Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic β-cell dysfunction. Reduced mitochondrial function is thought to ...be central to β-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in β-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D β-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D β-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their β-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of β-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D β-cells where we had little knowledge of which changes cause β-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to β-cell mitochondrial dysfunction in T2D.
Maintenance of a normal fetal nutrient supply requires major adaptations in maternal metabolic physiology, including of the islet beta-cell. The role of lipid signaling processes in the mechanisms of ...islet beta-cell adaptation to pregnancy has been minimally investigated.
To determine the effects of pregnancy on islet fatty acid (FA) metabolic partitioning and FA augmentation of glucose-stimulated insulin secretion (GSIS).
Age matched virgin, early pregnant (gestational day-11, G11) and late pregnant (G19) Sprague-Dawley rats were studied. Fasted and fed state biochemistry, oral glucose tolerance tests (OGTT), and fasted and post-OGTT liver glycogen, were determined to assess
metabolic characteristics. In isolated islets, FA (BSA-bound palmitate 0.25 mmol/l) augmentation of GSIS, FA partitioning into esterification and oxidation processes using metabolic tracer techniques, lipolysis by glycerol release, triacylglycerols (TG) content, and the expression of key beta-cell genes were determined.
Plasma glucose in pregnancy was lower, including during the OGTT (glucose area under the curve 0-120 min (AUC
); 655±24 versus 849±13 mmol.l
.min; G19
virgin;
<0.0001), with plasma insulin concentrations equivalent to those of virgin rats (insulin AUC
; 97±7 versus 83±7 ng.ml
.min; G19 vs virgin; not significant). Liver glycogen was depleted in fasted G19 rats with full recovery after oral glucose. Serum TG increased during pregnancy (4.4±0.4, 6.7±0.5; 17.1±1.5 mmol/l; virgin, G11, G19,
<0.0001), and islet TG content decreased (147±42, 172±27, 73±13 ng/µg protein; virgin, G11, G19;
<0.01). GSIS in isolated islets was increased in G19 compared to virgin rats, and this effect was augmented in the presence of FA. FA esterification into phospholipids, monoacylglycerols and TG were increased, whereas FA oxidation was reduced, in islets of pregnant compared to virgin rats, with variable effects on lipolysis dependent on gestational age. Expression of
, a key regulator of mitochondrial metabolism, was reduced by 51% in G11 and 64% in G19 pregnant rat islets compared to virgin rat islets (
<0.001).
A lowered set-point for islet and hepatic glucose homeostasis in the pregnant rat has been confirmed. Islet adaptation to pregnancy includes increased FA esterification, reduced FA oxidation, and enhanced FA augmentation of glucose-stimulated insulin secretion.
Chronic hyperglycemia contributes to β-cell dysfunction in diabetes and with islet transplantation, but the mechanisms remain unclear. Recent studies demonstrate that the unfolded protein response ...(UPR) is critical for β-cell function. Here, we assessed the influence of hyperglycemia on UPR gene expression in transplanted islets. Streptozotocin-induced diabetic or control nondiabetic mice were transplanted under the kidney capsule with syngeneic islets either sufficient or not to normalize hyperglycemia. Twenty-one days after transplantation, islet grafts were excised and RT-PCR was used to assess gene expression. In islet grafts from diabetic mice, expression levels of many UPR genes of the IRE1/ATF6 pathways, which are important for adaptation to endoplasmic reticulum stress, were markedly reduced compared with that in islet grafts from control mice. UPR genes of the PERK pathway were also downregulated. The normalization of glycemia restored the changes in mRNA expression, suggesting that chronic hyperglycemia contributes to the downregulation of multiple arms of UPR gene expression. Similar correlations were observed between blood glucose and mRNA levels of transcription factors involved in the maintenance of β-cell phenotype and genes implicated in β-cell function, suggesting convergent regulation of UPR gene expression and β-cell differentiation by hyperglycemia. However, the normalization of glycemia was not accompanied by restoration of antioxidant or pro-inflammatory cytokine mRNA levels, which were increased in islet grafts from diabetic mice. These studies demonstrate that chronic hyperglycemia contributes to the downregulation of multiple arms of UPR gene expression in transplanted mouse islets. Failure of the adaptive UPR may contribute to β-cell dedifferentiation and dysfunction in diabetes.
Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response (UPR) have been implicated in β-cell death in type 1 and type 2 diabetes. However, the UPR is also a fundamental ...mechanism required for β-cell adaptation and survival. The mechanisms regulating the transition from adaptive to apoptotic UPR remain to be clarified. Here, we investigated the relationships between XBP1, CHOP and JNK in the transition from adaptive to apoptotic UPR and β-cell death in models of type 1 and type 2 diabetes. XBP1 inhibition potentiated cell death induced by pro-inflammatory cytokines or the saturated fatty acid palmitate in MIN6 β-cells. This response was prevented by CHOP inhibition. IRE1/XBP1 inhibition led to alterations in islets from diabetes-resistant ob/ob mice that resemble those found in diabetes, including increases in cell death and inflammation and antioxidant gene expression. Similarly, IRE1/XBP1 inhibition increased cell death in islets from NOD mice. On the other hand, JNK inhibition: 1) increased adaptive UPR and reduced cell death in islets from diabetic db/db mice, and 2) restored adaptive UPR while protecting against apoptotic UPR gene expression and β-cell death and dysfunction following cytokine exposure. These findings suggest that the balance between XBP1-mediated adaptive and CHOP-dependent apoptotic UPR is critically important for β-cell survival during ER stress. JNK activation regulates the transition from adaptive to apoptotic UPR, thus providing a mechanism for β-cell propensity to cell death rather than ER stress adaptation in type 1 and type 2 diabetes.
•Balance between adaptive and apoptotic UPR regulates β-cell survival during ER stress.•XBP1 inhibition potentiates cytokine- and palmitate-induced β-cell death.•JNK regulates the transition from adaptive UPR to β-cell death in diabetes.
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
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.
Tau protein is implicated in the pathogenesis of Alzheimer's disease (AD) and other tauopathies, but its physiological function is in debate. Mostly explored in the brain, tau is also expressed in ...the pancreas. We further explored the mechanism of tau's involvement in the regulation of glucose-stimulated insulin secretion (GSIS) in islet β-cells, and established a potential relationship between type 2 diabetes mellitus (T2DM) and AD. We demonstrate that pancreatic tau is crucial for insulin secretion regulation and glucose homeostasis. Tau levels were found to be elevated in β-islet cells of patients with T2DM, and loss of tau enhanced insulin secretion in cell lines, drosophila, and mice. Pharmacological or genetic suppression of tau in the db/db diabetic mouse model normalized glucose levels by promoting insulin secretion and was recapitulated by pharmacological inhibition of microtubule assembly. Clinical studies further showed that serum tau protein was positively correlated with blood glucose levels in healthy controls, which was lost in AD. These findings present tau as a common therapeutic target between AD and T2DM.
The normal beta-cell response to obesity-associated insulin resistance is hypersecretion of insulin. Type 2 diabetes develops in subjects with beta-cells that are susceptible to failure. Here, we ...investigated the time-dependent gene expression changes in islets of diabetes-prone db/db and diabetes-resistant ob/ob mice. The expressions of adaptive unfolded protein response (UPR) genes were progressively induced in islets of ob/ob mice, whereas they declined in diabetic db/db mice. Genes important for beta-cell function and maintenance of the islet phenotype were reduced with time in db/db mice, whereas they were preserved in ob/ob mice. Inflammation and antioxidant genes displayed time-dependent upregulation in db/db islets but were unchanged in ob/ob islets. Treatment of db/db mouse islets with the chemical chaperone 4-phenylbutyric acid partially restored the changes in several beta-cell function genes and transcription factors but did not affect inflammation or antioxidant gene expression. These data suggest that the maintenance (or suppression) of the adaptive UPR is associated with beta-cell compensation (or failure) in obese mice. Inflammation, oxidative stress, and a progressive loss of beta-cell differentiation accompany diabetes progression. The ability to maintain the adaptive UPR in islets may protect against the gene expression changes that underlie diabetes development in obese mice.
Background
: Pancreatic beta cell dedifferentiation may be an important contributing factor to beta cell failure in type 2 diabetes, but the triggering stimuli are poorly understood. Macrophage ...levels are elevated in the islets of humans and mice with type 2 diabetes, but their effect on beta cell identity is not clear. 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, obese diabetes-prone
db/db
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 to controls. Macrophages from
db/db
and
ob/ob
islets displayed distinct changes in gene expression, suggesting differential shifts in functional state. Macrophages from
db/db
islets maintained pro-inflammatory gene expression, whereas macrophages from
ob/ob
islets shifted towards an anti-inflammatory gene expression pattern with elevated levels of transforming growth factor beta 1 (
Tgfb1
) and reduced interleukin 1 beta (
IL1b
). Clodronate-liposome treatment of islets depleted macrophages, as evidenced by reduced mRNA expression of
CD11b
and
F4/80
. The depletion of macrophages in
db/db
islets increased the expression of beta cell identity genes. The mRNA levels of islet-associated transcription factors (
Mafa
and
Pdx1
), glucose transporter (
Glut2
), ATP-sensitive potassium channel (
kcnj11
), incretin receptor (
Gipr
) and adaptive unfolded protein response 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. Furthermore, macrophage depletion in
db/db
islets reduced the expression of genes involved in inflammatory (
IL1b
,
Fas
and
Nfkbia
) and oxidative stress catalase (
Cat
) and heme oxygenase-1 (
Hmox1
). In contrast, depletion of macrophages in islets of
ob/ob
mice did not affect beta cell identity gene expression.
Conclusion
: These novel findings suggest that distinct alterations in islet macrophages of obese mice are critically important for the maintenance of beta cell compensation or the loss of beta cell identity in diabetes. Depleting islet macrophages could be beneficial for reducing beta cell stress and preserving beta cell identity in type 2 diabetes.
Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic beta-cell dysfunction. Reduced mitochondrial function is thought ...to be central to beta-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in beta-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D beta-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D beta-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their beta-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of beta-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D beta-cells where we had little knowledge of which changes cause beta-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to beta-cell mitochondrial dysfunction in T2D.
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