Glucose and fatty acids synergize to promote B-cell apoptosis through activation of glycogen synthase kinase 3β independent of JNK activation

Background

The combination of elevated glucose and free-fatty acids (FFA), prevalent in diabetes, has been suggested to be a major contributor to pancreatic β-cell death. This study examines the synergistic effects of glucose and FFA on β-cell apoptosis and the molecular mechanisms involved. Mouse insulinoma cells and primary islets were treated with palmitate at increasing glucose and effects on apoptosis, endoplasmic reticulum (ER) stress and insulin receptor substrate (IRS) signaling were examined.

Principal Findings

Increasing glucose (5–25 mM) with palmitate (400 µM) had synergistic effects on apoptosis. Jun NH2-terminal kinase (JNK) activation peaked at the lowest glucose concentration, in contrast to a progressive reduction in IRS2 protein and impairment of insulin receptor substrate signaling. A synergistic effect was observed on activation of ER stress markers, along with recruitment of SREBP1 to the nucleus. These findings were confirmed in primary islets. The above effects associated with an increase in glycogen synthase kinase 3β (Gsk3β) activity and were reversed along with apoptosis by an adenovirus expressing a kinase dead Gsk3β.

Conclusions/Significance

Glucose in the presence of FFA results in synergistic effects on ER stress, impaired insulin receptor substrate signaling and Gsk3β activation. The data support the importance of controlling both hyperglycemia and hyperlipidemia in the management of Type 2 diabetes, and identify pancreatic islet β-cell Gsk3β as a potential therapeutic target.     

Pharmacological analysis of CCK(2) receptor ligands using COS-7 and SK-N-MC cells, expressing the human CCK(2) receptor.

A series of CCK(2) receptor ligands were analysed with respect to their interaction with binding sites in the membranes of COS-7 cells and SK-N-MC cells transiently expressing the human CCK(2) receptor (short isoform). The ligands were YF476, YM022, AG041R, L-740,093, JB93182, PD134308, and PD136450. Their binding was analysed by radioligand competition using [3H]L-365,260 as the labelled ligand. Saturation binding analysis indicated that [3H]L-365,260 interacted with a single class of binding sites. In competition binding experiments using COS-7-cell membranes, all seven ligands were incubated together with 2 nM [3H]L-365,260. The data for four of the compounds fitted a one-site model (pK(i) values: YM022: 9.2+/-0.02; YF476: 9.6+/-0.04; L-740,093: 9.2+/-0.01; and AG041R: 8.3+/-0.06), while the data for the three others fitted a two-site model (pK(i) values: JB93182: 8.8+/-0.04 and 6.0+/-0.15; PD134308: 9.0+/-0.04 and 6.1+/-0.15; and PD136450: 9.0+/-0.02 and 5.4+/-0.41). SK-N-MC cell membranes and 2 nM [3H]L-365,260 were incubated together with YM022, YF476, JB93182, and PD134308. The data for YM022 and YF476 fitted a one-site model (pK(i) values: YM022: 9.3+/-0.06; YF476: 9.4+/-0.02), while the data for JB93182 and PD134308 fitted a two-site model (pK(i) values: JB93182: 8.7+/-0.06 and 6.2+/-0.06; PD134308: 9.1+/-0.06 and 7.0+/-0.17). Competition binding experiments in the presence of the GTP-analogue guanylylimidodiphosphate, using either of the two cell types, produced similar binding data for PD134308 and JB93182 as in the absence of GTP-analogue. The human receptor seems to exist in a low and/or high affinity state. The shift from low to high affinity does not seem to reflect the degree of G protein coupling.     

Joining forces in the quest for orthologs

A report of the 24th International Conference on Yeast Genetics and Molecular Biology, Manchester, UK, 19-24 July 2009.     

Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).     

Possible CaMKK-dependent regulation of AMPK phosphorylation and glucose uptake at the onset of mild tetanic skeletal muscle contraction

The Ca(2+)/calmodulin (CaM) competitive inhibitor KN-93 has previously been used to evaluate 5'-AMP-activated protein kinase (AMPK)-independent Ca(2+)-signaling to contraction-stimulated glucose uptake in muscle during intense electrical stimulation ex vivo. With the use of low-intensity tetanic contraction of mouse soleus and extensor digitorum longus (EDL) muscles ex vivo, this study demonstrates that KN-93 can potently inhibit AMPK phosphorylation and activity after 2 min but not 10 min of contraction while strongly inhibiting contraction-stimulated 2-deoxyglucose uptake at both the 2- and 10-min time points. These data suggest inhibition of Ca(2+)/CaM-dependent signaling events upstream of AMPK, the most likely candidate being the novel AMPK kinase CaM-dependent protein kinase kinase (CaMKK). CaMKK protein expression was detected in mouse skeletal muscle. Similar to KN-93, the CaMKK inhibitor STO-609 strongly reduced AMPK phosphorylation and activity at 2 min and less potently at 10 min. Pretreatment with STO-609 inhibited contraction-stimulated glucose uptake at 2 min in soleus, but not EDL, and in both muscles after 10 min. Neither KN-93 nor STO-609 inhibited 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside-stimulated glucose uptake, AMPK phosphorylation, or recombinant LKB1 activity, suggestive of an LKB1-independent effect. Finally, neither KN-93 nor STO-609 had effects on the reductions in glucose uptake seen in mice overexpressing a kinase-dead AMPK construct, indicating that the effects of KN-93 and STO-609 on glucose uptake require inhibition of AMPK activity. We propose that CaMKKs act in mouse skeletal muscle regulating AMPK phosphorylation and glucose uptake at the onset of mild tetanic contraction and that an intensity- and/or time-dependent switch occurs in the relative importance of AMPKKs during contraction.     

Tree Species Traits but Not Diversity Mitigate Stem Breakage in a Subtropical Forest following a Rare and Extreme Ice Storm

Future climates are likely to include extreme events, which in turn have great impacts on ecological systems. In this study, we investigated possible effects that could mitigate stem breakage caused by a rare and extreme ice storm in a Chinese subtropical forest across a gradient of forest diversity. We used Bayesian modeling to correct stem breakage for tree size and variance components analysis to quantify the influence of taxon, leaf and wood functional traits, and stand level properties on the probability of stem breakage. We show that the taxon explained four times more variance in individual stem breakage than did stand level properties; trees with higher specific leaf area (SLA) were less susceptible to breakage. However, a large part of the variation at the taxon scale remained unexplained, implying that unmeasured or undefined traits could be used to predict damage caused by ice storms. When aggregated at the plot level, functional diversity and wood density increased after the ice storm. We suggest that for the adaption of forest management to climate change, much can still be learned from looking at functional traits at the taxon level.     

Synthesis and incorporation of an alpha-hexofuranosyl thymidine into oligodeoxynucleotides via its two exocyclic OH-groups

1-(2,3-Dideoxy-3-amino-alpha-D-arabino-hexofuranosyl)thymine is considered as a conformationally restricted acyclic nucleoside using the furanose ring to link the diol backbone to the nucleobase. The appropriately substituted phosphoramidites were synthesised via 1-(5,6-di-O-acetyl-2,3-dideoxy-3-phthalimido-alpha-D-arabino-hexofuranosyl)thymine and used in oligodeoxynucleotide (ODN) synthesis. However, the binding affinity of the mixed ODNs towards complementary DNA and RNA was decreased compared to the wild-type oligos. The decrease was smaller when the monomer was inserted near the end of the sequence. The insertions into an alpha T sequence or in a beta T sequence gave nearly the same dropping in melting temperature per modification which indicates that the new nucleotide modifications behave both as alpha and beta nucleotides.     

A Multiplex Human Syndrome Implicates a Key Role for Intestinal Cell Kinase in Development of Central Nervous, Skeletal, and Endocrine Systems

Six infants in an Old Order Amish pedigree were observed to be affected with endocrine-cerebro-osteodysplasia (ECO). ECO is a previously unidentified neonatal lethal recessive disorder with multiple anomalies involving the endocrine, cerebral, and skeletal systems. Autozygosity mapping and sequencing identified a previously unknown missense mutation, R272Q, in ICK, encoding intestinal cell kinase (ICK). Our results established that R272 is conserved across species and among ethnicities, and three-dimensional analysis of the protein structure suggests protein instability due to the R272Q mutation. We also demonstrate that the R272Q mutant fails to localize at the nucleus and has diminished kinase activity. These findings suggest that ICK plays a key role in the development of multiple organ systems.     

A search for phylogenetically informative wood characters within Lecythidaceae s.l

The wood structure of 71 species representing 24 genera of the pantropical Lecythidaceae s.l., including the edible Brazil nuts (Bertholletia excelsa) and the spectacular cannon-ball tree (Couroupita guianensis), was investigated using light and scanning electron microscopy. This study focused on finding phylogenetically informative characters to help elucidate any obscure evolutionary patterns within the family. The earliest diverging subfamily Napoleonaeoideae has mixed simple/scalariform vessel perforations, scalariform vessel-ray pitting, and high multiseriate rays, all features that are also present in Scytopetaloideae. The wood structure of Napoleonaea is distinct, but its supposed close relative Crateranthus strongly resembles Scytopetaloideae. The isolated position of Foetidia (Foetidioideae) can be supported by a unique type of vessel-ray pitting that is similar in shape and size to intervessel pitting (distinctly bordered, <5 μm). The more derived Planchonioideae and Lecythidoideae share exclusively simple perforations and two types of vessel-ray pitting, but they can easily be distinguished from each other by the size of intervessel pitting, shape of body ray cells in multiseriate rays, and the type of crystalliferous axial parenchyma cells. The anatomical diversity observed is clearly correlated with differences in plant size (shrubs vs. tall trees): the percentage of scalariform perforations, as well as vessel density, and the length of vessel elements, fibers, and multiseriate rays are negatively correlated with increasing plant size, while the reverse is true for vessel diameter.     

Productivity, herbivory, and species traits rather than diversity influence invasibility of experimental phytoplankton communities

Biological invasions are a major threat to natural biodiversity; hence, understanding the mechanisms underlying invasibility (i.e., the susceptibility of a community to invasions by new species) is crucial. Invasibility of a resident community may be affected by a complex but hitherto hardly understood interplay of (1) productivity of the habitat, (2) diversity, (3) herbivory, and (4) the characteristics of both invasive and resident species. Using experimental phytoplankton microcosms, we investigated the effect of nutrient supply and species diversity on the invasibility of resident communities for two functionally different invaders in the presence or absence of an herbivore. With increasing nutrient supply, increased herbivore abundance indicated enhanced phytoplankton biomass production, and the invasion success of both invaders showed a unimodal pattern. At low nutrient supply (i.e., low influence of herbivory), the invasibility depended mainly on the competitive abilities of the invaders, whereas at high nutrient supply, the susceptibility to herbivory dominated. This resulted in different optimum nutrient levels for invasion success of the two species due to their individual functional traits. To test the effect of diversity on invasibility, a species richness gradient was generated by random selection from a resident species pool at an intermediate nutrient level. Invasibility was not affected by species richness; instead, it was driven by the functional traits of the resident and/or invasive species mediated by herbivore density. Overall, herbivory was the driving factor for invasibility of phytoplankton communities, which implies that other factors affecting the intensity of herbivory (e.g., productivity or edibility of primary producers) indirectly influence invasions.