An inducible model of chronic hyperglycemia.

Transgene driven expression of Escherichia coli nitroreductase (NTR1.0) renders animal cells susceptible to the antibiotic metronidazole (MTZ). Many NTR1.0/MTZ ablation tools have been reported in zebrafish, which have significantly impacted regeneration studies. However, NTR1.0-based tools are not appropriate for modeling chronic cell loss as prolonged application of the required MTZ dose (10 mM) is deleterious to zebrafish health. We established that this dose corresponds to the median lethal dose (LD50) of MTZ in larval and adult zebrafish and that it induced intestinal pathology. NTR2.0 is a more active nitroreductase engineered from Vibrio vulnificus NfsB that requires substantially less MTZ to induce cell ablation. Here, we report on the generation of two new NTR2.0-based zebrafish lines in which acute ß-cell ablation can be achieved without MTZ-associated intestinal pathology. For the first time, we were able to sustain ß-cell loss and maintain elevated glucose levels (chronic hyperglycemia) in larvae and adults. Adult fish showed significant weight loss, consistent with the induction of a diabetic state, indicating that this paradigm will allow the modeling of diabetes and associated pathologies.

A Receptor Story: Insulin Resistance Pathophysiology and Physiologic Insulin Resensitization's Role as a Treatment Modality.

Physiologic insulin secretion consists of an oscillating pattern of secretion followed by distinct trough periods that stimulate ligand and receptor activation. Apart from the large postprandial bolus release of insulin, ß cells also secrete small amounts of insulin every 4-8 min independent of a meal. Insulin resistance is associated with a disruption in the normal cyclical pattern of insulin secretion. In the case of type-2 diabetes, ß-cell mass is reduced due to apoptosis and ß cells secrete insulin asynchronously. When ligand/receptors are constantly exposed to insulin, a negative feedback loop down regulates insulin receptor availability to insulin, creating a relative hyperinsulinemia. The relative excess of insulin leads to insulin resistance (IR) due to decreased receptor availability. Over time, progressive insulin resistance compromises carbohydrate metabolism, and may progress to type-2 diabetes (T2D). In this review, we discuss insulin resistance pathophysiology and the use of dynamic exogenous insulin administration in a manner consistent with more normal insulin secretion periodicity to reverse insulin resistance. Administration of insulin in such a physiologic manner appears to improve insulin sensitivity, lower HgbA1c, and, in some instances, has been associated with the reversal of end-organ damage that leads to complications of diabetes. This review outlines the rationale for how the physiologic secretion of insulin orchestrates glucose metabolism, and how mimicking this secretion profile may serve to improve glycemic control, reduce cellular inflammation, and potentially improve outcomes in patients with diabetes.

Physiologic Insulin Resensitization as a Treatment Modality for Insulin Resistance Pathophysiology.

Prevalence of type 2 diabetes increased from 2.5% of the US population in 1990 to 10.5% in 2018. This creates a major public health problem, due to increases in long-term complications of diabetes, including neuropathy, retinopathy, nephropathy, skin ulcers, amputations, and atherosclerotic cardiovascular disease. In this review, we evaluated the scientific basis that supports the use of physiologic insulin resensitization. Insulin resistance is the primary cause of type 2 diabetes. Insulin resistance leads to increasing insulin secretion, leading to beta-cell exhaustion or burnout. This triggers a cascade leading to islet cell destruction and the long-term complications of type 2 diabetes. Concurrent with insulin resistance, the regular bursts of insulin from the pancreas become irregular. This has been treated by the precise administration of insulin more physiologically. There is consistent evidence that this treatment modality can reverse the diabetes-associated complications of neuropathy, diabetic ulcers, nephropathy, and retinopathy, and that it lowers HbA1c. In conclusion, physiologic insulin resensitization has a persuasive scientific basis, significant treatment potential, and likely cost benefits.

SOX9 modulates cancer biomarker and cilia genes in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer with high mortality. The cellular origins of PDAC are largely unknown; however, ductal cells, especially centroacinar cells (CACs), have several characteristics in common with PDAC, such as expression of SOX9 and components of the Notch-signaling pathway. Mutations in KRAS and alterations to Notch signaling are common in PDAC, and both these pathways regulate the transcription factor SOX9. To identify genes regulated by SOX9, we performed siRNA knockdown of SOX9 followed by RNA-seq in PANC-1s, a human PDAC cell line. We report 93 differentially expressed (DE) genes, with convergence on alterations to Notch-signaling pathways and ciliogenesis. These results point to SOX9 and Notch activity being in a positive feedback loop and SOX9 regulating cilia production in PDAC. We additionally performed ChIP-seq in PANC-1s to identify direct targets of SOX9 binding and integrated these results with our DE gene list. Nine of the top 10 downregulated genes have evidence of direct SOX9 binding at their promoter regions. One of these targets was the cancer stem cell marker EpCAM. Using whole-mount in situ hybridization to detect epcam transcript in zebrafish larvae, we demonstrated that epcam is a CAC marker and that Sox9 regulation of epcam expression is conserved in zebrafish. Additionally, we generated an epcam null mutant and observed pronounced defects in ciliogenesis during development. Our results provide a link between SOX9, EpCAM and ciliary repression that can be exploited in improving our understanding of the cellular origins and mechanisms of PDAC.