Hyperglucagonaemia in diabetes: altered amino acid metabolism triggers mTORC1 activation, which drives glucagon production.

AIM/HYPOTHESIS: Hyperglycaemia is associated with alpha cell dysfunction, leading to dysregulated glucagon secretion in type 1 and type 2 diabetes; however, the mechanisms involved are still elusive. The nutrient sensor mammalian target of rapamycin complex 1 (mTORC1) plays a major role in the maintenance of alpha cell mass and function. We studied the regulation of alpha cell mTORC1 by nutrients and its role in the development of hyperglucagonaemia in diabetes. METHODS: Alpha cell mTORC1 activity was assessed by immunostaining for phosphorylation of its downstream target, the ribosomal protein S6, and glucagon, followed by confocal microscopy on pancreatic sections and flow cytometry on dispersed human and mouse islets and the alpha cell line, αTC1-6. Metabolomics and metabolic flux were studied by 13C glucose labelling in 2.8 or 16.7 mmol/l glucose followed by LC-MS analysis. To study the role of mTORC1 in mediating hyperglucagonaemia in diabetes, we generated an inducible alpha cell-specific Rptor knockout in the Akita mouse model of diabetes and tested the effects on glucose tolerance by IPGTT and on glucagon secretion. RESULTS: mTORC1 activity was increased in alpha cells from diabetic Akita mice in parallel to the development of hyperglycaemia and hyperglucagonaemia (two- to eightfold increase). Acute exposure of mouse and human islets to amino acids stimulated alpha cell mTORC1 (3.5-fold increase), whereas high glucose concentrations inhibited mTORC1 (1.4-fold decrease). The mTORC1 response to glucose was abolished in human and mouse diabetic alpha cells following prolonged islet exposure to high glucose levels, resulting in sustained activation of mTORC1, along with increased glucagon secretion. Metabolomics and metabolic flux analysis showed that exposure to high glucose levels enhanced glycolysis, glucose oxidation and the synthesis of glucose-derived amino acids. In addition, chronic exposure to high glucose levels increased the expression of Slc7a2 and Slc38a4, which encode amino acid transporters, as well as the levels of branched-chain amino acids and methionine cycle metabolites (~1.3-fold increase for both). Finally, conditional Rptor knockout in alpha cells from adult diabetic mice inhibited mTORC1, thereby inhibiting glucagon secretion (~sixfold decrease) and improving diabetes, despite persistent insulin deficiency. CONCLUSIONS/INTERPRETATION: Alpha cell exposure to hyperglycaemia enhances amino acid synthesis and transport, resulting in sustained activation of mTORC1, thereby increasing glucagon secretion. mTORC1 therefore plays a major role in mediating alpha cell dysfunction in diabetes. DATA AVAILABILITY: All sequencing data are available from the Gene Expression Omnibus (GEO) repository (accession no. GSE154126; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE154126 ).

Induction of ß-cell replication by a synthetic HNF4α antagonist.

Increasing the number of ß cells is critical to a definitive therapy for diabetes. Previously, we discovered potent synthetic small molecule antagonists of the nuclear receptor transcription factor HNF4α. The natural ligands of HNF4α are thought to be fatty acids. Because obesity, in which there are high circulating levels of free fatty acids, is one of the few conditions leading to ß-cell hyperplasia, we tested the hypothesis that a potent HNF4α antagonist might stimulate ß-cell replication. A bioavailable HNF4α antagonist was injected into normal mice and rabbits and ß-cell ablated mice and the effect on ß-cell replication was measured. In normal mice and rabbits, the compound induced ß-cell replication and repressed the expression of multiple cyclin-dependent kinase inhibitors, including p16 that plays a critical role in suppressing ß-cell replication. Interestingly, in ß-cell ablated mice, the compound induced α- and δ-cell, in addition to ß-cell replication, and ß-cell number was substantially increased. Overall, the data presented here are consistent with a model in which the well-known effects of obesity and high fat diet on ß-cell replication occur by inhibition of HNF4α. The availability of a potent synthetic HNF4α antagonist raises the possibility that this effect might be a viable route to promote significant increases in ß-cell replication in diseases with reduced ß-cell mass, including type I and type II diabetes.

Par2-mediated responses in inflammation and regeneration: choosing between repair and damage.

The protease activated receptor 2 (Par2) plays a pivotal role in various damage models, influencing injury, proliferation, inflammation, and regeneration. Despite extensive studies, its binary roles- EITHER aggravating injury or promoting recovery-make a conclusive translational decision on its modulation strategy elusive. Analyzing two liver regeneration models, autoimmune hepatitis and direct hepatic damage, we discovered Par2's outcome depends on the injury's nature. In immune-mediated injury, Par2 exacerbates damage, while in direct tissue injury, it promotes regeneration. Subsequently, we evaluated the clinical significance of this finding by investigating Par2's expression in the context of autoimmune diabetes. We found that the absence of Par2 in all lymphocytes provided full protection against the autoimmune destruction of insulin-producing ß-cells in mice, whereas the introduction of a ß-cell-specific Par2 null mutation accelerated the onset of autoimmune diabetes. This pattern led us to hypothesize whether these observations are universal. A comprehensive review of recent Par2 publications across tissues and systems confirms the claim drafted above: Par2's initial activation in the immune system aggravates inflammation, hindering recovery, whereas its primary activation in the damaged tissue fosters regeneration. As a membrane-anchored receptor, Par2 emerges as an attractive drug target. Our findings highlight a crucial translational modulation strategy in regenerative medicine based on injury type.

Controlling autoimmune diabetes onset by targeting Protease-Activated Receptor 2.

BACKGROUND: Type 1 diabetes (T1D) is a challenging autoimmune disease, characterized by an immune system assault on insulin-producing ß-cells. As insulin facilitates glucose absorption into cells and tissues, ß-cell deficiency leads to elevated blood glucose levels on one hand and target-tissues starvation on the other. Despite efforts to halt ß-cell destruction and stimulate recovery, success has been limited. Our recent investigations identified Protease-Activated Receptor 2 (Par2) as a promising target in the battle against autoimmunity. We discovered that Par2 activation's effects depend on its initial activation site: exacerbating the disease within the immune system but fostering regeneration in affected tissue. METHODS: We utilized tissue-specific Par2 knockout mice strains with targeted Par2 mutations in ß-cells, lymphocytes, and the eye retina (as a control) in the NOD autoimmune diabetes model, examining T1D onset and ß-cell survival. RESULTS: We discovered that Par2 expression within the immune system accelerates autoimmune processes, while its presence in ß-cells offers protection against ß-cell destruction and T1D onset. This suggests a dual-strategy treatment for T1D: inhibiting Par2 in the immune system while activating it in ß-cells, offering a promising strategy for T1D. CONCLUSIONS: This study highlights Par2's potential as a drug target for autoimmune diseases, particularly T1D. Our results pave the way for precision medicine approaches in treating autoimmune conditions through targeted Par2 modulation.

Algorithm of myogenic differentiation in higher-order organisms.

Cell fate determination is governed by complex signaling molecules at appropriate concentrations that regulate the cell decision-making process. In vertebrates, however, concentration and kinetic parameters are practically unknown, and therefore the mechanism by which these molecules interact is obscure. In myogenesis, for example, multipotent cells differentiate into skeletal muscle as a result of appropriate interplay between several signaling molecules, which is not sufficiently characterized. Here we demonstrate that treatment of biochemical events with SAT (satisfiability) formalism, which has been primarily applied for solving decision-making problems, can provide a simple conceptual tool for describing the relationship between causes and effects in biological phenomena. Specifically, we applied the Lukasiewicz logic to a diffusible protein system that leads to myogenesis. The creation of an automaton that describes the myogenesis SAT problem has led to a comprehensive overview of this non-trivial phenomenon and also to a hypothesis that was subsequently verified experimentally. This example demonstrates the power of applying Lukasiewicz logic in describing and predicting any decision-making problem in general, and developmental processes in particular.

Resolving the conflicts around Par2 opposing roles in regeneration by comparing immune-mediated and toxic-induced injuries.

BACKGROUND: Different factors may lead to hepatitis. Among which are liver inflammation and poisoning. We chose two hepatitis models, typical for these two underlying causes. Thus, we aimed to characterize the role of protease-activated receptor 2 (Par2) in liver regeneration and inflammation to reconcile Par2 conflicting role in many damage models, which sometimes aggravates the induced damage and sometimes alleviates it. METHODS: WT and knockout (Par2KO) mice were injected with concanavalin A (ConA) to induce immune-mediated hepatitis or with carbon tetrachloride (CCl4) to elicit direct hepatic damage. To distinguish the immune component from the liver regenerative response, we conducted bone marrow (BM) replacements of WT and Par2KO mice and repeated the damage models. RESULTS: ConA injection caused limited damage in Par2KO mice livers, while in the WT mice severe damage followed by leukocyte infiltration was evident. Reciprocal BM replacement of WT and Par2KO showed that WT BM-reconstituted Par2KO mice displayed marked liver damage, while in Par2KO BM-reconstituted WT mice, the tissue was generally protected. In the CCl4 direct damage model, hepatocytes regenerated in WT mice, whereas Par2KO mice failed to recover. Reciprocal BM replacement did not show significant differences in hepatic regeneration. In Par2KO mice, hepatitis was more apparent, while WT recovered regardless of the BM origin. CONCLUSIONS: We conclude that Par2 activation in the immune system aggravates hepatitis and that Par2 activation in the damaged tissue promotes liver regeneration. When we incorporate this finding and revisit the literature reports, we reconciled the conflicts surrounding Par2's role in injury, recovery, and inflammation.

Pancreatic ß-cell neogenesis by direct conversion from mature α-cells.

Because type 1 and type 2 diabetes are characterized by loss of ß-cells, ß-cell regeneration has garnered great interest as an approach to diabetes therapy. Here, we developed a new model of ß-cell regeneration, combining pancreatic duct ligation (PDL) with elimination of pre-existing ß-cells with alloxan. In this model, in which virtually all ß-cells observed are neogenic, large numbers of ß-cells were generated within 2 weeks. Strikingly, the neogenic ß-cells arose primarily from α-cells. α-cell proliferation was prominent following PDL plus alloxan, providing a large pool of precursors, but we found that ß-cells could form from α-cells by direct conversion with or without intervening cell division. Thus, classical asymmetric division was not a required feature of the process of α- to ß-cell conversion. Intermediate cells coexpressing α-cell- and ß-cell-specific markers appeared within the first week following PDL plus alloxan, declining gradually in number by 2 weeks as ß-cells with a mature phenotype, as defined by lack of glucagon and expression of MafA, became predominant. In summary, these data revealed a novel function of α-cells as ß-cell progenitors. The high efficiency and rapidity of this process make it attractive for performing the studies required to gain the mechanistic understanding of the process of α- to ß-cell conversion that will be required for eventual clinical translation as a therapy for diabetes.

Current Approaches in Diabetes Treatment and Other Strategies to Reach Normoglycemia.

Cells are mainly dependent on glucose as their energy source. Multicellular organisms need to adequately control individual glucose uptake by the cells, and the insulin-glucagon endocrine system serves as the key glucose regulation mechanism. Insulin allows for effective glucose entry into the cells when blood glucose levels are high, and glucagon acts as its opponent, balancing low blood glucose levels. A lack of insulin will prevent glucose entry to the cells, resulting in glucose accumulation in the bloodstream. Diabetes is a disease which is characterized by elevated blood glucose levels. All diabetes types are characterized by an inefficient insulin signaling mechanism. This could be the result of insufficient insulin secretion, as in the case of type I diabetes and progressive incidents of type II diabetes or due to insufficient response to insulin (known as insulin resistance). We emphasize here, that Diabetes is actually a disease of starved tissues, unable to absorb glucose (and other nutrients), and not a disease of high glucose levels. Indeed, diabetic patients, prior to insulin discovery, suffered from glucose malabsorption. In this mini-review, we will define diabetes, discuss the current status of diabetes treatments, review the current knowledge of the different hormones that participate in glucose homeostasis and the employment of different modulators of these hormones. As this issue deals with peptide therapeutics, special attention will be given to synthetic peptide analogs, peptide agonists as well as antagonists.

Gestational diabetes induces behavioral and brain gene transcription dysregulation in adult offspring.

The etiology of Autism Spectrum Disorders (ASD) includes a strong genetic component and a complicated environmental component. Recent evidence indicates that maternal diabetes, including gestational diabetes, is associated with an increased prevalence of ASD. While previous studies have looked into possible roles for maternal diabetes in neurodevelopment, there are few studies into how gestational diabetes, with no previous diabetic or metabolic phenotype, may affect neurodevelopment. In this study, we have specifically induced gestational diabetes in mice, followed by behavioral and molecular phenotyping of the mice offspring. Pregnant mice were injected with STZ a day after initiation of pregnancy. Glucose levels increased to diabetic levels between E7 and E14 in pregnancy in a subset of the pregnant animals. Male offspring of Gestational Diabetic mothers displayed increased repetitive behaviors with no dysregulation in the three-chambered social interaction test. RNA-seq analysis revealed a dysregulation in genes related to forebrain development in the frontal cortex and a dysregulation of a network of neurodevelopment and immune related genes in the striatum. Together, these results give evidence that gestational diabetes can induce changes in adulthood behavior and gene transcription in the brain.

PAR2 regulates regeneration, transdifferentiation, and death.

Understanding the mechanisms by which cells sense and respond to injury is central to developing therapies to enhance tissue regeneration. Previously, we showed that pancreatic injury consisting of acinar cell damage+ß-cell ablation led to islet cell transdifferentiation. Here, we report that the molecular mechanism for this requires activating protease-activated receptor-2 (PAR2), a G-protein-coupled receptor. PAR2 modulation was sufficient to induce islet cell transdifferentiation in the absence of ß-cells. Its expression was modulated in an islet cell type-specific manner in murine and human type 1 diabetes (T1D). In addition to transdifferentiation, PAR2 regulated ß-cell apoptosis in pancreatitis. PAR2's role in regeneration is broad, as mice lacking PAR2 had marked phenotypes in response to injury in the liver and in digit regeneration following amputation. These studies provide a pharmacologically relevant target to induce tissue regeneration in a number of diseases, including T1D.

The COP9 signalosome-like complex in S. cerevisiae and links to other PCI complexes.

The COP9 signalosome (CSN), the lid subcomplex of the proteasome and translational initiation factor 3 (eIF3) share structural similarities and are often referred to as the PCI family of complexes. In multicellular eukaryotes, the CSN is highly conserved as an 8-subunit complex but in Saccharomyces cerevisiae the complex is rather divergent. We further characterize the composition and properties of the CSN in budding yeast and its interactions with these related complexes. Using the generalized profile method we identified CSN candidates, four with PCI domains: Csn9, Csn10, Pci8/Csn11, and Csn12, and one with an MPN domain, Csn5/Rri1. These proteins and an additional interactor, Csi1, were tested for pairwise interactions by yeast two-hybrid and were found to form a cluster surrounding Csn12. Csn5 and Csn12 cofractionate in a complexed form with an apparent molecular weight of roughly 250kDa. However, Csn5 migrates as a monomer in Deltacsn12 supporting the pivotal role of Csn12 in stabilizing the complex. Confocal fluorescence microscopy detects GFP-tagged Csn5 preferentially in the nucleus, whereas in absence of Csn12, Csn10, Pci8/Csn11, or Csi1, Csn5 is delocalized throughout the cell, indicating that multiple subunits are required for nuclear localization of Csn5. Two CSN subunits, Csn9 and Csi1, interact with the proteasome lid subunit Rpn5. Pci8/Csn11 has previously been shown to interact with eIF3. Together, these results point to a network of interactions between these three structurally similar, yet functionally diverse, complexes.

Biologically relevant molecular transducer with increased computing power and iterative abilities.

As computing devices, which process data and interconvert information, transducers can encode new information and use their output for subsequent computing, offering high computational power that may be equivalent to a universal Turing machine. We report on an experimental DNA-based molecular transducer that computes iteratively and produces biologically relevant outputs. As a proof of concept, the transducer accomplished division of numbers by 3. The iterative power was demonstrated by a recursive application on an obtained output. This device reads plasmids as input and processes the information according to a predetermined algorithm, which is represented by molecular software. The device writes new information on the plasmid using hardware that comprises DNA-manipulating enzymes. The computation produces dual output: a quotient, represented by newly encoded DNA, and a remainder, represented by E. coli phenotypes. This device algorithmically manipulates genetic codes.

Identification of alverine and benfluorex as HNF4α activators.

The principal finding of this study is that two drugs, alverine and benfluorex, used in vastly different clinical settings, activated the nuclear receptor transcription factor HNF4α. Both were hits in a high-throughput screen for compounds that reversed the inhibitory effect of the fatty acid palmitate on human insulin promoter activity. Alverine is used in the treatment of irritable bowel syndrome, while benfluorex (Mediator) was used to treat hyperlipidemia and type II diabetes. Benfluorex was withdrawn from the market recently because of serious cardiovascular side effects related to fenfluramine-like activity. Strikingly, alverine and benfluorex have a previously unrecognized structural similarity, consistent with a common mechanism of action. Gene expression and biochemical studies revealed that they both activate HNF4α. This novel mechanism of action should lead to a reinterpretation of previous studies with these drugs and suggests a path toward the development of therapies for diseases such as inflammatory bowel and diabetes that may respond to HNF4α activators.

COP9 signalosome components play a role in the mating pheromone response of S. cerevisiae.

A family of genetically and structurally homologous complexes, the proteasome lid, Cop9 signalosome (CSN) and eukaryotic translation initiation factor 3, mediate different regulatory pathways. The CSN functions in numerous eukaryotes as a regulator of development and signaling, yet until now no evidence for a complex has been found in Saccharomyces cerevisiae. We identified a group of proteins, including a homolog of Csn5/Jab1 and four uncharacterized PCI components, that interact in a manner suggesting they form a complex analogous to the CSN in S. cerevisiae. These newly identified subunits play a role in adaptation to pheromone signaling. Deletants for individual subunits enhance pheromone response and increase mating efficiency. Overexpression of individual subunits or a human homolog mitigates sst2-induced pheromone sensitivity. Csi1, a novel CSN interactor, exhibits opposite phenotypes. Deletants also accumulate Cdc53/cullin in a Rub1-modified form; however, this role of the CSN appears to be distinct from that in the mating pathway.