Higher Glucose Tolerance Research Articles

241-LB: Inhibition of TXNIP-Thioredoxin Redox Switch Improves Insulin Sensitivity on a High-Fat Diet

Thioredoxin interacting protein (TXNIP) is a regulator of glucose metabolism and oxidative stress. It is the only arrestin-domain protein known to bind to reduced thioredoxin (Trx) at a C247 residue. This binding site is conserved in mammals, but its specific purpose is unclear. Here, we studied TXNIP's metabolic roles through the binding of Trx in vivo. We hypothesized that the TXNIP-Trx interaction may regulate a High-Fat Diet (HFD) induced insulin resistance triggered by redox stress. To abolish this interaction, we generated a new mouse model with a single C247S amino acid substitution in the txnip gene using CRISPR-Cas9. Over 8 weeks, we fed male C57BL/6J WT, C247S, and TXNIP KO mice (n=10/group) chow and HFD and performed insulin and glucose tolerance tests. Notably, C247S mice on HFD exhibited improved insulin sensitivity compared to WT, but this was not observed on a chow diet (Figure 1). Additionally, these mice had lower glucose tolerance on a chow diet and higher glucose tolerance on a HFD compared to WT. Moreover, KO mice were hypoglycemic and hypersensitive to insulin compared to C247S and WT mice in both diets. Our data reveals that inhibiting the TXNIP-Trx interaction on a HFD (stressed condition) can improve insulin sensitivity, suggesting a protective effect against insulin resistance. Future studies are needed to unravel this relationship between redox and arrestin biology in regulating metabolism. Disclosure M. F. Hoang: None. S. Dagdeviren: None. V. Meier: None. J. Benoit: None. V. Y. Melnik: None. R. Lee: Advisory Panel; Elevian, Inc., Research Support; BlueRock Therapeutics. Funding National Institutes of Health (1F32DK126289, R01DK126688)

Metabolomic and transcriptomic analyses of Fmo5-/- mice reveal roles for flavin-containing monooxygenase 5 (FMO5) in NRF2-mediated oxidative stress response, unfolded protein response, lipid homeostasis, and carbohydrate and one-carbon metabolism.

Flavin-containing monooxygenase 5 (FMO5) is a member of the FMO family of proteins, best known for their roles in the detoxification of foreign chemicals and, more recently, in endogenous metabolism. We have previously shown that Fmo5-/- mice display an age-related lean phenotype, with much reduced weight gain from 20 weeks of age. The phenotype is characterized by decreased fat deposition, lower plasma concentrations of glucose, insulin and cholesterol, higher glucose tolerance and insulin sensitivity, and resistance to diet-induced obesity. In the present study we report the use of metabolomic and transcriptomic analyses of livers of Fmo5-/- and wild-type mice to identify factors underlying the lean phenotype of Fmo5-/- mice and gain insights into the function of FMO5. Metabolomics was performed by the Metabolon platform, utilising ultrahigh performance liquid chromatography-tandem mass spectroscopy. Transcriptomics was performed by RNA-Seq and results analysed by DESeq2. Disruption of the Fmo5 gene has wide-ranging effects on the abundance of metabolites and expression of genes in the liver. Metabolites whose concentration differed between Fmo5-/- and wild-type mice include several saturated and monounsaturated fatty acids, complex lipids, amino acids, one-carbon intermediates and ADP-ribose. Among the genes most significantly and/or highly differentially expressed are Apoa4, Cd36, Fitm1, Hspa5, Hyou1, Ide, Me1 and Mme. The results reveal that FMO5 is involved in upregulating the NRF2-mediated oxidative stress response, the unfolded protein response and response to hypoxia and cellular stress, indicating a role for the enzyme in adaptation to oxidative and metabolic stress. FMO5 also plays a role in stimulating a wide range of metabolic pathways and processes, particularly ones involved in lipid homeostasis, the uptake and metabolism of glucose, the generation of cytosolic NADPH, and in one-carbon metabolism. The results predict that FMO5 acts by stimulating the NRF2, XBP1, PPARA and PPARG regulatory pathways, while inhibiting STAT1 and IRF7 pathways.

Hepatic interleukin-1 receptor type 1 signalling regulates insulin sensitivity in the early phases of nonalcoholic fatty liver disease.

BackgroundNonalcoholic fatty liver disease (NAFLD) is associated with hepatic as well as systemic insulin resistance even in the absence of type 2 diabetes. The extent and pathways through which hepatic inflammation modulates insulin sensitivity in NAFLD are only partially understood. We explored the contribution of hepatic interleukin (IL)‐1 signalling in a novel conditional knockout mouse model and expand the knowledge on this signalling pathway with regard to its liver‐specific functions.MethodsA high‐fat, high‐carbohydrate diet (HFD) over 12 weeks was used in male hepatocyte‐specific IL‐1 receptor type 1 (IL‐1R1) knockout mice (Il1r1 Hep−/–) and wild‐type (WT) littermates.ResultsBoth genotypes developed an obese phenotype and accompanying macrovesicular hepatic steatosis. In contrast to WT mice, microvesicular steatosis and ballooning injury was less pronounced in HFD‐fed Il1r1 Hep−/– mice, and alanine aminotransferase remained in the normal range. This was paralleled by the suppression of injurious and proinflammatory hepatic c‐Jun N‐terminal kinases and extracellular signal‐regulated kinases signalling, stable peroxisome proliferator activated receptor gamma coactivator‐1alpha and farnesoid X receptor‐alpha expression and preservation of mitochondrial function. Strikingly, despite HFD‐feeding Il1r1 Hep−/– mice remained highly insulin sensitive as indicated by lower insulin levels, homeostatic model assessment for insulin resistance, higher glucose tolerance, more stable hepatic insulin signalling cascade, and less adipose tissue inflammation compared to the WT.ConclusionsThe current data highlights that hepatocyte IL‐1R1 contributes to hepatic and extrahepatic insulin resistance. Future liver‐directed therapies in NAFLD could have effects on insulin sensitivity when improving hepatic inflammation and IL‐1R1 signalling.

Mechanism of protection from insulin resistance by toll-like receptor 2 deficiency in high-fat diet fed mice: involvement in macrophage polarization.

Toll-like receptor 2 (TLR2) deficiency can increase insulin sensitivity and improves glucose tolerance. However, it is not yet fully understood about its underlying mechanism. The regulation of M1/M2 macrophage polarization has been verified to involve in insulin resistance. Here, we evaluated whether the beneficial effect of TLR2 deficiency is mediated by TLR2-associated macrophage polarization in mice fed with high-fat diet (HFD). Wild-type and TLR2 knockout (TLR2-/-) mice received HFD for two months. Following intraperitoneal glucose tolerance and insulin resistance tests, peripheral monocytes were isolated, and in vitro induced for differentiation into M1 and M2 macrophages, respectively. Macrophages polarization was evaluated using flow cytometry. The expression of macrophage polarization marker genes and cytokine production in visceral adipose tissue were measured by qRT-PCR and ELISA. Compared to wild-type mice, TLR2-/- mice showed higher glucose tolerance and insulin sensitivity, along with significantly reduced the population of M1 and increased M2 count in vitro. Additionally, TLR2-/- mice demonstrated higher expression of M2 marker iNOS mRNA and interleukin-10 level in adipose tissues. Our results reveal that TLR2 knockout-mediated macrophages M2 polarization is a crucial factor for preventing against diet-induced insulin resistance in mice. These findings deepen our knowledge about the mechanism underlying HFD-induced insulin resistance.

Crosstalk between Drp1 phosphorylation sites during mitochondrial remodeling and their impact on metabolic adaptation

SummaryMitochondria constantly undergo fusion and fission events, referred as mitochondrial dynamics, which determine mitochondrial architecture and bioenergetics. Cultured cell studies demonstrate that mitochondrial dynamics are acutely regulated by phosphorylation of the mitochondrial fission orchestrator dynamin-related protein 1 (Drp1) at S579 or S600. However, the physiological impact and crosstalk of these phosphorylation sites is poorly understood. Here, we describe the functional interrelation between S579 and S600 phosphorylation sites in vivo and their role on mitochondrial remodeling. Mice carrying a homozygous Drp1 S600A knockin (Drp1 KI) mutation display larger mitochondria and enhanced lipid oxidation and respiratory capacities, granting improved glucose tolerance and thermogenic response upon high-fat feeding. Housing mice at thermoneutrality blunts these differences, suggesting a role for the brown adipose tissue in the protection of Drp1 KI mice against metabolic damage. Overall, we demonstrate crosstalk between Drp1 phosphorylation sites and provide evidence that their modulation could be used in the treatment and prevention of metabolic diseases.

Beneficial effects of daytime high-intensity light exposure on daily rhythms, metabolic state and affect

While the importance of the circadian system to health and well-being is extensively studied, the role of daylight exposure in these interactions is relatively poorly understood. Here we show, using a diurnal animal model naturally exposed to daylight, that daily morning exposure to 3000 lux, full spectrum electric light has beneficial health effects. Compared with controls, sand rats (Psammomys obesus) subjected to morning light treatment demonstrate daily rhythms with high peak to trough difference in activity, blood glucose levels and per2 gene expression in the suprachiasmatic nucleus, pre-frontal cortex, kidney and liver. The treated animals were also healthier, being normoglycemic, having higher glucose tolerance, lower body and heart weight and lower anxiety- and depression-like behavior. Our results suggest that exposure to high intensity light is important for the proper function of the circadian system and well-being, and are important in face of human's low exposure to daylight and extensive use of artificial light at night.

Rho-kinase activity is upregulated in the skeletal muscle of aged exercised rats.

There is a gap in the knowledge regarding the regulation of glucose uptake in skeletal muscle during the development of insulin resistance in the elderly. Rho-Kinase (Rock) signaling has been demonstrated as a crucial mechanism related to glucose metabolism and insulin sensitivity in skeletal muscle. This kinase is involved in the insulin receptor substrate 1 (IRS1) phosphorylation, leading to glucose uptake stimulation in the skeletal muscle; however, the mechanisms elucidating the role of Rock regulation in the context of advanced ages are still limited. In this study, we submitted old Fischer 344 rats to short-term treadmill physical exercise protocol (5 days) and evaluated the glucose tolerance and proteins involved with Rock/insulin signaling in the skeletal muscle. Compared to young rats, the old rats showed glucose intolerance, hyperinsulinemia, and decreased phosphorylation in the proteins related to the insulin signaling pathway in the skeletal muscle, without changes in body mass and adiposity. Otherwise, when these rats were submitted to physical exercise, it was found decreased fasting glucose, higher glucose tolerance, decreased insulinemia, and upregulation of Rock2/pIRS1/pAkt/pGSK3β/GLUT4 pathway in the skeletal muscle. In summary, the aging process did not change Rock signaling, but the physical exercise was able to increase Rock2 content and insulin signaling pathway in the skeletal muscle. This finding suggests the benefic role of physical exercise to advanced ages, promoting insulin-sensitive effects with Rho-kinase contribution.

Retraction: Muscle-derived Satellite Cells for Treating Type 1 Diabetes in Rats (Rattus norvegicus).

Diabetes mellitus(DM) is a complicated metabolic disease, with the fundamental treatment nowadays being diet control, insulin injections, slet or pancreas transplantation, which is limited because exogenous insulin injections fail to simulate normal insulin secretion in islet beta cells successfully and islet transplantation lacks organ donors. So far, stem cells with highly self-renewal and multi-directional differentiation potential have become a new hope for the treatment of diabetes. In this research, rat Muscle-derived satellite cells *MDSCs*were separated and cultivated in vitro and inducted into insulin-producing cells with observation and identification using dithizone staining and so on. T1D rat model was built with Streptozocin(STZ), then MDSCs and induced insulin-producing cells from MDSCs labeled by Dil were transplanted respectively. Transplantation of MDSCs (group 1) and the induced insulin-producing cells (group 2) into streptozotocin (STZ)-induced diabetic rats resulted in lower blood glucose, lower urine glucose, higher body weight, higher glucose tolerance and less water intake and urine output than control rats (P < 0.05). Among the male rats, the blood glucose levels of 50% of group 1 rats were <16.7 mmol/L, and 40% of group 2 rats yielded similar results (P < 0.01). Among the female rats, the blood glucose levels of 66.67% of group 1 rats were <16.7 mmol/L, and 33.33% of group 2 rats showed similar blood glucose levels (P < 0.01). Histological examination revealed that the transplanted cells reached the pancreas and repaired damaged tissues. The above research conclusions provide theoretical basis, cell selection and good prospect for the treatment of diabetes. This article is protected by copyright. All rights reserved.

Energy metabolism couples hepatocyte integrin-linked kinase to liver glucoregulation and postabsorptive responses of mice in an age-dependent manner.

Integrin-linked kinase (ILK) is a critical intracellular signaling node for integrin receptors. Its role in liver development is complex, as ILK deletion at E10.5 (before hepatocyte differentiation) results in biochemical and morphological differences that resolve as mice age. Nevertheless, mice with ILK depleted specifically in hepatocytes are protected from the hepatic insulin resistance during obesity. Despite the potential importance of hepatocyte ILK to metabolic health, it is unknown how ILK controls hepatic metabolism or glucoregulation. The present study tested the role of ILK in hepatic metabolism and glucoregulation by deleting it specifically in hepatocytes, using a cre-lox system that begins expression at E15.5 (after initiation of hepatocyte differentiation). These mice develop the most severe morphological and glucoregulatory abnormalities at 6 wk, but these gradually resolve with age. After identifying when the deletion of ILK caused a severe metabolic phenotype, in depth studies were performed at this time point to define the metabolic programs that coordinate control of glucoregulation that are regulated by ILK. We show that 6-wk-old ILK-deficient mice have higher glucose tolerance and decreased net glycogen synthesis. Additionally, ILK was shown to be necessary for transcription of mitochondrial-related genes, oxidative metabolism, and maintenance of cellular energy status. Thus, ILK is required for maintaining hepatic transcriptional and metabolic programs that sustain oxidative metabolism, which are required for hepatic maintenance of glucose homeostasis.

Tributyrin in Inflammation: Does White Adipose Tissue Affect Colorectal Cancer?

Colorectal cancer affects the large intestine, leading to loss of white adipose tissue (WAT) and alterations in adipokine secretion. Lower incidence of colorectal cancer is associated with increased fibre intake. Fructooligosaccharides (FOS) are fibres that increase production of butyrate by the intestinal microbiota. Tributyrin, a prodrug of butyric acid, exerts beneficial anti-inflammatory effects on colorectal cancer. Our aim was to characterise the effects of diets rich in FOS and tributyrin within the context of a colon carcinogenesis model, and characterise possible support of tumorigenesis by WAT. C57/BL6 male mice were divided into four groups: a control group (CT) fed with chow diet and three colon carcinogenesis-induced groups fed either with chow diet (CA), tributyrin-supplemented diet (BUT), or with FOS-supplemented diet. Colon carcinogenesis decreased adipose mass in subcutaneous, epididymal, and retroperitoneal tissues, while also reducing serum glucose and leptin concentrations. However, it did not alter the concentrations of adiponectin, interleukin (IL)-6, IL-10, and tumour necrosis factor alpha (TNF)-α in WAT. Additionally, the supplements did not revert the colon cancer affected parameters. The BUT group exhibited even higher glucose tolerance and levels of IL-6, VEGF, and TNF-α in WAT. To conclude our study, FOS and butyrate supplements were not beneficial. In addition, butyrate worsened adipose tissue inflammation.

Endothelial-specific FoxO1 depletion prevents obesity-related disorders by increasing vascular metabolism and growth.

Impaired angiogenesis is a hallmark of metabolically dysfunctional adipose tissue in obesity. However, the underlying mechanisms restricting angiogenesis within this context remain ill-defined. Here, we demonstrate that induced endothelial-specific depletion of the transcription factor Forkhead Box O1 (FoxO1) in male mice led to increased vascular density in adipose tissue. Upon high-fat diet feeding, endothelial cell FoxO1-deficient mice exhibited even greater vascular remodeling in the visceral adipose depot, which was paralleled with a healthier adipose tissue expansion, higher glucose tolerance and lower fasting glycemia concomitant with enhanced lactate levels. Mechanistically, FoxO1 depletion increased endothelial proliferative and glycolytic capacities by upregulating the expression of glycolytic markers, which may account for the improvements at the tissue level ultimately impacting whole-body glucose metabolism. Altogether, these findings reveal the pivotal role of FoxO1 in controlling endothelial metabolic and angiogenic adaptations in response to high-fat diet and a contribution of the endothelium to whole-body energy homeostasis.

Molecular modification of β-glucosidase from the midgut of Macrotermes barneyi

Cellulose hydrolysis to glucose requires a series of cellulase enzymes, of which β-glucosidases play a crucial role. β-glucosidase (MbmgBG1) derived from the midgut of Macrotermes barneyi has higher glucose tolerance (maintaining more than 60% enzyme activity at 1.5 mol/L glucose). However, low enzyme activity and poor thermal stability limit the applications of β-glucosidase in food industries. Point mutants (F167L, T176C, E347I, R354K, N393G and V425M) were obtained by site-directed mutagenesis of non-conserved amino acids near conserved amino acids. Among them, the specific activities against to substrate pNPG of two mutants (F167L and R354K) were about 2-fold and 4-fold higher than that of MbmgBG1. Kcat/Km values were also higher than that of the wild-type, reflecting stronger affinity to the substrate and higher catalytic ability of mutants than MbmgBG1. When the glucose concentration was 1.5 mol/L, the enzyme activity of MbmgBG1 was about 60% of the original activity. F167L and R354K kept 60% enzymatic activity when the glucose concentrations of was 2.0 mol/L and 3.0 mol/L, respectively. These results lay a foundation for further studies on the catalytic efficiency of β-glucosidase.

Maternal parity affects placental development, growth and metabolism of foals until 1 year and a half

Primiparous mares are known to produce smaller foals than multiparous mares. This difference seems to be partly explained by the reduced exchange surface and volume of the placental villi in primiparous compared to multiparous placentas. The effect of maternal parity on foals' post-natal growth, metabolism and sexual maturation, however, has been given little consideration. The objectives of this work were to analyse placental biometry and structure at term, growth of foals and yearlings, their metabolism and testicular maturation at one year of age.Twenty multiparous mares (M), aged over 6 years and 12 primiparous mares (P), aged up to 5 years were artificially inseminated with the same stallion and monitored the same way until foaling. At birth, foals and placentas were measured and placentas were sampled above at the umbilical cord insertion, as well as in the pregnant and the non-pregnant horn to perform stereological analyses. Foals were weighed and measured until 540 days of age. At 120 and 360 days of age, an Intravenous Glucose Tolerance Test was performed on foals and yearlings. At 360 days of age, the males were castrated and testicular maturation analysed by RT-qPCR.At birth, P dams produced lighter and smaller foals and placentas. The foal birth weight to placental surface ratio was lower in the P compared to the M group. P Foals remained lighter than M foals until 360 days of age and smaller until at least 540 days of age. At 120 days of age, P foals had a higher glucose tolerance than M foals, and then may be less mature than M foals in terms of the control of their glucose homeostasis. At 360 days of age, the testicles of prepubertal P stallions were less mature in the P vs the M group.In conclusion, primiparous dams produce intrauterine growth restricted, less mature and smaller foals compared to multiparous dams with altered metabolism and growth until at least 540 days of age. These differences could affect the sport career of these foals, especially if it begins at an early age.

Reduced AMPKα2 protein expression restores glucose-induced insulin secretion in islets from calorie-restricted rats.

In a state of caloric restriction (CR), improved insulin action was associated with the activation of AMP-activated kinase (AMPK). Here, we verified whether AMPK was involved in impaired β-cell function in islets from rats subjected to CR for 21 days. Eight-week-old male rats were distributed into a control (CTL) group that was fed an isocaloric diet ad libitum or a CR group that received 60% of the food consumed by the CTL group. From days 18-21, CTL and CR rats were treated with sense (CTLS and CRS) or antisense (CTLAS and CRAS) AMPKα2 oligonucleotides. Caloric restriction was associated with decreased body weight, perigonadal fat pads and insulinaemia, while higher glucose tolerance was observed in CRS rats. Antisense treatment normalized insulinaemia and glucose tolerance in CRAS rats and increased cholesterolaemia in CRAS and CTLAS groups. These effects were associated with reduced pAMPK/AMPK protein expression in the liver of rats treated with antisense oligonucleotides. Additionally, CRS islets showed higher pAMPK/AMPK content and lower glucose-induced insulin release. As expected, antisense oligonucleotides against AMPKα2 efficiently reduced pAMPK/AMPK protein in CRAS and CTLAS islets. The lower AMPK content in CRAS islets normalized the insulin secretion in islets exposed to 16.7 mM glucose. In addition, CTLAS islets presented higher insulin secretion at 2.8 and 16.7 mM glucose. These findings support the hypothesis that higher AMPK protein expression is involved in impaired β-cell function in islets from rats subjected to CR for 21 days.

The effect of maternal and post-weaning low and high glycaemic index diets on glucose tolerance, fat deposition and hepatic function in rat offspring.

Clinical studies have reported beneficial effects of a maternal low glycaemic index (GI) diet on pregnancy and neonatal outcomes, but the impact of the diet on the offspring in later life, and the mechanisms underlying these effects, remain unclear. In this study, Albino Wistar rats were fed either a low GI (n=14) or high GI (n=14) diet during pregnancy and lactation and their offspring weaned onto either the low or high GI diet. Low GI dams had better glucose tolerance (AUC[glucose], 1322±55 v. 1523±72 mmol min/l, P&lt;0.05) and a lower proportion of visceral fat (19.0±2.9 v. 21.7±3.8% of total body fat, P&lt;0.05) compared to high GI dams. Female offspring of low GI dams had lower visceral adiposity (0.45±0.03 v. 0.53±0.03% body weight, P&lt;0.05) and higher glucose tolerance (AUC[glucose], 1243±29 v. 1351±39 mmol min/l, P&lt;0.05) at weaning, as well as lower hepatic PI3K-p85 mRNA at 12 weeks of age. No differences in glucose tolerance or hepatic gene expression were observed in male offspring, but the male low GI offspring did have reduced hepatic lipid content at weaning. These findings suggest that consuming a low GI diet during pregnancy and lactation can improve glucose tolerance and reduce visceral adiposity in the female offspring at weaning, and may potentially produce long-term reductions in the hepatic lipogenic capacity of these offspring.

Engineering a novel glucose-tolerant β-glucosidase as supplementation to enhance the hydrolysis of sugarcane bagasse at high glucose concentration

BackgroundMost β-glucosidases reported are sensitive to the end product (glucose), making it the rate limiting component of cellulase for efficient degradation of cellulose through enzymatic route. Thus, there are ongoing interests in searching for glucose-tolerant β-glucosidases, which are still active at high glucose concentration. Although many β-glucosidases with different glucose-tolerance levels have been isolated and characterized in the past decades, the effects of glucose-tolerance on the hydrolysis of cellulose are not thoroughly studied.ResultsIn the present study, a novel β-glucosidase (Bgl6) with the half maximal inhibitory concentration (IC50) of 3.5 M glucose was isolated from a metagenomic library and characterized. However, its poor thermostability at 50 °C hindered the employment in cellulose hydrolysis. To improve its thermostability, random mutagenesis was performed. A thermostable mutant, M3, with three amino acid substitutions was obtained. The half-life of M3 at 50 °C is 48 h, while that of Bgl6 is 1 h. The Kcat/Km value of M3 is 3-fold higher than that of Bgl6. The mutations maintained its high glucose-tolerance with IC50 of 3.0 M for M3. In a 10-h hydrolysis of cellobiose, M3 completely converted cellobiose to glucose, while Bgl6 reached a conversion of 80 %. Then their synergistic effects with the commercial cellulase (Celluclast 1.5 L) on hydrolyzing pretreated sugarcane bagasse (SCB) were investigated. The supplementation of Bgl6 or mutant M3 to Celluclast 1.5 L significantly improved the SCB conversion from 64 % (Celluclast 1.5 L alone) to 79 % (Bgl6) and 94 % (M3), respectively. To further evaluate the application potential of M3 in high-solids cellulose hydrolysis, such reactions were performed at initial glucose concentration of 20–500 mM. Results showed that the supplementation of mutant M3 enhanced the glucose production from SCB under all the conditions tested, improving the SCB conversion by 14–35 %.ConclusionsThese results not only clearly revealed the significant role of glucose-tolerance in cellulose hydrolysis, but also showed that mutant M3 may be a potent candidate for high-solids cellulose refining.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0383-z) contains supplementary material, which is available to authorized users.

Early differences in metabolic flexibility between obesity-resistant and obesity-prone mice.

Decreased metabolic flexibility, i.e. a compromised ability to adjust fuel oxidation to fuel availability supports development of adverse consequences of obesity. The aims of this study were (i) to learn whether obesity-resistant A/J and obesity-prone C57BL/6J mice differ in their metabolic flexibility right after weaning; and (ii) to characterize possible differences in control of glucose homeostasis in these animals using glucose tolerance tests (GTT). A/J and C57BL/6J mice of both genders were maintained at 20°C and weaned to standard low-fat diet at 30 days of age. During the first day after weaning, using several separate animal cohorts, (i) GTT was performed using 1 or 3mg glucose/g body weight (BW), while glucose was administered either orally (OGTT) or intraperitoneally (IPGTT) at 20°C; and (ii) indirect calorimetry (INCA) was performed, either in a combination with oral gavage of 1 or 7.5mg glucose/g BW, or during a fasting/re-feeding transition. INCA was conducted either at 20°C or 34°C. Results of both OGTT and IPGTT using 1mg glucose/g BW at 20°C, and INCA using 7.5mg glucose/g BW at 34°C, indicated higher glucose tolerance and higher metabolic flexibility to glucose, respectively, and lower fasting glycemia in A/J mice as compared with C57BL/6J mice. Thus, control of whole body glucose metabolism between A/J and C57BL/6J mice represents a phenotypic feature differentiating between the strains right after weaning.

Deletion of Kinin B2 Receptor Alters Muscle Metabolism and Exercise Performance.

Metabolic syndrome is a cluster of metabolic risk factors such as obesity, diabetes and cardiovascular diseases. Mitochondria is the main site of ATP production and its dysfunction leads to decreased oxidative phosphorylation, resulting in lipid accumulation and insulin resistance. Our group has demonstrated that kinins can modulate glucose and lipid metabolism as well as skeletal muscle mass. By using B2 receptor knockout mice (B2R-/-) we investigated whether kinin action affects weight gain and physical performance of the animals. Our results show that B2R-/- mice are resistant to high fat diet-induced obesity, have higher glucose tolerance as well as increased mitochondrial mass. These features are accompanied by higher energy expenditure and a lower feed efficiency associated with an increase in the proportion of type I fibers and intermediary fibers characterized by higher mitochondrial content and increased expression of genes related to oxidative metabolism. Additionally, the increased percentage of oxidative skeletal muscle fibers and mitochondrial apparatus in B2R-/- mice is coupled with a higher aerobic exercise performance. Taken together, our data give support to the involvement of kinins in skeletal muscle fiber type distribution and muscle metabolism, which ultimately protects against fat-induced obesity and improves aerobic exercise performance.

Kinin B1 and B2 receptor deficiency protects against obesity induced by a high-fat diet and improves glucose tolerance in mice.

The kallikrein-kinin system is well known for its role in pain and inflammation, and has been shown recently by our group to have a role also in the regulation of energy expenditure. We have demonstrated that B1 receptor knockout (B1KO) mice are resistant to obesity induced by a high-fat diet (HFD) and that B1 receptor expression in adipocytes regulates glucose tolerance and predisposition to obesity. However, it is also known that in the absence of B1 receptor, the B2 receptor is overexpressed and can take over the function of its B1 counterpart, rendering uncertain the role of each kinin receptor in these metabolic effects. Therefore, we investigated the impact of ablation of each kinin receptor on energy metabolism using double kinin receptor knockout (B1B2KO) mice. Our data show that B1B2KO mice were resistant to HFD-induced obesity, with lower food intake and feed efficiency when compared with wild-type mice. They also had lower blood insulin and leptin levels and higher glucose tolerance after treatment with an HFD. Gene expression for tumor necrosis factor-alpha and C-reactive protein, which are important genes for insulin resistance, was reduced in white adipose tissue, skeletal muscle, and the liver in B1B2KO mice after the HFD. In summary, our data show that disruption of kinin B1 and B2 receptors has a profound impact on metabolic homeostasis in mice, by improving glucose tolerance and preventing HFD-induced obesity. These novel findings could pave the way for development of new pharmacological strategies to treat metabolic disorders such as insulin resistance and obesity.

Molecular cloning and characterization of a novel β-glucosidase with high hydrolyzing ability for soybean isoflavone glycosides and glucose-tolerance from soil metagenomic library

A novel β-glucosidase (Bgl1269) was identified from a metagenomic library of mangrove soil by activity-based functional screening. Sequence analysis revealed that Bgl1269 encodes a protein of 422 amino acids. After being overexpressed in Escherichia coli and purified, the enzymatic properties of Bgl1269 were investigated. The recombinant enzyme displayed a pH optimum of 6.0 and a temperature optimum of 40°C, and the addition of most common metal ions (1 or 10mM) increased the enzymatic activity evidently. In addition, the enzyme showed high hydrolyzing ability for soybean isoflavone glycosides, and 0.8unit of enzyme could completely converted daidzin and genistin (0.5mg/mL) to daidzein and genistein at 40°C for 0.5h. Interestingly, Bgl1269 also exhibited a very high glucose-tolerance, with the highest inhibition constant Ki (4.28M) among β-glucosidases reported so far. These properties make it a good candidate in the production of soybean isoflavone aglycones after further study.