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BACKGROUND: A growing body of literature on Roux-en-Y gastric bypass surgery (RYGB) has generated inconclusive results on the mechanism underlying the beneficial effects on weight loss and glycaemia, partially due to the problems of designing clinical studies with the appropriate controls. Moreover, RYGB is only performed in obese individuals, in whom metabolism is perturbed and not completely understood.METHODS: In an attempt to isolate the effects of RYGB and its effects on normal metabolism, we investigated the effect of RYGB in lean pigs, using sham-operated pair-fed pigs as controls. Two weeks post-surgery, pigs were subjected to an intravenous glucose tolerance test (IVGTT) and circulating metabolites, hormones and lipids measured. Bile acid composition was profiled after extraction from blood, faeces and the gallbladder.RESULTS: A similar weight development in both groups of pigs validated our experimental model. Despite similar changes in fasting insulin, RYGB-pigs had lower fasting glucose levels. During an IVGTT RYGB-pigs had higher insulin and lower glucose levels. VLDL and IDL were lower in RYGB- than in sham-pigs. RYGB-pigs had increased levels of most amino acids, including branched-chain amino acids, but these were more efficiently suppressed by glucose. Levels of bile acids in the gallbladder were higher, whereas plasma and faecal bile acid levels were lower in RYGB- than in sham-pigs.CONCLUSION: In a lean model RYGB caused lower plasma lipid and bile acid levels, which were compensated for by increased plasma amino acids, suggesting a switch from lipid to protein metabolism during fasting in the immediate postoperative period.

Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.

The most frequently used, and effective, treatment for morbid obesity is Roux-en-Y gastric bypass surgery (RYGB), which results in rapid remission of T2D in most cases. To what extent this is accounted for by weight loss or other factors remains elusive. To gain insight into these mechanisms, we investigated the effects of RYGB on ß-cell function and ß-cell mass in the pig, a species highly reminiscent of the human. RYGB was performed using linear staplers during open surgery. Sham-operated pigs were used as controls. Both groups were fed a low calorie diet for 3 weeks after surgery. Intravenous glucose-tolerance tests were performed 2 weeks after surgery. Body weight in RYGB-pigs and sham-operated, pair-fed control pigs developed similarly. RYGB-pigs displayed improved glycaemic control, which was attributed to increases in ß-cell mass, islet number and number of extra-islet ß-cells. Pancreatic expression of insulin and glucagon was elevated, and cells expressing the GLP-1-receptor were more abundant in RYGB-pigs. Our data from a pig model of RYGB emphasize the key role of improved ß-cell function and ß-cell mass to explain the improved glucose tolerance after RYGB as food intake and body weight remained identical.

The myeloid translocation gene 16 product MTG16 is found in multiple transcription factor-containing complexes as a regulator of gene expression implicated in development and tumorigenesis. A stable Tet-On system for doxycycline-dependent expression of MTG16 was established in B-lymphoblastoid Raji cells to unravel its molecular functions in transformed cells. A noticeable finding was that expression of certain genes involved in tumor cell metabolism including 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 and 4 (PFKFB3 and PFKFB4), and pyruvate dehydrogenase kinase isoenzyme 1 (PDK1) was rapidly diminished when MTG16 was expressed. Furthermore, hypoxia-stimulated production of PFKFB3, PFKFB4 and PDK1 was inhibited by MTG16 expression. The genes in question encode key regulators of glycolysis and its coupling to mitochondrial metabolism and are commonly found to be overexpressed in transformed cells. The MTG16 Nervy Homology Region 2 (NHR2) oligomerization domain and the NHR3 protein-protein interaction domain were required intact for inhibition of PFKFB3, PFKFB4 and PDK1 expression to occur. Expression of MTG16 reduced glycolytic metabolism while mitochondrial respiration and formation of reactive oxygen species increased. The metabolic changes were paralleled by increased phosphorylation of mitogen-activated protein kinases, reduced levels of amino acids and inhibition of proliferation with a decreased fraction of cells in S-phase. Overall, our findings show that MTG16 can serve as a brake on glycolysis, a stimulator of mitochondrial respiration and an inhibitor of cell proliferation. Hence, elevation of MTG16 might have anti-tumor effect.

Diabetic patients have a much more widespread and aggressive form of atherosclerosis and therefore, higher risk for myocardial infarction, peripheral vascular disease and stroke, but the molecular mechanisms leading to accelerated damage are still unclear. Recently, we showed that hyperglycemia activates the transcription factor NFAT in the arterial wall, inducing the expression of the pro-atherosclerotic protein osteopontin. Here we investigate whether NFAT activation may be a link between diabetes and atherogenesis.

The influence of instrumental parameters affecting the ionization in continuous full filling capillary electrochromatography/electrospray ionization mass spectrometry (CFF-CEC/ESI-MS) was investigated. The investigated parameters were the BGE and sheath liquid ion strength and organic modifier content, the nebulizer gas pressure, and the concentration of nanoparticles in the BGE. It was found that the nebulizer pressure had the largest influence on the separation efficiency and apparent retention. It was shown that even the lowest pressure investigated was sufficient to guide the nanoparticle flow away from the mass spectrometer inlet. A nebulizer pressure of 5 psi was found to be optimal; increasing the pressure significantly decreased the separation efficiency due to the generation of a hydrodynamic flow. Generally, the ion strength of both the BGE and the sheath liquid were found to have very moderate effects on the separation of a homologous series of dialkyl phthalates, whereas the ionization efficiency was found to be unaffected by the nanoparticles and the separation efficiency was found to increase with increasing concentrations up to 3.8 mg/mL, whereafter it was observed to drop. The optimized method was linear over a wide concentration range and presented LOD and LOQ more than threefold lower than those previously reported using CFF-CEC/ESI-MS.

Insulin secretion from pancreatic beta-cells is controlled by complex metabolic and energetic changes provoked by exposure to metabolic fuels. Perturbations in these processes lead to impaired insulin secretion, the ultimate cause of T2D (Type 2 diabetes). To increase our understanding of stimulus secretion coupling and metabolic processes potentially involved in the pathogenesis of T2D, a comprehensive investigation of the metabolic response in the glucose-responsive INS-1 832/13 and glucose-unresponsive INS-1 832/2 beta-cell lines was performed. For this metabolomics analysis, we used GC/MS (gas chromatography/mass spectrometry) combined with multivariate statistics. We found that perturbed secretion in the 832/2 line was characterized by disturbed coupling of glycolytic and TCA (tricarboxylic acid)-cycle metabolism. The importance of this metabolic coupling was reinforced by our observation that insulin secretion partially could be reinstated by stimulation of the cells with mitochondrial fuels which bypass glycolytic metabolism. Furthermore, metabolic and functional profiling of additional beta-cell lines (INS-1, INS-1 832/1) confirmed the important role of coupled glycolytic and TCA-cycle metabolism in stimulus-secretion coupling. Dependence of the unresponsive clones on glycolytic metabolism was paralleled by increased stabilization of HIF-1 alpha (hypoxia-inducible factor 1 alpha). The relevance of a similar perturbation for human T2D was suggested by increased expression of HIF-1 alpha target genes in islets from T2D patients.

Genome-wide association studies have shown that variation in MTNR1B (melatonin receptor 1B) is associated with insulin and glucose concentrations. Here we show that the risk genotype of this SNP predicts future type 2 diabetes (T2D) in two large prospective studies. Specifically, the risk genotype was associated with impairment of early insulin response to both oral and intravenous glucose and with faster deterioration of insulin secretion over time. We also show that the MTNR1B mRNA is expressed in human islets, and immunocytochemistry confirms that it is primarily localized in beta cells in islets. Nondiabetic individuals carrying the risk allele and individuals with T2D showed increased expression of the receptor in islets. Insulin release from clonal beta cells in response to glucose was inhibited in the presence of melatonin. These data suggest that the circulating hormone melatonin, which is predominantly released from the pineal gland in the brain, is involved in the pathogenesis of T2D. Given the increased expression of MTNR1B in individuals at risk of T2D, the pathogenic effects are likely exerted via a direct inhibitory effect on beta cells. In view of these results, blocking the melatonin ligand-receptor system could be a therapeutic avenue in T2D.