GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation

GCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene (GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically 'normal' GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 β-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations.     

Human urinary excretion and metabolism of (82)Se-enriched selenite and selenate determined by LC-ICP-MS

Urinary excretion of selenium after ingestion of isotope labeled selenite and selenate was studied in seven healthy volunteers, 4 men and 3 women (age 28-50 years). An aqueous solution containing 330 μL (82)Se-selenate (corresponding to 74.3 μg (82)Se) was given orally and urine samples were subsequently collected during the following 24 hours. The scheme was repeated four weeks later with a 280 μL (82)Se-selenite solution (corresponding to 74.4 μg (82)Se). The amount of total Se in the urine samples was determined by inductively coupled mass spectrometry. The mean total urinary excretion of (82)Se following (82)Se-selenate administration was 33.7% (range 15.6-42.5%) while the mean total excretion of (82)Se after (82)Se-selenite administration was 3.2% (range 2.8-3.9%) of the ingested amount. LC-ICPMS analysis of the urine samples showed that the majority of the selenium excreted after selenate ingestion was unchanged selenate for 6 of the individuals while one individual had metabolized a fraction (approx. 20%) of the selenate to selenosugar. Ingestion of 10 times larger doses of selenite in two individuals resulted in 13-23% excretion primarily excreted as selenosugar. These results show that the human metabolic pathways of selenite and selenate are different and indicate that not all selenate, although well absorbed, may be available for the beneficial health effects.     

Endurance Training Per Se Increases Metabolic Health in Young,Moderately Overweight Men

Health benefits of physical activity may depend on a concomitant weight loss. In a randomized, controlled trial, we compared the effects of endurance training with or without weight loss to the effect of weight loss induced by an energy‐reduced diet in 48 sedentary, moderately overweight men who completed a 12‐week intervention program of training (T), energy‐reduced diet (D), training and increased diet (T‐iD), or control (C). An energy deficit of 600 kcal/day was induced by endurance training or diet in T and D and a similar training regimen plus an increased dietary intake of 600 kcal/day defined the T‐iD group. Primary end point was insulin sensitivity as evaluated by HOMA‐IR (mainly reflecting hepatic insulin sensitivity) and hyperinsulinemic, isoglycemic clamps (primarily reflecting peripheral insulin sensitivity). Body mass decreased in T and D by 5.9 ± 0.7 and 5.3 ± 0.7 kg, respectively, whereas T‐iD and C remained weight stable. Total and abdominal fat mass were reduced in an additive manner in the T‐iD, D, and T groups by 1.9 ± 0.3/0.2 ± 0.1, 4.4 ± 0.7/0.5 ± 0.1, and 7.7 ± 0.8/0.9 ± 0.1 kg, respectively. HOMA‐IR was improved in T, D, and T‐iD, whereas insulin‐stimulated glucose clearance and suppression of plasma nonesterified fatty acids (NEFAs) were increased only in the two training groups. Thus, loss of fat mass (diet or training induced) improves hepatic insulin sensitivity, whereas peripheral insulin sensitivity in skeletal muscle and adipose tissue is increased by endurance training only. This demonstrates that endurance training per se increases various metabolic health parameters and that endurance training should preferably always be included in any intervention regimen for improving metabolic health in moderately overweight men.     

Carnitine Deficiency in OCTN2?/? Newborn Mice Leads to a Severe Gut and Immune Phenotype with Widespread Atrophy,Apoptosis and a Pro-Inflammatory Response

We have investigated the gross, microscopic and molecular effects of carnitine deficiency in the neonatal gut using a mouse model with a loss-of-function mutation in the OCTN2 (SLC22A5) carnitine transporter. The tissue carnitine content of neonatal homozygous (OCTN2^−/−) mouse small intestine was markedly reduced; the intestine displayed signs of stunted villous growth, early signs of inflammation, lymphocytic and macrophage infiltration and villous structure breakdown. Mitochondrial β-oxidation was active throughout the GI tract in wild type newborn mice as seen by expression of 6 key enzymes involved in β-oxidation of fatty acids and genes for these 6 enzymes were up-regulated in OCTN2^−/− mice. There was increased apoptosis in gut samples from OCTN2^−/− mice. OCTN2^−/− mice developed a severe immune phenotype, where the thymus, spleen and lymph nodes became atrophied secondary to increased apoptosis. Carnitine deficiency led to increased expression of CD45-B220⁺ lymphocytes with increased production of basal and anti-CD3-stimulated pro-inflammatory cytokines in immune cells. Real-time PCR array analysis in OCTN2^−/− mouse gut epithelium demonstrated down-regulation of TGF-β/BMP pathway genes. We conclude that carnitine plays a major role in neonatal OCTN2^−/− mouse gut development and differentiation, and that severe carnitine deficiency leads to increased apoptosis of enterocytes, villous atrophy, inflammation and gut injury.     

Small angle X-ray scattering study of calreticulin reveals conformational plasticity

Calreticulin plays a central role in vital cell processes such as protein folding, Ca(2+) homeostasis and immunogenicity. Even so, only limited three-dimensional structural information is presently available. We present a series of Small-Angle X-ray Scattering data on human placenta calreticulin. The data from the calreticulin monomer reveal the shape of calreticulin in solution: The previously structurally un-described C-terminal is seen as a globular domain, and the P-domain beta-hairpin extends from the N-domain in a spiral like conformation. In the calreticulin solution dimer, the N-, C-, and P-domains are easily identified, and the P-domain is in an extended conformation connecting to the second calreticulin molecule. The SAXS solution data enables the construction of a medium-resolution model of calreticulin. In the light of the unresolved chaperone mechanism of calreticulin and calnexin, we discuss the functional consequences of the conformational plasticity of the calreticulin P-domain.     

Predicted changes in vegetation structure affect the susceptibility to invasion of bryophyte-dominated subarctic heath

Background and Aims

A meta-analysis of global change experiments in arctic tundra sites suggests that plant productivity and the cover of shrubs, grasses and dead plant material (i.e. litter) will increase and the cover of bryophytes will decrease in response to higher air temperatures. However, little is known about which effects these changes in vegetation structure will have on seedling recruitment of species and invasibility of arctic ecosystems.

Methods

A field experiment was done in a bryophyte-dominated, species-rich subarctic heath by manipulating the cover of bryophytes and litter in a factorial design. Three phases of seedling recruitment (seedling emergence, summer seedling survival, first-year recruitment) of the grass Anthoxanthum alpinum and the shrub Betula nana were analysed after they were sown into the experimental plots.

Key Results

Bryophyte and litter removal significantly increased seedling emergence of both species but the effects of manipulations of vegetation structure varied strongly for the later phases of recruitment. Summer survival and first-year recruitment were significantly higher in Anthoxanthum. Although bryophyte removal generally increased summer survival and recruitment, seedlings of Betula showed high mortality in early August on plots where bryophytes had been removed.

Conclusions

Large species-specific variation and significant effects of experimental manipulations on seedling recruitment suggest that changes in vegetation structure as a consequence of global warming will affect the abundance of grasses and shrubs, the species composition and the susceptibility to invasion of subarctic heath vegetation.     

Joint axes of rotation and body segment parameters of pig limbs

To enable a quantification of net joint moments and joint reaction forces, indicators of joint loading, this study aimed to locate the mediolateral joint axes of rotation and establish the body segment parameters of the limbs of pigs (Sus scrofa). To locate the joint axes of rotation the scapulohumeral, humeroradial, carpal complex, metacarpophalangeal, coxofemoral, femorotibial, tarsal, and metatarsophalangeal joints from 12 carcasses were studied. The joints were photographed in three positions, bisecting lines drawn at fixed landmarks with their intersection marking the joint axes of rotation. The body segment parameters, i.e. the segment mass, center of mass and moment of inertia were measured on the humerus, radius/ulna, metacarpus, forepastern, foretoe, femur, tibia, metatarsus, hindpastern, and hindtoe segments from five carcasses. The segments were weighed, and their center of mass was found by balancing them. The moments of inertia of the humerus, radius/ulna, femur and tibia were found by rotating the segments. The moments of inertia of the remaining segments were calculated. Generally, the joint axes of rotation were near the attachment site of the lateral collateral ligaments. The forelimb, with segments taken as one, was significantly lighter and shorter than the hindlimb (P < 0.001). In all segments the center of mass was located 31 to 50% distal to the proximal segment end. The segment mass decreased with distance from the trunk, as did the segment moment of inertia. The results may serve as reference on the location of the joint axes of rotation and on the body segment parameters for inverse dynamic modeling of pigs.     

A high-throughput method for measuring growth and loss rates in microalgal cultures

A miniaturized and low-cost assay for algal growth and loss rates, and estimation of compensation light was developed and optimized. Microalgal cultures were grown in white 96-well microplates to estimate specific growth rates at six temperatures, five salinities and eight light levels. Data from black 24-well microplates at six temperatures, five salinities and five light conditions were used in addition to estimate loss rates and compensation light. Absorption and reflection of light were different in the white and black microplates. Growth rates were estimated from daily in vivo fluorescence (IVF) measurements using a microplate reader fitted with a fluorometer. To validate the microplate algal growth assay, IVF was compared with cell counting by flow cytometry. Maximal growth rate for the test alga Pseudochattonella farcimen (Heterokonta) was estimated to 0.52?±?0.05 day^?1 at optimal temperatures ranging from 9 to 14°C and salinities 18–26 psu. Lowest value of compensation light as photosynthetic photon flux density (PPFD) was 4.2?±?1.2 μmol photons m^?2 s^?1, and lowest saturation light, 34.1?±?3.7 μmol photons m^?2 s^?1, was observed in the temperature range 5–11°C and salinity range 23–28 psu. Minimum loss rate was obtained at temperatures 5–8°C and salinities 26–31 psu. Blooms of P. farcimen have been recorded in nature under conditions similar to those minimizing loss rates rather than maximizing growth rates in this study. The microalgal assay described here allows for a large number of conditions to be tested, and accurate optimal conditions for growth and loss rates to be obtained.     

Stimulatory role for endogenous opioid peptides on postexercise insulin secretion in rats

Endogenous opioid peptides (EOP) and prior exercise may modulate the stimulatory effect of glucose on insulin secretion. To gain insights into these relationships, we studied male Wistar rats (187-245 g) during sustained hyperglycemia by use of the glucose clamp technique. Four groups of sedentary fed rats (n = 8/group) either ran (Ex) at 24 m/min, 0% grade, or rested (R) for 40 min. Thirty minutes after Ex or R, arterial blood glucose was elevated to and maintained at 11 mM for 2 h by a variable glucose infusion. At the start of Ex or R rats had saline (Sal) or naloxone (Nal, an opioid antagonist) intravenous infusions for 160 min (40 min Ex + 30 min R + 90 min of a 120-min glucose clamp). Steady-state glucose infusion rates (SSGIR) were approximately 55 mg.kg-1.min-1 at the start of the clamp and declined significantly over the 2nd h to approximately 45 mg.kg-1.min-1. No significant differences existed in SSGIR between groups. R-Sal and Ex-Sal groups did not differ in their insulin response to hyperglycemia. In contrast, when all groups were compared at the end of the Nal or Sal infusion, Ex-Nal had the lowest insulin concentration (749 +/- 174 pmol/l), whereas the R-Nal group had the highest (1,581 +/- 216 pmol/l, P less than 0.05). These data suggest a stimulatory role for EOP on insulin secretion that is expressed after a prior stress (Ex). Thus one function of exercise-induced activation of EOP may be to regulate insulin secretion in the immediate postexercise period.     

Postexercise dose-response relationship between plasma glucose and insulin secretion

To investigate whether exertion changes beta-cell reactivity to glucose stimulation and to characterize the beta-cell response to glucose in humans, we performed four sequential 90-min hyperglycemic clamps (7, 11, 20, and 35 mM). Concentrations of hormones and metabolites involved in glucoregulation were measured. Metabolic rate and substrate utilization were studied by indirect calorimetry. Studies were performed without prior exercise, as well as 2 and 48 h after 60 min of bicycle exercise at 150 W. We found 1) a progressive increase in insulin concentrations reaching 1,092 +/- 135 microU/ml with increasing glucose levels, 2) linear relationships between glucose concentrations and concentrations of C-peptide (r = 0.931 +/- 0.008) and proinsulin (r = 0.952 +/- 0.009),3) increased glucose oxidation with increasing glucose uptake, 4) increased plasma norepinephrine, O2 uptake, and beta-hydroxybutyrate at greater than or equal to 20 mM glucose, and 5) no change in beta-cell response or glucose-induced thermogenesis after one bout of exercise despite no compensating changes in plasma concentrations of hormones or metabolites. We conclude that the beta-cell has a very large secretory potential. Secretion of the beta-cell increases linearly with prolonged, graded hyperglycemia. The processing of proinsulin is unchanged during prolonged beta-cell stimulation. In addition, hyperglycemia and sympathetic nervous activity induced by hyperinsulinemia enhance metabolic rate and ketone body production. Finally, a single bout of exercise does not influence either the beta-cell response to intravenous glucose or glucose-induced thermogenesis.