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As global warming and the energy crisis receive increasing attention, the development of ecofriendly functional food packaging materials has also garnered significant interest. In this study, curdlan was combined with foaming agents (Cremodan and xanthan) and a crosslinking agent (sodium trimetaphosphate) to form a porous curdlan hydrogel. The material properties of the curdlan hydrogels were characterized using scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and a thermogravimetric analysis (TGA). Results showed that the 2 % curdlan hydrogel exhibited a high water content (96.2 %), and swelling (89.2 %) and reswelling abilities (89.5 %), with a distinctly porous structure and excellent thermostability. These properties make it suitable for loading carvacrol, resulting in the formation of a carvacrol/curdlan hydrogel. In the results of antimicrobial testing, the carvacrol/curdlan hydrogel showed greater antimicrobial properties against Shewanella putrefaciens (32.3 mm) and Vibrio harveyi (33.1 mm) compared to V. parahaemolyticus (24.5 mm) in inhibition zone test. Noteworthy was the certain degree of antimicrobial ability of the carvacrol/curdlan hydrogel, which continuously exhibited microbial inhibition of the total viable count (TVC) of the no-inoculated and V. parahaemolyticus-inoculated group on sea bass fillets during 6 days of storage. In addition, the carvacrol/curdlan hydrogel inhibited the S. putrefaciens-caused microbial spoilage of fish. However, the continuous antibacterial ability of carvacrol/curdlan hydrogel needs to be improved. In future work, changing the porous size of curdlan hydrogel to control the carvacrol release for elongating the durability and long-term usage will be accomplished to fit the need in functional packaging applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Egg sausages, an essential component of traditional Chinese hot pot cuisine, have specific storage requirements and are predominantly distributed through refrigerated channels. A significant consideration in the freezing of egg sausages pertains to syneresis and textural modifications that manifest in the protein gel structure upon thawing. This research investigated the efficacy of incorporating whey protein isolate, soy protein isolate (at concentrations of 0.5%, 1.0%, and 2.0%), and modified cassava starch (at concentrations of 1.0%, 2.0%, and 3.0%) to enhance the textural integrity and mitigate syneresis in frozen egg sausage gels. The research demonstrated that syneresis in frozen egg sausages could be significantly minimized from 9.01% to 1.16% through the incorporation of 3% modified cassava starch and 2% whey protein isolate, to 2.01% with 1.0% soy protein isolate, and to 3.05% with 1.0% whey protein isolate. Furthermore, the combination of modified cassava starch (3%) and whey protein isolate (2%) demonstrated enhanced textural characteristics in frozen egg sausages with 20% additional water content following a 15-day storage period. Notably, egg sausages formulated with 0.5% whey protein isolate exhibited superior sensory attributes, including springiness, texture, and overall acceptability, compared to other formulations. The incorporation of whey protein isolate yielded markedly improved sensory characteristics relative to soy protein isolate additions. The findings indicate that the incorporation of whey protein isolate (0.5-1.0%) in conjunction with modified cassava starch (3%) effectively improves textural properties while reducing syneresis in thawed egg sausages.

Essential oils (EOs), regarded as secondary metabolites from plants, possess effective antibacterial properties. This study investigates the antibacterial efficacy of seven citrus EOs against six spoilage bacteria: Vibrio parahaemolyticus , V. harveyi , Photobacterium damselae , Shewanella putrefaciens , Carnobacterium divergens , and Lactobacillus pentosus . The antibacterial activity of these EOs was evaluated using solid- and vapor-phase applications. All tested EOs demonstrated effective antibacterial activity at a concentration of 294 μL/L against Gram-negative bacteria. Notably, lemon and orange EOs exhibited dose-dependent inhibition in both solid- and vapor-phase applications, with minimum effective concentrations ranging from 29.4 to 58.8 μL/L. Following treatment with lemon and orange EOs for 6 h at 1/4 minimum inhibitory concentration, leakage of intracellular DNA and proteins was observed, indicating damage to the cell membrane/wall. Proteomic analysis revealed distinct mechanisms: lemon EO impaired bacterial antioxidant defenses, while orange EO disrupted cell division, leading to reduced bacterial viability. These findings provide valuable insights into the potential of different EO application forms in controlling spoilage bacteria.

Fried pork rind, a processed pork by-product, is popular as a snack globally, prized for its distinctive flavor and crisp texture achieved through frying. Although various studies have examined processing factors such as thickness, moisture content, and brine concentration, there is a scarcity of research addressing the effect of frying temperature on the quality of fried pork rinds. In the present study, the effects of varying hot air drying times (12, 18, and 24 h at 50°C), traditional deep-fat frying temperatures (180°C, 195°C, and 210°C), and frying durations (3, 4, and 5 min) on the oil content, moisture content, breaking force, color, puffing ratio, and microstructural appearance of pork rinds were evaluated. The results revealed a significant correlation between frying temperature and time with the oil content of the pork rinds. The oil content and puffing ratio peaked at approximately 195°C. Moreover, the breaking force of the pork rinds decreased with increased frying time at 180°C, while the opposite trend was observed at 210°C., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Food Science & Nutrition published by Wiley Periodicals LLC.)

Insulin-deficient (type 1) diabetes is treated by providing insulin to maintain euglycemia. The current standard of care is a quasi-closed loop integrating automated insulin delivery with a continuous glucose monitoring sensor. Cell replacement technologies are advancing as an alternative treatment and have been tested as surrogates to cadaveric islets in transplants. In addition, immunomodulatory treatments to delay the onset of type 1 diabetes in high-risk (stage 2) individuals have gained regulatory approval. We have pioneered a cell conversion approach to restore insulin production through pharmacological conversion of intestinal epithelial cells into insulin-producing cells. We have advanced this approach along a translational trajectory through the discovery of small molecule forkhead box protein O1 inhibitors. When administered to different rodent models of insulin-deficient diabetes, these inhibitors have resulted in robust glucose-lowering responses and generation of insulin-producing cells in the gut epithelium. We review past work and delineate a path to human clinical trials., (© 2024 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Traditionally, the processing of Gelidium seaweed into Gelidium jelly was very complicated, and involved repeated washing with water and sun drying for seven rounds. The seaweed, which is originally reddish-purple in color, turns yellow in color after the repeated washing and sun drying cycles. However, the sun drying process can only be used on sunny days. Therefore, this study evaluated an alternative method, halogen lamp drying, and compared the qualities of the product, Gelidium jelly, made using the halogen lamp drying and traditional sun drying methods. The properties investigated included the agar yield, gelling temperature, hardness, springiness, rheological parameters, sensory attributes, color, and volatile compounds. The results demonstrated that the halogen lamp drying method required 12 washing and drying cycles to achieve similar jelly properties to seven rounds of sun drying in the experimental conditions. Volatiles including heptanal, β-ionone, and (E)-2-decenal could be used as indicators to monitor the washing and drying processes. Halogen lamp drying could be an alternative processing method for seaweed drying, especially on rainy days.

The interactions among agar, gellan gum, gelatin, and modified waxy corn starch in the formation of mixed gels were examined in five different ratios. Binary hydrocolloid gels were prepared using three ingredients: two hydrocolloids (total hydrocolloid concentration: 0.5 wt%, ratios of mixture: 0/0.5, 0.1/0.4, 0.2/0.3, 0.3/0.2, 0.4/0.1, and 0.5/0) and water. The textural properties of the hydrocolloid gels were studied by measuring the gel strength, rigidity, breaking force, breaking point, and syneresis as functions of the mixing ratio. The higher syneresis percentage of binary modified waxy corn starch and gum gels than that of mixed gum gels after cold storage was mainly due to the retrogradation of amylopectin. Agar was shown be the most influential with regards to increasing the gel strength, breaking force, and rigidity among the three kinds of gum, while gellan gum was more effective against syneresis than agar and gelatin for storage periods of 7 and 14 days. In the mixed gels, a dramatic increase in the breaking point from 0 to 0.5% was only exhibited for gellan gum. The results provided useful information, including gel strength, rigidity, breaking force, breaking point, and syneresis, for gum and modified corn starch ingredients selected from refrigerated binary gum gels such as pudding for food product development.

This study examined the physicochemical effects of the fortification of noodles with 0.25-1.00% ( w / w ) calcium salts, viz. calcium acetate, calcium carbonate, calcium citrate, and calcium lactate. Fortification with calcium citrate, calcium acetate, and calcium carbonate increased the pH and breaking force of the dried noodles. However, the fortification of noodles with any concentration of calcium did not increase the extent of elongation of the control raw noodles. The L* and b* values of the raw and dried noodle color increased with increasing concentrations of calcium salts, except for noodles with added calcium citrate. Fortification with calcium citrate yielded no significant influence on color, texture, adhesiveness, springiness, flavor, and overall scores for cooked noodles. Noodles fortified with 0.5% calcium citrate made from oyster shells were compared with a control sample of noodles and noodles fortified with commercially available calcium citrate. The particle size of the calcium citrate made from oyster shells (258 nm) was smaller than that of the purchased calcium citrate (2631 nm). Noodles fortified with calcium citrate made from oyster shells showed no significantly difference compared to noodles fortified with commercially available calcium citrate. These results suggest that calcium citrate made from oyster shells may be used as the additive of choice for the manufacture of calcium-fortified noodles.

Targeting lineage-defined transcriptional dependencies has emerged as an effective therapeutic strategy in cancer treatment. Through screening for molecular vulnerabilities of mantle cell lymphoma (MCL), we identified a set of transcription factors (TFs) including FOXO1, EBF1, PAX5, and IRF4 that are essential for MCL propagation. Integrated chromatin immunoprecipitation and sequencing (ChIP-Seq) with transcriptional network reconstruction analysis revealed FOXO1 as a master regulator that acts upstream in the regulatory TF hierarchy. FOXO1 is both necessary and sufficient to drive MCL lineage commitment through supporting the lineage-specific transcription programs. We further show that FOXO1, but not its close paralog FOXO3, can reprogram myeloid leukemia cells and induce B-lineage gene expression. Finally, we demonstrate that cpd10, a small molecule identified from an enriched FOXO1 inhibitor library, induces a robust cytotoxic response in MCL cells in vitro and suppresses MCL progression in vivo. Our findings establish FOXO1 inhibition as a therapeutic strategy targeting lineage-driven transcriptional addiction in MCL.

β-lactam-resistant Vibrio strains are a significant clinical problem, and β-lactamase inhibitors are generally coadministered with β-lactam drugs to control drug-resistant bacteria. Seaweed is a rich source of natural bioactive compounds; however, their potential as β-lactamase inhibitors against bacterial pathogens remains unknown. Herein, we evaluated the potential β-lactamase inhibitory effect of the ethanolic extracts of the red seaweed Gracilaria sp. (GE) against four Vibrio strains. The minimum inhibitory concentration, half-maximal inhibitory concentration, checkerboard assay results, and time-kill study results indicate that GE has limited antibacterial activity but can potentiate the activity of the β-lactam antibiotic carbenicillin against Vibrio parahemolyticus and V . cholerae . We overexpressed and purified recombinant metallo-β-lactamase, VarG, from V . cholerae for in vitro studies and observed that adding GE reduced the carbenicillin and nitrocefin degradation by VarG by 20% and 60%, respectively. Angiotensin I-converting enzyme inhibition studies demonstrated that GE did not inhibit VarG via metal chelation. Toxicity assays indicated that GE exhibited mild toxicity against human cells. Through gas chromatography and mass spectrometry, we showed that GE comprises alkaloids, phenolic compounds, terpenoids, terpenes, and halogenated aromatic compounds. This study revealed that extracts of the red seaweed Gracillaria sp. can potentially inhibit β-lactamase activity., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Objective: Lifelong insulin replacement remains the mainstay of type 1 diabetes treatment. Genetic FoxO1 ablation promotes enteroendocrine cell (EECs) conversion into glucose-responsive β-like cells. Here, we tested whether chemical FoxO1 inhibitors can generate β-like gut cells., Methods: We used Ngn3-or Villin-driven FoxO1 ablation to capture the distinctive developmental effects of FoxO1 on EEC pool. We combined FoxO1 ablation with Notch inhibition to enhance the expansion of EEC pool. We tested the ability of an orally available small molecule of FoxO1 inhibitor, Cpd10, to phenocopy genetic ablation of FoxO1. We evaluated the therapeutic impact of genetic ablation or chemical inhibition of FoxO1 on insulin-deficient diabetes in Ins2 Akita/+ mice., Results: Pan-intestinal epithelial FoxO1 ablation expanded the EEC pool, induced β-like cells, and improved glucose tolerance in Ins2 Akita/+ mice. This genetic effect was phenocopied by Cpd10. Cpd10 induced β-like cells that released insulin in response to glucose in gut organoids, and this effect was enhanced by the Notch inhibitor, DBZ. In Ins2 Akita/+ mice, a five-day course of either Cpd10 or DBZ induced intestinal insulin-immunoreactive β-like cells, lowered glycemia, and increased plasma insulin levels without apparent adverse effects., Conclusion: These results provide proof of principle of gut cell conversion into β-like cells by a small molecule FoxO1 inhibitor, paving the way for clinical applications., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The objectives of this study were to evaluate the effect of 0.5% chitosan incorporation on acrylamide development in a food model solution containing 0.5% fructose and asparagine after heating for 30 min at 180 °C. All the solutions were investigated for the following characteristics: acrylamide, asparagine, reducing sugar content, color, kinematic viscosity, Maillard reaction products (MRPs), and pH every 10 min. After heating for 10 min, the viscosity of chitosan-containing solutions reduced significantly. The investigational data confirmed that chitosan may have decomposed into lower molecular structures, as demonstrated by the reduced viscosity of the solution at pH < 6 and a decrease in the acrylamide content during 30 min of heating in a fructose−asparagine system. This study also confirms that the formation of ultraviolet-absorbing intermediates and browning intensity of MRPs containing acrylamide prepared by fructose−asparagine was more than those of MRPs prepared by glucose−asparagine solution system. MRPs containing acrylamide resulted from the reaction of asparagine with fructose (ketose) rather than glucose (aldose). Acrylamide formation could be significantly mitigated in the fructose−asparagine−chitosan model system as compared to the fructose−asparagine model system for possible beverage and food application.

The World Health Organization indicated that antibiotic resistance is one of the greatest threats to health, food security, and development in the world. Drug resistance efflux pumps are essential for antibiotic resistance in bacteria. Here, we evaluated the plant phenolic compound ethyl 3,4-dihydroxybenzoate (EDHB) for its efflux pump inhibitory (EPI) activity against drug-resistant Escherichia coli . The half-maximal inhibitory concentration, modulation assays, and time-kill studies indicated that EDHB has limited antibacterial activity but can potentiate the activity of antibiotics for drug-resistant E. coli . Dye accumulation/efflux and MALDI-TOF studies showed that EDHB not only significantly increases dye accumulation and reduces dye efflux but also increases the extracellular amount of antibiotics in the drug-resistant E. coli , indicating its interference with substrate translocation via a bacterial efflux pump. Molecular docking analysis using AutoDock Vina indicated that EDHB putatively posed within the distal binding pocket of AcrB and in close interaction with the residues by H-bonds and hydrophobic contacts. Additionally, EDHB showed an elevated postantibiotic effect on drug-resistant E. coli . Our toxicity assays showed that EDHB did not change the bacterial membrane permeability and exhibited mild human cell toxicity. In summary, these findings indicate that EDHB could serve as a potential EPI for drug-resistant E. coli .

We have investigated different properties (thickness, moisture loss, oil uptake, breaking force, color, puffing ratio during 0.5-5 min frying, microstructure, and sensory evaluation) of raw pork skins with varying thickness (2, 3, and 4 mm) after drying, intended as deep-fried snacks. We have found that the oil content, breaking force, and puffing ratio of fried pork skin with different raw skin thickness have no significant difference under similar water content (1.68-1.98 g/100 g wet weight basis, wb) after 3-5 min of deep-frying at 180 °C. Additionally, sensory score results have shown that fried pork skins with 4 mm raw skin thickness had lower flavor, texture, and overall acceptability than those with 2 mm and 3 mm raw skin thickness. Scanning electron micrographs (SEM) have revealed less holes and irregular and crack microstructure in fried pork skins with 4 mm raw skin thickness than in other groups. Different thickness of raw pork skins resulted in different effects in microstructure and influenced water evaporation and oil uptake of fried pork skin. Finally, we have proposed the kinetic equations of water loss and oil uptake of fried pork skins. Fried pork skin from raw skin thicker than 4 mm need frying at temperature higher than 180 °C to improve their puffing ratio and sensory acceptability.

The fat content of fried pork rinds is high, and alternative frying helps reduce the oil content and maintain their texture and taste. Different frying methods such as microwave-assisted, traditional deep frying and vacuum frying on the breaking force, color, microstructure, water loss and oil absorption attributes of fried pork rinds were evaluated in this study. The fat content of microwave-assisted and vacuum-fried pork rinds was lower (24.2 g/100 g dry weight basis (db) and 17.1 g/100 g db, respectively) than that (35.6 g/100 g db) of traditional deep-fat frying. Non-uniform, holy and irregular surface microstructures were obtained by vacuum frying due to rapid mass transfer at low pressure. The first-order kinetic models of water loss and oil absorption of traditional and microwave-assisted frying of pork rinds were established. Microwave frying caused a faster moisture loss rate, shorter frying time and lower pork rind oil content, makes it an attractive substitute for traditional deep-fat frying.

Drug efflux pumps are one of the major elements used by antibiotic-resistant bacteria. Efflux pump inhibitors (EPIs) are potential therapeutic agents for adjunctive therapy, which can restore the activity of antibiotics that are no longer effective against pathogens. This study evaluated the seaweed compound diphenylmethane (DPM) for its EPI activity. The IC 50 and modulation results showed that DPM has no antibacterial activity but can potentiate the activity of antibiotics against drug-resistant E. coli . Time-kill studies reported that a combination of DPM and erythromycin exhibited greater inhibitory activity against drug-resistant Escherichia coli . Dye accumulation and dye efflux studies using Hoechst 33342 and ethidium bromide showed that the addition of DPM significantly increased dye accumulation and reduced dye efflux in drug-resistant E. coli , suggesting its interference with dye translocation by an efflux pump. Using MALDI-TOF, it was observed that the addition of DPM could continuously reduce antibiotic efflux in drug-resistant E. coli . Additionally, DPM did not seem to damage the E. coli membranes, and the cell toxicity test showed that it features mild human-cell toxicity. In conclusion, these findings showed that DPM could serve as a potential EPI for drug-resistant E. coli .

Vibrio vulnificus is a pathogen that accounts for one of the highest mortality rates and is responsible for most reported seafood-related illnesses and deaths worldwide. Owing to the threats of pathogens with β-lactamase activity, it is important to identify and characterize β-lactamases with clinical significance. In this study, the protein sequence of the metallo-β-lactamase (MBL) fold metallohydrolase from V. vulnificus (designated Vmh) was analyzed, and its oligomeric state, β-lactamase activity, and metal binding ability were determined. BLASTp analysis indicated that the V. vulnificus Vmh protein showed no significant sequence identity with any experimentally identified Ambler class B MBLs or enzymes containing the MBL protein fold; it was also predicted to have a signal peptide of 19 amino acids at its N terminus and an MBL protein fold from amino acid residues 23 to 216. Recombinant V. vulnificus Vmh protein was overexpressed and purified. Analytical ultracentrifugation and electrospray ionization-mass spectrometry (MS) data demonstrated its monomeric state in an aqueous solution. Recombinant V. vulnificus Vmh protein showed broad degrading activities against β-lactam antibiotics, such as penicillins, cephalosporins, and imipenems, with k cat / K m values ranging from 6.23 × 10 2 to 1.02 × 10 4 M -1 s -1 . The kinetic reactions of this enzyme exhibited sigmoidal behavior, suggesting the possibility of cooperativity. Zinc ions were required for the enzyme activity, which was abolished by adding the metal chelator EDTA. Inductively coupled plasma-MS indicated that this enzyme might bind two zinc ions per molecule as a cofactor.

Objective: Forkhead box protein O1 (FOXO1) plays a key role in regulating hepatic glucose production, but investigations of FOXO1 inhibition as a potential therapeutic approach have been hampered by a lack of selective chemical inhibitors. By profiling structurally diverse FOXO1 inhibitors, the current study validates FOXO1 as a viable target for the treatment of diabetes., Methods: Using reporter gene assays, hepatocyte gene expression studies, and in vivo studies in mice, we profiled our leading tool compound 10 and a previously characterized FOXO1 inhibitor, AS1842856 (AS)., Results: We show that AS has significant FOXO1-independent effects, as demonstrated by testing in FOXO1-deficient cell lines and animals, while compound 10 is highly selective for FOXO1 both in vitro and in vivo and fails to elicit any effect in genetic models of FOXO1 ablation. Chronic administration of compound 10 improved insulin sensitivity and glucose control in db/db mice without causing weight gain. Furthermore, chronic compound 10 treatment combined with FGF21 led to synergistic glucose lowering in lean, streptozotocin-induced diabetic mice., Conclusions: We show that the widely used AS compound has substantial off-target activities and that compound 10 is a superior tool molecule for the investigation of FOXO1 function. In addition, we provide preclinical evidence that selective FOXO1 inhibition has potential therapeutic benefits for diabetes as a monotherapy or in combination with FGF21., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The aim of this research was to investigate the effects of the addition of 0.5% hydroxymethylfurfural (HMF) and low molecular chitosan on acrylamide and HMF formation in a food model system, which contains 0.5% glucose, asparagine, and HMF within 30 min of heating at 180 °C. At an interval of 10 min, all solutions were evaluated in the following aspects: reducing sugar, asparagine, acrylamide, HMF content, pH, Maillard reaction products, kinematic viscosity, and color. After heating for 10 min, the kinematic viscosity of solutions containing chitosan reduced significantly. The values of the acrylamide, HMF, and absorbance increased at OD 294 and OD 420 (optical density measured at 294 nm and 420 nm) of solutions. Experimental results showed that low-molecular-weight chitosan might be hydrolyzed into much lower molecular weight, followed by the decrease in kinematic viscosity of the solution at pH lower than 6 and the increase in the formation of acrylamide after heating for 30 min.

This research focuses on the proteolytic capacity of sea bass byproduct (SB) and their hypocholesterolemic activity via the cholesterol micelle formation (CMF) inhibition. SB was fermented with seven mixed lactic acid bacteria for 5 h at 42 °C. The lactic fermented SB was hydrolyzed with Protease N for 6 h under HHP to obtain the SB hydrolysates (HHP-assisted Protease N hydrolysis after fermentation, F-HHP-PN6). The supernatant was separated from the SB hydrolysate and freeze-dried. As the hydrolysis time extended to 6 h, soluble protein content increased from 187.1 to 565.8 mg/g, and peptide content increased from 112.8 to 421.9 mg/g, while inhibition of CMF increased from 75.0% to 88.4%. Decreasing the CMF inhibitory activity from 88.4% to 42.1% by simulated gastrointestinal digestion (FHHP-PN6 was further hydrolyzed by gastrointestinal enzymes, F-HHP-PN6-PP) reduced the CMF inhibitory activity of F-HHP-PN6. Using gel filtration chromatography, the F-HHP-PN6-PP was fractioned into six fractions. The molecular weight of the fifth fraction from F-HHP-PN6-PP was between 340 and 290 Da, and the highest inhibitory efficiency ratio (IER) on CMF was 238.9%/mg/mL. Further purification and identification of new peptides with CMF inhibitory activity presented the peptide sequences in Ser-Ala-Gln, Pro-Trp, and Val-Gly-Gly-Thr; the IERs were 361.7, 3230.0, and 302.9%/mg/mL, respectively.

Periodontitis is an inflammatory disease that can lead to the periodontal pocket formation and tooth loss. This study was aimed to develop antimicrobials loaded hydrogels composed of cellulose nanofibers (CNF) and κ-carrageenan oligosaccharides (CO) nanoparticles for the treatment of periodontitis. Two antimicrobial agents such as surfactin and Herbmedotcin were selected as the therapeutic agents and the hydrogels were formulated based on the increasing concentration of surfactin. The proposed material has high thermal stability, controlled release, and water absorption capacity. This study was proceeded by investigating the in vitro antibacterial and anti-inflammatory properties of the hydrogels. This material has strong antibacterial activity against periodontal pathogens such as Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum, and Pseudomonas aeruginosa. Moreover, a significant increase in malondialdehyde (MDA) production and a decrease in biofilm formation and metabolic activity of the bacteria was observed in the presence of hydrogel. Besides, it reduced the reactive oxygen species (ROS) generation, transcription factor, and cytokines production in human gingival fibroblast cells (HGF) under inflammatory conditions. In conclusion, the hydrogels were successfully developed and proven to have antibacterial and anti-inflammatory properties for the treatment of periodontitis. Thus, it can be used as an excellent candidate for periodontitis treatment.

Multidrug efflux pumps play an essential role in antibiotic resistance. The conventional methods, including minimum inhibitory concentration and fluorescent assays, to monitor transporter efflux activity might have some drawbacks, such as indirect evidence or interference from color molecules. In this study, MALDI-TOF MS use was explored for monitoring drug efflux by a multidrug transporter, and the results were compared for validation with the data from conventional methods. Minimum inhibitory concentration was used first to evaluate the activity of Escherichia coli drug transporter AcrB, and this analysis showed that the E. coli overexpressing AcrB exhibited elevated resistance to various antibiotics and dyes. Fluorescence-based studies indicated that AcrB in E. coli could decrease the accumulation of intracellular dyes and display various efflux rate constants for different dyes, suggesting AcrB's efflux activity. The MALDI-TOF MS analysis parameters were optimized to maintain a detection accuracy for AcrB's substrates; furthermore, the MS data showed that E. coli overexpressing AcrB led to increased ions abundancy of various dyes and drugs in the extracellular space at different rates over time, illustrating continuous substrate efflux by AcrB. This study concluded that MALDI-TOF MS is a reliable method that can rapidly determine the drug pump efflux activity for various substrates.

Purpose: Periodontitis is a chronic inflammatory disease associated with microbial accumulation. The purpose of this study was to reuse the agricultural waste to produce cellulose nanofibers (CNF) and further modification of the CNF with κ-carrageenan oligosaccharides (CO) for drug delivery. In addition, this study is focused on the antimicrobial activity of surfactin-loaded CO-CNF towards periodontal pathogens., Materials and Methods: A chemo-mechanical method was used to extract the CNF and the modification was done by using CO. The studies were further proceeded by adding different quantities of surfactin [50 mg (50 SNPs), 100 mg (100 SNPs), 200 mg (200 SNPs)] into the carrier (CO-CNF). The obtained materials were characterized, and the antimicrobial activity of surfactin-loaded CO-CNF was evaluated., Results: The obtained average size of CNF and CO-CNF after ultrasonication was 263 nm and 330 nm, respectively. Microscopic studies suggested that the CNF has a short diameter with long length and CO became cross-linked to form as beads within the CNF network. The addition of CO improved the degradation temperature, crystallinity, and swelling property of CNF. The material has a controlled drug release, and the entrapment efficiency and loading capacity of the drug were 53.15 ± 2.36% and 36.72 ± 1.24%, respectively. It has antioxidant activity and inhibited the growth of periodontal pathogens such as Streptococcus mutans and Porphyromonas gingivalis by preventing the biofilm formation, reducing the metabolic activity, and promoting the oxidative stress., Conclusion: The study showed the successful extraction of CNF and modification with CO improved the physical parameters of the CNF. In addition, surfactin-loaded CO-CNF has potential antimicrobial activity against periodontal pathogens. The obtained biomaterial is economically valuable and has great potential for biomedical applications., Competing Interests: The authors declare no conflict of interest., (© 2020 Johnson et al.)

The discovery and optimization of a novel series of GPR142 agonists are described. These led to the identification of compound 21 (LY3325656), which demonstrated anti-diabetic benefits in pre-clinical studies and ADME/PK properties suitable for human dosing. Compound 21 is the first GPR142 agonist molecule advancing to phase 1 clinic trials for the treatment of Type 2 diabetes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this study, the low-molecular-weight (LMW) fucoidan, rich in fucose and sulfate, was extracted and purified from the edible brown seaweed, Laminaria japonica . In this study, we orally administered LMW fucoidan to mice for 6 weeks. We then examined fucoidan's effects on innate immunity, adaptive immunity, and Mycoplasma pneumoniae (MP)-antigen-stimulated immune responses. Our data showed that LMW fucoidan stimulated the innate immune system by increasing splenocyte proliferation, natural killer (NK) cell activity, and phagocytic activity. LMW fucoidan also increased interleukin (IL)-2, IL-4, and interferon (IFN)-γ secretion by splenocytes and immunoglobulin (Ig)-G and IgA content in serum, which help regulate adaptive immune cell functions, and decreased allergen-specific IgE. In MP-antigen-stimulated immune responses, the IgM and IgG content in the serum were significantly higher in the LMW fucoidan group after MP-antigen stimulation. Our study provides further information about the immunomodulatory effects of LMW fucoidan and highlights a potential role in preventing M. pneumoniae infection.

Multidrug-resistant pathogens are a significant clinical problem. Efflux pump inhibitors (EPIs) can restore the activities of existing antibiotics by interfering with drug efflux pumps located in bacterial cell membranes. Seaweeds are important sources of biologically active metabolites of natural origin; however, their potential as EPIs remains uninvestigated. Here, functional extracts from the brown seaweeds Laminaria japonica and Sargassum horneri and the red seaweeds Gracilaria sp. and Porphyra dentata were evaluated as potential EPIs against drug-resistant Escherichia coli . All these extracts were found to potentiate the activities of drugs in modulation tests, although not to the same extent. Synergistic effects of the extracts and the drug clarithromycin were observed from the onset of Time-kill assays, with no evidence of bacterial regrowth. Ethidium bromide accumulation studies revealed that the efflux decreased in the presence of each extract, as indicated by the presence of EPIs. Most identified EPIs that have been discovered to date have aromatic structures, and the seaweed extracts were found to contain various terpenes, terpenoids, phenolic compounds, indoles, pyrrole derivatives, alkaloids, and halogenated aromatic compounds. Our study highlights the potential of these compounds of the seaweeds as drug EPIs.

Objective: GPR142 agonists are being pursued as novel diabetes therapies by virtue of their insulin secretagogue effects. But it is undetermined whether GPR142's functions in pancreatic islets are limited to regulating insulin secretion. The current study expands research on its action., Methods and Results: We demonstrated by in situ hybridization and immunostaining that GPR142 is expressed not only in β cells but also in a subset of α cells. Stimulation of GPR142 by a selective agonist increased glucagon secretion in both human and mouse islets. More importantly, the GPR142 agonist also potentiated glucagon-like peptide-1 (GLP-1) production and its release from islets through a mechanism that involves upregulation of prohormone convertase 1/3 expression. Strikingly, stimulation of insulin secretion and increase in insulin content via GPR142 engagement requires intact GLP-1 receptor signaling. Furthermore, GPR142 agonist increased β cell proliferation and protected both mouse and human islets against stress-induced apoptosis., Conclusions: Collectively, we provide here evidence that local GLP-1 release from α cells defines GPR142's beneficial effects on improving β cell function and mass, and we propose that GPR142 agonism may have translatable and durable efficacy for the treatment of type 2 diabetes., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Vibrio cholerae ATP-binding cassette transporter VcaM ( V. cholerae ABC multidrug resistance pump) has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi) production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein), as well as cells lacking the outer membrane factor (OMF) TolC (Tolerance to colicins). Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV), however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency., Competing Interests: The authors declare no conflict of interest.

Transcripts of key enzymes in the Leloir pathway of galactose metabolism in mouse livers are significantly increased after chronic high-fat/high-sucrose feeding. UDP-galactose-4-epimerase (GALE) is the last enzyme in this pathway that converts UDP-galactose to UDP-glucose and was previously identified as a downstream target of the endoplasmic reticulum (ER) stress effector spliced X-box binding protein 1, suggesting an interesting cross talk between galactose and glucose metabolism in the context of hepatic ER stress and whole-body metabolic fitness. However, its specific role in glucose metabolism is not established. Using an inducible and tissue-specific mouse model, we report that hepatic overexpression of Gale increases gluconeogenesis from pyruvate and impairs glucose tolerance. Conversely, genetic reduction of Gale in liver improves glucose tolerance. Transcriptional profiling identifies trefoil factor 3 ( Tff3 ) as one of the downstream targets of GALE. Restoration of Tff3 expression corrects glucose intolerance in Gale -overexpressing mice. These studies reveal a new link between hepatic GALE activity and whole-body glucose homeostasis via regulation of hepatic Tff3 expression., (© 2017 by the American Diabetes Association.)

The genome sequence of V. cholerae O1 Biovar Eltor strain N16961 has revealed a putative antibiotic resistance (var) regulon that is predicted to encode a transcriptional activator (VarR), which is divergently transcribed relative to the putative resistance genes for both a metallo-β-lactamase (VarG) and an antibiotic efflux-pump (VarABCDEF). We sought to test whether these genes could confer antibiotic resistance and are organised as a regulon under the control of VarR. VarG was overexpressed and purified and shown to have β-lactamase activity against penicillins, cephalosporins and carbapenems, having the highest activity against meropenem. The expression of VarABCDEF in the Escherichia coli (ΔacrAB) strain KAM3 conferred resistance to a range of drugs, but most significant resistance was to the macrolide spiramycin. A gel-shift analysis was used to determine if VarR bound to the promoter regions of the resistance genes. Consistent with the regulation of these resistance genes, VarR binds to three distinct intergenic regions, varRG, varGA and varBC located upstream and adjacent to varG, varA and varC, respectively. VarR can act as a repressor at the varRG promoter region; whilst this repression was relieved upon addition of β-lactams, these did not dissociate the VarR/varRG-DNA complex, indicating that the de-repression of varR by β-lactams is indirect. Considering that the genomic arrangement of VarR-VarG is strikingly similar to that of AmpR-AmpC system, it is possible that V. cholerae has evolved a system for resistance to the newer β-lactams that would prove more beneficial to the bacterium in light of current selective pressures.

The combined effects of low-molecular-weight fucoidan (LMF) and fucoxanthin (Fx) in terms of antihyperglycemic, antihyperlipidemic, and hepatoprotective activities were investigated in a mouse model of type II diabetes. The intake of LMF, Fx, and LMF + Fx lowered the blood sugar and fasting blood sugar levels, and increased serum adiponectin levels. The significant decrease in urinary sugar was only observed in LMF + Fx supplementation. LMF and Fx had ameliorating effects on the hepatic tissue of db/db mice by increasing hepatic glycogen and antioxidative enzymes, and LMF was more effective than Fx at improving hepatic glucose metabolism. As for glucose and lipid metabolism in the adipose tissue, the expression of insulin receptor substrate (IRS)-1, glucose transporter (GLUT), peroxisome proliferator-activated receptor gamma (PPARγ), and uncoupling protein (UCP)-1 mRNAs in the adipose tissue of diabetic mice was significantly upregulated by Fx and LMF + Fx, and levels of inflammatory adipocytokines, such as adiponectin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), were significantly modulated only by LMF + Fx supplementation. The efficacy of LMF + Fx supplementation on the decrease in urinary sugar and on glucose and lipid metabolism in the white adipose tissue of db/db mice was better than that of Fx or LMF alone, indicating the occurrence of a synergistic effect of LMF and Fx.

Hollow, poly(L-lactic acid) microtube array membranes (MTAM) were used in preparing membranes that contained immobilized yeast cells. To evaluate the performance of the developed system for continuous and fed-batch fermentation, a gas chromatography/milli-whistle device was used to on-line monitor the production of ethanol. The milli-whistle was connected to the outlet of a GC capillary, and when the fermentation gases and the GC carrier gas passed through it, a sound with a fundamental frequency was produced. The online data obtained for frequency-change vs. retention time can be recorded after a fast Fourier transform. In typical bioethanol fermentation, the yeast cells cannot be recycled, whereas the artificial yeast-MTAMs can be. The hollow-MTAM containing immobilized yeast cells significantly enhanced to bioethanol productivity, and represent a novel, promising technology for bioethanol fermentation. Our data indicate that the gas chromatography/milli-whistle device, which is economical and stable, is a very useful detector for long-term monitoring.

"Beige" adipocytes reside in white adipose tissue (WAT) and dissipate energy as heat. Several studies have shown that cold temperature can activate pro-opiomelanocortin-expressing (POMC) neurons and increase sympathetic neuronal tone to regulate WAT beiging. WAT, however, is traditionally known to be sparsely innervated. Details regarding the neuronal innervation and, more importantly, the propagation of the signal within the population of "beige" adipocytes are sparse. Here, we demonstrate that beige adipocytes display an increased cell-to-cell coupling via connexin 43 (Cx43) gap junction channels. Blocking of Cx43 channels by 18α-glycyrrhetinic acid decreases POMC-activation-induced adipose tissue beiging. Adipocyte-specific deletion of Cx43 reduces WAT beiging to a level similar to that observed in denervated fat pads. In contrast, overexpression of Cx43 is sufficient to promote beiging even with mild cold stimuli. These data reveal the importance of cell-to-cell communication, effective in cold-induced WAT beiging, for the propagation of limited neuronal inputs in adipose tissue., Competing Interests: none., (Copyright © 2016 Elsevier Inc. All rights reserved.)

GPR142, a putative amino acid receptor, is expressed in pancreatic islets and the gastrointestinal tract, but the ligand affinity and physiological role of this receptor remain obscure. In this study, we show that in addition to L-Tryptophan, GPR142 signaling is also activated by L-Phenylalanine but not by other naturally occurring amino acids. Furthermore, we show that Tryptophan and a synthetic GPR142 agonist increase insulin and incretin hormones and improve glucose disposal in mice in a GPR142-dependent manner. In contrast, Phenylalanine improves in vivo glucose disposal independently of GPR142. Noteworthy, refeeding-induced elevations in insulin and glucose-dependent insulinotropic polypeptide are blunted in Gpr142 null mice. In conclusion, these findings demonstrate GPR142 is a Tryptophan receptor critically required for insulin and incretin hormone regulation and suggest GPR142 agonists may be effective therapies that leverage amino acid sensing pathways for the treatment of type 2 diabetes.

GPR142 is an islet-enriched G protein-coupled receptor that has been investigated as a novel therapeutic target for the treatment of type 2 diabetes by virtue of its insulin secretagogue activity. However, the signaling pathways downstream of GPR142 and whether its stimulation of insulin release is glucose-dependent remain poorly characterized. In this study, we show that both native and synthetic GPR142 agonists can activate Gq as well as Gi signaling when GPR142 is recombinantly expressed in HEK293 cells. However, in primary pancreatic islets, a native cellular system, the insulin secretagogue activity of GPR142 agonists only requires Gq activation. In addition, our results show that stimulation of insulin secretion by GPR142 in pancreatic islets is strictly glucose-dependent.

Inhibition of Diacylglycerol O-acyltransferase 1 (DGAT1) has been a mechanism of interest for metabolic disorders. DGAT1 inhibition has been shown to be a key regulator in an array of metabolic pathways; however, based on the DGAT1 KO mouse phenotype the anticipation is that pharmacological inhibition of DGAT1 could potentially lead to skin related adverse effects. One of the aims in developing small molecule DGAT1 inhibitors that target key metabolic tissues is to avoid activity on skin-localized DGAT1 enzyme. In this report we describe a modeling-based approach to identify molecules with physical properties leading to differential exposure distribution. In addition, we demonstrate histological and RNA based biomarker approaches that can detect sebaceous gland atrophy pre-clinically that could be used as potential biomarkers in a clinical setting.

Diacylglycerol acyltransferase-1 (DGAT1) is a potential therapeutic target for treatment of obesity and related metabolic diseases. However, the degree of DGAT1 inhibition required for metabolic benefits is unclear. Here we show that partial DGAT1 deficiency in mice suppressed postprandial triglyceridemia, led to elevations in glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) only following meals with very high lipid content, and did not protect from diet-induced obesity. Maximal DGAT1 inhibition led to enhanced GLP-1 and PYY secretion following meals with physiologically relevant lipid content. Finally, combination of DGAT1 inhibition with dipeptidyl-peptidase-4 (DPP-4) inhibition led to further enhancements in active GLP-1 in mice and dogs. The current study suggests that targeting DGAT1 to enhance postprandial gut hormone secretion requires maximal inhibition, and suggests combination with DPP-4i as a potential strategy to develop DGAT1 inhibitors for treatment of metabolic diseases.

Diabetes is associated with β cell failure. But it remains unclear whether the latter results from reduced β cell number or function. FoxO1 integrates β cell proliferation with adaptive β cell function. We interrogated the contribution of these two processes to β cell dysfunction, using mice lacking FoxO1 in β cells. FoxO1 ablation caused hyperglycemia with reduced β cell mass following physiologic stress, such as multiparity and aging. Surprisingly, lineage-tracing experiments demonstrated that loss of β cell mass was due to β cell dedifferentiation, not death. Dedifferentiated β cells reverted to progenitor-like cells expressing Neurogenin3, Oct4, Nanog, and L-Myc. A subset of FoxO1-deficient β cells adopted the α cell fate, resulting in hyperglucagonemia. Strikingly, we identify the same sequence of events as a feature of different models of murine diabetes. We propose that dedifferentiation trumps endocrine cell death in the natural history of β cell failure and suggest that treatment of β cell dysfunction should restore differentiation, rather than promoting β cell replication., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Complications of atherosclerosis are the leading cause of death of patients with type 2 (insulin-resistant) diabetes. Understanding the mechanisms by which insulin resistance and hyperglycemia contribute to atherogenesis in key target tissues (liver, vessel wall, hematopoietic cells) can assist in the design of therapeutic approaches. We have shown that hyperglycemia induces FoxO1 deacetylation and that targeted knock-in of alleles encoding constitutively deacetylated FoxO1 in mice (Foxo1(KR/KR)) improves hepatic lipid metabolism and decreases macrophage inflammation, setting the stage for a potential anti-atherogenic effect of this mutation. Surprisingly, we report here that when Foxo1(KR/KR) mice are intercrossed with low density lipoprotein receptor knock-out mice (Ldlr(-/-)), they develop larger aortic root atherosclerotic lesions than Ldlr(-/-) controls despite lower plasma cholesterol and triglyceride levels. The phenotype is unaffected by transplanting bone marrow from Ldlr(-/-) mice into Foxo1(KR/KR) mice, indicating that it is independent of hematopoietic cells and suggesting that the primary lesion in Foxo1(KR/KR) mice occurs in the vessel wall. Experiments in isolated endothelial cells from Foxo1(KR/KR) mice indicate that deacetylation favors FoxO1 nuclear accumulation and exerts target gene-specific effects, resulting in higher Icam1 and Tnfα expression and increased monocyte adhesion. The data indicate that FoxO1 deacetylation can promote vascular endothelial changes conducive to atherosclerotic plaque formation.

Short-chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate, are metabolites formed by gut microbiota from complex dietary carbohydrates. Butyrate and acetate were reported to protect against diet-induced obesity without causing hypophagia, while propionate was shown to reduce food intake. However, the underlying mechanisms for these effects are unclear. It was suggested that SCFAs may regulate gut hormones via their endogenous receptors Free fatty acid receptors 2 (FFAR2) and 3 (FFAR3), but direct evidence is lacking. We examined the effects of SCFA administration in mice, and show that butyrate, propionate, and acetate all protected against diet-induced obesity and insulin resistance. Butyrate and propionate, but not acetate, induce gut hormones and reduce food intake. As FFAR3 is the common receptor activated by butyrate and propionate, we examined these effects in FFAR3-deficient mice. The effects of butyrate and propionate on body weight and food intake are independent of FFAR3. In addition, FFAR3 plays a minor role in butyrate stimulation of Glucagon-like peptide-1, and is not required for butyrate- and propionate-dependent induction of Glucose-dependent insulinotropic peptide. Finally, FFAR3-deficient mice show normal body weight and glucose homeostasis. Stimulation of gut hormones and food intake inhibition by butyrate and propionate may represent a novel mechanism by which gut microbiota regulates host metabolism. These effects are largely intact in FFAR3-deficient mice, indicating additional mediators are required for these beneficial effects.

Insulin receptor (InsR) signaling through transcription factor FoxO1 is important in the development of hypothalamic neuron feeding circuits, but knowledge about underlying mechanisms is limited. To investigate the role of InsR/FoxO1 signaling in the development and maintenance of these circuits, we surveyed the pool of hypothalamic neurons expressing Pomc mRNA in different mouse models of impaired hypothalamic InsR signaling. InsR ablation in the entire hypothalamus did not affect Pomc-neuron number at birth, but resulted in a 25% increase, most notably in the middle arcuate nucleus region, in young adults. Selective restoration of InsR expression in POMC neurons in these mice partly reversed the abnormality, resulting in a 10% decrease compared to age-matched controls. To establish whether FoxO1 signaling plays a role in this process, we examined POMC neuron number in mice with POMC-specific deletion of FoxO1, and detected a 23% decrease in age-matched animals, consistent with a cell-autonomous role of InsR/FoxO1 signaling in regulating POMC neuron number, distinct from its established role to activate Pomc transcription. These changes in Pomc cells occurred in the absence of marked changes in humoral factors or hypothalamic NPY neurons.

Dyslipidemia and atherosclerosis are associated with reduced insulin sensitivity and diabetes, but the mechanism is unclear. Gain of function of the gene encoding deacetylase SirT1 improves insulin sensitivity and could be expected to protect against lipid abnormalities. Surprisingly, when transgenic mice overexpressing SirT1 (SirBACO) are placed on atherogenic diet, they maintain better glucose homeostasis, but develop worse lipid profiles and larger atherosclerotic lesions than controls. We show that transcription factor cAMP response element binding protein (Creb) is deacetylated in SirBACO mice. We identify Lys136 is a substrate for SirT1-dependent deacetylation that affects Creb activity by preventing its cAMP-dependent phosphorylation, leading to reduced expression of glucogenic genes and promoting hepatic lipid accumulation and secretion. Expression of constitutively acetylated Creb (K136Q) in SirBACO mice mimics Creb activation and abolishes the dyslipidemic and insulin-sensitizing effects of SirT1 gain of function. We propose that SirT1-dependent Creb deacetylation regulates the balance between glucose and lipid metabolism, integrating fasting signals., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We review mechanisms that regulate production of glucose by the liver, focusing on areas of budding consensus, and endeavoring to provide a candid assessment of lingering controversies. We also attempt to reconcile data from tracer studies in humans and large animals with the growing compilation of mouse knockouts that display changes in glucose production. A clinical hallmark of diabetes, excessive glucose production remains key to its treatment. Hence, we attempt to integrate emerging pathways into the broader goal to rejuvenate the staid antidiabetic pharmacopeia., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Type 2 diabetes results from an impairment of insulin action. The first demonstrable abnormality of insulin signaling is a decrease of insulin-dependent glucose disposal followed by an increase in hepatic glucose production. In an attempt to dissect the relative importance of these two changes in disease progression, we have employed genetic knock-outs/knock-ins of the insulin receptor. Previously, we demonstrated that insulin receptor knock-out mice (Insr(-/-)) could be rescued from diabetes by reconstitution of insulin signaling in liver, brain, and pancreatic β cells (L1 mice). In this study, we used a similar approach to reconstitute insulin signaling in tissues that display insulin-dependent glucose uptake. Using GLUT4-Cre mice, we restored InsR expression in muscle, fat, and brain of Insr(-/-) mice (GIRKI (Glut4-insulin receptor knock-in line 1) mice). Unlike L1 mice, GIRKI mice failed to thrive and developed diabetes, although their survival was modestly extended when compared with Insr(-/-). The data underscore the role of developmental factors in the presentation of murine diabetes. The broader implication of our findings is that diabetes treatment should not necessarily target the same tissues that are responsible for disease pathogenesis.

Objective: Impaired insulin-dependent glucose disposal in muscle and fat is a harbinger of type 2 diabetes, but murine models of selective insulin resistance at these two sites are conspicuous by their failure to cause hyperglycemia. A defining feature of muscle and fat vis-à-vis insulin signaling is that they both express the insulin-sensitive glucose transporter Glut4. We hypothesized that diabetes is the result of impaired insulin signaling in all Glut4-expressing tissues., Research Design and Methods: To test the hypothesis, we generated mice lacking insulin receptors at these sites ("GIRKO" mice), including muscle, fat, and a subset of Glut4-positive neurons scattered throughout the central nervous system., Results: GIRKO mice develop diabetes with high frequency because of reduced glucose uptake in peripheral organs, excessive hepatic glucose production, and β-cell failure., Conclusions: The conceptual advance of the present findings lies in the identification of a tissue constellation that melds cell-autonomous mechanisms of insulin resistance (in muscle/fat) with cell-nonautonomous mechanisms (in liver and β-cell) to cause overt diabetes. The data are consistent with the identification of Glut4 neurons as a distinct neuroanatomic entity with a likely metabolic role.

Objective: The sites of insulin action in the central nervous system that regulate glucose metabolism and energy expenditure are incompletely characterized. We have shown that mice with hypothalamic deficiency (L1) of insulin receptors (InsRs) fail to regulate hepatic glucose production (HGP) in response to insulin., Research Design and Methods: To distinguish neurons that mediate insulin's effects on HGP from those that regulate energy homeostasis, we used targeted knock-ins to express InsRs in agouti-related protein (AgRP) or proopiomelanocortin (POMC) neurons of L1 mice., Results: Restoration of insulin action in AgRP neurons normalized insulin suppression of HGP. Surprisingly, POMC-specific InsR knock-in increased energy expenditure and locomotor activity, exacerbated insulin resistance and increased HGP, associated with decreased expression of the ATP-sensitive K(+) channel (K(ATP) channel) sulfonylurea receptor 1 subunit, and decreased inhibitory synaptic contacts on POMC neurons., Conclusions: The contrasting phenotypes of InsR knock-ins in POMC and AgRP neurons suggest a branched-pathway model of hypothalamic insulin signaling in which InsR signaling in AgRP neurons decreases HGP, whereas InsR activation in POMC neurons promotes HGP and activates the melanocortinergic energy expenditure program.

We review mechanisms of beta-cell failure in type 2 diabetes. A wealth of information indicates that it is caused by impaired insulin secretion and decreased beta-cell mass. Interestingly, there appears to be a link between these two mechanisms. The earliest reaction to peripheral insulin resistance is an increase in insulin production, owing primarily to increased secretion, and to a lesser extent to decreased clearance. Experimental animal models indicate that hyperinsulinaemia promotes an increase in beta-cell mass, largely via increased beta-cell replication. In contrast, following the onset of overt diabetes, there is a slowly progressive loss of beta-cell function and mass, both in animal models and in diabetic humans. It is of great interest that most diabetes-associated genes identified in genome-wide association studies appear to be enriched in the beta-cell and to have the potential to regulate mass and/or function. Here, we review evidence derived from experimental animal models to unravel the mechanisms underlying beta-cell dysfunction. We focus primarily on signalling pathways, as opposed to nutrient sensing, and specifically on the notion that insulin and growth factor signalling via Foxo1 in pancreatic beta-cells links insulin secretion with cellular proliferation and survival.

Reduced food intake brings about an adaptive decrease in energy expenditure that contributes to the recidivism of obesity after weight loss. Insulin and leptin inhibit food intake through actions in the central nervous system that are partly mediated by the transcription factor FoxO1. We show that FoxO1 ablation in pro-opiomelanocortin (Pomc)-expressing neurons in mice (here called Pomc-Foxo1(-/-) mice) increases Carboxypeptidase E (Cpe) expression, resulting in selective increases of alpha-melanocyte-stimulating hormone (alpha-Msh) and carboxy-cleaved beta-endorphin, the products of Cpe-dependent processing of Pomc. This neuropeptide profile is associated with decreased food intake and normal energy expenditure in Pomc-Foxo1(-/-) mice. We show that Cpe expression is downregulated by diet-induced obesity and that FoxO1 deletion offsets the decrease, protecting against weight gain. Moreover, moderate Cpe overexpression in the arcuate nucleus phenocopies features of the FoxO1 mutation. The dissociation of food intake from energy expenditure in Pomc-Foxo1(-/-) mice represents a model for therapeutic intervention in obesity and raises the possibility of targeting Cpe to develop weight loss medications.