Incretin dysregulation of lysyl oxidase: a new mechanism for diabetic bone disease

The American Diabetes Association has determined that the number of people with diabetes in America was 30.3 million in 2015, and it is becoming increasingly more prevalent. Diabetes is a condition characterized by chronic hyperglycemia due to either insufficient insulin or reduced insulin sensitivity. Diabetes comes with a host of complications; one major complication is osteopenia, which increases fracture risks in both type 1 and type 2 diabetics14. The changes in diabetic bone may be due to reductions in lysyl oxidase (LOX) levels leading to decreased amounts of insoluble type 1 collagen fibers, which are necessary for bone strength28. LOX catalyzes oxidative deamination of the hydroxylysine and lysine side chains of the collagen molecules to create reactive aldehyde groups9,14,17. These aldehyde groups rapidly react with the lysine or hydroxylysine on the helical region of neighboring molecules of collagen fibrils, creating crosslinks between molecules leading to a mature cross-linked collagen matrix9,15,18. Our lab has now linked the LOX reductions in diabetes to incretins such as glucose-dependent insulinotropic peptide (GIP), which directly increases LOX, and anti-incretins such as dopamine, which reduces LOX by inhibiting GIP. Incretins and anti-incretins are gastric hormones released by the intestine in response to nutrient consumption4. Generally, incretins are hormones that stimulate insulin secretion from the pancreas where as anti-incretins inhibit that insulinotropic effect13,23. GIP is an incretin that also has an anabolic effect on bone in addition to its insulinotropic effect23. In diabetes there is an impaired cellular response to GIP in the pancreas, but its effects on the bone as related to diabetes are unknown. Additionally, it was observed in our lab that bone-derived LOX levels were decreased in diabetes while the serum level of anti-incretin gut-derived dopamine was vastly increased. Dopamine is known to inhibit GIP in the pancreas, but its effect on the bone was unknown. Prior to this experiment our lab had already started exploring a potential mechanism for diabetic osteopenia, where diabetes leads to an increase in gut-derived anti-incretin (dopamine), which interferes with GIP-stimulated LOX in osteoblasts. The decreased levels of LOX lead to lowered collagen integrity and a loss in trabecular bone structure. The current work is a sub-study within the larger aim to elucidate the mechanism for diabetic osteopenia. The aim of this study was to further verify the effects of dopamine by using amisulpride, a dopamine receptor antagonist, to determine if amisulpride could restore bone health in streptozotocin (STZ) induced diabetic mice. For this study C57BL/6 wild type mice were used. The mice were divided at random into three groups (N=10 per group) as follows: control (no STZ, no amisulpride), STZ diabetic (STZ, no amisulpride), and STZ + amisulpride. Twenty of the mice had low dose intraperitoneal injections of STZ for five days to induce diabetes and ten mice received vehicle injections for the control group. The mice were maintained diabetic or normal for 8 weeks, after which ten of the STZ mice received intraperitoneal injections of amisulpride daily for a month. It was found that STZ induced diabetic mice had a significant decrease in LOX levels and insulin levels. Amisulpride was able to rescue the decreases in LOX levels but did not alter insulin levels. Furthermore, micro-CT analysis and picrosirius red histology of long bones indicated that there is a decrease in bone volume, impaired trabecular structure, and disorganized collagen matrix in the STZ diabetic group. These impairments could be rescued by amisulpride administration, giving further evidence to the new mechanism for diabetic osteopenia. The increase in anti-incretin gut-derived dopamine signaling could be the cause of diabetic osteopenia. Inhibition of the anti-incretin gut-derived dopamine may possibly provide a therapeutic target for diabetic osteopenia

MOLECULAR MECHANISM OF GLP-1 POTENTIATED INSULIN GRANULE EXOCYTOSIS

Ph.DDOCTOR OF PHILOSOPH

Mesoporous silicon systems for oral protein/peptide-based diabetes mellitus therapy

Regardless of the considerable efforts, there have been no major breakthroughs in the development of effective oral protein/peptide delivery. When compared to parenteral administration, oral delivery can significantly improve the patients quality of life, especially in chronic conditions, such as diabetes mellitus (DM), which requires multiple injections daily. However, oral absorption of proteins/peptides is severely limited by their physico-chemical properties and various physiological barriers in the gastrointestinal tract. Porous silicon (PSi) has emerged as a promising drug delivery system, owing to its beneficial properties, such as top-down production, customizable particle and pore morphology, easy surface modification, simple drug loading, biodegradability and biocompatibility. Thus, the aim of this dissertation was to develop a multifunctional PSi based platforms that would be able to overcome the physiological barriers and efficiently deliver insulin and glucagon-like peptide-1 (GLP-1) orally. First, the influence of different PSi surface chemistries was evaluated on the intestinal transport of insulin. Due to the negatively charged surface of PSi, there was minimal interactions with the intestinal cells. Thus, chitosan, a polycationic mucoadhesive biopolymer with permeation enhancing effect, was used to modify the surface of the PSi microparticles. When comparing different surface modification techniques, chemical conjugation of chitosan to PSi exhibited strongest cellular interaction, and the highest insulin permeation and uptake across the intestinal cell monolayers. Secondly, three different nanoparticles (NPs) were developed based on lipids, polymers and PSi, with and without chitosan coating, and evaluated as potential oral GLP-1 delivery system. The results showed that the chitosan-modified PSi NPs were the most efficient nanosystem with the best loading degree and the highest GLP-1 permeation across the cellular monolayer. To overcome several physiological barriers, the next step was to develop a multistage nanocomposite comprising of chitosan-conjugated PSi NPs that were coated with a pH responsive polymer, in order to deliver GLP-1 and dipeptidylpeptidase-4 (DPP4) inhibitor simultaneously via the oral route. This multistage nanosystem showed enhanced GLP-1 transport across the intestinal cell monolayers and across the rat intestinal tissue. Furthermore, the nanosystem also demonstrated hypoglycemic effect in vivo after the oral administration in diabetic rats. The efficacy of the nanosystem could be attributed to the combined effect of the permeation enhancing chitosan-modified PSi NPs, the presence of DPP4 inhibitor that prevented GLP-1 degradation, and the pH responsive coating that helped in avoiding premature GLP-1 release/degradation in the stomach. Moreover, it was shown that the mucoadhesivity and permeation enhancing ability of chitosan-modified PSi NPs could be significantly increased by further surface modification of NPs with either L-cysteine or cell-penetrating peptide (CPP). It was disclosed that electrostatic interactions between the NPs and the glycocalyx were the most prominent pathway for the transport and uptake of insulin from the NPs, together with the contribution of active transport, adsorptive endocytosis and clathrin-mediated endocytosis. Overall, advanced PSi-based systems were developed which successfully overcame several limitations associated with the oral delivery of biomacromolecules, and thus, showed high clinical potential as oral protein/peptide delivery systems for DM therapySuun kautta annettavaa proteiini- tai peptidilääkettä ei ole suurista ponnistuksista huolimatta vielä onnistuttu kehittämään. Pitkäaikaisissa sairauksissa, kuten diabeteksessa, joudutaan antamaan useita insuliini-injektioita päivittäin, vaikka potilaan kannalta olisi mukavinta ottaa lääke suun kautta. Imeytyminen suun kautta annosteltuna, erityisesti proteiini- ja peptidilääkkeillä, on hyvin rajallista niiden fysikaalisten ja kemiallisten ominaisuuksien sekä ruuansulatuskanavan rakenteen takia. Huokoinen pii on lupaava materiaali lääkeaineiden annossa; ne on melko helppo valmistaa, niiden kokoa on helppo säädellä, huokosten kautta sisään voidaan kapseloida lääkeainetta, materiaalina se on hyvin siedettyä ja se on biohajoava. Tämän väitöskirjan tavoitteena oli kehittää sopiva huokoisiin piihiukkasiin perustuva lääkkeenkuljetin, jotta insuliinia ja GLP-1 peptidiä voidaan antaa suun kautta. Aluksi piihiukkasten pinnan kemiallisia ominaisuuksia tutkittiin, jotta saataisiin selville miten erilaiset piihiukkaset vaikuttaisivat insuliinin kulkeutumiseen suolistossa. Koska piihiukkasten pinta on negatiivisesti varautunut, niillä oli vähäinen vuorovaikutus ohutsuolen solujen kanssa. Lisäämällä kitosaania piihiukkasten pintaan, niiden varaus voitiin muuttaa positiiviseksi ja näin parantaa soluun kulkeutumista. Paras tapa liittää kitosaania hiukkasten pintaan oli kemiallinen konjugaatio. Näin valmistetut piihiukkaset aikaansaivat vahvimmat vuorovaikutukset solujen kanssa ja paransivat eniten solujen kykyä ottaa hiukkasia sisään. Jatkossa kehitettiin kolme erilaista nanohiukkasta lipidipohjainen, polymeeripohjainen ja huokoinen pii kitosaanilla muokattuna tai ilman sitä joiden soveltuvuutta oraalisessa GLP-1 proteiinin annossa tutkittiin. Näistä kitosaanilla muokatut piihiukkaset osoittautuivat tehokkaimmiksi kuljettamaan GLP-1 proteiinia solukerroksen läpi. Kitosaanilla päällystettyjä piihiukkasia kehitettiin lisää siten, että niihin lisättiin polymeeri, joka liukenee vain tietyssä pH:ssa. Näitä hiukkasia käytettiin kuljettamaan yhdessä GLP-1 peptidiä sekä DPP4 entsyymin estäjää. Tämä paransi GLP-1 peptidin kulkeutumista ohutsuolta mallintavien solujen läpi ja toimi samoin myös rotilla. Diabetesta sairastaville rotille annettuna nämä hiukkaset alensivat verensokeria. Tämä vaikutus voitiin selittää sillä, että kitosaanin vaikutuksesta hiukkaset läpäisivät soluja paremmin ja DPP4 entsyymin estäjän vaikutuksesta GLP-1 peptidi ei hajonnut niin helposti. Lisäksi pH herkkä polymeeri piihiukkasen pinnalla esti GLP-1 peptidin enneaikaisen vapautumisen ja hajoamisen mahassa. Muuttamalla kitosaanilla päällystettyjä huokoisia piihiukkasia lisää siten, että pinnallle kiinnitettiin L-kysteiini aminohappoa tai soluunottoa parantavaa CPP-peptidiä, voitiin parantaa lisää hiukkasten ominaisuutta kiinnittyä limakalvoon ja parantaa solujen läpäisevyyttä. Sähköstaattiset vuorovaikutukset nanohiukkasten ja solujen välillä oli tärkein mekanismi insuliinin soluihin kulkeutumisessa yhdessä aktiivisen kuljetuksen ja endosytoosin (adsorptio ja klatriinivälitteinen) kanssa. Väitöskirjatutkimuksessa onnistuttiin kehittämään huokoisiin piihiukkasiin perustuvia kehittyneitä insuliinin ja GLP-1 peptidin kuljetussysteemejä, joilla pystyttiin parantamaan näiden lääkeaineide

Spray drying of fenofibrate loaded nanostructured lipid carriers

AbstractThe conversion of aqueous dispersion of nanostructured lipid carriers (NLCs) into dry powder by spray drying could be a useful approach to render NLCs with better physical chemical stability than the aqueous dispersion. In this study, aqueous NLC dispersion containing fenofibrate was converted into dry, easily reconstitutable powder using spray drying. A central composite face centered design (CCFD) was used to investigate the influence of the ratio of lipid to protectant (mannitol and trehalose) and crystallinity of spray-dried powder on the particle size, yield and residual moisture content of the dried powder. A linear relationship (R2 = 0.9915) was established between the crystalline content of the spray-dried powders against the ratio of mannitol to trehalose from 3:7 to 10:0 (w/w). Spray drying of NLC aqueous dispersion using a mannitol and trehalose mixture resulted in an increase in particle size of the NLCs after reconstitution in water as compared to that in the initial aqueous dispersion. The decrease in crystallinity of the dry powder by reducing the ratio of mannitol to trehalose could improve the reconstitution of the NLCs in water. However the yield and residual moisture content of dry powder decreased with an increase in the ratio of mannitol to trehalose. Lipid nanoparticles were able to retain the drug incorporation and the prolonged drug release profile after spray drying. The experimental model was robust, and suggested that spray drying is a viable technique for the conversion of NLCs into dry powder

Strengthening Supply Chains for a Sustainable Housing Sector in Nepal : Factors Influencing the Organization, Management, Relationships and the Adoption and Use of Green Practices, Products and Services

EU’s main approach to sustainable housing is promoting green practices/products within the building construction sectors SCs. EU Switch Asia program financed research conducted in Nepal, 2015, to understand the organization of SCs, identify factors/barriers affecting SCM and existing relationships, the reasons to adopt green concepts/approaches and recommend support for SMEs to strengthen the sector’s management and sustainability. 109 companies, non-probabilistic snowball sampling and semi-closed questionnaires used in the survey. Conclusions: Companies create SCs to increase customer satisfaction, maintain long-term sales and retain clients. Size of company influences SCs organisation, SCM and using/producing green products/services; majority of participants had a person in the company to manage SC’s and had very good knowledge of SC, SCM and greening issues. SMEs need support with: information/communication technology, promotion and improvement/development of green products/services. Government should: increases awareness of green building products

Validating an Agency-based Tool for Measuring Women's Empowerment in a Complex Public Health Trial in Rural Nepal.

Despite the rising popularity of indicators of women's empowerment in global development programmes, little work has been done on the validity of existing measures of such a complex concept. We present a mixed methods validation of the use of the Relative Autonomy Index for measuring Amartya Sen's notion of agency freedom in rural Nepal. Analysis of think-aloud interviews (n = 7) indicated adequate respondent understanding of questionnaire items, but multiple problems of interpretation including difficulties with the four-point Likert scale, questionnaire item ambiguity and difficulties with translation. Exploratory Factor Analysis of a calibration sample (n = 511) suggested two positively correlated factors (r = 0.64) loading on internally and externally motivated behaviour. Both factors increased with decreasing education and decision-making power on large expenditures and food preparation. Confirmatory Factor Analysis on a validation sample (n = 509) revealed good fit (Root Mean Square Error of Approximation 0.05-0.08, Comparative Fit Index 0.91-0.99). In conclusion, we caution against uncritical use of agency-based quantification of women's empowerment. While qualitative and quantitative analysis revealed overall satisfactory construct and content validity, the positive correlation between external and internal motivations suggests the existence of adaptive preferences. High scores on internally motivated behaviour may reflect internalized oppression rather than agency freedom

Autoimmune Hepatitis Leading to Liver Cirrhosis: A Case Report

Autoimmune hepatitis is a rare form of chronic liver inflammation that begins as acute hepatitis and progresses to chronic liver disease. It presents with varied clinical features from acute hepatitis to chronic liver diseases like chronic viral hepatitis and alcoholic liver disease, making it difficult to diagnose in the absence of a high index of suspicion and adequate laboratory support. Autoimmune hepatitis is divided into two categories autoimmune hepatitis-1 and autoimmune hepatitis-2 based on the antibodies involved. We discuss the case of a 37-year-old woman who developed autoimmune hepatitis-1, with swelling and epigastric pain. These symptoms later progressed to liver cirrhosis leading to the death of the patient. Autoimmune hepatitis is extremely sensitive to immunosuppressive medication, it is necessary to maintain a high suspicion index for the disease because a prompt diagnosis can be an integral step toward a better prognosis of the disease

In vivo dual-delivery of glucagon like peptide -1 (GLP-1) and dipeptidyl peptidase-4 (DPP4) inhibitor through composites prepared by microfluidics for diabetes therapy

Oral delivery of proteins is still a challenge in the pharmaceutical field. Nanoparticles are among the most promising carrier systems for the oral delivery of proteins by increasing their oral bioavailability. However, most of the existent data regarding nanosystems for oral protein delivery is from in vitro studies, lacking in vivo experiments to evaluate the efficacy of these systems. Herein, a multifunctional composite system, tailored by droplet microfluidics, was used for dual delivery of glucagon like peptide-1 (GLP-1) and dipeptidyl peptidase-4 inhibitor (iDPP4) in vivo. Oral delivery of GLP-1 with nano- or micro-systems has been studied before, but the simultaneous nanodelivery of GLP-1 with iDPP4 is a novel strategy presented here. The type 2 diabetes mellitus (T2DM) rat model, induced through the combined administration of streptozotocin and nicotinamide, a non-obese model of T2DM, was used. The combination of both drugs resulted in an increase in the hypoglycemic effects in a sustained, but prolonged manner, where the iDPP4 improved the therapeutic efficacy of GLP-1. Four hours after the oral administration of the system, blood glucose levels were decreased by 44%, and were constant for another 4 h, representing half of the glucose area under the curve when compared to the control. An enhancement of the plasmatic insulin levels was also observed 6 h after the oral administration of the dual-drug composite system and, although no statistically significant differences existed, the amount of pancreatic insulin was also higher. These are promising results for the oral delivery of GLP-1 to be pursued further in a chronic diabetic model study.Peer reviewe

Microfluidic assembly of multistage porous silicon-lipid vesicles for controlled drug release

A reliable microfluidic platform for the generation of stable and monodisperse multistage drug delivery systems is reported. A glass-capillary flow-focusing droplet generation device was used to encapsulate thermally hydrocarbonized porous silicon (PSi) microparticles into the aqueous cores of double emulsion drops, yielding the formation of a multistage PSi–lipid vesicle. This composite system enables a large loading capacity for hydrophobic drugs.Peer reviewe

Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome