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Gene editing of human pluripotent stem cells is a promising approach for developing targeted gene therapies for metabolic diseases. Here, we present a protocol for generating a CRISPR-Cas12a gene knockout of protein tyrosine phosphatases in human embryonic stem cells. We describe steps for differentiating the edited clones into pancreatic islet-like spheroids rich in β-like cells. We then detail procedures for implanting these spheroids under the murine kidney capsule for in vivo maturation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

An altered gut microbiota is associated with type 1 diabetes (T1D), affecting the production of short-chain fatty acids (SCFA) and glucose homeostasis. We previously demonstrated that enhancing serum acetate and butyrate using a dietary supplement (HAMSAB) improved glycemia in non-obese diabetic (NOD) mice and patients with established T1D. The effects of SCFA on immune-infiltrated islet cells remain to be clarified. Here, we performed single-cell RNA sequencing on islet cells from NOD mice fed an HAMSAB or control diet. HAMSAB induced a regulatory gene expression profile in pancreas-infiltrated immune cells. Moreover, HAMSAB maintained the expression of β-cell functional genes and decreased cellular stress. HAMSAB-fed mice showed preserved pancreatic endocrine cell identity, evaluated by decreased numbers of poly-hormonal cells. Finally, SCFA increased insulin levels in human β-like cells and improved transplantation outcome in NOD/SCID mice. Our findings support the use of metabolite-based diet as attractive approach to improve glucose control in T1D., Competing Interests: E.M. is an inventor on a patent WO2018027274A1 submitted by Monash University that covers methods and compositions of metabolites for treatment and prevention of autoimmune disease related to this paper and stock ownership for ImmunoBiota Therapeutics Pty Ltd. E.N.G. declares that there are no other relationships or activities that might bias, or be perceived to bias, the present work., (© 2023 The Author(s).)

Obesity-related complications such as non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) are well-established risk factors for the development of hepatocellular carcinoma (HCC). This review provides insights into the molecular mechanisms that underlie the role of steatosis, hyperinsulinemia and hepatic inflammation in HCC development and progression. We focus on recent findings linking intracellular pathways and transcription factors that can trigger the reprogramming of hepatic cells. In addition, we highlight the role of enzymes in dysregulated metabolic activity and consequent dysfunctional signalling. Finally, we discuss the potential uses and challenges of novel therapeutic strategies to prevent and treat NAFLD/T2D-associated HCC., (© 2023 The Author(s).)

Objectives: Type 1 diabetes (T1D) is caused by progressive immune-mediated loss of insulin-producing β-cells. Inflammation is detrimental to β-cell function and survival, moreover, both apoptosis and necrosis have been implicated as mechanisms of β-cell loss in T1D. The receptor interacting serine/threonine protein kinase 1 (RIPK1) promotes inflammation by serving as a scaffold for NF-κB and MAPK activation, or by acting as a kinase that triggers apoptosis or necroptosis. It is unclear whether RIPK1 kinase activity is involved in T1D pathology. In the present study, we investigated if absence of RIPK1 activation would affect the susceptibility to immune-mediated diabetes or diet induced obesity (DIO)., Methods: The RIPK1 knockin mouse line carrying a mutation mimicking serine 25 phosphorylation (Ripk1 S25D/S25D ), which abrogates RIPK1 kinase activity, was utilized to assess the in vivo role of RIPK1 in immune-mediated diabetes or diet induced obesity (DIO). In vitro, β-cell death and RIPK1 kinase activity was analysed in conditions known to induce RIPK1-dependent apoptosis/necroptosis., Results: We demonstrate that Ripk1 S25D/S25D mice presented normal glucose metabolism and β-cell function. Furthermore, immune-mediated diabetes and DIO were not different between Ripk1 S25D/S25D and Ripk1 +/+ mice. Despite strong activation of RIPK1 kinase and other necroptosis effectors (RIPK3 and MLKL) by TNF+BV6+zVAD, no cell death was observed in mouse islets nor human β-cells., Conclusion: Our results contrast recent literature showing that most cell types undergo necroptosis following RIPK1 kinase activation. This peculiarity may reflect an adaptation to the inability of β-cells to proliferate and self-renewal., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The loss of functional pancreatic β-cell mass is an important hallmark of both type 1 and type 2 diabetes. The RNA-binding protein NOVA1 is expressed in human and rodent pancreatic β-cells. Previous in vitro studies indicated that NOVA1 is necessary for glucose-stimulated insulin secretion and its deficiency-enhanced cytokine-induced apoptosis. Moreover, Bim, a proapoptotic protein, is differentially spliced and potentiates apoptosis in NOVA1-deficient β-cells in culture. We generated two novel mouse models by Cre-Lox technology lacking Nova1 (βNova1 -/- ) or Bim (βBim -/- ) in β-cells. To test the impact of Nova1 or Bim deletion on β-cell function, mice were subjected to multiple low-dose streptozotocin (MLD-STZ)-induced diabetes or high-fat diet-induced insulin resistance. β-cell-specific Nova1 or Bim deficiency failed to affect diabetes development in response to MLD-STZ-induced β-cell dysfunction and death evidenced by unaltered blood glucose levels and pancreatic insulin content. In addition, body composition, glucose and insulin tolerance test, and pancreatic insulin content were indistinguishable between control and βNova1 -/- or βBim -/- mice on a high fat diet. Thus, Nova1 or Bim deletion in β-cells does not impact on glucose homeostasis or diabetes development in mice. Together, these data argue against an in vivo role for the Nova1-Bim axis in β-cells.

The transcription factor nuclear factor-κB (NF-κB) has a key role in the pathogenesis of diabetes and its complications. Although activation of the canonical NF-κB pathway in β-cells is generally deleterious, little is known about the role of the non-canonical NF-κB signalling and its main regulator, the NF-κB-inducing kinase (NIK), on pancreatic β-cell survival and function. Previous studies based on models of NIK overexpression in pancreatic islet cells showed that NIK induced either spontaneous β-cell death due to islet inflammation or glucose intolerance during diet-induced obesity (DIO) in mice. Therefore, NIK has been proposed as a potential target for diabetes therapy. However, no clear studies showed whether inhibition of NIK improves diabetes development. Here we show that genetic silencing of NIK in pancreatic β-cells neither modifies diabetes incidence nor inflammatory responses in a mouse model of immune-mediated diabetes. Moreover, NIK silencing in DIO mice did not influence body weight gain, nor glucose metabolism. In vitro studies corroborated the in vivo findings in terms of β-cell survival, function, and downstream gene regulation. Taken together, our data suggest that NIK activation is dispensable for the development of diabetes., (© 2022. The Author(s).)

Type 1 diabetes (T1D) results from autoimmune destruction of β-cells in the pancreas. Protein tyrosine phosphatases (PTPs) are candidate genes for T1D and play a key role in autoimmune disease development and β-cell dysfunction. Here, we assessed the global protein and individual PTP profiles in the pancreas from nonobese mice with early-onset diabetes (NOD) mice treated with an anti-CD3 monoclonal antibody and interleukin-1 receptor antagonist. The treatment reversed hyperglycemia, and we observed enhanced expression of PTPN2, a PTP family member and T1D candidate gene, and endoplasmic reticulum (ER) chaperones in the pancreatic islets. To address the functional role of PTPN2 in β-cells, we generated PTPN2-deficient human stem cell-derived β-like and EndoC-βH1 cells. Mechanistically, we demonstrated that PTPN2 inactivation in β-cells exacerbates type I and type II interferon signaling networks and the potential progression toward autoimmunity. Moreover, we established the capacity of PTPN2 to positively modulate the Ca2+-dependent unfolded protein response and ER stress outcome in β-cells. Adenovirus-induced overexpression of PTPN2 partially protected from ER stress-induced β-cell death. Our results postulate PTPN2 as a key protective factor in β-cells during inflammation and ER stress in autoimmune diabetes., (© 2022 by the American Diabetes Association.)

Background: Short-chain fatty acids (SCFAs) produced by the gut microbiota have beneficial anti-inflammatory and gut homeostasis effects and prevent type 1 diabetes (T1D) in mice. Reduced SCFA production indicates a loss of beneficial bacteria, commonly associated with chronic autoimmune and inflammatory diseases, including T1D and type 2 diabetes. Here, we addressed whether a metabolite-based dietary supplement has an impact on humans with T1D. We conducted a single-arm pilot-and-feasibility trial with high-amylose maize-resistant starch modified with acetate and butyrate (HAMSAB) to assess safety, while monitoring changes in the gut microbiota in alignment with modulation of the immune system status., Results: HAMSAB supplement was administered for 6 weeks with follow-up at 12 weeks in adults with long-standing T1D. Increased concentrations of SCFA acetate, propionate, and butyrate in stools and plasma were in concert with a shift in the composition and function of the gut microbiota. While glucose control and insulin requirements did not change, subjects with the highest SCFA concentrations exhibited the best glycemic control. Bifidobacterium longum, Bifidobacterium adolescentis, and vitamin B7 production correlated with lower HbA1c and basal insulin requirements. Circulating B and T cells developed a more regulatory phenotype post-intervention., Conclusion: Changes in gut microbiota composition, function, and immune profile following 6 weeks of HAMSAB supplementation were associated with increased SCFAs in stools and plasma. The persistence of these effects suggests that targeting dietary SCFAs may be a mechanism to alter immune profiles, promote immune tolerance, and improve glycemic control for the treatment of T1D., Trial Registration: ACTRN12618001391268. Registered 20 August 2018, https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=375792 Video Abstract., (© 2022. The Author(s).)

Type 1 diabetes develops in childhood and adolescence, with peak incidence in the early teenage years. There is an urgent need for an accurate method to detect insulin-producing β-cells in patients that is not affected by alterations in β-cell function. As part of our research program to design specific probes to measure β-cell mass, we recently developed a novel insulin-binding peptide probe (IBPP) for the detection of β-cells in vivo. Here, we applied our innovative method to show specific labeling of this IBPP to human and mouse fixed β-cells in pancreatic islets. Importantly, we showed staining of human and mouse islets in culture without any negative functional or cell viability impact. Moreover, the IBPP-stained mouse islets after tail vein injection in vivo, albeit with batch differences in staining efficiency. In conclusion, we provide evidence showing that the IBPP can be used for future accurate detection of β-cell mass in a variety of preclinical models of diabetes.