Determination of Postprandial Glycemic Responses by Continuous Glucose Monitoring in a Real-World Setting.

BACKGROUND: Self-monitoring of blood glucose using capillary glucose testing (C) has a number of shortcomings compared to continuous glucose monitoring (CGM). We aimed to compare these two methods and used blood glucose measurements in venous blood (IV) as a reference. Postprandial blood glucose levels were measured after 50 g oral glucose load and after the consumption of a portion of different foods containing 50 g of carbohydrates. We also evaluated the associations between postprandial glucose responses and the clinical characteristics of the participants at the beginning of the study. METHODS: 12 healthy volunteers (age: 36 ± 17 years, BMI: 24.9 ± 3.5 kg/m²) ate white bread (WB) and whole grain (WG) bread and drank a 50 g glucose drink as reference. Postprandial glucose responses were evaluated by CGM, IV and C blood glucose measurements. Incremental area under the curve (AUCi) of postprandial blood glucose was calculated for 1 h (AUCi 0-60) and 2 h (AUCi 0-120). RESULTS: After the consumption of white bread and whole grain bread, the AUCi 0-60 min did not differ between CGM and IV or C. AUCi 0-120 min of CGM showed no difference compared to C. Correlation analyses revealed a positive association of age with glucose AUCi 0-120 (r = 0.768; P = 0.004) and WG AUCi 0-120 (r = 0.758; P = 0.004); fasting blood glucose correlated with WG AUCi 0-120 (r = 0.838; P < 0.001). CONCLUSION: Despite considerable inter-individual variability of postprandial glycemic responses, CGM evaluated postprandial glycemic excursions which had comparable results compared to standard blood glucose measurements under real-life conditions. Associations of AUCi 0-60 and AUCi 0-120 postprandial glucose response with age or fasting blood glucose could be shown.

Deletion of the RabGAP TBC1D1 Leads to Enhanced Insulin Secretion and Fatty Acid Oxidation in Islets From Male Mice.

The Rab guanosine triphosphatase-activating protein (RabGAP) TBC1D1 has been shown to be a key regulator of glucose and lipid metabolism in skeletal muscle. Its function in pancreatic islets, however, is not yet fully understood. Here, we aimed to clarify the specific impact of TBC1D1 on insulin secretion and substrate use in pancreatic islets. We analyzed the dynamics of glucose-stimulated insulin secretion (GSIS) and lipid metabolism in isolated islets from Tbc1d1-deficient (D1KO) mice. To further investigate the underlying cellular mechanisms, we conducted pharmacological studies in these islets. In addition, we determined morphology and number of both pancreatic islets and insulin vesicles in ß-cells using light and transmission electron microscopy. Isolated pancreatic islets from D1KO mice exhibited substantially increased GSIS compared with wild-type (WT) controls. This was attributed to both enhanced first and second phase of insulin secretion, and this enhanced secretion persisted during repetitive glucose stimuli. Studies with sulfonylureas or KCl in isolated islets demonstrated that TBC1D1 exerts its function via a signaling pathway at the level of membrane depolarization. In line, ultrastructural analysis of isolated pancreatic islets revealed both higher insulin-granule density and number of docked granules in ß-cells from D1KO mice compared with WT controls. Like in skeletal muscle, lipid use in isolated islets was enhanced upon D1KO, presumably as a result of a higher mitochondrial fission rate and/or higher mitochondrial activity. Our results clearly demonstrate a dual role of TBC1D1 in controlling substrate metabolism of the pancreatic islet.

The biomechanical properties of an epithelial tissue determine the location of its vasculature.

An important question is how growing tissues establish a blood vessel network. Here we study vascular network formation in pancreatic islets, endocrine tissues derived from pancreatic epithelium. We find that depletion of integrin-linked kinase (ILK) in the pancreatic epithelial cells of mice results in glucose intolerance due to a loss of the intra-islet vasculature. In turn, blood vessels accumulate at the islet periphery. Neither alterations in endothelial cell proliferation, apoptosis, morphology, Vegfa expression and VEGF-A secretion nor 'empty sleeves' of vascular basement membrane are found. Instead, biophysical experiments reveal that the biomechanical properties of pancreatic islet cells, such as their actomyosin-mediated cortex tension and adhesive forces to endothelial cells, are significantly changed. These results suggest that a sorting event is driving the segregation of endothelial and epithelial cells and indicate that the epithelial biomechanical properties determine whether the blood vasculature invades or envelops a growing epithelial tissue.

Sheep primary cells as in vitro models to investigate Mycoplasma agalactiae host cell interactions.

Appropriate infection models are imperative for the understanding of pathogens like mycoplasmas that are known for their strict host and tissue specificity, and lack of suitable cell and small animal models has hindered pathogenicity studies. This is particularly true for the economically important group of ruminant mycoplasmas whose virulence factors need to be elucidated for designing effective intervention strategies. Mycoplasma agalactiae serves as a useful role model especially because it is phylogenetically very close to M. bovis and causes similar symptoms by as yet unknown mechanisms. Here, we successfully prepared and characterized four different primary sheep cell lines, namely the epithelial and stromal cells from the mammary gland and uterus, respectively. Using immunohistochemistry, we identified vimentin and cytokeratin as specific markers to confirm the typical cell phenotypes of these primary cells. Furthermore, M. agalactiae's consistent adhesion and invasion into these primary cells proves the reliability of these cell models. Mimicking natural infections, mammary epithelial and stromal cells showed higher invasion and adhesion rates compared to the uterine cells as also seen via double immunofluorescence staining. Altogether, we have generated promising in vitro cell models to study host-pathogen interactions of M. agalactiae and related ruminant pathogens in a more authentic manner.

Characterization of pancreatic NMDA receptors as possible drug targets for diabetes treatment.

In the nervous system, NMDA receptors (NMDARs) participate in neurotransmission and modulate the viability of neurons. In contrast, little is known about the role of NMDARs in pancreatic islets and the insulin-secreting beta cells whose functional impairment contributes to diabetes mellitus. Here we found that inhibition of NMDARs in mouse and human islets enhanced their glucose-stimulated insulin secretion (GSIS) and survival of islet cells. Further, NMDAR inhibition prolonged the amount of time that glucose-stimulated beta cells spent in a depolarized state with high cytosolic Ca(2+) concentrations. We also noticed that, in vivo, the NMDAR antagonist dextromethorphan (DXM) enhanced glucose tolerance in mice, and that in vitro dextrorphan, the main metabolite of DXM, amplified the stimulatory effect of exendin-4 on GSIS. In a mouse model of type 2 diabetes mellitus (T2DM), long-term treatment with DXM improved islet insulin content, islet cell mass and blood glucose control. Further, in a small clinical trial we found that individuals with T2DM treated with DXM showed enhanced serum insulin concentrations and glucose tolerance. Our data highlight the possibility that antagonists of NMDARs may provide a useful adjunct treatment for diabetes.

High level protein-purification allows the unambiguous polypeptide determination of latent isoform PPO4 of mushroom tyrosinase.

Tyrosinases catalyze two initial reaction steps in the formation of melanin. Purification of tyrosinases had always been a process accompanied with various problems caused by enzymatic browning processes. Here, an approach is presented for the purification of the latent enzyme from mushrooms which averts and removes interfering compounds (e.g. polyphenols) in advance to the extraction process. The described method is supposed being well suitable as a general protein purification protocol from natural sources like fungi and plants. The purified enzyme was investigated in detail by means of mass spectrometry: its intact protein mass was determined as 64,247.3 Da and it was identified as number four of in total six isoforms (PPO1-6) by means of sequence analysis. Some PTMs, strain specific sequence disparities and several cleavage sites including the one causing enzyme-activation (Ser³8³) were determined, thus, providing insights on the maturation process of this latent tyrosinase zymogen. Based on these sequence data it can be concluded that the polypeptide backbone of the latent form of the tyrosinase PPO4 ranges from Ser² to Thr565, missing when compared to the gene-derived sequence a small part (46 amino acids) of the C-terminal tail. The high content on hydrophobic amino acids within this missing tail gives rise to speculations whether this part might have a function as a membrane anchor.

Muscle and connective tissue progenitor populations show distinct Twist1 and Twist3 expression profiles during axolotl limb regeneration.

Limb regeneration involves re-establishing a limb development program from cells within adult tissues. Identifying molecular handles that provide insight into the relationship between cell differentiation status and cell lineage is an important step to study limb blastema cell formation. Here, using single cell PCR, focusing on newly isolated Twist1 sequences, we molecularly profile axolotl limb blastema cells using several progenitor cell markers. We link their molecular expression profile to their embryonic lineage via cell tracking experiments. We use in situ hybridization to determine the spatial localization and extent of overlap of different markers and cell types. Finally, we show by single cell PCR that the mature axolotl limb harbors a small but significant population of Twist1(+) cells.

Calcineurin/NFATc signaling: role in postnatal ß cell development and diabetes mellitus.

Pancreatic islets are aggregates of endocrine cells required for blood glucose control and diabetes prevention after birth. In this issue of Developmental Cell, Goodyer et al. (2012) reveal a function of calcineurin, a calcium-activated serine/threonine phosphatase, in postnatal NFATc-regulated expression of genes that help ß cells to form insulin-containing vesicles and enter the cell cycle.

'Giving and taking': endothelial and beta-cells in the islets of Langerhans.

The beta-cells of the islets of Langerhans are embedded in a dense capillary network. The blood vessels supply the islet cells with nutrients and oxygen, and in turn take up the secreted islet hormones to deliver them to target tissues. In addition, vessels provide a basement membrane, which optimizes islet function. In this review we focus on the dynamic interactions between blood vessels and beta-cells, which are pivotal for enhancing insulin expression and beta-cell proliferation in response to increased insulin demand during body growth, pregnancy, and virtually all conditions associated with insulin resistance. Importantly, a failure in this adaptive response might contribute to the onset of type 2 diabetes mellitus.

Basement membrane in pancreatic islet function.

Clinical treatment of diabetic patients by islet transplantation faces various complications. At present, in vitro expansion of islets occurs at the cost of their essential features, which are insulin production and release. However, the recent discovery of blood vessel/beta-cell interactions as an important aspect of insulin transcription, secretion, and proliferation might point us to ways of how this problem could be overcome. The correct function of beta-cells depends on the presence of a basement membrane, a specialized extracellular matrix located around the blood vessel wall in mouse and human pancreatic islets. In this chapter, we summarize how the vascular basement membrane influences insulin transcription, insulin secretion, and beta-cell proliferation. In addition, a brief overview about basement membrane components and their interactions with cell surface receptors is given.

Cells keep a memory of their tissue origin during axolotl limb regeneration.

During limb regeneration adult tissue is converted into a zone of undifferentiated progenitors called the blastema that reforms the diverse tissues of the limb. Previous experiments have led to wide acceptance that limb tissues dedifferentiate to form pluripotent cells. Here we have reexamined this question using an integrated GFP transgene to track the major limb tissues during limb regeneration in the salamander Ambystoma mexicanum (the axolotl). Surprisingly, we find that each tissue produces progenitor cells with restricted potential. Therefore, the blastema is a heterogeneous collection of restricted progenitor cells. On the basis of these findings, we further demonstrate that positional identity is a cell-type-specific property of blastema cells, in which cartilage-derived blastema cells harbour positional identity but Schwann-derived cells do not. Our results show that the complex phenomenon of limb regeneration can be achieved without complete dedifferentiation to a pluripotent state, a conclusion with important implications for regenerative medicine.

Hedgehog signaling controls dorsoventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration.

Tail regeneration in urodeles requires the coordinated growth and patterning of the regenerating tissues types, including the spinal cord, cartilage and muscle. The dorsoventral (DV) orientation of the spinal cord at the amputation plane determines the DV patterning of the regenerating spinal cord as well as the patterning of surrounding tissues such as cartilage. We investigated this phenomenon on a molecular level. Both the mature and regenerating axolotl spinal cord express molecular markers of DV progenitor cell domains found during embryonic neural tube development, including Pax6, Pax7 and Msx1. Furthermore, the expression of Sonic hedgehog (Shh) is localized to the ventral floor plate domain in both mature and regenerating spinal cord. Patched1 receptor expression indicated that hedgehog signaling occurs not only within the spinal cord but is also transmitted to the surrounding blastema. Cyclopamine treatment revealed that hedgehog signaling is not only required for DV patterning of the regenerating spinal cord but also had profound effects on the regeneration of surrounding, mesodermal tissues. Proliferation of tail blastema cells was severely impaired, resulting in an overall cessation of tail regeneration, and blastema cells no longer expressed the early cartilage marker Sox9. Spinal cord removal experiments revealed that hedgehog signaling, while required for blastema growth is not sufficient for tail regeneration in the absence of the spinal cord. By contrast to the cyclopamine effect on tail regeneration, cyclopamine-treated regenerating limbs achieve a normal length and contain cartilage. This study represents the first molecular localization of DV patterning information in mature tissue that controls regeneration. Interestingly, although tail regeneration does not occur through the formation of somites, the Shh-dependent pathways that control embryonic somite patterning and proliferation may be utilized within the blastema, albeit with a different topography to mediate growth and patterning of tail tissues during regeneration.

Vitamin B2 (riboflavin) and a mixture of vitamin B2 and C affects MMC efficiency in aerated media under irradiation.

Vitamin B2 (Riboflavin) acts as a strong radiation protecting agent in Escherichia coli bacteria (AB1157) in aerated media. This ability is reinforced by the addition of vitamin C. Under the influence of gamma-radiation, vitamin B2 completely suppresses the cytostatic activity of mitomycin C (MMC). In the presence of both vitamins, B2 and C, MMC is converted from an efficient cytostatic to a rather strong radiation protecting agent. This effect opens a new pathway for specific protection of normal mammalian cells (with a high O2-content) under treatment with ionizing radiation.