxidation of palmitate caused excess electron flux in the mitochondrial respiratory chain, resulting in increased ROS generation. Thus, mitochondria-derived ROS induced by palmitate may be major contributors to JNK activation and cellular insulin resistance.Insulin is the major hormone that inhibits gluconeogenesis in the liver. Visceral adiposity in obesity causes hepatic steatosis and insulin resistance. In an insulin-resistant state, impaired insulin action allows enhancement of glucose production in the liver, resulting in systemic hyperglycemia (1) and contributing to the development of type 2 diabetes. In addition, we have demonstrated experimentally that insulin resistance accelerated the pathology of steatohepatitis in genetically obese diabetic OLETF rats (2). In contrast, lipid-induced oxidative stress caused steatohepatitis and hepatic insulin resistance in mice (3). In fact, steatosis of the liver is an independent predictor of insulin resistance in patients with nonalcoholic fatty liver disease (4).It remains unclear whether hepatic steatosis causally contributes to insulin resistance or whether it is merely a resulting pathology. Excessive dietary free fatty acid (FFA)² flux into the liver via the portal vein may cause fatty liver disease and hepatic insulin resistance. Indeed, elevated plasma FFA concentrations correlate with obesity and decreased target tissue insulin sensitivity (5).Experimentally, lipid infusion or a high fat diet that increases circulating FFA levels promotes insulin resistance in the liver. Candidate events linking FFA to insulin resistance in vivo are the up-regulation of SREBP-1c (6), inflammation caused by activation of c-Jun amino-terminal kinase (JNK) (7) or IKKβ (8), endoplasmic reticulum (ER) stress (9), ceramide (10, 11), and TRB3 (12).However, which event is the direct and initial target of FFA in the liver is unclear. Insulin resistance induced by lipid infusion or a high fat diet is complex and may be accompanied by alterations not restricted to the liver, making it difficult to determine the contribution of FFAs to hepatic insulin resistance. For example, hyperinsulinemia and hyperglycemia secondary to the initial event also may contribute to the development of diet-induced insulin resistance in vivo (6).To address the early event(s) triggering the development of high fat diet- or obesity-induced insulin resistance, we investigated the molecular mechanism(s) underlying the direct action of FFA on hepatocytes to cause insulin resistance in vitro, using the rat hepatocyte cell line H4IIEC3. We found that mitochondria-derived reactive oxygen species (ROS) were a cause of palmitate-induced insulin resistance in hepatocytes.     

Structural Studies of Soybean Calmodulin Isoform 4 Bound to the Calmodulin-binding Domain of Tobacco Mitogen-activated Protein Kinase Phosphatase-1 Provide Insights into a Sequential Target Binding Mode

The calcium regulatory protein calmodulin (CaM) binds in a calcium-dependent manner to numerous target proteins. The calmodulin-binding domain (CaMBD) region of Nicotiana tabacum MAPK phosphatase has an amino acid sequence that does not resemble the CaMBD of any other known Ca²⁺-CaM-binding proteins. Using a unique fusion protein strategy, we have been able to obtain a high resolution solution structure of the complex of soybean Ca²⁺-CaM4 (SCaM4) and this CaMBD. Complete isotope labeling of both parts of the complex in the fusion protein greatly facilitated the structure determination by NMR. The 12-residue CaMBD region was found to bind exclusively to the C-lobe of SCaM4. A specific Trp and Leu side chain are utilized to facilitate strong binding through a novel “double anchor” motif. Moreover, the orientation of the helical peptide on the surface of Ca²⁺-SCaM4 is distinct from other known complexes. The N-lobe of Ca²⁺-SCaM4 in the complex remains free for additional interactions and could possibly act as a calcium-dependent adapter protein. Signaling through the MAPK pathway and increases in intracellular Ca²⁺ are both hallmarks of the plant stress response, and our data support the notion that coordination of these responses may occur through the formation of a unique CaM-MAPK phosphatase multiprotein complex.Calmodulin (CaM)² is a ubiquitous intracellular Ca²⁺ sensor protein that plays an essential role in various Ca²⁺ signaling pathways. Contiguous and unique CaM-binding domain (CaMBD) regions are found widely distributed in many different types of CaM target proteins including protein phosphatases and kinases, cytoskeletal proteins, ion channels, and pumps (1, 2). Even though the CaMBD from various proteins share relatively poor amino acid sequence similarity, the majority of CaMBD become α-helical upon binding to CaM, and they can be grouped into either the 1-5-10 or the 1-8-14 motif, where the first and the last number indicate the position of two hydrophobic anchor residues that attach the CaMBD to the two binding pockets of the bilobal Ca²⁺-CaM. However, several noncanonical classes of CaMBD have also been identified. For example, in the CaMBD of the MARCKS protein, the two anchor residues are separated by a single amino acid residue (3). On the other hand, in the recently determined crystal structure of Ca²⁺-CaM complexed with the CaMBD of the skeletal muscle ryanodine receptor RYR1, they are separated by 15 residues (1–17 motif) (4). Bulky hydrophobic side chains of residues such as Trp, Leu, Phe, and Ile are most commonly utilized as anchor residues (see Fig. 1a), and these are usually deeply inserted into the hydrophobic target-binding pocket of either the N- or C-lobe of Ca²⁺-CaM. However, in several cases, including the N-methyl-d-aspartate receptors (NMDAR) (5) and the voltage-gated Ca²⁺ channels (Cav1–2) (6, 7), a polar side chain from Thr or Tyr has also been found to act as an anchor residue. In almost all Ca²⁺-CaM complexes studied to date, the two lobes of Ca²⁺-CaM become collapsed on the helical CaMBD, and they form a globular complex structure. An exception was found in the case of the Ca²⁺-CaM complex with an incomplete CaMBD from the plasma membrane Ca²⁺ pump (C20W), where only the C-lobe of Ca²⁺-CaM binds to the CaMBD, and the N-lobe was free in solution (8). The versatility of CaM target protein binding has been discussed in many recent reviews (for example, Refs. 2, 9–11).Open in a separate windowFIGURE 1.a, amino acid sequences of CaMBD from tobacco NtMKP1 (residues 438–449), Arabidopsis AtMKP1 (residues 451–462), and rice OsMKP1 (residues 456–467) are compared with various CaMBDs. The sequences are aligned at the position of the first hydrophobic anchor residue. The hydrophobic anchor residues are colored in red, while the other hydrophobic residues are shown in green. The basic residues and acidic residues are colored in cyan and pink, respectively. The residue numbers of the NtMKP1 sequence in SCaM4-NtMKP1/NtMKP1 protein are also indicated. b, schematic drawing of the two fusion proteins, SCaM4-NtMKP1 and SCaM4CT-NtMKP1.In plant cells, Ca²⁺ signals, arising from various extracellular stimuli such as abiotic stresses, hormones, or phytopathogens are mediated by multiple CaM isoforms to create specific cellular responses. In contrast, only a single CaM protein exists in animal cells. For example, the model plant Arabidopsis thaliana has nine CaM genes (CaM1–9) encoding seven different CaM isoforms (12, 13). Five distinct CaM genes (SCaM1–5) encoding four different CaM proteins have been identified so far in the soybean genome (14). Despite the relatively high amino acid sequence identity among these CaM isoforms (50–90%), each isoform is utilized to regulate different target enzymes related to specific cellular responses (15, 16). For example, the expression of two soybean CaM isoforms, SCaM4 and 5 is markedly up-regulated after pathogen infection, and these two proteins can activate the enzyme nitric-oxide synthase (NOS) (17). Production of nitric oxide is thought to be one of the early events in the plant defense reactions (18, 19). On the other hand, SCaM1 is incapable of activation of the NOS enzyme, and it does in fact act as a competitive inhibitor. Likewise, in Nicotiana tabacum (tobacco), the CaM isoforms NtCaM1 and NtCaM13 are overexpressed in tobacco leaf tissue in response to wounding and the TMV-triggered hypersensitive reaction, respectively (20). Recently, we have addressed the question as to how distinct CaM isoforms can give rise to selectivity in their target regulation by determining the solution structures of the two soybean CaM isoforms, SCaM1 and SCaM4 (21). However, there are currently no structures available for plant CaM isoforms in a complex with a target CaMBD peptide, although many such complex structures have been determined for animal CaM. Therefore, determining the structure of plant CaM-target complexes and uncovering their unique features relative to those of animal CaM or other plant CaM isoforms will undoubtedly enhance our understanding of the CaM-target regulation mechanisms in plants and mammals.The mitogen-activated protein kinase cascade (MAPK cascade) is an important signal transduction pathway in animals, yeast as well as in plants (22). The activity of MAPK is regulated via phosphorylation by its immediate upstream regulator, MAPK kinase (MAPKK). Following activation, MAPKs are dephosphorylated and inactivated by MAPK phosphatases. Recently, the MAPK phosphatase from tobacco (NtMKP1) was shown to be a novel plant-specific CaM-binding protein (23). This finding indicated a possible link between the MAPK cascade and Ca²⁺ signaling pathways in plant cells. The MAPK cascade is thought to play an important role in plant defense signaling, and the accumulation of Ca²⁺ in plant cells is also a well-known response to pathogens and other stresses (for recent reviews, see Refs. 24, 25). The putative CaMBD reported for NtMKP1 (residues 396–447) is located directly upstream of the conserved Ser-rich domain in the middle of the protein. Mutagenesis studies have revealed that Trp⁴⁴⁰ and Leu⁴⁴³ are indispensable for Ca²⁺-CaM binding. More recently, we have tested various truncated versions of the CaMBD of NtMKP1 and narrowed it down to 12 residues (residues 438–449) (Fig. 1a) that are sufficient for Ca²⁺-CaM binding (26). Interestingly, the NtMKP1 CaMBD does not belong to any of the typical CaM-binding motif classes. Binding assays using isothermal titration calorimetry (ITC) as well as NMR titration studies for various SCaM isoforms, and their half-lobe fragments have revealed a novel sequential target binding mechanism for the Ca²⁺-CaM isoforms. The first strong binding event involves the C-lobe of Ca²⁺-CaM and the reported binding constant for NtMKP1 peptide was 10⁷–10⁸ m⁻¹. On the other hand, the binding of a second CaMBD of NtMKP1 occurs only through the N-lobe of Ca²⁺-CaM, and the binding constant was around 10⁵ m⁻¹ (26). To date, structural information about the manner in which Ca²⁺-CaM binds sequentially to the unusual amino acid sequence of the CaMBD of NtMKP1 is unavailable. Here, we have determined the solution NMR structure of the C-lobe fragment of SCaM4 (SCaM4CT) fused with the CaMBD of NtMKP1 (Fig. 1b). We have chosen SCaM4 over other SCaM isoforms, as the stress-induced SCaM4 would provide a more important connection between stress MAPK response and Ca²⁺ signaling. The interaction of the α-helical CaMBD of NtMKP1 with SCaM4CT domain is stabilized by hydrophobic interactions mainly through Trp⁴⁴⁰ and Leu⁴⁴³ in the NtMKP1 sequence. Moreover, the basic residue, Arg⁴⁴⁴ that is unusual at position 5 (Fig. 1a) stays outside of the hydrophobic patch, and it seems to form a unique hydrogen bond to Glu⁸⁴ of the SCaM4CT domain. The resulting orientation of the α-helical CaMBD relative to the SCaM4CT domain is therefore very different from those seen in the other previously reported Ca²⁺-CaM-target complexes. We have also studied binding of a synthetic CaMBD peptide to intact Ca²⁺-SCaM4 fused at the C-terminal end with the CaMBD of NtMKP1 (Fig. 1b) providing additional information about the role of the N-lobe of Ca²⁺-SCaM4 in NtMKP1 binding. Furthermore, we have studied the interactions between the N-lobe of Ca²⁺-SCaM4 and a second potential CaMBD in the C-terminal region of NtMKP1.     

Reactions of Lotus japonicus ecotypes and mutants to root parasitic plants

Witchweeds (Striga spp.) and broomrapes (Orobanche spp.) are obligate root parasitic plants on economically important field and horticultural crops. The parasites' seeds are induced to germinate by root-derived chemical signals. The radicular end is transformed into a haustorium which attaches, penetrates the host root and establishes connection with the vascular system of the host. Reactions of Lotus japonicus, a model legume for functional genomics, were studied for furthering the understanding of host-parasite interactions. Lotus japonicus was compatible with Orobanche aegyptiaca, but not with Orobanche minor, Striga hermonthica and Striga gesnerioides. Orobanche minor successfully penetrated Lotus japonicus roots, but failed to establish connections with the vascular system. Haustoria in Striga hermonthica attached to the roots, but penetration and subsequent growth of the endophyte in the cortex were restricted. Striga gesnerioides did not parasitize Lotus japonicus. Among seven mutants of Lotus japonicus (castor-5, har1-5, alb1-1, ccamk-3, nup85-3, nfr1-3 and nsp2-1) with altered characteristics in relation to rhizobial nodulation and mycorrhizal colonization, castor-5 and har1-5 were parasitized by Orobanche aegyptiaca with higher frequency than the wild type. In contrast, Orobanche aegyptiaca tubercle development was delayed on the mutants nup85-3, nfr1-3 and nsp2-1. These results suggest that nodulation, mycorrhizal colonization and infection by root parasitic plants in Lotus japonicus may be modulated by similar mechanisms and that Lotus japonicus is a potential model legume for studying plant-plant parasitism.     

Copper(II) nitrato and chloro complexes with sterically hindered tridentate ligands: Influence of ligand framework and charge on their structure and physicochemical properties

Copper(II) coordination complexes of the neutral ligand, tris(3-tert-butyl-5-methyl-1-pyrazolyl)methane (L2′), i.e. the copper(II) nitrato complexes [Cu(L2′)(NO₃)][Cu(NO₃)₄]_1/2 (1) and [Cu(L2′)(NO₃)](ClO₄) (2) and the copper(II) chloro complex [Cu(L2′)(Cl)](ClO₄) (3), and its anionic borate analogue, hydrotris(3-tert-butyl-5-methyl-1-pyrazolyl)borate (L2⁻), i.e. the copper(II) nitrato complex [Cu(L2)(NO₃)] (4) and the copper(II) chloro complex [Cu(L2)(Cl)] (5), were synthesized in order to investigate the influence of ligand framework and charge on their structure and physicochemical properties. While X-ray crystallography did not show any definitive trends in terms of copper(II) atom geometry in four-coordinate copper(II) chloro complexes 3 and 5, different structural trends were observed in five-coordinate copper(II) nitrato complexes 1, 2, and 4. These complexes were also characterized by spectroscopic techniques, namely, UV-Vis, ESR, IR/far-IR, and X-ray absorption spectroscopy.     

Requirement of RNA Binding of Mammalian Eukaryotic Translation Initiation Factor 4GI (eIF4GI) for Efficient Interaction of eIF4E with the mRNA Cap

Eukaryotic mRNAs possess a 5′-terminal cap structure (cap), m⁷GpppN, which facilitates ribosome binding. The cap is bound by eukaryotic translation initiation factor 4F (eIF4F), which is composed of eIF4E, eIF4G, and eIF4A. eIF4E is the cap-binding subunit, eIF4A is an RNA helicase, and eIF4G is a scaffolding protein that bridges between the mRNA and ribosome. eIF4G contains an RNA-binding domain, which was suggested to stimulate eIF4E interaction with the cap in mammals. In Saccharomyces cerevisiae, however, such an effect was not observed. Here, we used recombinant proteins to reconstitute the cap binding of the mammalian eIF4E-eIF4GI complex to investigate the importance of the RNA-binding region of eIF4GI for cap interaction with eIF4E. We demonstrate that chemical cross-linking of eIF4E to the cap structure is dramatically enhanced by eIF4GI fragments possessing RNA-binding activity. Furthermore, the fusion of RNA recognition motif 1 (RRM1) of the La autoantigen to the N terminus of eIF4GI confers enhanced association between the cap structure and eIF4E. These results demonstrate that eIF4GI serves to anchor eIF4E to the mRNA and enhance its interaction with the cap structure.The cap structure, m⁷GpppN, is present at the 5′ terminus of all nuclear transcribed eukaryotic mRNAs. Cap-dependent binding of the ribosome to mRNA is mediated by the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E), which forms a complex termed eIF4F together with eIF4G and eIF4A. Mammalian eIF4G, which has two isoforms, eIF4GI and eIF4GII, is a modular, multifunctional protein that binds to poly(A)-binding protein (PABP) (14) and eIF4E (18, 20) via the N-terminal third region. Mammalian eIF4G binds to eIF4A and eIF3 (15) via the middle third region and to eIF4A and Mnk protein kinase at the C-terminal region. eIF4GI also possesses an RNA-binding sequence (2, 9, 33) in the middle region. There are two RNA-binding sites on eIF4GI; one is located amino terminal to the first HEAT domain, and the other is located within the first HEAT domain (23). Mammalian and Saccharomyces cerevisiae eIF4E are similar in size (24 kDa), but mammalian eIF4GI (220 kDa) is larger than its yeast counterpart (150 kDa), as the latter lacks a C-terminal domain corresponding to mammalian eIF4GI (38).The affinity of eIF4E for the cap structure has been a matter of dispute for some time. The earlier works of Carberry et al. (4) and Ueda et al. (39) estimated the equilibrium dissociation constant (K_d) of the eIF4E-cap complex by fluorescence titration to be 2 × 10⁻⁶ to 5 × 10⁻⁶ M depending on the nature of the cap analog. Later on, development of a new methodology for the fluorescence titration experiments yielded K_d values of 10⁻⁷ to 10⁻⁸ (29, 41). The source of the difference with the previous reports was thoroughly analyzed (29, 30). The interaction between the cap structure and eIF4E is dramatically enhanced by eIF4GI. This was first reported by showing that cross-linking of mammalian eIF4E to the cap structure is more efficient when it is a subunit of the eIF4F complex (19) or when it is complexed to eIF4GI (11). A similar enhancement of the binding of eIF4E to the cap structure was observed in yeast (40). However, two very different mechanisms were proposed to explain these observations. For the mammalian system, it was postulated that the middle segment of eIF4GI, which binds RNA, stabilizes the eIF4E interaction with the cap structure (11). This model was based primarily on the finding that in poliovirus-infected cells, eIF4GI is cleaved between its N-terminal third and the middle third, and consequently, eIF4E remains attached to the N-terminal eIF4GI fragment lacking the RNA-binding region. Under these conditions, cross-linking of eIF4E to the cap structure was poor (19, 31). In contrast, in yeast, a strong interaction between the cap structure and eIF4E was achieved using an eIF4G fragment containing the eIF4E-binding site that lacks the RNA-binding region (34, 40). Also, the yeast eIF4G fragment from amino acids 393 to 490 (fragment 393-490), which does not contain the RNA-binding site, forms a right-handed helical ring that wraps around the N terminus of eIF4E. This conformational change was suggested in turn to engender an allosteric enhancement of the association of eIF4E with the cap structure (10). Such an interaction between mammalian eIF4GI and eIF4E has not been reported.To understand the mechanism by which eIF4GI stimulates the interaction of eIF4E with the cap structure in mammals, we reconstituted the eIF4E-cap recognition activity in vitro with purified eIF4E and eIF4GI recombinant proteins. Using a chemical cross-linking assay, we demonstrate that only mammalian eIF4GI fragments possessing RNA-binding activity enhance the cross-linking of eIF4E to the cap structure. Our data provide new insight into the mechanism of cap recognition by the eIF4E-eIF4GI complex.     

Antitrypanosomal activity of some pregnane glycosides isolated from Caralluma species

Pregnane glycosides previously isolated from genus Caralluma (C. Penicillata, C. tuberculata and C. russelliana) were tested for their antitrypanosomal activity. Penicilloside E showed the highest antitrypanosomal activity (IC₅₀ 1.01 μg/ml) followed by caratuberside C (IC₅₀ 1.85 μg/ml), which exhibited the highest selectivity index (SI 12.04). It was noticed that acylation is required for the antitrypanosomal activity while glycosylation at C-20 has no significant effect on the activity.     

Identification of novel meristem factors involved in shoot regeneration through the analysis of temperature-sensitive mutants of Arabidopsis

Adventitious organogenesis in plant tissue culture involves de novo formation of apical meristems and should therefore provide important information about the fundamentals of meristem gene networks. We identified novel factors required for neoformation of the shoot apical meristem (SAM) through an analysis of shoot regeneration in root initiation defective3 ( rid3 ) and root growth defective3 ( rgd3 ) temperature-sensitive mutants of Arabidopsis. After induction of callus to regenerate shoots, cell division soon ceased and was then reactivated locally in the surface region, resulting in formation of mounds of dense cells in which adventitious-bud SAMs were eventually constructed. The rgd3 mutation inhibited reactivation of cell division and suppressed expression of CUP-SHAPED COTYLEDON1 ( CUC1 ), CUC2 and SHOOT MERISTEMLESS ( STM ). In contrast, the rid3 mutation caused excess ill-controlled cell division on the callus surface. This was intimately related to enhanced and broadened expression of CUC1 . Positional cloning revealed that the RGD3 and RID3 genes encode BTAF1 (a kind of TATA-binding protein-associated factor) and an uncharacterized WD-40 repeat protein, respectively. In the early stages of shoot regeneration, RGD3 was expressed (as was CUC1 ) in the developing cell mounds, whereas RID3 was expressed outside the cell mounds. When RID3 was over-expressed artificially, the expression levels of CUC1 and STM were significantly reduced. Taken together, these findings show that both negative regulation by RID3 and positive regulation by RGD3 of the CUC–STM pathway participate in proper control of cell division as a prerequisite for SAM neoformation.