Impact of dietary protein on postprandial glycaemic control and insulin requirements in Type 1 diabetes: a systematic review.

AIM: Postprandial hyperglycaemia is a challenge for people living with Type 1 diabetes. In addition to carbohydrate, dietary protein has been shown to contribute to postprandial glycaemic excursions with recommendations to consider protein when calculating mealtime insulin doses. The aim of this review is to identify and synthesize evidence about the glycaemic impact of dietary protein and insulin requirements for individuals with Type 1 diabetes. METHODS: A systematic literature search of relevant biomedical databases was performed to identify research on the glycaemic impact of dietary protein when consumed alone, and in combination with other macronutrients in individuals with Type 1 diabetes. RESULTS: The review included 14 published studies dated from 1992 to 2018, and included studies that researched the impact of protein alone (n = 2) and protein in a mixed meal (n = 12). When protein was consumed alone a glycaemic effect was not seen until ≥ 75 g. In a carbohydrate-containing meal ≥ 12.5 g of protein impacted the postprandial glucose. Inclusion of fat in a high-protein meal enhanced the glycaemic response and further increased insulin requirements. The timing of the glycaemic effect from dietary protein ranged from 90 to 240 min. Studies indicate that the postprandial glycaemic response and insulin requirements for protein are different when protein is consumed alone or with carbohydrate and/or fat. CONCLUSIONS: This systematic review provides evidence that dietary protein contributes to postprandial glycaemic excursions and insulin requirements. These insights have important implications for the education of people with Type 1 diabetes and highlights the need for more effective insulin dosing strategies for mixed macronutrient meals.

Phosphorylation of the voltage-gated potassium channel Kv2.1 by AMP-activated protein kinase regulates membrane excitability.

Firing of action potentials in excitable cells accelerates ATP turnover. The voltage-gated potassium channel Kv2.1 regulates action potential frequency in central neurons, whereas the ubiquitous cellular energy sensor AMP-activated protein kinase (AMPK) is activated by ATP depletion and protects cells by switching off energy-consuming processes. We show that treatment of HEK293 cells expressing Kv2.1 with the AMPK activator A-769662 caused hyperpolarizing shifts in the current-voltage relationship for channel activation and inactivation. We identified two sites (S440 and S537) directly phosphorylated on Kv2.1 by AMPK and, using phosphospecific antibodies and quantitative mass spectrometry, show that phosphorylation of both sites increased in A-769662-treated cells. Effects of A-769662 were abolished in cells expressing Kv2.1 with S440A but not with S537A substitutions, suggesting that phosphorylation of S440 was responsible for these effects. Identical shifts in voltage gating were observed after introducing into cells, via the patch pipette, recombinant AMPK rendered active but phosphatase-resistant by thiophosphorylation. Ionomycin caused changes in Kv2.1 gating very similar to those caused by A-769662 but acted via a different mechanism involving Kv2.1 dephosphorylation. In cultured rat hippocampal neurons, A-769662 caused hyperpolarizing shifts in voltage gating similar to those in HEK293 cells, effects that were abolished by intracellular dialysis with Kv2.1 antibodies. When active thiophosphorylated AMPK was introduced into cultured neurons via the patch pipette, a progressive, time-dependent decrease in the frequency of evoked action potentials was observed. Our results suggest that activation of AMPK in neurons during conditions of metabolic stress exerts a protective role by reducing neuronal excitability and thus conserving energy.

Selective expression in carotid body type I cells of a single splice variant of the large conductance calcium- and voltage-activated potassium channel confers regulation by AMP-activated protein kinase.

Inhibition of large conductance calcium-activated potassium (BKCa) channels mediates, in part, oxygen sensing by carotid body type I cells. However, BKCa channels remain active in cells that do not serve to monitor oxygen supply. Using a novel, bacterially derived AMP-activated protein kinase (AMPK), we show that AMPK phosphorylates and inhibits BKCa channels in a splice variant-specific manner. Inclusion of the stress-regulated exon within BKCa channel α subunits increased the stoichiometry of phosphorylation by AMPK when compared with channels lacking this exon. Surprisingly, however, the increased phosphorylation conferred by the stress-regulated exon abolished BKCa channel inhibition by AMPK. Point mutation of a single serine (Ser-657) within this exon reduced channel phosphorylation and restored channel inhibition by AMPK. Significantly, RT-PCR showed that rat carotid body type I cells express only the variant of BKCa that lacks the stress-regulated exon, and intracellular dialysis of bacterially expressed AMPK markedly attenuated BKCa currents in these cells. Conditional regulation of BKCa channel splice variants by AMPK may therefore determine the response of carotid body type I cells to hypoxia.

The effect of different doses of an arginine-containing supplement on the healing of pressure ulcers.

OBJECTIVE: To investigate if a lower dose of arginine in the form of an oral nutritional supplement can show similar benefit in the healing rate of pressure ulcers compared with the current evidence for 9g of arginine. METHOD: Twenty-three inpatients with category II, III or IV pressure ulcers were randomised to receive daily, for 3 weeks, the standard hospital diet plus 4.5 or 9g arginine in the form of a commercial supplement. Pressure ulcer size and severity was measured weekly (by PUSH tool; pressure ulcer scale for healing; 0= completely healed, 17= greatest severity). Nutritional status was determined by Subjective Global Assessment. RESULTS: There were no significant differences in patients' age, gender, BMI, haemoglobin levels, albumin levels and diagnosis of diabetes between treatment groups. There was a significant decrease in pressure ulcer severity over time (p < 0.001), with no evidence of a difference in healing rate between the two arginine dosages (p=0.991). Based on expected healing time, patients in both treatment groups were estimated to achieve an almost 2-fold improvement compared with the historical control group. Patients categorised as malnourished showed clinically significant impaired healing rates compared with well nourished patients (p=0.057), although this was unaffected by arginine dosage (p=0.727). CONCLUSION: Similar clinical benefits in healing of pressure ulcers can be achieved with a lower dosage of arginine, which can translate into improved concordance and significant cost-savings for both the health-care facilities and for patients.

Development and validation of predictive risk models for sight threatening diabetic retinopathy in patients with type 2 diabetes to be applied as triage tools in resource limited settings.

Background: Delayed diagnosis and treatment of sight threatening diabetic retinopathy (STDR) is a common cause of visual impairment in people with Type 2 diabetes. Therefore, systematic regular retinal screening is recommended, but global coverage of such services is challenging. We aimed to develop and validate predictive models for STDR to identify 'at-risk' population for retinal screening. Methods: Models were developed using datasets obtained from general practices in inner London, United Kingdom (UK) on adults with type 2 Diabetes during the period 2007-2017. Three models were developed using Cox regression and model performance was assessed using C statistic, calibration slope and observed to expected ratio measures. Models were externally validated in cohorts from Wales, UK and India. Findings: A total of 40,334 people were included in the model development phase of which 1427 (3·54%) people developed STDR. Age, gender, diabetes duration, antidiabetic medication history, glycated haemoglobin (HbA1c), and history of retinopathy were included as predictors in the Model 1, Model 2 excluded retinopathy status, and Model 3 further excluded HbA1c. All three models attained strong discrimination performance in the model development dataset with C statistics ranging from 0·778 to 0·832, and in the external validation datasets (C statistic 0·685 - 0·823) with calibration slopes closer to 1 following re-calibration of the baseline survival. Interpretation: We have developed new risk prediction equations to identify those at risk of STDR in people with type 2 diabetes in any resource-setting so that they can be screened and treated early. Future testing, and piloting is required before implementation. Funding: This study was funded by the GCRF UKRI (MR/P207881/1) and supported by the NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology.

Development and validation of resource-driven risk prediction models for incident chronic kidney disease in type 2 diabetes.

Prediction models for population-based screening need, for global usage, to be resource-driven, involving predictors that are affordably resourced. Here, we report the development and validation of three resource-driven risk models to identify people with type 2 diabetes (T2DM) at risk of stage 3 CKD defined by a decline in estimated glomerular filtration rate (eGFR) to below 60 mL/min/1.73m2. The observational study cohort used for model development consisted of data from a primary care dataset of 20,510 multi-ethnic individuals with T2DM from London, UK (2007-2018). Discrimination and calibration of the resulting prediction models developed using cox regression were assessed using the c-statistic and calibration slope, respectively. Models were internally validated using tenfold cross-validation and externally validated on 13,346 primary care individuals from Wales, UK. The simplest model was simplified into a risk score to enable implementation in community-based medicine. The derived full model included demographic, laboratory parameters, medication-use, cardiovascular disease history (CVD) and sight threatening retinopathy status (STDR). Two less resource-intense models were developed by excluding CVD and STDR in the second model and HbA1c and HDL in the third model. All three 5-year risk models had good internal discrimination and calibration (optimism adjusted C-statistics were each 0.85 and calibration slopes 0.999-1.002). In Wales, models achieved excellent discrimination(c-statistics ranged 0.82-0.83). Calibration slopes at 5-years suggested models over-predicted risks, however were successfully updated to accommodate reduced incidence of stage 3 CKD in Wales, which improved their alignment with the observed rates in Wales (E/O ratios near to 1). The risk score demonstrated similar model performance compared to direct evaluation of the cox model. These resource-driven risk prediction models may enable universal screening for Stage 3 CKD to enable targeted early optimisation of risk factors for CKD.

Crystal structure of DNA recombination protein RuvA and a model for its binding to the Holliday junction.

The Escherichia coli DNA binding protein RuvA acts in concert with the helicase RuvB to drive branch migration of Holliday intermediates during recombination and DNA repair. The atomic structure of RuvA was determined at a resolution of 1.9 angstroms. Four monomers of RuvA are related by fourfold symmetry in a manner reminiscent of a four-petaled flower. The four DNA duplex arms of a Holliday junction can be modeled in a square planar configuration and docked into grooves on the concave surface of the protein around a central pin that may facilitate strand separation during the migration reaction. The model presented reveals how a RuvAB-junction complex may also accommodate the resolvase RuvC.

A mechanism of drug action revealed by structural studies of enoyl reductase.

Enoyl reductase (ENR), an enzyme involved in fatty acid biosynthesis, is the target for antibacterial diazaborines and the front-line antituberculosis drug isoniazid. Analysis of the structures of complexes of Escherichia coli ENR with nicotinamide adenine dinucleotide and either thienodiazaborine or benzodiazaborine revealed the formation of a covalent bond between the 2' hydroxyl of the nicotinamide ribose and a boron atom in the drugs to generate a tight, noncovalently bound bisubstrate analog. This analysis has implications for the structure-based design of inhibitors of ENR, and similarities to other oxidoreductases suggest that mimicking this molecular linkage may have generic applications in other areas of medicinal chemistry.

Alkylpurine-DNA-N-glycosylase knockout mice show increased susceptibility to induction of mutations by methyl methanesulfonate.

Alkylpurine-DNA-N-glycosylase (APNG) null mice have been generated by homologous recombination in embryonic stem cells. The null status of the animals was confirmed at the mRNA level by reverse transcription-PCR and by the inability of cell extracts of tissues from the knockout (ko) animals to release 3-methyladenine (3-meA) or 7-methylguanine (7-meG) from 3H-methylated calf thymus DNA in vitro. Following treatment with DNA-methylating agents, increased persistence of 7-meG was found in liver sections of APNG ko mice in comparison with wild-type (wt) mice, demonstrating an in vivo phenotype for the APNG null animals. Unlike other null mutants of the base excision repair pathway, the APNG ko mice exhibit a very mild phenotype, show no outward abnormalities, are fertile, and have an apparently normal life span. Neither a difference in the number of leukocytes in peripheral blood nor a difference in the number of bone marrow polychromatic erythrocytes was found when ko and wt mice were exposed to methylating or chloroethylating agents. These agents also showed similar growth-inhibitory effects in primary embryonic fibroblasts isolated from ko and wt mice. However, treatment with methyl methanesulfonate resulted in three- to fourfold more hprt mutations in splenic T lymphocytes from APNG ko mice than in those from wt mice. These mutations were predominantly single-base-pair changes; in the ko mice, they consisted primarily of AT-->TA and GC-->TA transversions, which most likely are caused by 3-meA and 3- or 7-meG, respectively. These results clearly show an important role for APNG in attenuating the mutagenic effects of N-alkylpurines in vivo.

Insights into the mechanisms of homologous recombination from the structure of RuvA.

The recent structure determination of RuvA has provided the first insights into the structural basis for its interaction with Holliday junction DNA. Multiple copies of a helix-hairpin-helix motif which line the four grooves between the monomers in the tetrameric structure are thought to be involved in the interaction of the protein with its DNA target. This suggests that the four arms of the junction are held by RuvA in a fourfold symmetric arrangement and has fuelled ideas on the way in which components of the Ruv complex combine to catalyse the process of homologous recombination.

The X-ray structure of Brassica napus beta-keto acyl carrier protein reductase and its implications for substrate binding and catalysis.

BACKGROUND: beta-Keto acyl carrier protein reductase (BKR) catalyzes the pyridine-nucleotide-dependent reduction of a 3-oxoacyl form of acyl carrier protein (ACP), the first reductive step in de novo fatty acid biosynthesis and a reaction often performed in polyketide biosynthesis. The Brassica napus BKR enzyme is NADPH-dependent and forms part of a dissociable type II fatty acid synthetase (FAS). Significant sequence similarity is observed with enoyl acyl carrier protein reductase (ENR), the other reductase of FAS, and the short-chain alcohol dehydrogenase (SDR) family. RESULTS: The first crystal structure of BKR has been determined at 2.3 A resolution in a binary complex with an NADP(+) cofactor. The structure reveals a homotetramer in which each subunit has a classical dinucleotide-binding fold. A triad of Ser154, Tyr167 and Lys171 residues is found at the active site, characteristic of the SDR family. Overall BKR has a very similar structure to ENR with good superimposition of catalytically important groups. Modelling of the substrate into the active site of BKR indicates the need for conformational changes in the enzyme. CONCLUSIONS: A catalytic mechanism can be proposed involving the conserved triad. Helix alpha6 must shift its position to permit substrate binding to BKR and might act as a flexible lid on the active site. The similarities in fold, mechanism and substrate binding between BKR, which catalyzes a carbon-oxygen double-bond reduction, and ENR, the carbon-carbon double-bond oxidoreductase in FAS, suggest a close evolutionary link during the development of the fatty acid biosynthetic pathway.

Common themes in redox chemistry emerge from the X-ray structure of oilseed rape (Brassica napus) enoyl acyl carrier protein reductase.

BACKGROUND: Enoyl acyl carrier protein reductase (ENR) catalyzes the NAD(P)H-dependent reduction of trans-delta 2-enoyl acyl carrier protein, an essential step in de novo fatty acid biosynthesis. Plants contain both NADH-dependent and separate NADPH-dependent ENR enzymes which form part of the dissociable type II fatty acid synthetase. Highly elevated levels of the NADH-dependent enzyme are found during lipid deposition in maturing seeds of oilseed rape (Brassica napus). RESULTS: The crystal structure of an ENR-NAD binary complex has been determined at 1.9 A resolution and consists of a homotetramer in which each subunit forms a single domain comprising a seven-stranded parallel beta sheet flanked by seven alpha helices. The subunit has a topology highly reminiscent of a dinucleotide-binding fold. The active site has been located by difference Fourier analysis of data from crystals equilibrated in NADH. CONCLUSIONS: The structure of ENR shows a striking similarity with the epimerases and short-chain alcohol dehydrogenases, in particular, 3 alpha,20 beta-hydroxysteroid dehydrogenase (HSD). The similarity with HSD extends to the conservation of a catalytically important lysine that stabilizes the transition state and to the use of a tyrosine as a base--with subtle modifications arising from differing requirements of the reduction chemistry.

Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase.

BACKGROUND: Glycerol-3-phosphate (1)-acyltransferase(G3PAT) catalyzes the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acyl-CoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol-3-phosphate. G3PATs can either be selective, preferentially using the unsaturated fatty acid, oleate (C18:1), as the acyl donor, or nonselective, using either oleate or the saturated fatty acid, palmitate (C16:0), at comparable rates. The differential substrate specificity for saturated versus unsaturated fatty acids seen within this enzyme family has been implicated in the sensitivity of plants to chilling temperatures. RESULTS: The three-dimensional structure of recombinant G3PAT from squash chloroplast has been determined to 1.9 A resolution by X-ray crystallography using the technique of multiple isomorphous replacement and provides the first representative structure of an enzyme of this class. CONCLUSIONS: The tertiary structure of G3PAT comprises two domains, the larger of which, domain II, features an extensive cleft lined by hydrophobic residues and contains at one end a cluster of positively charged residues flanked by a H(X)(4)D motif, which is conserved amongst many glycerolipid acyltransferases. We predict that these hydrophobic and positively charged residues represent the binding sites for the fatty acyl substrate and the phosphate moiety of the glycerol 3-phosphate, respectively, and that the H(X)(4)D motif is a critical component of the enzyme's catalytic machinery.

Identification of the cross-link between human O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea-treated DNA.

Chloroethylnitrosoureas induce reactive O6-guanine adducts in DNA that can form either interstrand cross-links or a covalent complex with the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). To test our hypothesis that these end-products are formed from the common precursor, 1-O6-ethanoguanine, we compared the kinetics of interstrand cross-link formation with those of decay of MGMT complex forming capacity. The half-lives of these processes were identical. Our hypothesis also predicts that the linkage between DNA and MGMT is 1-(guan-1-yl)-2-(cystein-S-yl)ethane. This notion was tested by forming the complex with 35S-labeled recombinant human MGMT and a chloroethylnitrosourea-treated oligodeoxynucleotide. After degradation by depurination and proteolytic digestion, the identity of the [35S]cysteine-guanine linkage was confirmed by comparison with the synthetic marker compound using high performance liquid chromatography and UV spectrometry. These results strengthen the hypothesis that DNA interstrand cross-links and DNA-MGMT complex both arise from the same precursor. The data also suggest that 1-O6-ethanoguanine is a good substrate for MGMT such that, under certain conditions in vivo, DNA-MGMT complex formation may constitute a significant secondary lesion.

Detection of workplace sedentary behavior using thermal sensors.

Sedentary behavior has been linked to leading causes of morbidity and mortality, including cancer, cardiovascular disease and diabetes. Those who work in office workplaces are susceptible to higher levels of sedentary behavior during the working day. This paper introduces a novel approach to the detection of sedentary behavior through the use of a thermal sensor mounted on the ceiling above a busy workspace. This solution was found to more accurately record 7 out of 10 activity metrics in comparison to self-assessment, when compared to chair pressure sensor recordings.

Induction of murine O6-alkylguanine-DNA-alkyltransferase in response to ionising radiation is p53 gene dose dependent.

Expression of both the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase) and the p53 tumour suppressor protein are inducible by a number of DNA damaging agents. It is probable that DNA strand breaks are the common inducing signals. This similarity, and the function of p53 as a transcription factor lead us to reason that p53 might be involved in ATase inducibility. We now report that the induction of ATase activity in mouse tissues following gamma-radiation is p53 gene dose dependent. While the extent and kinetics of induction in p53 wildtype mice are consistent with previous reports (a 2-3-fold peak increase at 36 h), no induction is observed in p53 null animals. Importantly the heterozygous mice show an intermediate response but the same kinetics. The basal levels of expression in all tissues examined are unaffected by p53 status. These data represent the first report of a discrete DNA repair function being p53 regulated in vivo and their potential clinical implications are discussed.

Cloning, characterisation and crystallisation of a diadenosine 5',5"'-P(1),P(4)-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans.

Asymmetrically cleaving diadenosine 5',5"'-P(1),P(4)-tetraphosphate (Ap4A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coli. As expected, sequence analysis shows the enzyme to be a member of the Nudix hydrolase family. The purified recombinant enzyme behaves as a typical animal Ap4A hydrolase. It hydrolyses Ap4A with a K(m) of 7 microM and k(cat) of 27 s(-1) producing AMP and ATP as products. It is also active towards other adenosine and diadenosine polyphosphates with four or more phosphate groups, but not diadenosine triphosphate, always generating ATP as one of the products. It is inhibited non-competitively by fluoride (K(i)=25 microM) and competitively by adenosine 5'-tetraphosphate with Ap4A as substrate (K(i)=10 nM). Crystals of diffraction quality with the morphology of rectangular plates were readily obtained and preliminary data collected. These crystals diffract to a minimum d-spacing of 2 A and belong to either space group C222 or C222(1). Phylogenetic analysis of known and putative Ap4A hydrolases of the Nudix family suggests that they fall into two groups comprising plant and Proteobacterial enzymes on the one hand and animal and archaeal enzymes on the other. Complete structural determination of the C. elegans Ap4A hydrolase will help determine the basis of this grouping.

The potential role of glycine-160 of human O6-alkylguanine-DNA alkyltransferase in reaction with O6-benzylguanine as determined by site-directed mutagenesis and molecular modelling comparisons.

O6-Alkylguanine DNA-alkyltransferase (ATase) repairs toxic, mutagenic and carcinogenic O6-alkylguanine (O6-alkG) lesions in DNA by a highly conserved reaction involving the stoichiometric transfer of the alkyl group to the active centre cysteine residue of the ATase protein. In the Escherichia coli Ada ATase, which is effectively refactory to inhibition by O6-benzylguanine (O6-BzG), the residue corresponding to glycine-160 (G160) for the mammalian proteins of this class is replaced by a tryptophan (W). Therefore, to investigate the potential role of the G160 of the human ATase (hAT) protein in determining sensitivity to O6-BzG, site-directed mutagenesis was used to produce a mutant protein (hATG160W) substituted at position 160 with a W residue. The hATG160W mutant was found to be stably expressed and was 3- and 5-fold more sensitive than hAT to inactivation by O6-BzG, in the absence and presence of additional calf-thymus DNA respectively. A similar, DNA dependent increased sensitivity of the hATG160W mutant relative to wild-type was also found for O6-methylguanine mediated inactivation. The potential role of the W160 residue in stabilising the binding of the O6-alkG to the protein is discussed in terms of a homology model of the structure of hAT. The region occupied by G/W-160 forms the site of a putative hinge that could be important in the conformational change that is likely to occur on DNA binding. Three sequence motifs have been identified in this region which may influence O6-BzG access to the active site; YSGG or YSGGG in mammals (YAGG in E. coli Ogt, YAGS in Dat from Bacillus subtilis), YRWG in E. coli Ada and Salmonella typhimurium (but YKWS in Saccharomyces cerevisiae) or YRGGF in AdaB from B. Subtilis. Finally,conformational and stereoelectronic analysis of the putative transition states for the alkyl transfer from a series of inactivators of hAT, including O6-BzG was undertaken to rationalise the unexpected weak inhibition shown by the alpha-pi-unsaturated electrophiles.

Crystallization of the NADH-specific enoyl acyl carrier protein reductase from Brassica napus.

The tetrameric, NADH-dependent enoyl acyl carrier protein reductase from developing seeds of Brassica napus (oil seed rape) has been crystallized from solutions containing ammonium sulphate as the precipitant in the presence of NAD+ or NADH using the hanging drop method of vapour diffusion. The crystals belong to the tetragonal system and are in space group P4(2)2(1)2 with cell dimensions a = b = 70.5 A, c = 117.8 A. Considerations of the possible values of Vm indicate that the asymmetric unit contains a single subunit. The crystals are resistant to radiation damage and X-ray diffraction photographs taken with synchrotron radiation show measurable reflections to beyond 1.9 A resolution. Determination of the structure of this enzyme will advance the understanding of the mechanisms of lipid biosynthesis in plants and provide an opportunity to study the interactions between this enzyme and its acyl carrier protein substrate.