Synaptotagmin-7 as a positive regulator of glucose-induced glucagon-like peptide-1 secretion in mice.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1), a hormone with potent antihyperglycaemic effects, is produced and secreted from highly specialised gut endocrine L-cells. It regulates glucose homeostasis by promoting glucose-dependent insulin secretion, suppressing glucagon secretion and enhancing insulin sensitivity. Similar to islet alpha and beta cells, L-cells are electrically excitable, and express calcium channels and ATP-sensitive potassium channels. GLP-1 is also stored in secretory granules, the exocytosis of which is triggered by increased intracellular calcium levels. Although the calcium dependence of GLP-1 granule exocytosis is well established, the identities of calcium-sensing proteins in GLP-1 secretion remain elusive. Here we tested whether synaptotagmin-7, a calcium sensor in pancreatic alpha and beta cells, regulates GLP-1 secretion. METHODS: We studied GLP-1 secretion using synaptotagmin-7 knockout (KO) mice and GLUTag cells with lentiviral-mediated synaptotagmin-7 silencing. RESULTS: We found that synaptotagmin-7 was co-localised with GLP-1 in intestinal L-cells. GLP-1 secretion was impaired in synaptotagmin-7 KO mice when they were challenged by glucose ingestion. Lentiviral knockdown (KD) of synaptotagmin-7 in GLUTag cells led to similar reductions in GLP-1 secretion, as determined by biochemical assays and by membrane capacitance measurements. Calcium response was not altered in synaptotagmin-7 KD cells. CONCLUSIONS/INTERPRETATION: These results demonstrate that synaptotagmin-7 functions as a positive regulator of GLP-1 secretion in intestinal L-cells and GLUTag cells, consistent with its proposed role as a calcium sensor in GLP-1 secretion.

Delayed onset of hyperglycaemia in a mouse model with impaired glucagon secretion demonstrates that dysregulated glucagon secretion promotes hyperglycaemia and type 2 diabetes.

AIMS/HYPOTHESIS: Type 2 diabetes is caused by relative deficiency of insulin secretion and is associated with dysregulation of glucagon secretion during the late stage of diabetes development. Like insulin secretion from beta cells, glucagon secretion is dependent on calcium signals and a calcium sensing protein, synaptotagmin-7. In this study, we tested the relative contribution of dysregulated glucagon secretion and reduced insulin release in the development of hyperglycaemia and type 2 diabetes by using synaptotagmin-7 knockout (KO) mice, which exhibit glucose intolerance, reduced insulin secretion and nearly abolished Ca(2+)-stimulated glucagon secretion. METHODS: We fed the synaptotagmin-7 KO and control mice with a high-fat diet (HFD) for 14 weeks, and compared their body weight, glucose levels, glucose and insulin tolerance, and insulin and glucagon secretion. RESULTS: On the HFD, synaptotagmin-7 KO mice showed progressive impairment of glucose tolerance and insulin secretion, along with continued maintenance of a low glucagon level. The control mice were less affected in terms of glucose intolerance, and showed enhanced insulin secretion with a concurrent increase in glucagon levels. Unexpectedly, after 14 weeks of HFD feeding, only the control mice displayed resting hyperglycaemia, whereas in synaptotagmin-7 KO mice defective insulin secretion and reduced insulin sensitivity were not sufficient to cause hyperglycaemia in the absence of enhanced glucagon secretion. CONCLUSIONS/INTERPRETATION: Our data uncover a previously overlooked role of dysregulated glucagon secretion in promoting hyperglycaemia and the ensuing diabetes, and strongly suggest maintenance of adequate regulation of glucagon secretion as an important therapeutic target in addition to the preservation of beta cell function and mass in the prevention and treatment of diabetes.

Timing of Ca2+ response in pancreatic beta-cells is related to mitochondrial mass.

The timing and magnitude of calcium response are cell-specific in individual beta-cells. This may indicate that the cells have different roles in the intact islet. It is unknown what mechanisms determine these characteristics. We previously found that the mechanisms setting cell-specific response timing are disturbed in beta-cells from hyperglycemic mice and one of the causes is likely to be an altered mitochondrial metabolism. Mitochondria play a key role in the control of nutrient-induced insulin secretion. Here, we used confocal microscopy with the fluorescent probe MitoTracker Red CMXRos and Fluo-3 to study how the amount of active mitochondria is related to the lag-time and the magnitude of calcium response to 20mM glucose in isolated beta-cells and in cells within intact lean and ob/ob mouse islets. Results show that the mitochondrial mass is inversely correlated with the lag-times for calcium response both in lean and ob/ob mouse beta-cells (r=-0.73 and r=-0.43, respectively, P<0.05). Thus, the state of mitochondria may determine the timing of calcium response.

The universal stress protein paralogues of Escherichia coli are co-ordinately regulated and co-operate in the defence against DNA damage.

We have cloned, characterized and inactivated genes encoding putative UspA paralogues in Escherichia coli. The yecG (uspC), yiiT (uspD) and ydaA (uspE) genes were demonstrated to encode protein pro-ducts and these were mapped to spots in the E. coli proteomic database. Expression analysis using chromosomal transcriptional lacZ fusions and two-dimensional gels revealed that all usp genes analysed are regulated in a similar fashion. Thus, uspC, D and E are all induced in stationary phase and by a variety of stresses causing growth arrest of cells. Induction is independent of rpoS but is abolished in a deltarelA deltaspoT (ppGpp0) background and rescued by suppressor mutations rendering the beta-subunit of RNA polymerase to behave like a stringent polymerase. Ectopic elevation of ppGpp levels in growing cells, by overproducing the RelA protein, triggered the induction of all usp genes. The expression of all usp genes was also elevated by a mutation in the ftsK cell division gene, and this super-induction could be suppressed by inactivating recA indicating that the usp paralogues are involved in the management of DNA. Indeed, uspC, uspD and uspE deletion mutants were all found to be sensitive to UV exposure. Overexpression of UspD could compensate for the lack of a chromosomal uspD gene but not a uspA gene. Similarly, UspA overproduction could only compensate for the lack of chromosomal uspA. Moreover, combination of usp mutations had no additive effect on UV sensitivity indicating that they are all co-operating and required in the same pathway, which could explain the co-ordinated regulation of the genes.

Substitution of conserved methionines by leucines in chloroplast small heat shock protein results in loss of redox-response but retained chaperone-like activity.

During evolution of land plants, a specific motif occurred in the N-terminal domain of the chloroplast-localized small heat shock protein, Hsp21: a sequence with highly conserved methionines, which is predicted to form an amphipathic alpha-helix with the methionines situated along one side. The functional role of these conserved methionines is not understood. We have found previously that treatment, which causes methionine sulfoxidation in Hsp21, also leads to structural changes and loss of chaperone-like activity. Here, mutants of Arabidopsis thaliana Hsp21 protein were created by site-directed mutagenesis, whereby conserved methionines were substituted by oxidation-resistant leucines. Mutants lacking the only cysteine in Hsp21 were also created. Protein analyses by nondenaturing electrophoresis, size exclusion chromatography, and circular dichroism proved that sulfoxidation of the four highly conserved methionines (M49, M52, M55, and M59) is responsible for the oxidation-induced conformational changes in the Hsp21 oligomer. In contrast, the chaperone-like activity was not ultimately dependent on the methionines, because it was retained after methionine-to-leucine substitution. The functional role of the conserved methionines in Hsp21 may be to offer a possibility for redox control of chaperone-like activity and oligomeric structure dynamics.

The chaperone-like activity of a small heat shock protein is lost after sulfoxidation of conserved methionines in a surface-exposed amphipathic alpha-helix.

The small heat shock proteins (sHsps) possess a chaperone-like activity which prevents aggregation of other proteins during transient heat or oxidative stress. The sHsps bind, onto their surface, molten globule forms of other proteins, thereby keeping them in a refolding competent state. In Hsp21, a chloroplast-located sHsp in all higher plants, there is a highly conserved region forming an amphipathic alpha-helix with several methionines on the hydrophobic side according to secondary structure prediction. This paper describes how sulfoxidation of the methionines in this amphipathic alpha-helix caused conformational changes and a reduction in the Hsp21 oligomer size, and a complete loss of the chaperone-like activity. Concomitantly, there was a loss of an outer-surface located alpha-helix as determined by limited proteolysis and circular dichroism spectroscopy. The present data indicate that the methionine-rich amphipathic alpha-helix, a motif of unknown physiological significance which evolved during the land plant evolution, is crucial for binding of substrate proteins and has rendered the chaperone-like activity of Hsp21 very dependent on the chloroplast redox state.

The universal stress protein A of Escherichia coli is required for resistance to DNA damaging agents and is regulated by a RecA/FtsK-dependent regulatory pathway.

The link between cell division defects and the induction of the universal stress response is demonstrated to operate via the RecA regulator of the SOS response. An insertion in the cell division gene ftsK upregulates uspA in a recA-dependent manner. Unlike true SOS response genes, this upregulation only occurs in growth-arrested cells and is LexA independent. Thus, besides ppGpp-dependent starvation signals, DNA aberrations transduce RecA-dependent signals to the uspA promoter, which only affect the promoter during stasis. Further, we show that ftsK itself, like uspA, is induced in stationary phase and that this induction requires the stringent control modulon rather than activated RecA. Thus, ftsK, like uspA, is regulated by at least two global regulators: ppGpp of the stringent control network and RecA of the SOS modulon. We suggest that UspA is a new bona fide member of the RecA-dependent DNA protection and repair system, as mutants lacking functional UspA were found to be sensitive to UV irradiation and mitomycin C exposure. Moreover, the UV sensitivity of uspA mutants is enhanced in an additive manner by the ftsK1 mutation.

Methionine sulfoxidation of the chloroplast small heat shock protein and conformational changes in the oligomer.

The small heat shock proteins (sHsps), which counteract heat and oxidative stress in an unknown way, belong to a protein family of sHsps and alpha-crystallins whose members form large oligomeric complexes. The chloroplast-localized sHsp, Hsp21, contains a conserved methionine-rich sequence, predicted to form an amphipatic helix with the methionines situated along one of its sides. Here, we report how methionine sulfoxidation was detected by mass spectrometry in proteolytically cleaved peptides that were produced from recombinant Arabidopsis thaliana Hsp21, which had been treated with varying concentrations of hydrogen peroxide. Sulfoxidation of the methionine residues in the conserved amphipatic helix coincided with a significant conformational change in the Hsp21 protein oligomer.

Exposure to natural fluoride in well water and hip fracture: a cohort analysis in Finland.

In the retrospective cohort study based on record linkage, the authors studied a cohort of persons born in 1900-1930 (n = 144,627), who had lived in the same rural location at least from 1967 to 1980. Estimates for fluoride concentrations (median, 0.1 mg/liter; maximum, 2.4 mg/liter) in well water in each member of the cohort were obtained by a weighted median smoothing method based on ground water measurements. Information on hip fractures was obtained from the Hospital Discharge Registry for 1981-1994. No association was observed between hip fractures and estimated fluoride concentration in the well water in either men or women when all age groups were analyzed together. However, the association was modified by age and sex so that among younger women, those aged 50-64 years, higher fluoride levels increased the risk of hip fractures. Among older men and women and younger men, no consistent association was seen. The adjusted rate ratio was 2.09 (95% confidence interval: 1.16, 3.76) for younger women who were the most exposed (>1.5 mg/liter) when compared with those who were the least exposed (< or =0.1 mg/liter). The results suggest that fluoride increases the risk of hip fractures only among women.

Maintenance energy requirement: what is required for stasis survival of Escherichia coli?

Little is known about how the energy of maintenance is generated in a cell supporting its persistence solely on endogenous carbon material, and what this energy is used for. However, it is clear that the endogenous metabolism of Escherichia coli cells held in the absence of exogenous carbon includes de novo protein synthesis, and that this synthesis is required for the maintenance of the growth-arrested cell. Recent findings suggest that several genes/proteins responding to carbon starvation are themselves involved in reorganizing and modulating catabolic flux, while others form an integral part of a defense system aimed at avoiding the damaging effects of ongoing respiratory activity. A significant fraction of the energy of maintenance is suggested to be required to prevent the denaturation and spontaneous aging of proteins during stasis.

Placental transfer of cadmium.

Cadmium levels in blood have been determined in mother-newborn pairs from the surroundings of a copper smelter and a control area in Northern Sweden. The smelter's cadmium emissions to the air have decreased substantially since the 1970s, and in 1989 the emission was one ton. Venous blood was sampled from the mothers during delivery and from the umbilical cords, and analysed for cadmium by flameless atomic absorption spectrophotometry. There were no significant differences in cadmium levels, as between exposed women and controls, and blood levels were low, even in an industrial area. The most important environmental exposure seemed to be smoking. There was, however, a significant increase in cadmium levels during pregnancy among non-smoking women in both groups, p < 0.01. The cadmium levels in the newborn babies were about 70% of those in the mothers. Cadmium levels in the babies of non-smoking mothers were significantly higher in the vicinity of the smelter than in the control area (p < 0.05).

Drug exposed infants and their mothers: facts, myths, and needs.

There is much interest in the use and abuse of chemicals by pregnant women. Although maternal chemical use is believed to be harmful to the fetus and has become the basis for criminal prosecution of mothers, obtaining accurate information about the incidence and consequences of prenatal exposure is difficult. This article critically examines the research on incidence and consequences of chemical use and offers suggestions for providing services to this vulnerable population for health care social workers in policy and practice positions.