The number of people suffering from Diabetes mellitus in both developed and developing nations has increased dramatically over the past two-three decades and this increase is predicted. The condition can result in numerous complications in the patients suffering from the disease if it is not with long-term effects including retinopathy with potential blindness, nephropathy that may lead to renal failure, and/or neuropathy and an increased risk of cardiovascular, peripheral vascular and cerebrovascular disease. Camel milk consumption has been reported to have a positive effect with respect to diabetic severity and complications and as a treatment of diabetes with regular camel milk drinkers requiring lower doses of insulin to control their blood glucose levels. A number of studies have now suggested a direct link between camel milk insulin and the effect of drinking camel milk on diabetic suffers. In this study, insulin from dromedary camel (Camelus dromedaries) milk was isolated and characterized in order to investigate the stability and nature of the protein to begin to understand why it may have positive benefits on patients consuming camel milk Initia1ly the camel insulin gene was amplified and sequenced from 32 camels using primers used to amplify the camel pro insulin cDNA. The sequenced fragment was aligned using a BLAST search and the sequence matched a sequence from lama with 96% homology. A number of SNPs were identified in key regions of the camel gene sequence, although none in the coding sequence with 3 located in the C-peptide intron, 2 in the 3' -UTR and 1 after the termination signal. When the gene sequence was converted to the corresponding amino acid sequence this revealed that across the 13 species compared the camel insulin has two unique amino_ acid changes in the signal peptide, one unique amino acid change in the B-chain and two changes in the A-chain compared to human (which are observed in some other species). There is much variation across species in the C-peptide region and in the camel there were various changes compared to other species. Using the sequence, 3D homology modeling of camel proinsulin was undertaken which suggests the formation of different secondary structure compared to that for human insulin which may impact upon the stability of the molecule. Using FISH, the camel insulin gene was also mapped to the distal (Telomeric) end of the q-arm of camel chromosome 10. In addition to investigating camel insulin at the genomic level, studies were undertaken at the protein level. For protein analysis, fresh camel milk samples were collected from the Emirates dairy farm. The samples were initially defatted and then treated with ethanol in order to precipitate casein which is the major milk protein and other large molecular weight proteins, before the supernatant was passed through an ion-exchange chromatography column of A-25 DEAE Sephadex beads. The bound camel milk insulin was then eluted and further purified by immune-affinity chromatography using an anti -human insulin antibody. The resulting insulin concentration and activity was measured by radioimmunoassay using a human insulin kit. The camel insulin gene was also cloned into a mammalian expression vector and transfected into a mammalian cell line and attempts made to produce cell lines stably expressing camel insulin. The thermostability of the insulin purified from camel milk was then compared with human and bovine insulin in camel and bovine milk. The results suggest that camel insulin appears to be stabilised due to the unique both amino acid variants in comparison to human insulin but these studies also suggested that camel milk itself provides protection against thermostability which bovine milk does not and this property contributed the most towards the enhanced thermostability of camel milk insulin. The exact nature of this protection and the agents responsible are not currently known, but further elucidation of the mechanism(s) may provide possible routes for the thermostabilisation of other proteins.

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Obesity, metabolic syndrome and type 2 diabetes are all characterised by insulin resistance and in most cases large intra-abdominal fat stores. Insulin resistance is associated with the dysregulation of adipose tissue (AT) Metab including a reduced effectiveness for insulin to suppress lipolysis. Although patients with type 2 diabetes arc treated with metformin initially. the majority of patients eventually need treatment with insulin to maintain glucose control. Insulin treatment with most types of insulin therapy is associated with weight gain. However the insulin analogue detemir is weight neutral or causes weight loss and it has been suggested that this may be related to a reduced effect of this' insulin in peripheral tissues such as AT. A high sugar intake (fructose and sucrose) is associated with insulin resistance but the effects' on AT Metab are not established. Methods were developed to measure cell sizes, in vitro basal, stimulated and insulin inhibited lipolysis and lipoprotein lipase (LPL) activity in AT biopsies, These methods were then applied to study a) subcutaneous AT (SCAT) and omental AT (OMAT) biopsies from twelve participants, seven non-diabetic subjects and five patients with type 2 diabetes treated with metformin (T20H) undergoing abdominal surgery and b) the effect of treatment of patients with type 2 diabetes with insulin detemir (n=7) or insulin neutral protamine hagedorn (NPH) (n~5), in a 16 week parallel group study. The effect of sugar intake on the in vivo rate of lipolysis and post-heparin lipolysis was investigated in 25 men at risk of metabolic syndrome, In a cross-over study design participants were studied after a 12 week diet high in extrinsic sugars and low in extrinsic sugars Adipocyte size of SCAT and OMAT was significantly larger in T20H than healthy participants (p<0.001, p<0.05 respectively). In healthy subjects OMAT had higher basal, isoproterenol stimulated and insulin inhibited lipolysis than SCAT (p<0.0I, p<0.001, p<0.05 respectively). In participants with T20R, stimulated lipolysis with isoproterenol in SCAT was significantly higher than OMAT. but there was no significant difference in basal and insulin inhibited lipolysis. There was no significant difference between healthy and T20H in SCAT lipolytic activity, but OMAT has higher basal, isoproterenol stimulated and insulin inhibited lipolysis than SCAT (p<0.05, p<0.001, p<0.01 respectively) in healthy participants. LPL activity in OMAT was significantly higher than SCAT in both healthy and T20H group (p<0.001,p<0.05 respectively). In the insulin detemir clinical trial, fasting non-esterified fatty acids (NEFA) decreased significantly with detemir compared to NPH (p<0.05) . Basal lipolysis in the fat biopsies showed a significant reduction with detemir treatment (p<0.05) with no significant change following NPH treatment. LPL mass and activity increased significantly after 16 weeks treatment with detemir compared to NPH (p=0.006, p=0.005). No significant differences were observed on stimulated lipolysis or adipocyte size following either of the treatments. In the dietary intervention study after the high sugar diet compared with low 'sugar diet, liver fat, fasting plasma glucose and NEF A concentrations were significantly higher in men with higher liver fat at screening (all p<0.05). In the low-liver fat group, plasma TG (p=0.07) NEF A (p=0.04) and LPL activity (p<0.05) were higher after the low versus high sugar diet, whereas, in the high-liver fat group, hepatic lipase (HL) activity was higher after the high versus the low sugar diet (p<0.05). The palmitate (P A) concentration decreased significantly after the high sugar diet compared with low sugar diet (p<0.001) in the high liver fat group. In this group, the percentage of palmitate to total NEFA in the circulation (P A:NEF A) decreased significantly after the high sugar diet, this value was significantly lower than the value after the high sugar diet in the low liver fat group (p<0.05). However, the palmitate rate of appearance increased after the high sugar diet in the high liver fat group, but did not reach significance (p=0.06). Conclusion: This study showed that OMAT had less sensitivity to insulin action than SCAT in T20H. In the clinical trial of insulin detemir, the decrease in basal NEFA levels and basal lipolysis and the increased LPL mass and activity suggests that detemir improves fasting insulin sensitivity. This may be due to a greater hepatoselective action of detemir reducing over-insulinisation of adipose tissue and skeletal muscle. The change in plasma lipid Metab and pattern of response in lipase activities in response to a diet high in extrinsic sugars increased the availability of NEFA from peripheral and endothelial lipolysis; this may be due to reduced insulin sensitivity in participants with high liver fat.

Amphibian skin secretions are considered to be one of the richest resources of bioactive molecules in the animal kingdom for pharmaceutical prospecting. This thesis has investigated amphibian skin peptides of eight different species of anurans (frogs and toads) for insulin-releasing and anti-diabetic properties. The skins of Pseudis paradoxa, Hylarana guntheri, Hylomantis lemur and Leptodactylus laticeps, were tested for the presence of peptides with stimulatory effects on insulin-release from the clonal BRIN-BDll cell line. Upon purification of the skin secretions/extract, multiple insulin-releasing peptides were isolated and fully characterised by mass spectrometry along with N-terminal amino acid sequencing by Edman degradation. A number of synthetic peptides demonstrated potent in vitro biological activities, possessing the ability to stimulate insulin release up to a concentration of 3 flM without any association of cellular toxicity. For example, brevinin-2GUb isolated from the skin extract of H. guntheri stimulated insulin release 3.7 fold compared to 5.6 mmol/l glucose control (P<O.OOI). Under similar experimental conditions, the insulin output of several synthetic peptides identified, were akin to the well-characterised incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-l (GLP-I). Structure/function studies presented suggest the insulin-releasing potency of a peptide was related to charge and hydrophobicity. Mechanistic studies on the following synthetic peptides; pseudin-2 (ps-2), [Lys-18]_ps_2, [Phe 8]_ps_2, brevinin-2GUb, phylloseptin-L2, and [Asp23]ocellatin-Ll suggest that their stimulatory effects on insulin secretion were mediated via calcium independent pathways. Additionally, when tested in vivo each of these peptides, displayed valuable anti-diabetic properties, lowering blood glucose and increasing insulin following an intraperitoneal glucose load (18 mmol/kg). The skin extract of Rana luteiventris has previously been established as a rich source of antimicrobial peptides. Fourteen purified fractions with varying degrees of antimicrobial activity were tested for their ability to stimulate insulin-release. Serial dilutions gave an insight to the potency of the peptides. Multiple insulin- releasing non-toxic peptides belonging to ranatuerin, brevinin, temporin and esculentin families were identified. Mechanistic studies indicated that, ranatuerin- 2Lb, esculentin-2L, brevinin-1Lb, and 3 unclassified peptides stimulated insulin release via Ca2+ independent pathways. In contrast, bradykinin, ternporin-I La and temporin-l Lb appeared to stimulate insulin secretion via a calcium dependent mechanism. The most effective insulinotropic peptide identified from the skin extract of R. luteiventris was esculentin-2L. Skin extracts of Bufo arabicus, Buergeria buergeri and Hoplobatrachus rugulosus were also investigated. Following reverse-phase HPLC purification, a number of non-toxic insulin-releasing peptides were identified. This included peptides isolated from the skin of B. arabicus and B. buergeri as well as two from H. rugulosus (Molecular mass; 745.1, 1758.3, 1305.8 and 1368.5 Da, respectively). In conclusion, this work has identified novel peptides from the skins of various amphibians with stimulatory effects on insulin release and, in some cases, promising glucose lowering effects in vivo. Such an approach into nature's untapped resources may ultimately offer novel agents with valuable anti-diabetic properties for the future treatment of type 2 diabetes.

Leptin and insulin act as adiposity signals signalling in the brain to regulate energy homeostasis. However, in contrast to leptin, the precise details of the cell types and signalling pathways involved in the actions of insulin have not been well defined. The dominant view in the field at the commencement of this work was largely extrapolated from studies on leptin action. It was therefore suggested that insulin exerted its effects on energy balance by inhibiting orexigenic NPY/AgRP and activating anorexigenic POMC/CART neurons of the arcuate nucleus region of the hypothalamus, thereby co-ordinately regulating energy homeostasis. Mouse genetic studies have demonstrated that insulin receptor substrate (IRS) 2, a major downstream effector of insulin signalling, plays a key role in the regulation of glucose and energy homeostasis. Mice lacking Irs2 in all tissues exhibit insulin resistance, hyperglycaemia and (3-cell failure. In addition, Irs2 null female mice are hyperphagjc, obese and infertile. However, the precise contribution of CNS IRS2 signalling in the actions of insulin and leptin, and the identity of the neuronal circuits in which IRS2 acts to regulate energy homeostasis, are unclear. The PI3K signalling pathway has been implicated in mediating the effects of insulin and leptin, in part acting downstream of IRS signalling. However, the precise hypothalamic cell types in which PI3K signalling acts also remain to be defined. To address these issues, mice with deletion of Irs2 in all neurons (NesCreIrs2KO POMC neurons (POMCCreIrs2KO) and AgRP neurons (AgRPCreIrs2KO) were generated. Animals lacking the PI3K pi 10(3 catalytic subunit in POMC (POMCCrepllOfiKO) and AgRP neurons (AgRPCrepllOfiKO) were also generated. NesCreIrs2KO animals were obese, hyperphagic and long, suggesting altered melanocortin function. Despite hyperleptinaemia, NesCreIrs2KO animals were leptin sensitive suggesting that IRS2 pathways are not required for leptin action. Reproductive function in NesCreIrs2KO females was normal. In addition, NesCreIrs2KO mice displayed hyperglycaemia, mild glucose intolerance and hyperinsulinaemia. In contrast, POMCCreIrs2KO and AgRPCreIrs2KO mice exhibited normal hypothalamic function and glucose homeostasis. AgRPCrepl 10/3KO mice were lean and hypophagic. Conversely, POMCCrepllOfiKO animals demonstrated increased adiposity and are hyperphagia. Taken together, these studies highlight a key role for CNS IRS2 pathways in the regulation of energy homeostasis but demonstrate that CNS IRS2 pathways act in neuronal populations distinct from POMC and AgRP/NPY neurons to regulate energy homeostasis. In contrast, pllOp-mediated signals in POMC and AgRP neurons play a key role in the regulation of energy homeostasis. Overall, these studies have provided new insights into the role insulin receptor substrate signalling mechanisms in the hypothalamic regulation of energy homeostasis.

Background - The alternatively spliced insulin receptor (IR) exon 11 (36 nucleotides) and the constitutively spliced Apolipoprotein B (APOB) exon 26 (7572 nucleotides) are examples of the shortest and longest exons in the genome. Aim - The aim of this study was to investigate the regulation of splicing of these 2 exons in cell culture using 2′-O-methyl RNA antisense oligonucleotides (ASOs) and peptide nucleic acid (PNA)-peptide hybrid ASOs. Methods - ASOs were designed to target key sequences involved in the splicing of the IR exon 11 and exonic splicing silencer sequences (ESS) in APOB exon 26. HepG2 cells were reverse-transfected with the ASOs for 48 hours, mRNA harvested and RT-PCR was performed to amplify the IR isoform and APOB cDNAs which were separated by PAGE and quantified. Results Insulin receptor exon 11 - 2′-O-methyl RNA ASOs targeted to two intronic sites, the 3′ half of exon 11 and spanning the entire exon caused significant exon skipping. PNA-peptide hybrids predicted to increase exon 11 splicing, paradoxically caused exon skipping. PNA-peptide hybrids with 3′ tails caused exon 11 skipping more effectively than hybrids with 5′ tails. Apolipoprotein B exon 26 - Only combinations of 2′-O-methyl RNA ASOs targeting multiple ESSs in APOB exon 26 caused a small proportion of aberrant splicing. This consisted of complete exon 26 skipping and the selection of a downstream cryptic 3′ splice site in preference to the native 3′ splice site. Discussion - Exclusion of the IR exon 11 can be induced by targeting a combination of intronic or exonic sequences. PNA-peptide hybrid ASOs were unable to increase exon 11 splicing. The aberrant splicing of large constitutive exons such as APOB exon 26 can be induced by targeting multiple ESS sites along its course.

The metabolic syndrome is highly prevalent in western society, and the numbers affected by obesity and diabetes continue to rise. This thesis reviews the mechanisms at play and the gaps in the literature that, if filled, may increase knowledge of treatment regimes for affected individuals. Experimentally, it was demonstrated that the oral glucose tolerance test can be a reliable tool to measure insulin sensitivity following adequate dietary and exercise control. Acute and chronic cinnamon ingestion was shown to improve insulin sensitivity. Feeding frequency was found to alter insulin and ghrelin responses and relationships following mixed-meal ingestion. And finally, postprandial lipaemia was found to be attenuated for up to 24 hours following moderate-intensity exercise, illustrating the requirement of daily exercise. In summary, oral glucose tolerance tests are suitable for experimental interventions; and the clinical management of factors associated with the metabolic syndrome should perhaps consider dietary supplements, meal frequency, and exercise timing in addition to the traditional dietary and physical activity guidelines currently in practice.