Autophagy is required for gamete differentiation in the moss Physcomitrella patens

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.     

Human liver epigenetic alterations in non-alcoholic steatohepatitis are related to insulin action

Both genetic and lifestyle factors contribute to the risk of non-alcoholic steatohepatitis (NASH). Additionally, epigenetic modifications may also play a key role in the pathogenesis of NASH. We therefore investigated liver DNA methylation, as a marker for epigenetic alterations, in individuals with simple steatosis and NASH, and further tested if these alterations were associated with clinical phenotypes. Liver biopsies obtained from 95 obese individuals (age: 49.5 ± 7.7 years, BMI: 43 ± 5.7 kg/m², type 2 diabetes [T2D]: 35) as a wedge biopsy during a Roux-en-Y gastric bypass operation were investigated. Thirty-four individuals had a normal liver phenotype, 35 had simple steatosis, and 26 had NASH. Genome-wide DNA methylation pattern was analyzed using the Infinium HumanMethylation450 BeadChip. mRNA expression was analyzed from 42 individuals using the HumanHT-12 Expression BeadChip. We identified 1,292 CpG sites representing 677 unique genes differentially methylated in liver of individuals with NASH (q < 0.001), independently of T2D, age, sex, and BMI. Focusing on the top-ranking 30 and another 37 CpG sites mapped to genes enriched in pathways of metabolism (q = 0.0036) and cancer (q = 0.0001) all together, 59 NASH-associated CpG sites correlated with fasting insulin levels independently of age, fasting glucose, or T2D. From these, we identified 30 correlations between DNA methylation and mRNA expression, for example LDHB (r = ?0.45, P = 0.003). We demonstrated that NASH, more than simple steatosis, associates with differential DNA methylation in the human liver. These epigenetic alterations in NASH are linked with insulin metabolism.     

Insulin: a small protein with a long journey

Insulin is a hormone that is essential for regulating energy storage and glucose metabolism in the body. Insulin in liver, muscle, and fat tissues stimulates the cell to take up glucose from blood and store it as glycogen in liver and muscle. Failure of insulin control causes diabetes mellitus (DM). Insulin is the unique medicine to treat some forms of DM. The population of diabetics has dramatically increased over the past two decades, due to high absorption of carbohydrates (or fats and proteins), lack of physical exercise, and development of new diagnostic techniques. At present, the two largest developing countries (India and China) and the largest developed country (United States) represent the top three countries in terms of diabetic population. Insulin is a small protein, but contains almost all structural features typical of proteins: α-helix, β-sheet, β-turn, high order assembly, allosteric T®R-transition, and conformational changes in amyloidal fibrillation. More than ten years’ efforts on studying insulin disulfide intermediates by NMR have enabled us to decipher the whole picture of insulin folding coupled to disulfide pairing, especially at the initial stage that forms the nascent peptide. Two structural switches are also known to regulate insulin binding to receptors and progress has been made to identify the residues involved in binding. However, resolving the complex structure of insulin and its receptor remains a challenge in insulin research. Nevertheless, the accumulated knowledge of insulin structure has allowed us to specifically design a new ultra-stable and active single-chain insulin analog (SCI-57), and provides a novel way to design super-stable, fast-acting and cheaper insulin formulations for DM patients. Continuing this long journey of insulin study will benefit basic research in proteins and in pharmaceutical therapy.     

Human deciduous mandibular molar incremental enamel development

Quantitative studies of incremental markings retained within human enamel have reconstructed the duration and rate (crown and cusp formation times, initiation and completion, daily enamel secretion rates) of permanent tooth development. This approach has provided one way of estimating human age‐at‐death, and facilitated comparative dental studies of primate evolution. Similar applications from deciduous enamel are inhibited because developmental reconstructions from incremental markings for these teeth are less frequently reported in the literature. This study quantified the duration and rate of enamel development for mesial (protoconid, metaconid) and distal cusps (hypoconid, entoconid) for first (dm1) and second (dm2) deciduous mandibular molars from an archaeological sample of modern human juveniles. Crown formation time can be calculated from the dm1 protoconid because growth initiates and completes in this cusp, and from the dm2 protoconid combined with the final period of hypoconid growth. The dm1 postnatal crown formation time included the time taken for the tubercle of Zuckerkandl to develop, and differed slightly compared to radiographic methods. The majority of dm1 protoconid cuspal (occlusal region) enamel formed before birth. The dm2 entoconid enamel formed mainly after birth. Birth reduced daily enamel secretion rates, changed the visibility of incremental markings, and disrupted enamel growth for 3 to 8 days. Findings presented here can contribute to age‐at‐death estimates for human infants aged 13‐postnatal months or less, and should facilitate comparisons of primate deciduous incremental enamel development in an evolutionary context. Regression equations are included so that cuspal formation time can be estimated from enamel thickness. Am J Phys Anthropol, 2011. © 2010 Wiley‐Liss, Inc.     

The evolution of insulin resistance in muscle of the glucose infused rat

Glucose infusion into rats causes skeletal muscle insulin resistance that initially occurs without changes in insulin signaling. The aim of the current study was to prolong glucose infusion and evaluate other events associated with the transition to muscle insulin resistance. Hyperglycemia was produced in rats by glucose infusion for 3, 5 and 8 h. The rate of infusion required to maintain hyperglycemia was reduced at 5 and 8 h. Glucose uptake into red quadriceps (RQ) and its incorporation into glycogen decreased between 3 and 5 h, further decreasing at 8 h. The earliest observed change in RQ was decreased AMPKα2 activity associated with large increases in muscle glycogen content at 3 h. Activation of the mTOR pathway occurred at 5 h. Akt phosphorylation (Ser⁴⁷³) was decreased at 8 h compared to 3 and 5, although no decrease in phosphorylation of downstream GSK-3β (Ser⁹) and AS160 (Thr⁶⁴²) was observed. White quadriceps showed a similar but delayed pattern, with insulin resistance developing by 8 h and decreased AMPKα2 activity at 5 h. These results indicate that, in the presence of a nutrient overload, alterations in muscle insulin signaling occur, but after insulin resistance develops and appropriate changes in energy/nutrient sensing pathways occur.     

Interaction of plasma membrane Ca-ATPase isoform 4 with calcineurin A: Implications for catecholamine secretion by PC12 cells

PMCA1–4 isoforms have been recently recognised as regulators of various signalling pathways in mammalian cells. PMCAs were found to interact with calcineurin A in an isoform specific manner. In this study we focus on the interaction of calcineurin A with PMCA4 and its effect on catecholamine secretion in PC12 cells with reduced PMCA2 or PMCA3 content. Reduction of synthesis of PMCA2 or PMCA3 led to upregulation of PMCA4 manifested by preferential interaction of PMCA4 with calcineurin A. On the other hand, we observed a significant reduction of dopamine secretion, which did not correspond with an increased [Ca²⁺]_c. This result indicates that the interaction of PMCA4 with calcineurin A plays a regulatory role in the signalling during catecholamine secretion.     

Berberine exerts anti-adipogenic activity through up-regulation of C/EBP inhibitors, CHOP and DEC2

Berberine exerts an anti-adipogenic activity that is associated with the down-regulation of C/EBPα and PPARγ. Stimulation of AMP-activated kinase (AMPK) caused by inhibition of mitochondrial respiration has been suggested to underlie such molecular regulation. In the present study, we show that berberine up-regulated the expression of two different sets of C/EBP inhibitors, CHOP and DEC2, while down-modulating C/EBPα, PPARγ and other adipogenic markers and effectors in differentiating 3T3-L1 preadipocytes and mature adipocytes. Data also suggested that the berberine-induced up-regulation of CHOP and DEC2 was attributable to selective activation of an unfolded protein response (UPR) and modified extracellular environment, respectively. As a result, the anti-adipogenic activity of berberine was diminished remarkably by adjusting the differentiation culture media and limitedly but consistently by knockdown of CHOP expression. Together, up-regulation of C/EBP inhibitors appears to underlie the berberine-induced repression of C/EBPα and PPARγ and, so, the inhibition of adipogenesis.     

Icilin induces G1 arrest through activating JNK and p38 kinase in a TRPM8-independent manner

Mechanistic models of glucose stimulated insulin secretion (GSIS) established in minimal media in vitro, may not accurately describe the complexity of coupling metabolism with insulin secretion that occurs in vivo. As a first approximation, we have evaluated metabolic pathways in a typical growth media, DMEM as a surrogate in vivo medium, for comparison to metabolic fluxes observed under the typical experimental conditions using the simple salt-buffer of KRB. Changes in metabolism in response to glucose and amino acids and coupling to insulin secretion were measured in INS-1 832/13 cells. Media effects on mitochondrial function and the coupling efficiency of oxidative phosphorylation were determined by fluorometrically measured oxygen consumption rates (OCRs) combined with ³¹P NMR measured rates of ATP synthesis. Substrate preferences and pathways into the TCA cycle, and the synthesis of mitochondrial 2nd messengers by anaplerosis were determined by ¹³C NMR isotopomer analysis of the fate of [U-¹³C] glucose metabolism.Despite similar incremental increases in insulin secretion, the changes of OCR in response to increasing glucose from 2.5 to 15 mM were blunted in DMEM relative to KRB. Basal and stimulated rates of insulin secretion rates were consistently higher in DMEM, while ATP synthesis rates were identical in both DMEM and KRB, suggesting greater mitochondrial uncoupling in DMEM. The relative rates of anaplerosis, and hence synthesis and export of 2nd messengers from the mitochondria were found to be similar in DMEM to those in KRB. And, the correlation of total PC flux with insulin secretion rates in DMEM was found to be congruous with the correlation in KRB. Together, these results suggest that signaling mechanisms associated with both TCA cycle flux and with anaplerotic flux, but not ATP production, may be responsible for the enhanced rates of insulin secretion in more complex, and physiologically-relevant media.     

Characterization and biological actions of N-terminal truncated forms of glucose-dependent insulinotropic polypeptide

The N-terminal domain of glucose-dependent insulinotropic polypeptide (GIP) plays an important role in regulating biological activity. This study examined biological properties of several N-terminal truncated forms of GIP and two novel forms with substitutions at Phe position-6 with Arg or Val. GIP(6-42), GIP(R6-42), GIP(V6-42), GIP(7-42) and GIP(9-42) stimulated cAMP production in BRIN-BD11 cells similar to native GIP, whereas responses to GIP(3-42), GIP(4-42), GIP(5-42) and GIP(8-42) were reduced (P < 0.01 to P < 0.001). GIP-induced cyclic AMP production was significantly inhibited by GIP(3-42), GIP(4-42), GIP(5-42), GIP(6-42), GIP(R6-42), GIP(7-42) and GIP(8-42) (P < 0.001). Compared with native GIP, in vitro insulinotropic activity of GIP(3-42), GIP(4-42), GIP(5-42), GIP(7-42) and GIP(8-42) was reduced (P < 0.05 to P < 0.001), with GIP(4-42), GIP(5-42), GIP(7-42) and GIP(8-42) also potently inhibiting GIP-stimulated insulin secretion (P < 0.001). In ob/ob mice, GIP(4-42) and GIP(8-42) increased (P < 0.05 to P < 0.01) plasma glucose concentrations compared to the glucose-lowering action of native GIP. When GIP(8-42) was co-administered with native GIP it countered the ability of the native peptide to lower plasma glucose and increase circulating insulin concentrations. These data confirm the importance of the N-terminal region of GIP in regulating bioactivity and reveal that sequential truncation of the peptide yields novel GIP receptor antagonists which may have functional significance.     

An efficient delivery of DAMPs on the cell surface by the unconventional secretion pathway

Damage-associated molecular patterns (DAMPs) are signals released from dying cells evoking the immune system response in several inflammatory disorders. In normal situations, many of DAMPs are nuclear or cytosolic proteins with defined intracellular function, but they could be found on the cell surface following tissue injury. The biological function of the translocated DAMPs is still not well known and an efficient delivery of these molecules on the cell surface is required to clarify their biological effects. In this study, we demonstrated that an unclassical secretory signal peptide, N-terminal 18 amino acids of HASPB (HASPB-N18), could efficiently deliver Hsp60, Hsp70, and HMGB1 on the cell surface. Furthermore, the delivery of these molecules on the cell surface by HASPB-N18 is not limited to a special cell line because several cell lines could use this delivery signal to deliver these molecules on the cell surface. Moreover, we demonstrated that Hsp60 on the cell surface delivered by HASPB-N18 could be recognized by a soluble form of LOX-1, which implies that DAMPs on the cell surface delivered by HASPB-N18 have a proper conformation during transport. Therefore, delivery of DAMPs by HASPB-N18 is a reliable model to further understand the biological significance of DAMPs on the cell surface.