There is strong evidence for an involvement of different classes of non-coding RNAs, including microRNAs and long non-coding RNAs, in the regulation of β-cell activities and in diabetes development. ...Circular RNAs were recently discovered to constitute a substantial fraction of the mammalian transcriptome but the contribution of these non-coding RNAs in physiological and disease processes remains largely unknown. The goal of this study was to identify the circular RNAs expressed in pancreatic islets and to elucidate their possible role in the control of β-cells functions.
We used a microarray approach to identify circular RNAs expressed in human islets and searched their orthologues in RNA sequencing data from mouse islets. We then measured the level of four selected circular RNAs in the islets of different Type 1 and Type 2 diabetes models and analyzed the role of these circular transcripts in the regulation of insulin secretion, β-cell proliferation, and apoptosis.
We identified thousands of circular RNAs expressed in human pancreatic islets, 497 of which were conserved in mouse islets. The level of two of these circular transcripts, circHIPK3 and ciRS-7/CDR1as, was found to be reduced in the islets of diabetic db/db mice. Mimicking this decrease in the islets of wild type animals resulted in impaired insulin secretion, reduced β-cell proliferation, and survival. ciRS-7/CDR1as has been previously proposed to function by blocking miR-7. Transcriptomic analysis revealed that circHIPK3 acts by sequestering a group of microRNAs, including miR-124-3p and miR-338-3p, and by regulating the expression of key β-cell genes, such as Slc2a2, Akt1, and Mtpn.
Our findings point to circular RNAs as novel regulators of β-cell activities and suggest an involvement of this novel class of non-coding RNAs in β-cell dysfunction under diabetic conditions.
•β-cells express thousands of abundant circRNAs.•ciRS-7 and circHIPK3 display altered expression in Type 2 diabetes models.•ciRS-7 controls β-cell proliferation and insulin secretion.•circHIPK3 silencing impairs β-cell proliferation, β-cell survival and insulin secretion.•circHIPK3 appears to control β-cell gene expression by sequestering a group of miRNAs.
Aims/hypothesis
Mild islet inflammation has been suggested as a contributing factor to beta cell failure in type 2 diabetes. Macrophage levels are elevated in the islets of humans and mice with type ...2 diabetes, but their effects on beta cells are not understood. Our goal was to examine the gene expression changes in islet-associated macrophages in obesity models with opposing disposition to diabetes development and to assess their potential contribution to beta cell (mal)adaptation.
Methods
Islets were isolated from lean control mice, obese diabetes-prone
db/db
mice and obese diabetes-resistant
ob/ob
mice. Macrophages were sorted using flow cytometry. Islets were treated ex vivo with clodronate-containing liposomes to deplete macrophages. Gene expression was assessed by real-time RT-PCR.
Results
Macrophage levels were increased in islets from
db/db
mice but not in islets from
ob/ob
mice compared with lean control mice. Macrophages from
db/db
and
ob/ob
islets displayed distinct changes in gene expression compared with control islet macrophages, suggesting differential shifts in functional state. Macrophages from
db/db
islets displayed increased expression of interferon regulatory factor 5 (
Irf5
), IL-1 receptor antagonist (
Il1rn
) and mannose receptor C-type 1 (
Mrc1
), whereas macrophages from
ob/ob
islets showed elevated levels of transforming growth factor beta 1 (
Tgfb1
) and reduced IL-1β (
Il1b
). Clodronate-liposome treatment of islets depleted macrophages, as evidenced by reduced mRNA expression of
Cd11b
(also known as
Itgam
) and
F4/80
(also known as
Adgre1
) compared with PBS-liposome-treated islets. The depletion of macrophages in
db/db
islets increased the expression of genes related to beta cell identity. The mRNA levels of islet-associated transcription factors (
Mafa
and
Pdx1
), glucose transporter (
Glut2
also known as
Slc2a2
), ATP-sensitive K
+
channel (
Kcnj11
), incretin receptor (
Gipr
) and adaptive unfolded protein response (UPR) genes (
Xbp1
,
Hspa5
,
Pdia4
and
Fkbp11
) were increased in
db/db
islets after macrophage depletion, whereas the mRNA levels of the deleterious UPR effector,
Ddit3
, were reduced. In contrast, depletion of macrophages in islets of
ob/ob
mice did not affect beta cell identity gene expression.
Conclusions/interpretation
The findings of this study suggest that distinct alterations in islet macrophages of obese mice are critically important for the disruption of beta cell gene expression in diabetes.
Obesity is caused by an imbalance between food intake and energy expenditure (EE). Here we identify a conserved pathway that links signalling through peripheral Y1 receptors (Y1R) to the control of ...EE. Selective antagonism of peripheral Y1R, via the non-brain penetrable antagonist BIBO3304, leads to a significant reduction in body weight gain due to enhanced EE thereby reducing fat mass. Specifically thermogenesis in brown adipose tissue (BAT) due to elevated UCP1 is enhanced accompanied by extensive browning of white adipose tissue both in mice and humans. Importantly, selective ablation of Y1R from adipocytes protects against diet-induced obesity. Furthermore, peripheral specific Y1R antagonism also improves glucose homeostasis mainly driven by dynamic changes in Akt activity in BAT. Together, these data suggest that selective peripheral only Y1R antagonism via BIBO3304, or a functional analogue, could be developed as a safer and more effective treatment option to mitigate diet-induced obesity.
Insulin secretion is tightly controlled through coordinated actions of a number of systemic and local factors. Peptide YY (PYY) is expressed in α-cells of the islet, but its role in control of islet ...function such as insulin release is not clear. In this study, we generated a transgenic mouse model (Pyytg/+/Rip-Cre) overexpressing the Pyy gene under the control of the rat insulin 2 gene promoter and assessed the impact of islet-released PYY on β-cell function, insulin release, and glucose homeostasis in mice. Our results show that up-regulation of PYY in islet β-cells leads to an increase in serum insulin levels as well as improved glucose tolerance. Interestingly, PYY-overproducing mice show increased lean mass and reduced fat mass with no significant changes in food intake or body weight. Energy expenditure is also increased accompanied by increased respiratory exchange ratio. Mechanistically, the enhanced insulin levels and improved glucose tolerance are primarily due to increased β-cell mass and secretion. This is associated with alterations in the expression of genes important for β-cell proliferation and function as well as the maintenance of the β-cell phenotype. Taken together, these data demonstrate that pancreatic islet-derived PYY plays an important role in controlling glucose homeostasis through the modulation of β-cell mass and function.
The loss of functional beta cell mass characterises all forms of diabetes. Beta cells are highly susceptible to stress, including cytokine, endoplasmic reticulum (ER) and oxidative stress. This study ...examined the role of pleckstrin homology-like, domain family A, member 3 (Phlda3) in beta cell survival under stress conditions and the regulatory basis. We found that the mRNA levels of Phlda3 were markedly upregulated in vivo in the islets of diabetic humans and mice. In vitro, exposure of MIN6 cells or islets to cytokines, palmitate, thapsigargin or ribose upregulated Phlda3 mRNA and protein levels, concurrent with the induction of ER stress (Ddit3 and Trb3) and antioxidant (Hmox1) genes. Furthermore, H
O
treatment markedly increased PHLDA3 immunostaining in human islets. Phlda3 expression was differentially regulated by adaptive (Xbp1) and apoptotic (Ddit3) unfolded protein response (UPR) mediators. siRNA-mediated knockdown of Xbp1 inhibited the induction of Phlda3 by cytokines and palmitate, whereas knockdown of Ddit3 upregulated Phlda3. Moreover, knockdown of Phlda3 potentiated cytokine-induced apoptosis in association with upregulation of inflammatory genes (iNos, IL1β and IκBα) and NFκB phosphorylation and downregulation of antioxidant (Gpx1 and Srxn1) and adaptive UPR (Xbp1, Hspa5 and Fkbp11) genes. Knockdown of Phlda3 also potentiated apoptosis under oxidative stress conditions induced by ribose treatment. These findings suggest that Phlda3 is crucial for beta cell survival under stress conditions. Phlda3 regulates the cytokine, oxidative and ER stress responses in beta cells via the repression of inflammatory gene expression and the maintenance of antioxidant and adaptive UPR gene expression. Phlda3 may promote beta cell survival in diabetes.
Failure to secrete sufficient quantities of insulin is a pathological feature of type-1 and type-2 diabetes, and also reduces the success of islet cell transplantation. Here we demonstrate that Y1 ...receptor signaling inhibits insulin release in β-cells, and show that this can be pharmacologically exploited to boost insulin secretion. Transplanting islets with Y1 receptor deficiency accelerates the normalization of hyperglycemia in chemically induced diabetic recipient mice, which can also be achieved by short-term pharmacological blockade of Y1 receptors in transplanted mouse and human islets. Furthermore, treatment of non-obese diabetic mice with a Y1 receptor antagonist delays the onset of diabetes. Mechanistically, Y1 receptor signaling inhibits the production of cAMP in islets, which via CREB mediated pathways results in the down-regulation of several key enzymes in glycolysis and ATP production. Thus, manipulating Y1 receptor signaling in β-cells offers a unique therapeutic opportunity for correcting insulin deficiency as it occurs in the pathological state of type-1 diabetes as well as during islet transplantation.Islet transplantation is considered one of the potential treatments for T1DM but limited islet survival and their impaired function pose limitations to this approach. Here Loh et al. show that the Y1 receptor is expressed in β- cells and inhibition of its signalling, both genetic and pharmacological, improves mouse and human islet function.
Intermittent severe energy restriction is popular for weight management. To investigate whether intermittent moderate energy restriction may improve this approach by enhancing weight loss efficiency, ...we conducted a study in mice, where energy intake can be controlled.
Male C57/Bl6 mice that had been rendered obese by an ad libitum diet high in fat and sugar for 22 weeks were then fed one of two energy-restricted normal chow diets for a 12-week weight loss phase. The continuous diet (CD) provided 82% of the energy intake of age-matched ad libitum chow-fed controls. The intermittent diet (ID) provided cycles of 82% of control intake for 5-6 consecutive days, and ad libitum intake for 1-3 days. Weight loss efficiency during this phase was calculated as (total weight change) ÷ (total energy intake of mice on CD or ID)-(total average energy intake of controls). Subsets of mice then underwent a 3-week weight regain phase involving ad libitum re-feeding.
Mice on the ID showed transient hyperphagia relative to controls during each 1-3-day ad libitum feeding period, and overall ate significantly more than CD mice (91.1±1.0 versus 82.2±0.5% of control intake respectively, n = 10, P<0.05). There were no significant differences between CD and ID groups at the end of the weight loss or weight regain phases with respect to body weight, fat mass, circulating glucose or insulin concentrations, or the insulin resistance index. Weight loss efficiency was significantly greater with ID than with CD (0.042±0.007 versus 0.018±0.001 g/kJ, n = 10, P<0.01). Mice on the CD exhibited significantly greater hypothalamic mRNA expression of proopiomelanocortin (POMC) relative to ID and control mice, with no differences in neuropeptide Y or agouti-related peptide mRNA expression between energy-restricted groups.
Intermittent moderate energy restriction may offer an advantage over continuous moderate energy restriction, because it induces significantly greater weight loss relative to energy deficit in mice.
Aims/hypothesis
Pancreatic beta cell dedifferentiation, transdifferentiation into other islet cells and apoptosis have been implicated in beta cell failure in type 2 diabetes, although the mechanisms ...are poorly defined. The endoplasmic reticulum stress response factor X-box binding protein 1 (XBP1) is a major regulator of the unfolded protein response. XBP1 expression is reduced in islets of people with type 2 diabetes, but its role in adult differentiated beta cells is unclear. Here, we assessed the effects of
Xbp1
deletion in adult beta cells and tested whether XBP1-mediated unfolded protein response makes a necessary contribution to beta cell compensation in insulin resistance states.
Methods
Mice with inducible beta cell-specific
Xbp1
deletion were studied under normal (chow diet) or metabolic stress (high-fat diet or obesity) conditions. Glucose tolerance, insulin secretion, islet gene expression, alpha cell mass, beta cell mass and apoptosis were assessed. Lineage tracing was used to determine beta cell fate.
Results
Deletion of
Xbp1
in adult mouse beta cells led to beta cell dedifferentiation, beta-to-alpha cell transdifferentiation and increased alpha cell mass. Cell lineage-specific analyses revealed that
Xbp1
deletion deactivated beta cell identity genes (insulin,
Pdx1
,
Nkx6.1
,
Beta2
,
Foxo1
) and derepressed beta cell dedifferentiation (
Aldh1a3
) and alpha cell (glucagon,
Arx
,
Irx2
) genes.
Xbp1
deletion in beta cells of obese
ob/ob
or high-fat diet-fed mice triggered diabetes and worsened glucose intolerance by disrupting insulin secretory capacity. Furthermore,
Xbp1
deletion increased beta cell apoptosis under metabolic stress conditions by attenuating the antioxidant response.
Conclusions/interpretation
These findings indicate that XBP1 maintains beta cell identity, represses beta-to-alpha cell transdifferentiation and is required for beta cell compensation and prevention of diabetes in insulin resistance states.
Graphical abstract
Pancreatic β-cell expansion throughout the neonatal period is essential to generate the appropriate mass of insulin-secreting cells required to maintain blood glucose homeostasis later in life. ...Hence, defects in this process can predispose to diabetes development during adulthood. Global profiling of transcripts in pancreatic islets of newborn and adult rats revealed that the transcription factor E2F1 controls expression of the long noncoding RNA H19, which is profoundly downregulated during the postnatal period. H19 silencing decreased β-cell expansion in newborns, whereas its re-expression promoted proliferation of β-cells in adults via a mechanism involving the microRNA let-7 and the activation of Akt. The offspring of rats fed a low-protein diet during gestation and lactation display a small β-cell mass and an increased risk of developing diabetes during adulthood. We found that the islets of newborn rats born to dams fed a low-protein diet express lower levels of H19 than those born to dams that did not eat a low-protein diet. Moreover, we observed that H19 expression increases in islets of obese mice under conditions of increased insulin demand. Our data suggest that the long noncoding RNA H19 plays an important role in postnatal β-cell mass expansion in rats and contributes to the mechanisms compensating for insulin resistance in obesity.
The mechanisms underpinning beta‐cell compensation for obesity‐associated insulin resistance and beta‐cell failure in type 2 diabetes remain poorly understood. We used a large‐scale strategy to ...determine the time‐dependent transcriptomic changes in islets of diabetes‐prone db/db and diabetes‐resistant ob/ob mice at 6 and 16 weeks of age. Differentially expressed genes were subjected to cluster, gene ontology, pathway and gene set enrichment analyses. A distinctive gene expression pattern was observed in 16 week db/db islets in comparison to the other groups with alterations in transcriptional regulators of islet cell identity, upregulation of glucose/lipid metabolism, and various stress response genes, and downregulation of specific amino acid transport and metabolism genes. In contrast, ob/ob islets displayed a coordinated downregulation of metabolic and stress response genes at 6 weeks of age, suggestive of a preemptive reconfiguration in these islets to lower the threshold of metabolic activation in response to increased insulin demand thereby preserving beta‐cell function and preventing cellular stress. In addition, amino acid transport and metabolism genes were upregulated in ob/ob islets, suggesting an important role of glutamate metabolism in beta‐cell compensation. Gene set enrichment analysis of differentially expressed genes identified the enrichment of binding motifs for transcription factors, FOXO4, NFATC1, and MAZ. siRNA‐mediated knockdown of these genes in MIN6 cells altered cell death, insulin secretion, and stress gene expression. In conclusion, these data revealed novel gene regulatory networks involved in beta‐cell compensation and failure. Preemptive metabolic reconfiguration in diabetes‐resistant islets may dampen metabolic activation and cellular stress during obesity.
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