Efficacy of fibre additions to flatbread flour mixes for reducing post-meal glucose and insulin responses in healthy Indian subjects.

The incidence of type 2 diabetes mellitus (T2DM) is increasing worldwide, including in developing countries, particularly in South Asia. Intakes of foods generating a high postprandial glucose (PPG) response have been positively associated with T2DM. As part of efforts to identify effective and feasible strategies to reduce the glycaemic impact of carbohydrate-rich staples, we previously found that addition of guar gum (GG) and chickpea flour (CPF) to wheat flour could significantly reduce the PPG response to flatbread products. On the basis of the results of an exploratory study with Caucasian subjects, we have now tested the effect of additions of specific combinations of CPF with low doses of GG to a flatbread flour mix for their impacts on PPG and postprandial insulin (PPI) responses in a South-Asian population. In a randomised, placebo-controlled full-cross-over design, fifty-six healthy Indian adults consumed flatbreads made with a commercial flatbread mix (100 % wheat flour) with no further additions (control) or incorporating 15 % CPF in combination with 2, 3 or 4 % GG. The flatbreads with CPF and 3 or 4 % GG significantly reduced PPG (both ≥15 % reduction in positive incremental AUC, P<0·01) and PPI (both ≥28 % reduction in total AUC, P<0·0001) compared with flatbreads made from control flour. These results confirm the efficacy and feasibility of the addition of CPF with GG to flatbread flour mixes to achieve significant reductions in both PPG and PPI in Indian subjects.

Efficacy of different fibres and flour mixes in South-Asian flatbreads for reducing post-prandial glucose responses in healthy adults.

PURPOSE: Type 2 diabetes (T2DM) is increasing, particularly in South-East Asia. Intake of high-glycaemic foods has been positively associated with T2DM, and feasible routes to reduce the glycaemic response to carbohydrate-rich staple foods are needed. The research question was whether different fibre and legume flour mixes in flatbreads lower postprandial glucose (PPG) responses. METHODS: Using a balanced incomplete block design, we tested the inclusion of guar gum (GG), konjac mannan (KM) and chickpea flour (CPF) in 10 combinations (2/4/6 g GG; 2/4 g KM; 15 g CPF, and 10 or 15 g CPF plus 2 or 4 g GG) in 100 g total of a control commercial high-fibre flatbread flour mix ("atta") on PPG in 38 normal-weight adults. Self-reported appetite was an additional exploratory outcome. An in vitro digestion assay was adapted for flatbreads and assessed for prediction of in vivo PPG. RESULTS: Flatbreads with 6 g GG, 4 g KM, and 15 g CPF plus 2 or 4 g GG reduced PPG ≥30 % (p < 0.01), while no other combinations differed significantly from the control. A statistical model with four in vitro parameters (rate of digestion, %RDS, AUC, carbohydrate level) was highly predictive of PPG results (adjusted R 2 = 0.89). Test products were similar to the control for appetite-related measures. CONCLUSIONS: The results confirm the efficacy of specific additions to flatbread flour mixes for reducing PPG and the value of the in vitro model as a predictive tool with these ingredients and product format. This trial is registered at ClinicalTrials.gov with identifier NCT02671214.

Plant-rich mixed meals based on Palaeolithic diet principles have a dramatic impact on incretin, peptide YY and satiety response, but show little effect on glucose and insulin homeostasis: an acute-effects randomised study.

There is evidence for health benefits from 'Palaeolithic' diets; however, there are a few data on the acute effects of rationally designed Palaeolithic-type meals. In the present study, we used Palaeolithic diet principles to construct meals comprising readily available ingredients: fish and a variety of plants, selected to be rich in fibre and phyto-nutrients. We investigated the acute effects of two Palaeolithic-type meals (PAL 1 and PAL 2) and a reference meal based on WHO guidelines (REF), on blood glucose control, gut hormone responses and appetite regulation. Using a randomised cross-over trial design, healthy subjects were given three meals on separate occasions. PAL2 and REF were matched for energy, protein, fat and carbohydrates; PAL1 contained more protein and energy. Plasma glucose, insulin, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP) and peptide YY (PYY) concentrations were measured over a period of 180 min. Satiation was assessed using electronic visual analogue scale (EVAS) scores. GLP-1 and PYY concentrations were significantly increased across 180 min for both PAL1 (P= 0·001 and P< 0·001) and PAL2 (P= 0·011 and P= 0·003) compared with the REF. Concomitant EVAS scores showed increased satiety. By contrast, GIP concentration was significantly suppressed. Positive incremental AUC over 120 min for glucose and insulin did not differ between the meals. Consumption of meals based on Palaeolithic diet principles resulted in significant increases in incretin and anorectic gut hormones and increased perceived satiety. Surprisingly, this was independent of the energy or protein content of the meal and therefore suggests potential benefits for reduced risk of obesity.

Glycogen synthase kinase 3alpha-specific regulation of murine hepatic glycogen metabolism.

Glycogen synthase kinase 3 comprises two isoforms (GSK-3alpha and GSK-3beta) that are implicated in type II diabetes, neurodegeneration, and cancer. GSK-3 activity is elevated in human and rodent models of diabetes, and various GSK-3 inhibitors improve glucose tolerance and insulin sensitivity in rodent models of obesity and diabetes. Here, we report the generation of mice lacking GSK-3alpha. Unlike GSK-3beta mutants, which die before birth, GSK-3alpha knockout (GSK-3alpha KO) animals are viable but display enhanced glucose and insulin sensitivity accompanied by reduced fat mass. Fasted and glucose-stimulated hepatic glycogen content was enhanced in GSK-3alpha KO mice, whereas muscle glycogen was unaltered. Insulin-stimulated protein kinase B (PKB/Akt) and GSK-3beta phosphorylation was higher in GSK-3alpha KO livers compared to wild-type littermates, and IRS-1 expression was markedly increased. We conclude that GSK-3 isoforms exhibit tissue-specific physiological functions and that GSK-3alpha KO mice are insulin sensitive, reinforcing the potential of GSK-3 as a therapeutic target for type II diabetes.

Deletion of GSK-3beta in mice leads to hypertrophic cardiomyopathy secondary to cardiomyoblast hyperproliferation.

Based on extensive preclinical data, glycogen synthase kinase-3 (GSK-3) has been proposed to be a viable drug target for a wide variety of disease states, ranging from diabetes to bipolar disorder. Since these new drugs, which will be more powerful GSK-3 inhibitors than lithium, may potentially be given to women of childbearing potential, and since it has controversially been suggested that lithium therapy might be linked to congenital cardiac defects, we asked whether GSK-3 family members are required for normal heart development in mice. We report that terminal cardiomyocyte differentiation was substantially blunted in Gsk3b(-/-) embryoid bodies. While GSK-3alpha-deficient mice were born without a cardiac phenotype, no live-born Gsk3b(-/-) pups were recovered. The Gsk3b(-/-) embryos had a double outlet RV, ventricular septal defects, and hypertrophic myopathy, with near obliteration of the ventricular cavities. The hypertrophic myopathy was caused by cardiomyocyte hyperproliferation without hypertrophy and was associated with increased expression and nuclear localization of three regulators of proliferation - GATA4, cyclin D1, and c-Myc. These studies, which we believe are the first in mammals to examine the role of GSK-3alpha and GSK-3beta in the heart using loss-of-function approaches, implicate GSK-3beta as a central regulator of embryonic cardiomyocyte proliferation and differentiation, as well as of outflow tract development. Although controversy over the teratogenic effects of lithium remains, our studies suggest that caution should be exercised in the use of newer, more potent drugs targeting GSK-3 in women of childbearing age.

Tissue-specific analysis of glycogen synthase kinase-3α (GSK-3α) in glucose metabolism: effect of strain variation.

BACKGROUND: Over-activity and elevated expression of glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology of insulin resistance and Type 2 diabetes. Administration of specific GSK-3 inhibitors to diabetic or obese rodent models improves glycaemic control and insulin sensitivity. However, due to the indiscriminatory nature of these inhibitors, the relative contribution of the two isoforms of GSK-3 (GSK-3α and GSK-3ß) is not known. Recently, we demonstrated that an out-bred strain of mice (ICR) lacking expression of GSK-3α in all tissues displayed improved insulin sensitivity and enhanced hepatic glucose metabolism. We also found that muscle (but not liver) inactivation of GSK-3ß conferred insulin and glucose sensitization in an in-bred strain of mice (C57BL/6). METHODOLOGY/PRINCIPAL FINDINGS: Here, we have employed tissue-specific deletion of GSK-3α, to examine the relative contribution of two insulin-sensitive tissues, muscle and liver, towards the insulin sensitization phenotype originally observed in the global GSK-3α KO animals. We found that mice in which GSK-3α has been inactivated in either skeletal-muscle or liver displayed no differences in glucose tolerance or insulin sensitivity compared to wild type littermates. Given the strain differences in our original analyses, we examined the insulin and glucose sensitivity of global GSK-3α KO animals bred onto a C57BL/6 background. These animals also revealed no significant differences in glucose metabolism/insulin sensitivity compared to their wild type littermates. Furthermore, deletion of hepatic GSK-3α on the out-bred, ICR background failed to reproduce the insulin sensitivity manifested by the global deletion of this isoform. CONCLUSIONS/SIGNIFICANCE: From these data we conclude that the improved insulin sensitivity and hepatic glucose homeostasis phenotype observed upon global inactivation of GSK-3α is strain-specific. We surmise that the insulin-sensitization observed in the out-bred strain of mice lacking GSK-3α is mediated by indirect means that do not require intrinsic function of GSK-3α in skeletal muscle and liver tissues.

GSK-3alpha directly regulates beta-adrenergic signaling and the response of the heart to hemodynamic stress in mice.

The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases consists of 2 highly related isoforms, alpha and beta. Although GSK-3beta has an important role in cardiac development, much remains unknown about the function of either GSK-3 isoform in the postnatal heart. Herein, we present what we believe to be the first studies defining the role of GSK-3alpha in the mouse heart using gene targeting. Gsk3a(-/-) mice over 2 months of age developed progressive cardiomyocyte and cardiac hypertrophy and contractile dysfunction. Following thoracic aortic constriction in young mice, we observed enhanced hypertrophy that rapidly transitioned to ventricular dilatation and contractile dysfunction. Surprisingly, markedly impaired beta-adrenergic responsiveness was found at both the organ and cellular level. This phenotype was reproduced by acute treatment of WT cardiomyocytes with a small molecule GSK-3 inhibitor, confirming that the response was not due to a chronic adaptation to LV dysfunction. Thus, GSK-3alpha appears to be the central regulator of a striking range of essential processes, including acute and direct positive regulation of beta-adrenergic responsiveness. In the absence of GSK-3alpha, the heart cannot respond effectively to hemodynamic stress and rapidly fails. Our findings identify what we believe to be a new paradigm of regulation of beta-adrenergic signaling and raise concerns given the rapid expansion of drug development targeting GSK-3.

Abnormalities in brain structure and behavior in GSK-3alpha mutant mice.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded by two genes that generate two related proteins: GSK-3alpha and GSK-3beta. Mice lacking a functional GSK-3alpha gene were engineered in our laboratory; they are viable and display insulin sensitivity. In this study, we have characterized brain functions of GSK-3alpha KO mice by using a well-established battery of behavioral tests together with neurochemical and neuroanatomical analysis. RESULTS: Similar to the previously described behaviours of GSK-3beta(+/-) mice, GSK-3alpha mutants display decreased exploratory activity, decreased immobility time and reduced aggressive behavior. However, genetic inactivation of the GSK-3alpha gene was associated with: decreased locomotion and impaired motor coordination, increased grooming activity, loss of social motivation and novelty; enhanced sensorimotor gating and impaired associated memory and coordination. GSK-3alpha KO mice exhibited a deficit in fear conditioning, however memory formation as assessed by a passive avoidance test was normal, suggesting that the animals are sensitized for active avoidance of a highly aversive stimulus in the fear-conditioning paradigm. Changes in cerebellar structure and function were observed in mutant mice along with a significant decrease of the number and size of Purkinje cells. CONCLUSION: Taken together, these data support a role for the GSK-3alpha gene in CNS functioning and possible involvement in the development of psychiatric disorders.

Targeting glycogen synthase kinase-3 (GSK-3) in the treatment of Type 2 diabetes.

BACKGROUND: In spite of its rather specific name, glycogen synthase kinase-3 (GSK-3) is an eclectic cellular regulator that modulates an array of processes from nuclear transcription, to neurological functions and metabolism. The enzyme is also a focal point for diverse signaling pathways that act to suppress its activity. OBJECTIVES: To review recent evidence supporting the important role GSK-3 plays in glucose homeostasis and discuss the therapeutic potential of inhibiting this enzyme in the treatment of diabetes and insulin resistance. RESULTS/CONCLUSION: Despite its pleiotropic nature, GSK-3 has significant promise as a target for diabetes due to functional partitioning of the enzyme, tissue-selectivity and acute dosage-dependency of effects of inhibition, suggesting useful therapeutic windows.

Tissue-specific role of glycogen synthase kinase 3beta in glucose homeostasis and insulin action.

Dysregulation of the protein kinase glycogen synthase kinase 3 (GSK-3) has been implicated in the development of type 2 diabetes mellitus. GSK-3 protein expression and kinase activity are elevated in diabetes, while selective GSK-3 inhibitors have shown promise as modulators of glucose metabolism and insulin sensitivity. There are two GSK-3 isoforms in mammals, GSK-3alpha and GSK-3beta. Mice engineered to lack GSK-3beta die in late embryogenesis from liver apoptosis, whereas mice engineered to lack GSK-3alpha are viable and exhibit improved insulin sensitivity and hepatic glucose homeostasis. To assess the potential role of GSK-3beta in insulin function, a conditional gene-targeting approach whereby mice in which expression of GSK-3beta was specifically ablated within insulin-sensitive tissues were generated was undertaken. Liver-specific GSK-3beta knockout mice are viable and glucose and insulin tolerant and display "normal" metabolic characteristics and insulin signaling. Mice lacking expression of GSK-3beta in skeletal muscle are also viable but, in contrast to the liver-deleted animals, display improved glucose tolerance that is coupled with enhanced insulin-stimulated glycogen synthase regulation and glycogen deposition. These data indicate that there are not only distinct roles for GSK-3alpha and GSK-3beta within the adult but also tissue-specific phenotypes associated with each of these isoforms.

Distinct sensor pathways in the hierarchical control of SNAT2, a putative amino acid transceptor, by amino acid availability.

Mammalian nutrient sensors are novel targets for therapeutic intervention in disease states such as insulin resistance and muscle wasting; however, the proteins responsible for this important task are largely uncharacterized. To address this issue we have dissected an amino acid (AA) sensor/effector regulon that controls the expression of the System A amino acid transporter SNAT2 in mammalian cells, a paradigm nutrient-responsive process, and found evidence for the convergence of at least two sensor/effector pathways. During AA withdrawal, JNK is activated and induces the expression of SNAT2 in L6 myotubes by stimulating an intronic nutrient-sensitive domain. A sensor for large neutral AA (e.g. Tyr, Gln) inhibits JNK activation and SNAT2 up-regulation. Additionally, shRNA and transporter chimeras demonstrate that SNAT2 provides a repressive signal for gene transcription during AA sufficiency, thus echoing AA sensing by transceptor (transporter-receptor) orthologues in yeast (Gap1/Ssy1) and Drosophila (PATH). Furthermore, the SNAT2 protein is stabilized during AA withdrawal.

Constitutive activation of GSK3 down-regulates glycogen synthase abundance and glycogen deposition in rat skeletal muscle cells.

The effects of inhibition or constitutive activation of glycogen synthase kinase-3 (GSK3) on glycogen synthase (GS) activity, abundance, and glycogen deposition in L6 rat skeletal muscle cells were investigated. GS protein expression increased approximately 5-fold during differentiation of L6 cells (comparing cells at the end of day 5 with those at the beginning of day 3). However, exposure of undifferentiated myoblasts (day 3) to 50 microM SB-415286, a GSK3 inhibitor, led to a significant elevation in GS protein that was not accompanied by changes in the abundance of GLUT4, another late differentiation marker. In contrast, stable expression of a constitutively active form of GSK3beta (GSK3S9A) led to a significant reduction (approximately 80%) in GS protein that was antagonized by SB-415286. Inhibition of GSK3 or expression of the constitutively active GSK3S9A did not result in any detectable changes in GS mRNA abundance. However, the increase in GS protein in undifferentiated myoblasts or that seen following incubation of cells expressing GSK3S9A with GSK3 inhibitors was blocked by cycloheximide suggesting that GSK3 influences GS abundance possibly via control of mRNA translation. Consistent with the reduction in GS protein, cells expressing GSK3S9A were severely glycogen depleted as judged using a specific glycogen-staining antibody. Inhibiting GSK3 in wild-type or GSK3S9A-expressing cells using SB-415286 resulted in an attendant activation of GS, but not that of glucose transport. However, GS activation alone was insufficient for stimulating glycogen deposition. Only when muscle cells were incubated simultaneously with insulin and SB-415286 or with lithium (which stimulates GS and glucose transport) was an increase in glycogen accretion observed. Our findings suggest that GSK3 activity is an important determinant of GS protein expression and that while glycogen deposition in muscle cells is inherently dependent upon the activity/expression of GS, glucose transport is a key rate-determining step in this process.

Use of lithium and SB-415286 to explore the role of glycogen synthase kinase-3 in the regulation of glucose transport and glycogen synthase.

Glycogen synthase kinase 3 (GSK3) is inactivated by insulin and lithium and, like insulin, Li also activates glycogen synthase (GS) via inhibition of GSK3. Li also mimics insulin's ability to stimulate glucose transport (GT), an observation that has led to the suggestion that GSK3 may coordinate hormonal increases in GT and glycogen synthesis. Here we have used Li and SB-415286, a selective GSK3 inhibitor, to establish the importance of GSK3 in the hormonal activation of GT in terms of its effect on GS in L6 myotubes and 3T3-L1 adipocytes. Insulin, Li and SB-415286 all induced a significant inhibition of GSK3, which was associated with a marked dephosphorylation and activation of GS. In L6 myotubes, SB-415286 induced a much greater activation of GS (6.8-fold) compared to that elicited by insulin (4.2-fold) or Li (4-fold). In adipocytes, insulin, Li and SB-415286 all caused a comparable activation of GS despite a substantial differentiation-linked reduction in GSK3 expression ( approximately 85%) indicating that GSK3 remains an important determinant of GS activation in fat cells. Whilst Li and SB-415286 both inhibit GSK3 in muscle and fat cells, only Li stimulated GT. This increase in GT was not sensitive to inhibitors of PI3-kinase, MAP kinase or mTOR, but was suppressed by the p38 MAP kinase inhibitor, SB-203580. Consistent with this, phosphorylation of p38 MAP kinase induced by Li correlated with its stimulatory effect on GT. Our findings support a crucial role for GSK3 in the regulation of GS, but based on the differential effects of Li and SB-415286, it is unlikely that acute inhibition of GSK3 contributes towards the rapid stimulation of GT by insulin in muscle and fat cells.