Optogenetic ß cell interrogation in vivo reveals a functional hierarchy directing the Ca2+ response to glucose supported by vitamin B6.

Coordination of cellular activity through Ca2+ enables ß cells to secrete precise quantities of insulin. To explore how the Ca2+ response is orchestrated in space and time, we implement optogenetic systems to probe the role of individual ß cells in the glucose response. By targeted ß cell activation/inactivation in zebrafish, we reveal a hierarchy of cells, each with a different level of influence over islet-wide Ca2+ dynamics. First-responder ß cells lie at the top of the hierarchy, essential for initiating the first-phase Ca2+ response. Silencing first responders impairs the Ca2+ response to glucose. Conversely, selective activation of first responders demonstrates their increased capability to raise pan-islet Ca2+ levels compared to followers. By photolabeling and transcriptionally profiling ß cells that differ in their thresholds to a glucose-stimulated Ca2+ response, we highlight vitamin B6 production as a signature pathway of first responders. We further define an evolutionarily conserved requirement for vitamin B6 in enabling the Ca2+ response to glucose in mammalian systems.

Simultaneous Calcium Imaging and Glucose Stimulation in Living Zebrafish to Investigate In Vivo ß-Cell Function.

The pancreatic ß-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in ß-cell function are associated with hyperglycemia that can lead to diabetes. During the process of insulin secretion, ß-cells experience an influx of Ca2+. Thus, imaging the glucose-stimulated Ca2+ influx using genetically encoded calcium indicators (GECIs) provides an avenue to studying ß-cell function. Previously, studies showed that isolated zebrafish islets expressing GCaMP6s exhibit significant Ca2+ activity upon stimulation with defined glucose concentrations. However, it is paramount to study how ß-cells respond to glucose not in isolation, but in their native environment, where they are systemically connected, vascularized, and densely innervated. To this end, the study leveraged the optical transparency of the zebrafish larvae at early stages of development to illuminate ß-cell activity in vivo. Here, a detailed protocol for Ca2+ imaging and glucose stimulation to investigate ß-cell function in vivo is presented. This technique allows to monitor the coordinated Ca2+ dynamics in ß-cells with single-cell resolution. Additionally, this method can be applied to work with any injectable solution such as small molecules or peptides. Altogether, the protocol illustrates the potential of the zebrafish model to investigate islet coordination in vivo and to characterize how environmental and genetic components might affect ß-cell function.

The expression signatures in liver and adipose tissue from obese Göttingen Minipigs reveal a predisposition for healthy fat accumulation.

BACKGROUND: Model animals are valuable resources for dissecting basic aspects of the regulation of obesity and metabolism. The translatability of results relies on understanding comparative aspects of molecular pathophysiology. Several studies have shown that despite the presence of overt obesity and dyslipidemia in the pig key human pathological hepatic findings such as hepatocellular ballooning and abundant steatosis are lacking in the model. OBJECTIVES: The aim of this study was to elucidate why these histopathological characteristics did not occur in a high fat, fructose and cholesterol (FFC) diet-induced obese Göttingen Minipig model. METHODS: High-throughput expression profiling of more than 90 metabolically relevant genes was performed in liver, subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) of male minipigs diet fed: standard chow (SD, n = 7); FFC diet (n = 14); FFC diet in streptozotocin-induced diabetic pigs (FFCDIA, n = 8). Moreover, histopathological assessment of SAT and VAT was performed. RESULTS: 12, 4 and 1 genes were highly significantly differentially expressed in liver, SAT and VAT when comparing the FFC and SD groups whereas the corresponding numbers were 15, 2, and 1 when comparing the FFCDIA and SD groups. Although the minipigs in both FFC groups developed sever obesity and dyslipidemia, the insulin-signaling pathways were not affected. Notably, four genes involved in lipid acquisition and removal, were highly deregulated in the liver: PPARG, LPL, CD36 and FABP4. These genes have been reported to play a major role in promoting hepatic steatosis in rodents and humans. Since very little macrophage-associated pro-inflammatory response was detected in the adipose tissues the expansion appears to have no adverse impact on adipose tissue metabolism. CONCLUSION: The study shows that morbidly obese Göttingen Minipigs are protected against many of the metabolic and hepatic abnormalities associated with obesity due to a remarkable ability to expand the adipose compartments to accommodate excess calories.

Gene expression of the two developmentally regulated dermatan sulfate epimerases in the Xenopus embryo.

Chondroitin sulfate (CS)/dermatan sulfate (DS) proteoglycans are abundant on the cell surface and in the extracellular matrix and have important functions in matrix structure, cell-matrix interaction and signaling. The DS epimerases 1 and 2, encoded by Dse and Dsel, respectively, convert CS to a CS/DS hybrid chain, which is structurally and conformationally richer than CS, favouring interaction with matrix proteins and growth factors. We recently showed that Xenopus Dse is essential for the migration of neural crest cells by allowing cell surface CS/DS proteoglycans to adhere to fibronectin. Here we investigate the expression of Dse and Dsel in Xenopus embryos. We show that both genes are maternally expressed and exhibit partially overlapping activity in the eyes, brain, trigeminal ganglia, neural crest, adenohypophysis, sclerotome, and dorsal endoderm. Dse is specifically expressed in the epidermis, anterior surface ectoderm, spinal nerves, notochord and dermatome, whereas Dsel mRNA alone is transcribed in the spinal cord, epibranchial ganglia, prechordal mesendoderm and myotome. The expression of the two genes coincides with sites of cell differentiation in the epidermis and neural tissue. Several expression domains can be linked to previously reported phenotypes of knockout mice and clinical manifestations, such as the Musculocontractural Ehlers-Danlos syndrome and psychiatric disorders.