Type 1 diabetes could begin with alterations in innate anti-viral immunity, which are already at this stage associated with HLA risk haplotypes.

AIMS: To investigate if HLA risk haplotypes and HbA1c levels are associated with the expression levels of innate anti-viral immune pathway genes in type 1 diabetes. MATERIALS AND METHODS: We investigated RNA expression levels of innate anti-viral immune pathway genes in laser-dissected islets from two to five tissue sections per donor from the Diabetes Virus Detection study and the network of Pancreatic Organ Donors in relation to HLA risk haplotypes (non-predisposed and predisposed) and HbA1c levels (normal, elevated, and high). RESULTS: The expression of innate anti-viral immune genes (TLR7, OAS1, OAS3 etc.) was significantly increased in individuals with predisposing vs non-predisposing HLA haplotypes. Also, the expression of several of the innate anti-viral immune genes from the HLA risk haplotype analysis was significantly increased in the group with high vs normal HbA1c. Furthermore, the gene expression of OAS2 was significantly increased in the group with high HbA1c vs elevated HbA1c. CONCLUSIONS: Expression of innate anti-viral immune pathway genes was increased in individuals with predisposing HLA risk haplotypes and those with high HbA1c. This indicates that type 1 diabetes might well begin with alterations in innate anti-viral immunity, and already at this stage be associated with HLA risk haplotypes.

Genetic predisposition in the 2'-5'A pathway in the development of type 1 diabetes: potential contribution to dysregulation of innate antiviral immunity.

AIMS/HYPOTHESIS: The incidence of type 1 diabetes is increasing more rapidly than can be explained by genetic drift. Viruses may play an important role in the disease, as they seem to activate the 2'-5'-linked oligoadenylate (2'-5'A) pathway of the innate antiviral immune system. Our aim was to investigate this possibility. METHODS: Innate antiviral immune pathways were searched for type 1 diabetes-associated polymorphisms using genome-wide association study data. SNPs within ±250kb flanking regions of the transcription start site of 64 genes were examined. These pathways were also investigated for type 1 diabetes-associated RNA expression profiles using laser-dissected islets from two to five tissue sections per donor from the Diabetes Virus Detection (DiViD) study and the network of Pancreatic Organ Donors (nPOD). RESULTS: We found 27 novel SNPs in genes nominally associated with type 1 diabetes. Three of those SNPs were located upstream of the 2'-5'A pathway, namely SNP rs4767000 (p = 1.03 × 10-9, OR 1.123), rs1034687 (p = 2.16 × 10-7, OR 0.869) and rs739744 (p = 1.03 × 10-9, OR 1.123). We also identified a large group of dysregulated islet genes in relation to type 1 diabetes, of which two were novel. The most aberrant genes were a group of IFN-stimulated genes. Of those, the following distinct pathways were targeted by the dysregulation (compared with the non-diabetic control group): OAS1 increased by 111% (p < 1.00 × 10-4, 95% CI -0.43, -0.15); MX1 increased by 142% (p < 1.00 × 10-4, 95% CI -0.52, -0.22); and ISG15 increased by 197% (p = 2.00 × 10-4, 95% CI -0.68, -0.18). CONCLUSIONS/INTERPRETATION: We identified a genetic predisposition in the 2'-5'A pathway that potentially contributes to dysregulation of the innate antiviral immune system in type 1 diabetes. This study describes a potential role for the 2'-5'A pathway and other components of the innate antiviral immune system in beta cell autoimmunity.

Abnormal islet sphingolipid metabolism in type 1 diabetes.

AIMS/HYPOTHESIS: Sphingolipids play important roles in beta cell physiology, by regulating proinsulin folding and insulin secretion and in controlling apoptosis, as studied in animal models and cell cultures. Here we investigate whether sphingolipid metabolism may contribute to the pathogenesis of human type 1 diabetes and whether increasing the levels of the sphingolipid sulfatide would prevent models of diabetes in NOD mice. METHODS: We examined the amount and distribution of sulfatide in human pancreatic islets by immunohistochemistry, immunofluorescence and electron microscopy. Transcriptional analysis was used to evaluate expression of sphingolipid-related genes in isolated human islets. Genome-wide association studies (GWAS) and a T cell proliferation assay were used to identify type 1 diabetes related polymorphisms and test how these affect cellular islet autoimmunity. Finally, we treated NOD mice with fenofibrate, a known activator of sulfatide biosynthesis, to evaluate the effect on experimental autoimmune diabetes development. RESULTS: We found reduced amounts of sulfatide, 23% of the levels in control participants, in pancreatic islets of individuals with newly diagnosed type 1 diabetes, which were associated with reduced expression of enzymes involved in sphingolipid metabolism. Next, we discovered eight gene polymorphisms (ORMDL3, SPHK2, B4GALNT1, SLC1A5, GALC, PPARD, PPARG and B4GALT1) involved in sphingolipid metabolism that contribute to the genetic predisposition to type 1 diabetes. These gene polymorphisms correlated with the degree of cellular islet autoimmunity in a cohort of individuals with type 1 diabetes. Finally, using fenofibrate, which activates sulfatide biosynthesis, we completely prevented diabetes in NOD mice and even reversed the disease in half of otherwise diabetic animals. CONCLUSIONS/INTERPRETATION: These results indicate that islet sphingolipid metabolism is abnormal in type 1 diabetes and suggest that modulation may represent a novel therapeutic approach. DATA AVAILABILITY: The RNA expression data is available online at https://www.dropbox.com/s/93mk5tzl5fdyo6b/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%2C%20RNA%20expression.xlsx?dl=0 . A list of SNPs identified is available at https://www.dropbox.com/s/yfojma9xanpp2ju/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%20SNP.xlsx?dl=0 .

Retinal pigment epithelium and microglia express the CD5 antigen-like protein, a novel autoantigen in age-related macular degeneration.

We report on a novel autoantigen expressed in human macular tissues, identified following an initial Western blot (WB)-based screening of sera from subjects with age-related macular degeneration (AMD) for circulating auto-antibodies (AAbs) recognizing macular antigens. Immunoprecipitation, 2D-gel electrophoresis (2D-GE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), direct enzyme-linked immunosorbent assays (ELISA), WBs, immunohistochemistry (IHC), human primary and ARPE-19 immortalized cell cultures were used to characterize this novel antigen. An approximately 40-kDa autoantigen in AMD was identified as the scavenger receptor CD5 antigen-like protein (CD5L), also known as apoptosis inhibitor of macrophage (AIM). CD5L/AIM was localized to human RPE by IHC and WB methods and to retinal microglial cells by IHC. ELISAs with recombinant CD5L/AIM on a subset of AMD sera showed a nearly 2-fold higher anti-CD5L/AIM reactivity in AMD vs. Control sera (p = 0.000007). Reactivity ≥0.4 was associated with 18-fold higher odds of having AMD (χ2 = 21.42, p = 0.00063). Circulating CD5L/AIM levels were also nearly 2-fold higher in AMD sera compared to controls (p = 0.0052). The discovery of CD5L/AIM expression in the RPE and in retinal microglial cells adds to the known immunomodulatory roles of these cells in the retina. The discovery of AAbs recognizing CD5L/AIM identifies a possible novel disease biomarker and suggest a potential role for CD5L/AIM in the pathogenesis of AMD in situ. The possible mechanisms via which anti-CD5L/AIM AAbs may contribute to AMD pathogenesis are discussed. In particular, since CD5L is known to stimulate autophagy and to participate in oxidized LDL uptake in macrophages, we propose that anti-CD5L/AIM auto-antibodies may play a role in drusen biogenesis and inflammatory RPE damage in AMD.

Islet cell hyperexpression of HLA class I antigens: a defining feature in type 1 diabetes.

AIMS/HYPOTHESIS: Human pancreatic beta cells may be complicit in their own demise in type 1 diabetes, but how this occurs remains unclear. One potentially contributing factor is hyperexpression of HLA class I antigens. This was first described approximately 30 years ago, but has never been fully characterised and was recently challenged as artefactual. Therefore, we investigated HLA class I expression at the protein and RNA levels in pancreases from three cohorts of patients with type 1 diabetes. The principal aims were to consider whether HLA class I hyperexpression is artefactual and, if not, to determine the factors driving it. METHODS: Pancreas samples from type 1 diabetes patients with residual insulin-containing islets (n = 26) from the Network for Pancreatic Organ donors with Diabetes (nPOD), Diabetes Virus Detection study (DiViD) and UK recent-onset type 1 diabetes collections were immunostained for HLA class I isoforms, signal transducer and activator of transcription 1 (STAT1), NLR family CARD domain containing 5 (NLRC5) and islet hormones. RNA was extracted from islets isolated by laser-capture microdissection from nPOD and DiViD samples and analysed using gene-expression arrays. RESULTS: Hyperexpression of HLA class I was observed in the insulin-containing islets of type 1 diabetes patients from all three tissue collections, and was confirmed at both the RNA and protein levels. The expression of ß2-microglobulin (a second component required for the generation of functional HLA class I complexes) was also elevated. Both 'classical' HLA class I isoforms (i.e. HLA-ABC) as well as a 'non-classical' HLA molecule, HLA-F, were hyperexpressed in insulin-containing islets. This hyperexpression did not correlate with detectable upregulation of the transcriptional regulator NLRC5. However, it was strongly associated with increased STAT1 expression in all three cohorts. Islet hyperexpression of HLA class I molecules occurred in the insulin-containing islets of patients with recent-onset type 1 diabetes and was also detectable in many patients with disease duration of up to 11 years, declining thereafter. CONCLUSIONS/INTERPRETATION: Islet cell HLA class I hyperexpression is not an artefact, but is a hallmark in the immunopathogenesis of type 1 diabetes. The response is closely associated with elevated expression of STAT1 and, together, these occur uniquely in patients with type 1 diabetes, thereby contributing to their selective susceptibility to autoimmune-mediated destruction.

Atrophied cardiomyocytes and their potential for rescue and recovery of ventricular function.

Cardiomyocytes must be responsive to demands placed on the heart's contractile work as a muscular pump. In turn, myocyte size is largely dependent on the workload they perform. Both hypertrophied and atrophic myocytes are found in the normal and diseased ventricle. Individual myocytes become atrophic when encumbered by fibrillar collagen, such as occurs at sites of fibrosis. The mechanisms include: (a) being immobilized and subject to disuse with ensuing protein degradation mediated by redox-sensitive, proteolytic ligases of the ubiquitin-proteasome system and (b) dedifferentiated re-expressing fetal genes induced by low intracellular triiodothyronine (T3) via thyroid hormone receptor ß1. This myocyte-selective, low T3 state is a consequence of heterocellular signaling emanating from juxtaposed scar tissue myofibroblasts and their secretome with its de novo generation of angiotensin II. In a paracrine manner, angiotensin II promotes myocyte Ca(2+) entry and subsequent Ca(2+) overload with ensuing oxidative stress that overwhelms antioxidant defenses to activate deiodinase-3 and its enzymatic degradation of T3. In the failing heart, atrophic myocytes represent an endogenous population of viable myocytes which could be rescued to augment contractile mass, reduce systolic wall stress (afterload) and recover ventricular function. Experimental studies have shown the potential for the rescue and recovery of atrophic myocytes in rebuilding the myocardium--a method complementary to today's quest in regenerating myocardium using progenitor cells.

Phosphoproteome mapping of cardiomyocyte mitochondria in a rat model of heart failure.

Mitochondria are complex organelles essential to cardiomyocyte survival. Protein phosphorylation is emerging as a key regulator of mitochondrial function. In the study reported here, we analyzed subsarcolemmal (SSM) mitochondria harvested from rats who have received 4 weeks of aldosterone/salt treatment to simulate the neurohormonal profile of human congestive heart failure. Our objective was to obtain an initial qualitative inventory of the phosphoproteins in this biologic system. SSM mitochondria were harvested, and the phosphoproteome was analyzed with a gel-free bioanalytical platform. Mitochondrial proteins were digested with trypsin, and the digests were enriched for phosphopeptides with immobilized metal ion affinity chromatography. The phosphopeptides were analyzed by ion trap liquid chromatography-tandem mass spectrometry, and the phosphoproteins identified via database searches. Based on MS/MS and MS(3) data, we characterized a set of 42 phosphopeptides that encompassed 39 phosphorylation sites. These peptides mapped to 26 proteins, for example, long-chain specific acyl-CoA dehydrogenase, Complex III subunit 6, and mitochondrial import receptor TOM70. Collectively, the characterized phosphoproteins belong to diverse functional modules, including bioenergetic pathways, protein import machinery, and calcium handling. The phosphoprotein panel discovered in this study provides a foundation for future differential phosphoproteome profiling toward an integrated understanding of the role of mitochondrial phosphorylation in heart failure.

Zinc and the prooxidant heart failure phenotype.

Neurohormonal activation with attendant aldosteronism contributes to the clinical appearance of congestive heart failure (CHF). Aldosteronism is intrinsically coupled to Zn and Ca dyshomeostasis, in which consequent hypozincemia compromises Zn homeostasis and Zn-based antioxidant defenses that contribute to the CHF prooxidant phenotype. Ionized hypocalcemia leads to secondary hyperparathyroidism with parathyroid hormone-mediated Ca overloading of diverse cells, including cardiomyocytes. When mitochondrial Ca overload exceeds a threshold, myocyte necrosis follows. The reciprocal regulation involving cytosolic free [Zn]i as antioxidant and [Ca]i as prooxidant can be uncoupled in favor of Zn-based antioxidant defenses. Increased [Zn]i acts as a multifaceted antioxidant by: (1) inhibiting Ca entry through L-type channels and hence cardioprotectant from the Ca-driven mitochondriocentric signal-transducer effector pathway to nonischemic necrosis, (2) serving as catalytic regulator of Cu/Zn-superoxide dismutase, and (3) activating its cytosolic sensor, metal-responsive transcription factor that regulates the expression of relevant antioxidant defense genes. Albeit present in subnanomolar range, increased cytosolic free [Zn]i enhances antioxidant capacity that confers cardioprotection. It can be achieved exogenously by ZnSO4 supplementation or endogenously using a ß3-receptor agonist (eg, nebivolol) that enhances NO generation to release inactive cytosolic Zn bound to metallothionein. By recognizing the pathophysiologic relevance of Zn dyshomeostasis in the prooxidant CHF phenotype and by exploiting the pharmacophysiologic potential of [Zn]i as antioxidant, vulnerable cardiomyocytes under assault from neurohormonal activation can be protected and the myocardium spared from adverse structural remodeling.

Small dedifferentiated cardiomyocytes bordering on microdomains of fibrosis: evidence for reverse remodeling with assisted recovery.

With the perspective of functional myocardial regeneration, we investigated small cardiomyocytes bordering on microdomains of fibrosis, where they are dedifferentiated re-expressing fetal genes, and determined: (1) whether they are atrophied segments of the myofiber syncytium, (2) their redox state, (3) their anatomic relationship to activated myofibroblasts (myoFb), given their putative regulatory role in myocyte dedifferentiation and redifferentiation, (4) the relevance of proteolytic ligases of the ubiquitin-proteasome system as a mechanistic link to their size, and (5) whether they could be rescued from their dedifferentiated phenotype. Chronic aldosterone/salt treatment (ALDOST) was invoked, where hypertensive heart disease with attendant myocardial fibrosis creates the fibrillar collagen substrate for myocyte sequestration, with propensity for disuse atrophy, activated myoFb, and oxidative stress. To address phenotype rescue, 4 weeks of ALDOST was terminated followed by 4 weeks of neurohormonal withdrawal combined with a regimen of exogenous antioxidants, ZnSO4, and nebivolol (assisted recovery). Compared with controls, at 4 weeks of ALDOST, we found small myocytes to be: (1) sequestered by collagen fibrils emanating from microdomains of fibrosis and representing atrophic segments of the myofiber syncytia, (2) dedifferentiated re-expressing fetal genes (ß-myosin heavy chain and atrial natriuretic peptide), (3) proximal to activated myoFb expressing α-smooth muscle actin microfilaments and angiotensin-converting enzyme, (4) expressing reactive oxygen species and nitric oxide with increased tissue 8-isoprostane, coupled to ventricular diastolic and systolic dysfunction, and (5) associated with upregulated redox-sensitive proteolytic ligases MuRF1 and atrogin-1. In a separate study, we did not find evidence of myocyte replication (BrdU labeling) or expression of stem cell antigen (c-Kit) at weeks 1-4 ALDOST. Assisted recovery caused complete disappearance of myoFb from sites of fibrosis with redifferentiation of these myocytes, loss of oxidative stress, and ubiquitin-proteasome system activation, with restoration of nitric oxide and improved ventricular function. Thus, small dedifferentiated myocytes bordering on microdomains of fibrosis can re-differentiate and represent a potential source of autologous cells for functional myocardial regeneration.

Mesenchymal stem cell-based therapy.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells which have self-renewal capacity and differentiation potential into several mesenchymal lineages including bones, cartilages, adipose tissues and tendons. MSCs may repair tissue injuries and prevent immune cell activation and proliferation. Immunomodulation and secretion of growth factors by MSCs have led to realizing the true potential of MSC-based cell therapy. The use of MSCs as immunomodulators has been explored in cell/organ transplant, tissue repair, autoimmune diseases, and prevention of graft vs host disease (GVHD). This review focuses on the clinical applications of MSC-based cell therapy, with particular emphasis on islet transplantation for treating type I diabetes.

Modification of oxidative stress on gene expression profiling in the rat infarcted heart.

Cardiac oxidative stress is developed following myocardial infarction (MI) particularly in the first week of MI. The influence of reactive oxygen species (ROS) on gene expression profiling and molecular pathways in the infarcted myocardium remains uncertain and is explored in the present study. Rats with MI were treated with or without antioxidants for 1 week. Normal rats served as controls. Cardiac oxidative stress and gene profiling were investigated. Compared to normal hearts, malondialdehyde, a marker of oxidative stress, was significantly increased in the infarcted myocardium, which was significantly suppressed by antioxidants. Microarray assay showed that over a thousand genes were differentially expressed in the infarcted myocardium. Antioxidants significantly altered the expression of 159 genes compared to untreated MI rats. Ingenuity pathway analysis indicated that multiple pathway networks were affected by antioxidants, including those related to cell movement, growth/development, death, and inflammatory/fibrotic responses. IPA further identified that these changes were primarily related to NFκB, p38 MAPK, and ERκ1/2 pathways. Hub genes were identified in the associated gene networks. This study reveals the gene networks associated with cardiac oxidative stress postMI. These observations indicate that ROS regulate various molecular and cellular actions related to cardiac repair/remodeling through multiple gene networks.

Nebivolol: a multifaceted antioxidant and cardioprotectant in hypertensive heart disease.

Cardiomyocyte necrosis with attendant microscopic scarring is a pathological feature of human hypertensive heart disease (HHD). Understanding the pathophysiological origins of necrosis is integral to its prevention. In a rat model of HHD associated with aldosterone/salt treatment (ALDOST), myocyte necrosis is attributable to oxidative stress induced by cytosolic-free [Ca]i and mitochondrial [Ca]m overloading in which the rate of reactive oxygen species generation overwhelms their rate of detoxification by endogenous Zn-based antioxidant defenses. We hypothesized that nebivolol (Neb), unlike another ß1 adrenergic receptor antagonist atenolol (Aten), would have a multifaceted antioxidant potential based on its dual property as a ß3 receptor agonist, which activates endothelial nitric oxide synthase to stimulate nitric oxide (NO) generation. NO promotes the release of cytosolic Zn sequestered inactive by its binding protein, metallothionein. Given the reciprocal regulation between these cations, increased [Zn]i reduces Ca entry and attendant rise in [Ca]i and [Ca]m. Herein, we examined the antioxidant and cardioprotectant properties of Neb and Aten in rats receiving 4 weeks ALDOST. Compared with untreated age-/sex-matched controls, ALDOST alone or ALDOST with Aten, Neb cotreatment induced endothelial nitric oxide synthase activation, NO generation and a marked increase in [Zn]i with associated decline in [Ca]i and [Ca]m. Attendant antioxidant profile at subcellular and cellular levels included attenuation of mitochondrial H2O2 production and lipid peroxidation expressed as reduced 8-isoprostane concentrations in both mitochondria and cardiac tissue. Myocyte salvage was expressed as reduced microscopic scarring and tissue collagen volume fraction. Neb is a multifaceted antioxidant with unique properties as cardioprotectant in HHD.

Atrophic cardiomyocyte signaling in hypertensive heart disease.

Cardinal pathological features of hypertensive heart disease (HHD) include not only hypertrophied cardiomyocytes and foci of scattered microscopic scarring, a footprint of prior necrosis, but also small myocytes ensnared by fibrillar collagen where disuse atrophy with protein degradation would be predicted. Whether atrophic signaling is concordant with the appearance of HHD and involves oxidative and endoplasmic reticulum (ER) stress remains unexplored. Herein, we examine these possibilities focusing on the left ventricle and cardiomyocytes harvested from hypertensive rats receiving 4 weeks aldosterone/salt treatment (ALDOST) alone or together with ZnSO4, a nonvasoactive antioxidant, with the potential to attenuate atrophy and optimize hypertrophy. Compared with untreated age-/sex-/strain-matched controls, ALDOST was accompanied by (1) left ventricle hypertrophy with preserved systolic function; (2) concordant cardiomyocyte atrophy (<1000 µm²) found at sites bordering on fibrosis where they were reexpressing ß-myosin heavy chain; and (3) upregulation of ubiquitin ligases, muscle RING-finger protein-1 and atrogin-1, and elevated 8-isoprostane and unfolded protein ER response with messenger RNA upregulation of stress markers. ZnSO4 cotreatment reduced lipid peroxidation, fibrosis, and the number of atrophic myocytes, together with a further increase in cell area and width of atrophied and hypertrophied myocytes, and improved systolic function but did not attenuate elevated blood pressure. We conclude that atrophic signaling, concordant with hypertrophy, occurs in the presence of a reparative fibrosis and induction of oxidative and ER stress at sites of scarring where myocytes are atrophied. ZnSO4 cotreatment in HHD with ALDOST attenuates the number of atrophic myocytes, optimizes size of atrophied and hypertrophied myocytes, and improves systolic function.

Mitochondria play a central role in nonischemic cardiomyocyte necrosis: common to acute and chronic stressor states.

The survival of cardiomyocytes must be ensured as the myocardium adjusts to a myriad of competing physiological and pathophysiological demands. A significant loss of these contractile cells, together with their replacement by stiff fibrillar collagen in the form of fibrous tissue accounts for a transition from a usually efficient muscular pump into one that is failing. Cellular and subcellular mechanisms involved in the pathogenic origins of cardiomyocyte cell death have long been of interest. This includes programmed molecular pathways to either necrosis or apoptosis, which are initiated from ischemic or nonischemic origins. Herein, we focus on the central role played by a mitochondriocentric signal-transducer-effector pathway to nonischemic cardiomyocyte necrosis, which is common to acute and chronic stressor states. We begin by building upon the hypothesis advanced by Albrecht Fleckenstein and coworkers some 40 years ago based on the importance of calcitropic hormone-mediated intracellular Ca(2+) overloading, which predominantly involves subsarcolemmal mitochondria and is the signal to pathway activation. Other pathway components, which came to be recognized in subsequent years, include the induction of oxidative stress and opening of the mitochondrial inner membrane permeability transition pore. The ensuing loss of cardiomyocytes and consequent replacement fibrosis, or scarring, represents a disease of adaptation and a classic example of when homeostasis begets dyshomeostasis.

Reverse remodeling and recovery from cachexia in rats with aldosteronism.

The congestive heart failure (CHF) syndrome with soft tissue wasting, or cachexia, has its pathophysiologic origins rooted in neurohormonal activation. Mechanical cardiocirculatory assistance reveals the potential for reverse remodeling and recovery from CHF, which has been attributed to device-based hemodynamic unloading whereas the influence of hormonal withdrawal remains uncertain. This study addresses the signaling pathways induced by chronic aldosteronism in normal heart and skeletal muscle at organ, cellular/subcellular, and molecular levels, together with their potential for recovery (Recov) after its withdrawal. Eight-week-old male Sprague-Dawley rats were examined at 4 wk of aldosterone/salt treatment (ALDOST) and following 4-wk Recov. Compared with untreated, age-/sex-/strain-matched controls, ALDOST was accompanied by 1) a failure to gain weight, reduced muscle mass with atrophy, and a heterogeneity in cardiomyocyte size across the ventricles, including hypertrophy and atrophy at sites of microscopic scarring; 2) increased cardiomyocyte and mitochondrial free Ca(2+), coupled to oxidative stress with increased H(2)O(2) production and 8-isoprostane content, and increased opening potential of the mitochondrial permeability transition pore; 3) differentially expressed genes reflecting proinflammatory myocardial and catabolic muscle phenotypes; and 4) reversal to or toward recovery of these responses with 4-wk Recov. Aldosteronism in rats is accompanied by cachexia and leads to an adverse remodeling of the heart and skeletal muscle at organ, cellular/subcellular, and molecular levels. However, evidence presented herein implicates that these tissues retain their inherent potential for recovery after complete hormone withdrawal.

Development of Type 1 Diabetes may occur through a Type 2 Diabetes mechanism.

Background: At diagnosis of Type 1 Diabetes (T1D), 30% of the beta cells are dormant, i.e. alive, but inactive. This could reduce beta cell destruction, as cellular stress contributes to beta cell damage. However, the beta cells, that are still active, must produce more insulin and are therefore more vulnerable. The inactive beta cells represent a potential for restoring the insulin secretion. Methods: We analyzed the expression of selected genes in islets from live, newly diagnosed T1D patients from the DiViD study and organ doners with longer duration of T1D, type 2 diabetes (T2D), or no diabetes from the nPOD study. Additionally, analysis of polymorphisms was performed on all the investigated genes. Findings: Various possibilities were considered for the inactivity of the beta cells: secretion defect, fetal state, hibernation, and insulin resistance. We analyzed genes related to the ceramide and sphingomyelin synthesis and degradation, secretion, circadian rhythm and insulin action, and found changes in T1D islets that resemble fetal dedifferentiation and asynchrony. Furthermore, we found low levels of insulin receptor mRNA in the islets. No polymorphisms were found. Interpretation: Our findings suggest a secretion defect, but also fetal dedifferentiation and desynchronization in the inactive beta cells. Together with previous evidence, that predisposing factors for T2D are also present for T1D development, we raise the idea to treat individuals with ongoing T1D development prophylactically with T2D medicine like GLP-1 receptor agonists, metformin, or others, combined with anti-inflammatory compounds, in order to reactivate the dormant beta cells, and to prevent autoimmune destruction. T2D mechanisms during T1D development should be investigated further.

Pancreas Whole Tissue Transcriptomics Highlights the Role of the Exocrine Pancreas in Patients With Recently Diagnosed Type 1 Diabetes.

Although type 1 diabetes (T1D) is primarily a disease of the pancreatic beta-cells, understanding of the disease-associated alterations in the whole pancreas could be important for the improved treatment or the prevention of the disease. We have characterized the whole-pancreas gene expression of patients with recently diagnosed T1D from the Diabetes Virus Detection (DiViD) study and non-diabetic controls. Furthermore, another parallel dataset of the whole pancreas and an additional dataset from the laser-captured pancreatic islets of the DiViD patients and non-diabetic organ donors were analyzed together with the original dataset to confirm the results and to get further insights into the potential disease-associated differences between the exocrine and the endocrine pancreas. First, higher expression of the core acinar cell genes, encoding for digestive enzymes, was detected in the whole pancreas of the DiViD patients when compared to non-diabetic controls. Second, In the pancreatic islets, upregulation of immune and inflammation related genes was observed in the DiViD patients when compared to non-diabetic controls, in line with earlier publications, while an opposite trend was observed for several immune and inflammation related genes at the whole pancreas tissue level. Third, strong downregulation of the regenerating gene family (REG) genes, linked to pancreatic islet growth and regeneration, was observed in the exocrine acinar cell dominated whole-pancreas data of the DiViD patients when compared with the non-diabetic controls. Fourth, analysis of unique features in the transcriptomes of each DiViD patient compared with the other DiViD patients, revealed elevated expression of central antiviral immune response genes in the whole-pancreas samples, but not in the pancreatic islets, of one DiViD patient. This difference in the extent of antiviral gene expression suggests different statuses of infection in the pancreas at the time of sampling between the DiViD patients, who were all enterovirus VP1+ in the islets by immunohistochemistry based on earlier studies. The observed features, indicating differences in the function, status and interplay between the exocrine and the endocrine pancreas of recent onset T1D patients, highlight the importance of studying both compartments for better understanding of the molecular mechanisms of T1D.

Detection of Antiviral Tissue Responses and Increased Cell Stress in the Pancreatic Islets of Newly Diagnosed Type 1 Diabetes Patients: Results From the DiViD Study.

Aims/hypothesis: The Diabetes Virus Detection (DiViD) study has suggested the presence of low-grade enteroviral infection in pancreatic tissue collected from six of six live adult patients newly diagnosed with type 1 diabetes. The present study aimed to compare the gene and protein expression of selected virally induced pathogen recognition receptors and interferon stimulated genes in islets from these newly diagnosed type 1 diabetes (DiViD) subjects vs age-matched non-diabetic (ND) controls. Methods: RNA was extracted from laser-captured islets and Affymetrix Human Gene 2.0 ST arrays used to obtain gene expression profiles. Lists of differentially expressed genes were subjected to a data-mining pipeline searching for enrichment of canonical pathways, KEGG pathways, Gene Ontologies, transcription factor binding sites and other upstream regulators. In addition, the presence and localisation of specific viral response proteins (PKR, MxA and MDA5) were examined by combined immunofluorescent labelling in sections of pancreatic tissue. Results: The data analysis and data mining process revealed a significant enrichment of gene ontologies covering viral reproduction and infectious cycles; peptide translation, elongation and initiation, as well as oxidoreductase activity. Enrichment was identified in the KEGG pathways for oxidative phosphorylation; ribosomal and metabolic activity; antigen processing and presentation and in canonical pathways for mitochondrial dysfunction, oxidative phosphorylation and EIF2 signaling. Protein Kinase R (PKR) expression did not differ between newly diagnosed type 1 diabetes and ND islets at the level of total RNA, but a small subset of ß-cells displayed markedly increased PKR protein levels. These PKR+ ß-cells correspond to those previously shown to contain the viral protein, VP1. RNA encoding MDA5 was increased significantly in newly diagnosed type 1 diabetes islets, and immunostaining of MDA5 protein was seen in α- and certain ß-cells in both newly diagnosed type 1 diabetes and ND islets, but the expression was increased in ß-cells in type 1 diabetes. In addition, an uncharacterised subset of synaptophysin positive, but islet hormone negative, cells expressed intense MDA5 staining and these were more prevalent in DiViD cases. MxA RNA was upregulated in newly diagnosed type 1 diabetes vs ND islets and MxA protein was detected exclusively in newly diagnosed type 1 diabetes ß-cells. Conclusion/interpretation: The gene expression signatures reveal that pathways associated with cellular stress and increased immunological activity are enhanced in islets from newly diagnosed type 1 diabetes patients compared to controls. The increases in viral response proteins seen in ß-cells in newly diagnosed type 1 diabetes provide clear evidence for the activation of IFN signalling pathways. As such, these data strengthen the hypothesis that an enteroviral infection of islet ß-cells contributes to the pathogenesis of type 1 diabetes.

Adaptation to chronic ER stress enforces pancreatic ß-cell plasticity.

Pancreatic ß-cells are prone to endoplasmic reticulum (ER) stress due to their role in insulin secretion. They require sustainable and efficient adaptive stress responses to cope with this stress. Whether episodes of chronic stress directly compromise ß-cell identity is unknown. We show here under reversible, chronic stress conditions ß-cells undergo transcriptional and translational reprogramming associated with impaired expression of regulators of ß-cell function and identity. Upon recovery from stress, ß-cells regain their identity and function, indicating a high degree of adaptive plasticity. Remarkably, while ß-cells show resilience to episodic ER stress, when episodes exceed a threshold, ß-cell identity is gradually lost. Single cell RNA-sequencing analysis of islets from type 1 diabetes patients indicates severe deregulation of the chronic stress-adaptation program and reveals novel biomarkers of diabetes progression. Our results suggest ß-cell adaptive exhaustion contributes to diabetes pathogenesis.