ARDS Acute respiratory distress syndrome BSL Biosafety level 3CLpro 3C-like serine protease COVID-19 Coronavirus disease 2019 This article has not been copyedited and formatted. The final version may differ from this version.

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Islet β ‐cell membrane excitability is a well‐established regulator of mammalian insulin secretion, and defects in β ‐cell excitability are linked to multiple forms of diabetes. Evolutionary conservation of islet excitability in lower organisms is largely unexplored. Here we show that adult zebrafish islet calcium levels rise in response to elevated extracellular [glucose], with similar concentration–response relationship to mammalian β ‐cells. However, zebrafish islet calcium transients are nor well coupled, with a shallower glucose‐dependence of cytoplasmic calcium concentration. We have also generated transgenic zebrafish that conditionally express gain‐of‐function mutations in ATP‐sensitive K + channels (K ATP ‐GOF) in β ‐cells. Following induction, these fish become profoundly diabetic, paralleling features of mammalian diabetes resulting from equivalent mutations. K ATP ‐GOF fish become severely hyperglycemic, with slowed growth, and their islets lose glucose‐induced calcium responses. These results indicate that, although lacking tight cell‐cell coupling of intracellular Ca 2+ , adult zebrafish islets recapitulate similar excitability‐driven β ‐cell glucose responsiveness to those in mammals, and exhibit profound susceptibility to diabetes as a result of inexcitability. While illustrating evolutionary conservation of islet excitability in lower vertebrates, these results also provide important validation of zebrafish as a suitable animal model in which to identify modulators of islet excitability and diabetes.

Introduction: 441Discussion: 3727 List of abbreviationsORF, open reading frame; NSP, non-structural protein; 3CLpro, 3-chymotrypsin-like protease; PLpro, papain-like protease; ACE2, angiotensin-converting enzyme 2; TMPRSS2, transmembrane protease serine 2; RdRp, RNA-dependent RNA polymerase; ISG15, interferon-stimulated gene 15 protein; CPE, cytopathic effect; HTS, high throughput screening; FRET, fluorescence resonance energy transfer; AMC, 7-amino-4-methylcoumarin This article has not been copyedited and formatted. The final version may differ from this version.

Persistent hyperglycemia is causally associated with pancreatic β-cell dysfunction and loss of pancreatic insulin. Glucose normally enhances β-cell excitability through inhibition of K ATP channels, opening of voltage-dependent calcium channels, increased [Ca 2+ ] i , which triggers insulin secretion. Glucose-dependent excitability is lost in islets from K ATP -knockout (K ATP -KO) mice, in which β-cells are permanently hyperexcited, [Ca 2+ ] i, is chronically elevated and insulin is constantly secreted. Mouse models of human neonatal diabetes in which K ATP gain-of-function mutations are expressed in β-cells (K ATP -GOF) also lose the link between glucose metabolism and excitation-induced insulin secretion, but in this case K ATP -GOF β-cells are chronically underexcited, with permanently low [Ca 2+ ] i and lack of glucose-dependent insulin secretion. We used K ATP -GOF and K ATP -KO islets to examine the role of altered-excitability in glucotoxicity. Wild-type islets showed rapid loss of insulin content when chronically incubated in high-glucose, an effect that was reversed by subsequently switching to low glucose media. In contrast, hyperexcitable K ATP -KO islets lost insulin content in both low- and high-glucose, while underexcitable K ATP -GOF islets maintained insulin content in both conditions. Loss of insulin content in chronic excitability was replicated by pharmacological inhibition of K ATP by glibenclamide, The effects of hyperexcitable and underexcitable islets on glucotoxicity observed in in vivo animal models are directly opposite to the effects observed in vitro : we clearly demonstrate here that in vitro , hyperexcitability is detrimental to islets whereas underexcitability is protective.

Progressive loss of pancreatic β-cell functional mass and anti-diabetic drug responsivity are classic findings in diabetes, frequently attributed to compensatory insulin hypersecretion and β-cell exhaustion. However, loss of β-cell mass and identity still occurs in mouse models of human KATP-gain-of-function induced Neonatal Diabetes Mellitus (NDM), in the absence of insulin secretion. Here we studied the temporal progression and mechanisms underlying glucotoxicity-induced loss of functional β-cell mass in NDM mice, and the effects of sodium-glucose transporter 2 inhibitors (SGLT2i) therapy. Upon tamoxifen induction of transgene expression, NDM mice rapidly developed severe diabetes followed by an unexpected loss of insulin content, decreased proinsulin processing and increased proinsulin at 2-weeks of diabetes. These early events were accompanied by a marked increase in β-cell oxidative and ER stress, without changes in islet cell identity. Strikingly, treatment with the SGLT2 inhibitor dapagliflozin restored insulin content, decreased proinsulin:insulin ratio and reduced oxidative and ER stress. However, despite reduction of blood glucose, dapagliflozin therapy was ineffective in restoring β-cell function in NDM mice when it was initiated at >40 days of diabetes, when loss of β-cell mass and identity had already occurred. Our data from mouse models demonstrate that: i) hyperglycemia per se, and not insulin hypersecretion, drives β-cell failure in diabetes, ii) recovery of β-cell function by SGLT2 inhibitors is potentially through reduction of oxidative and ER stress, iii) SGLT2 inhibitors revert/prevent β-cell failure when used in early stages of diabetes, but not when loss of β-cell mass/identity already occurred, iv) common execution pathways may underlie loss and recovery of β-cell function in different forms of diabetes. These results may have important clinical implications for optimal therapeutic interventions in individuals with diabetes, particularly for those with long-standing diabetes.

Together, our results suggest that restriction of dietary carbohydrates and caloric replacement by fat can induce metabolic changes that are beneficial in reducing glucotoxicity and secondary consequences of diabetes in a mouse model of insulin-secretory deficiency.

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