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Item Changes to insulin sensitivity in glucose clearance systems and redox following dietary supplementation with a novel cysteine-rich protein: A pilot randomized controlled trial in humans with type-2 diabetes.(Elsevier B.V, 2023-10-07) Peeters WM; Gram M; Dias GJ; Vissers MCM; Hampton MB; Dickerhof N; Bekhit AE; Black MJ; Oxbøll J; Bayer S; Dickens M; Vitzel K; Sheard PW; Danielson KM; Hodges LD; Brønd JC; Bond J; Perry BG; Stoner L; Cornwall J; Rowlands DSWe recently developed a novel keratin-derived protein (KDP) rich in cysteine, glycine, and arginine, with the potential to alter tissue redox status and insulin sensitivity. The KDP was tested in 35 human adults with type-2 diabetes mellitus (T2DM) in a 14-wk randomised controlled pilot trial comprising three 2×20 g supplemental protein/day arms: KDP-whey (KDPWHE), whey (WHEY), non-protein isocaloric control (CON), with standardised exercise. Outcomes were measured morning fasted and following insulin-stimulation (80 mU/m2/min hyperinsulinaemic-isoglycaemic clamp). With KDPWHE supplementation there was good and very-good evidence for moderate-sized increases in insulin-stimulated glucose clearance rate (GCR; 26%; 90% confidence limits, CL 2%, 49%) and skeletal-muscle microvascular blood flow (46%; 16%, 83%), respectively, and good evidence for increased insulin-stimulated sarcoplasmic GLUT4 translocation (18%; 0%, 39%) vs CON. In contrast, WHEY did not effect GCR (-2%; -25%, 21%) and attenuated HbA1c lowering (14%; 5%, 24%) vs CON. KDPWHE effects on basal glutathione in erythrocytes and skeletal muscle were unclear, but in muscle there was very-good evidence for large increases in oxidised peroxiredoxin isoform 2 (oxiPRX2) (19%; 2.2%, 35%) and good evidence for lower GPx1 concentrations (-40%; -4.3%, -63%) vs CON; insulin stimulation, however, attenuated the basal oxiPRX2 response (4%; -16%, 24%), and increased GPx1 (39%; -5%, 101%) and SOD1 (26%; -3%, 60%) protein expression. Effects of KDPWHE on oxiPRX3 and NRF2 content, phosphorylation of capillary eNOS and insulin-signalling proteins upstream of GLUT4 translocation AktSer437 and AS160Thr642 were inconclusive, but there was good evidence for increased IRSSer312 (41%; 3%, 95%), insulin-stimulated NFκB-DNA binding (46%; 3.4%, 105%), and basal PAK-1Thr423/2Thr402 phosphorylation (143%; 66%, 257%) vs WHEY. Our findings provide good evidence to suggest that dietary supplementation with a novel edible keratin protein in humans with T2DM may increase glucose clearance and modify skeletal-muscle tissue redox and insulin sensitivity within systems involving peroxiredoxins, antioxidant expression, and glucose uptake.Item Thromboelastography in obese horses with insulin dysregulation compared to healthy controls.(John Wiley and Sons, Inc., 2022-05-01) Lovett AL; Gilliam LL; Sykes BW; McFarlane DBACKGROUND: Both obesity and metabolic syndrome are associated with hypercoagulability in people, increasing the risk of cardiovascular disease and thromboembolic events. Whether hypercoagulability exists in obese, insulin-dysregulated horses is unknown. HYPOTHESIS/OBJECTIVES: To determine if coagulation profiles differ between healthy horses and those with obesity and insulin dysregulation. ANIMALS: Fifteen healthy horses (CON) and 15 obese, insulin-dysregulated horses (OBID). Individuals were university or client owned. METHODS: Case-control study. Obesity was defined as a body condition score (BCS) ≥7.5/9 (modified Henneke scale). Insulin dysregulation status was assessed by an oral sugar test (OST). Kaolin-thromboelastography and traditional coagulation variables were compared between groups. The direction and strength of the association between coagulation variables and BCS and OST results were determined using Spearman's correlation. RESULTS: Thromboelastography variables MA (OBID: 69.5 ± 4.5 mm; CON: 64.8 ± 4.3 mm; P = .007) and G-value (OBID: 11749 ± 2536 dyn/m2 ; CON: 9319 ± 1650 dyn/m2 ; P = .004) were higher in OBID compared to CON. Positive correlations between MA and BCS (R = 0.45, P = .01) and serum insulin (T0 : R = 0.45, P = .01; T60 : R = 0.39, P = .03), and G-value and BCS (R = 0.46, P = .01), and serum insulin (T0 : R = 0.48, P = .007; T60 : R = 0.43, P = .02; T90 : R = 0.38, P = .04) were present. CONCLUSIONS AND CLINICAL IMPORTANCE: Obese, insulin-dysregulated horses are hypercoagulable compared to healthy controls.Item Plasma metabolomic response to high-carbohydrate meals of differing glycaemic load in overweight women.(Springer Nature, 2023-04-21) Durainayagam B; Mitchell CJ; Milan AM; Kruger MC; Roy NC; Fraser K; Cameron-Smith DBACKGROUND: Metabolomic dysregulation following a meal in overweight individuals with the Metabolic Syndrome (MetS) involves multiple pathways of nutrient storage and oxidation. OBJECTIVE: The aim of the current study was to perform an acute cross-over intervention to examine the interactive actions of meal glycaemic load (GL) on the dynamic responses of the plasma metabolome in overweight females. METHODS: Postmenopausal women [63 ± 1.23y; Healthy (n = 20) and MetS (n = 20)] ingested two differing high-carbohydrate test meals (73 g carbohydrate; 51% energy) composed of either low glycemic index (LGI) or high (HGI) foods in a randomised sequence. Plasma metabolome was analysed using liquid chromatography-mass spectrometry (LC-MS). RESULTS: In the overweight women with MetS, there were suppressed postprandial responses for several amino acids (AAs), including phenylalanine, leucine, valine, and tryptophan, p < 0.05), irrespective of the meal type. Meal GL exerted a limited impact on the overall metabolomic response, although the postprandial levels of alanine were higher with the low GL meal and uric acid was greater following the high GL meal (p < 0.05). CONCLUSIONS: MetS participants exhibited reduced differences in the concentrations of a small set of AAs and a limited group of metabolites implicated in energy metabolism following the meals. However, the manipulation of meal GL had minimal impact on the postprandial metabolome. This study suggests that the GL of a meal is not a major determinant of postprandial response, with a greater impact exerted by the metabolic health of the individual. Trial registration Australia New Zealand Clinical Trials Registry: ACTRN12615001108505 (21/10/2015).Item A role for β-catenin in diet-induced skeletal muscle insulin resistance.(Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society, 2023-02-17) Masson SWC; Dissanayake WC; Broome SC; Hedges CP; Peeters WM; Gram M; Rowlands DS; Shepherd PR; Merry TLA central characteristic of insulin resistance is the impaired ability for insulin to stimulate glucose uptake into skeletal muscle. While insulin resistance can occur distal to the canonical insulin receptor-PI3k-Akt signaling pathway, the signaling intermediates involved in the dysfunction are yet to be fully elucidated. β-catenin is an emerging distal regulator of skeletal muscle and adipocyte insulin-stimulated GLUT4 trafficking. Here, we investigate its role in skeletal muscle insulin resistance. Short-term (5-week) high-fat diet (HFD) decreased skeletal muscle β-catenin protein expression 27% (p = 0.03), and perturbed insulin-stimulated β-cateninS552 phosphorylation 21% (p = 0.009) without affecting insulin-stimulated Akt phosphorylation relative to chow-fed controls. Under chow conditions, mice with muscle-specific β-catenin deletion had impaired insulin responsiveness, whereas under HFD, both mice exhibited similar levels of insulin resistance (interaction effect of genotype × diet p < 0.05). Treatment of L6-GLUT4-myc myocytes with palmitate lower β-catenin protein expression by 75% (p = 0.02), and attenuated insulin-stimulated β-catenin phosphorylationS552 and actin remodeling (interaction effect of insulin × palmitate p < 0.05). Finally, β-cateninS552 phosphorylation was 45% lower in muscle biopsies from men with type 2 diabetes while total β-catenin expression was unchanged. These findings suggest that β-catenin dysfunction is associated with the development of insulin resistance.Item Postexercise muscle glycogen synthesis with glucose, galactose, and combined galactose-glucose ingestion.(American Physiological Society, 2023-12-01) Podlogar T; Shad BJ; Seabright AP; Odell OJ; Lord SO; Civil R; Salgueiro RB; Shepherd EL; Lalor PF; Elhassan YS; Lai Y-C; Rowlands DS; Wallis GAIngested galactose can enhance postexercise liver glycogen repletion when combined with glucose but effects on muscle glycogen synthesis are unknown. In this double-blind randomized study participants [7 men and 2 women; V̇o2max: 51.1 (8.7) mL·kg-1·min-1] completed three trials of exhaustive cycling exercise followed by a 4-h recovery period, during which carbohydrates were ingested at the rate of 1.2 g·kg-1·h-1 comprising glucose (GLU), galactose (GAL) or galactose + glucose (GAL + GLU; 1:2 ratio). The increase in vastus lateralis skeletal-muscle glycogen concentration during recovery was higher with GLU relative to GAL + GLU [contrast: +50 mmol·(kg DM)-1; 95%CL 10, 89; P = 0.021] and GAL [+46 mmol·(kg DM)-1; 95%CL 8, 84; P = 0.024] with no difference between GAL + GLU and GAL [-3 mmol·(kg DM)-1; 95%CL -44, 37; P = 0.843]. Plasma glucose concentration in GLU was not significantly different vs. GAL + GLU (+ 0.41 mmol·L-1; 95%CL 0.13, 0.94) but was significantly lower than GAL (-0.75 mmol·L-1; 95%CL -1.34, -0.17) and also lower in GAL vs. GAL + GLU (-1.16 mmol·-1; 95%CL -1.80, -0.53). Plasma insulin was higher in GLU + GAL and GLU compared with GAL but not different between GLU + GAL and GLU. Plasma galactose concentration was higher in GAL compared with GLU (3.35 mmol·L-1; 95%CL 3.07, 3.63) and GAL + GLU (3.22 mmol·L-1; 95%CL 3.54, 2.90) with no difference between GLU + GAL (0.13 mmol·L-1; 95%CL -0.11, 0.37) and GLU. Compared with galactose or a galactose + glucose blend, glucose feeding was more effective in postexercise muscle glycogen synthesis. Comparable muscle glycogen synthesis was observed with galactose-glucose coingestion and exclusive galactose-only ingestion. NEW & NOTEWORTHY Postexercise galactose-glucose coingestion or exclusive galactose-only ingestion resulted in a lower rate of skeletal-muscle glycogen replenishment compared with exclusive glucose-only ingestion. Comparable muscle glycogen synthesis was observed with galactose-glucose coingestion and exclusive galactose-only ingestion.Item A higher-protein nut-based snack product suppresses glycaemia and decreases glycaemic response to co-ingested carbohydrate in an overweight prediabetic Asian Chinese cohort: the Tū Ora postprandial RCT(Cambridge University Press on behalf of The Nutrition Society, 2021-04-23) Lu LW; Silvestre MP; Sequeira IR; Plank LD; Foster M; Middleditch N; Acevedo-Fani A; Hollingsworth KG; Poppitt SDNut-based products may aid low-glycaemic dietary strategies that are important for diabetes prevention in populations at increased risk of dysglycaemia, such as Asian Chinese. This randomised cross-over trial assessed the postprandial glycaemic response (0-120 min) of a higher-protein nut-based (HP-NB) snack formulation, in bar format (1009 kJ, Nutrient Profiling Score, NPS, -2), when compared with an iso-energetic higher-carbohydrate (CHO) cereal-based bar (HC-CB, 985 kJ, NPS +3). It also assessed the ability to suppress glucose response to a typical CHO-rich food (white bread, WB), when co-ingested. Ten overweight prediabetic Chinese adults (mean, sd: age 47⋅9, 15⋅7 years; BMI 25⋅5, 1⋅6 kg/m2), with total body fat plus ectopic pancreas and liver fat quantified using dual-energy X-ray absorptiometry and magnetic resonance imaging and spectroscopy, received the five meal treatments in random order: HP-NB, HC-CB, HP-NB + WB (50 g available CHO), HC-CB + WB and WB only. Compared with HC-CB, HP-NB induced a significantly lower 30-120 min glucose response (P < 0⋅05), with an approximately 10-fold lower incremental area under the glucose curve (iAUC0-120; P < 0⋅001). HP-NB also attenuated glucose response by approximately 25 % when co-ingested with WB (P < 0⋅05). Half of the cohort had elevated pancreas and/or liver fat, with 13-21 % greater suppression of iAUC0-120 glucose in the low v. high organ fat subgroups across all five treatments. A nut-based snack product may be a healthier alternative to an energy equivalent cereal-based product with evidence of both a lower postprandial glycaemic response and modulation of CHO-induced hyperglycaemia even in high-risk, overweight, pre-diabetic adults.Item Comparative Effects of Co-Ingesting Whey Protein and Glucose Alone and Combined on Blood Glucose, Plasma Insulin and Glucagon Concentrations in Younger and Older Men(MDPI (Basel, Switzerland), 2022-08) Oberoi A; Giezenaar C; Rigda RS; Lange K; Horowitz M; Jones KL; Chapman I; Soenen S; Gropper SThe ingestion of dietary protein with, or before, carbohydrate may be a useful strategy to reduce postprandial hyperglycemia, but its effect in older people, who have an increased predisposition for type 2 diabetes, has not been clarified. Blood glucose, plasma insulin and glucagon concentrations were measured for 180 min following a drink containing either glucose (120 kcal), whey-protein (120 kcal), whey-protein plus glucose (240 kcal) or control (~2 kcal) in healthy younger (n = 10, 29 ± 2 years; 26.1 ± 0.4 kg/m2) and older men (n = 10, 78 ± 2 years; 27.3 ± 1.4 kg/m2). Mixed model analysis was used. In both age groups the co-ingestion of protein with glucose (i) markedly reduced the increase in blood glucose concentrations following glucose ingestion alone (p < 0.001) and (ii) had a synergistic effect on the increase in insulin concentrations (p = 0.002). Peak insulin concentrations after protein were unaffected by ageing, whereas insulin levels after glucose were lower in older than younger men (p < 0.05) and peak insulin concentrations were higher after glucose than protein in younger (p < 0.001) but not older men. Glucagon concentrations were unaffected by age. We conclude that the ability of whey-protein to reduce carbohydrate-induced postprandial hyperglycemia is retained in older men and that protein supplementation may be a useful strategy in the prevention and management of type 2 diabetes in older people.Item An investigation of the extractable insulin levels and pancreas weights of New Zealand sheep : a thesis presented in partial fulfilment of the requirements for the degree Master of Technology in Biotechnology at Massey University, Palmerston North, New Zealand(Massey University, 1971) Swan, Janis ElizabethIn 1921 Banting and Best were the first workers to successfully obtain the active substance insulin from the pancreas. The dramatic clinical benefits of the hormone were immediately apparent and insulin has become the most important commercially produced protein in the medical field. It also has a unique place in protein chemistry investigations as it was the first protein to be crystallised, the amino acid sequence determined and a biologically active molecule synthesized chemically. Methods for producing insulin were developed by Scott and Best at the Connaught Laboratories, Toronto, in the early 1920's. The Insulin Committee set up by the Connaught Laboratories selected Eli Lilly and Company as the first pharmaceutical company to manufacture insulin. Scott and Best developed methods for the preparation of insulin, including large-scale production methods and these methods were sent to interested manufacturers throughout the world at regular intervals (Best 1960). Active work on insulin has continued both at Connaught Laboratories and at the Banting and Best Institute in Toronto. The more recent work from these groups has been on the physiological effects of insulin, modes of insulin action, and factors affecting secretion of insulin by the pancreas. Many other laboratories throughout the world are also investigating similar aspects of insulin. [From Introduction]Item Explorations into the nature of insulin binding to oxidized dextran : this thesis was presented in partial fulfillment of the requirements for the degree of Master of Science in Chemistry at Massey University(Massey University, 1998) Li, YumingThe results reported in this thesis comprise an investigation into the conjugation of insulin to oxidized dextran, various release studies from the conjugates, and an attempt to interpret the binding nature of the conjugates. A model system involving the sustained release from insulin-dextran conjugates has been employed in this study. For insulin, up to 3 potential sites only (A1-Gly. B1-Phe and B29-Lys) were expected to bind to oxidized dextran. The rate of release and the maintenance of activity of the released protein are vital to such systems. Success in the interpretation of the binding nature of the conjugate will allow us to investigate its relationship to the rate of release. The desired rate of release for the sustained release of protein could then be achieved, once the projected binding could be obtained. Activation of dextran was achieved by periodate oxidation to give levels of 8%, 16% and 27% oxidized dextran. Insulin was chosen for its relatively 'uncomplicated' structure and few possible sites available for binding with activated dextran. Insulin was bound to the dextran through imine bonds. Complex formation was examined under a wide range of conditions. Initial studies were begun with the determination of a desirable basic molar ratio. A molar ratio of insulin to 8% activated dextran of 10 : 1 arose from this set of experiments. Insulin was bound to 27% activated dextran at pH 7.4, pH 9 and pH 10. In the cases of pH 9 and pH 10, many more lower MW complexes were formed than at pH 7.4. It seemed that the higher the pH of formation, the more crosslinks occurred between an insulin molecule and dextran molecules in the lower MW range. Approximate physiological pHs (pH 7.1-7.8) were used for complex formation in all subsequent experiments. Release studies were carried out under approximate physiological conditions (pH 7.4, 37°C). Immediate release was observed upon isolation by size exclusion chromatography. The greatest release occurred in the first 24 hours for all three activation levels. The higher the activation level of dextran, the lower the level of release. An equilibrium was established after several days' release and studies at 37°C produced the expected result: greater release relative to ambient. A number of studies were carried out with complex after sodium cyanoborohydride had been used to reduce the imine bonds. The first set of experiments on the reduced complexes was enzymatic cleavage studies, which employed trypsin and α-chvmotrypsin. The results for trypsin digestion of the reduced insulin-27% oxidized dextran complex indicated partial binding had occurred at B29-Lys, in combination with full binding at B1 and/or Al. Amino acid analysis results of the isolated complex after trypsin digestion indicated about 90% binding occurred at B29-Lys for the complex, which formed at pH 7.1. The results of α-chymotrypsin digestion study were shown questionable due to its incomplete cleavage. The reduced complexes were analyzed by amino acid analysis. The insulin-27% activated dextran complexes formed at pH 7.4, pH 9 and pH 10 showed similar extents of binding at B1-Phe, indicating B1 might be the prime binding site. There was more binding at B29 and A1 for the pH 9 than at pH 7.4 case. At pH 10 abnormal values arose. The studies for the complexes of insulin with 16% and 27% activated dextran indicated the more highly activated the dextran, the greater the binding at B29 and A1. Trials with the 2, 4-dinitrophenyl-derivativatization method proved to be a useful way to examine the degree of B1 and B29 binding from the amino acid analysis results of complex. The insulin-16% activated dextran complex formed at pH 7.1 was found to be about 100% binding at B1, 60% at A1 and 50% at B29. Oxidative and reductive cleavage studies of A and B chains of insulin and the complex were carried out to investigate the level of A1 binding. After chemical cleavage of the three disulfide bonds in insulin and subsequent chromatography, the amino acid analysis results for the treated complexes indicated a significant proportion of A chain had bound to dextran, i.e. at A1. An estimation of 60-70% of A1 binding was achieved for this study. This exploratory study has shown that varied complex formation conditions such as the level of activation of dextran, pH, and temperature could alter the extent of binding between insulin and dextran molecules. Amino acid analysis of the reduced complex was a useful method to interpret the binding.Item A study of the regulation of hepatic microsomal glycerol phosphate acyltransferase (GPAT) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University(Massey University, 1986) Stevens, Earl Victor JohnExperiments described in this thesis were conducted to examine the possibility that hepatic microsomal GPAT activity in rats is regulated by insulin. Hepatic microsomal fractions were prepared by a procedure based on published methods and it was established by assay of cytochrome oxidase, monoamine oxidase and NADPH cytochrome Creductase that there was less than 11% mitochondrial impurity. A butanol extraction of GPAT assays was adopted to separate the butanol-soluble [14cJ-lipid products from the unreacted aqueous-soluble [ 1 4C ) -glycerol 3-phosphate substrate . Methods were developed to simplify the determination of [14 c J-radioactivity. The kinetics of the response of the assay system to changes in the concentrations of glycerol 3-phosphate and palmitoyl-CoA were similar to those published for the microsomal GPAT. The products of the assay were identified as phosphatidic acid and lysophosphatidic acid by their chromatographic properties before and after hydrolysis with chicken liver phosphatidate phosphohydrolase. These products are consistent with literature reports. Male Sprague-Dawley rats were treated with insulin (4i.u./kg body weight) orsaline, and extracts of hind-limb muscle were prepared and fractionated on Sephadex G-25 in 5 0 mM formic acid. Fractions which eluted subsequent to the void volume were assayed with hepatic microsomal GPAT and the effect of insulin-treatment fractions were compared with the effect of saline-treatment fractions. Fractions containing material of approximately 3000 and 1000 daltons molecular weight enhanced GPAT activity in an insulin-dependent manner by 0.46 and 0.64 nmol/min/mg of microsomal protein, respectively (both P<0.01), compared to the effect of the saline controls. Control rates were approximately 3.5nmol/min/mg of microsomal protein. It was calculated that these insulin-dependent increases in hepatic microsomal GPAT activity would be sufficient to account for the difference between the estimated hepatic triacylglycerol production of fed and fasted rats. Furthermore , published studies suggest that insulin-dependent changes in activities of enzymes, demonstrated with in vitro systems utilising low molecular weight fractions from rat muscle, may parallel sensitivity of the same enzymes to insulin in vivo. The low molecular weight stimulator or stimulators of hepatic microsomal GPAT have an apparent molecular weight within the range 1000-3000 daltons, appear to be heat and acid stable, are soluble in aqueous solution, have very low absorbance at 220nm (or a very high specific activity) and may be sensitive to oxygen. These properties suggest that the low molecular weight stimulator or stimulators of hepatic microsomal GPAT activity may be related to the putative insulin mediator substance (IMS). In initial experiments, where rats were heparinised prior to treatment with insulin or saline, it was observed that some fractions were able to stimulate hepatic microsomal GPAT activity in an insulin-independent manner. Experiments to resolve this suggested that the treatment of rats with heparin alone led to the presence of low molecular weight material, in the fractions of muscle extracts, with the potential to enhance GPAT activity. It was found that low molecular weight fractions of the saline treatment muscle extracts did not enhance GPAT activity. This supported the suggestion that heparin was responsible for the ability of low molecular weight fractions of muscle extracts to stimulate GPAT activity in an insulin-independent manner. Experiments were also conducted in which impure hepatic plasma membranes were treated with insulin (20-1000 units/ml). However, when hepatic microsomal GPAT was assayed with material from these incubations a stimulator of GPAT was not detected. The results of experiments presented in this thesis provide further evidence in favour of the hypothesis that hepatic microsomal GPAT activity can be modified by insulin and may contribute to the overall regulation of glycerolipid biosynthesis in liver.