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.
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
Aims/hypothesis
Hypoxia may contribute to beta cell failure in type 2 diabetes and islet transplantation. The adaptive unfolded protein response (UPR) is required for endoplasmic reticulum (ER) ...homeostasis. Here we investigated whether or not hypoxia regulates the UPR in beta cells and the role the adaptive UPR plays during hypoxic stress.
Methods
Mouse islets and MIN6 cells were exposed to various oxygen (O
2
) tensions. DNA-damage inducible transcript 3 (DDIT3), hypoxia-inducible transcription factor (HIF)1α and HSPA5 were knocked down using small interfering (si)RNA;
Hspa5
was also overexpressed.
db/db
mice were used.
Results
Hypoxia-response genes were upregulated in vivo in the islets of diabetic, but not prediabetic,
db/db
mice. In isolated mouse islets and MIN6 cells, O
2
deprivation (1–5% vs 20%; 4–24 h) markedly reduced the expression of adaptive UPR genes, including
Hspa5
,
Hsp90b1
,
Fkbp11
and spliced
Xbp1
. Coatomer protein complex genes (
Copa
,
Cope
,
Copg
also known as
Copg1
,
Copz1
and
Copz2
) and ER-to-Golgi protein trafficking were also reduced, whereas apoptotic genes (
Ddit3
,
Atf3
and
Trb3
also known as
Trib3
), c-Jun N-terminal kinase (JNK) phosphorylation and cell death were increased. Inhibition of JNK, but not HIF1α, restored adaptive UPR gene expression and ER-to-Golgi protein trafficking while protecting against apoptotic genes and cell death following hypoxia. DDIT3 knockdown delayed the loss of the adaptive UPR and partially protected against hypoxia-induced cell death. The latter response was prevented by HSPA5 knockdown. Finally,
Hspa5
overexpression significantly protected against hypoxia-induced cell death.
Conclusions/interpretation
Hypoxia inhibits the adaptive UPR in beta cells via JNK and DDIT3 activation, but independently of HIF1α. Downregulation of the adaptive UPR contributes to reduced ER-to-Golgi protein trafficking and increased beta cell death during hypoxic stress.
Aims/hypothesis
Oxidative stress is implicated in beta cell glucotoxicity in type 2 diabetes. Inhibitor of differentiation (ID) proteins are transcriptional regulators induced by hyperglycaemia in ...islets, but the mechanisms involved and their role in beta cells are not clear. Here we investigated whether or not oxidative stress regulates ID levels in beta cells and the role of ID proteins in beta cells during oxidative stress.
Methods
MIN6 cells were cultured in H
2
O
2
or ribose to induce oxidative stress. ID1, ID3 and small MAF proteins (MAFF, MAFG and MAFK) were inhibited using small interfering RNA. Isolated islets from
Id1
−/−
,
Id3
−/−
and diabetic
db/db
mice were used.
Results
ID1–4 expression was upregulated in vivo in the islets of diabetic
db/db
mice and stimulated in vitro by ribose and H
2
O
2
.
Id1/3
inhibition reduced the expression of multiple antioxidant genes and potentiated oxidative stress-induced apoptosis. This finding was associated with increased levels of intracellular reactive oxygen species, altered mitochondrial morphology and reduced expression of
Tfam
, which encodes a mitochondrial transcription factor, and respiratory chain components.
Id1/3
inhibition also reduced the expression of small MAF transcription factors (
MafF
,
MafG
and
MafK
), interacting partners of nuclear factor, erythroid 2-like 2 (NFE2L2), master regulator of the antioxidant response. Inhibition of small MAFs reduced the expression of antioxidant genes and potentiated oxidative stress-induced apoptosis, thus recapitulating the effects of
Id1/3
inhibition.
Conclusions/interpretation
Our study identifies IDs as a novel family of oxidative stress-responsive proteins in beta cells. IDs are crucial regulators of the adaptive antioxidant–mitochondrial response that promotes beta cell survival during oxidative stress through a novel link to the NFE2L2–small MAF pathway.
Aims/hypothesis
The mechanisms responsible for beta cell compensation in obesity and for beta cell failure in type 2 diabetes are poorly defined. The mRNA levels of several metallothionein (MT) genes ...are upregulated in islets from individuals with type 2 diabetes, but their role in beta cells is not clear. Here we examined: (1) the temporal changes of islet
Mt1
and
Mt2
gene expression in mouse models of beta cell compensation and failure; and (2) the role of
Mt1
and
Mt2
in beta cell function and glucose homeostasis in mice.
Methods
Mt1
and
Mt2
expression was assessed in islets from: (1) control lean (chow diet-fed) and diet-induced obese (high-fat diet-fed for 6 weeks) mice; (2) mouse models of diabetes (
db/db
mice) at 6 weeks old (prediabetes) and 16 weeks old (after diabetes onset) and age-matched
db/+
(control) mice; and (3) obese non-diabetic
ob/ob
mice (16-week-old) and age-matched
ob/+
(control) mice.
MT1E
,
MT1X
and
MT2A
expression was assessed in islets from humans with and without type 2 diabetes.
Mt1-Mt2
double-knockout (KO) mice, transgenic mice overexpressing
Mt1
under the control of its natural promoter (Tg-
Mt1
) and corresponding control mice were also studied. In MIN6 cells, MT1 and MT2 were inhibited by small interfering RNAs. mRNA levels were assessed by real-time RT-PCR, plasma insulin and islet MT levels by ELISA, glucose tolerance by i.p. glucose tolerance tests and overnight fasting-1 h refeeding tests, insulin tolerance by i.p
.
insulin tolerance tests, insulin secretion by RIA, cytosolic free Ca
2+
concentration with Fura-2 leakage resistant (Fura-2 LR), cytosolic free Zn
2+
concentration with Fluozin-3, and NAD(P)H by autofluorescence.
Results
Mt1
and
Mt2
mRNA levels were reduced in islets of murine models of beta cell compensation, whereas they were increased in diabetic
db/db
mice. In humans,
MT1X
mRNA levels were significantly upregulated in islets from individuals with type 2 diabetes in comparison with non-diabetic donors, while
MT1E
and
MT2A
mRNA levels were unchanged. Ex vivo, islet
Mt1
and
Mt2
mRNA and MT1 and MT2 protein levels were downregulated after culture with glucose at 10–30 mmol/l vs 2–5 mmol/l, in association with increased insulin secretion. In human islets, mRNA levels of
MT1E
,
MT1X
and
MT2A
were downregulated by stimulation with physiological and supraphysiological levels of glucose. In comparison with wild-type (WT) mice,
Mt1-Mt2
double-KO mice displayed improved glucose tolerance in association with increased insulin levels and enhanced insulin release from isolated islets. In contrast, isolated islets from Tg-
Mt1
mice displayed impaired glucose-stimulated insulin secretion (GSIS). In both
Mt1-Mt2
double-KO and Tg-
Mt1
models, the changes in GSIS occurred despite similar islet insulin content, rises in cytosolic free Ca
2+
concentration and NAD(P)H levels, or intracellular Zn
2+
concentration vs WT mice. In MIN6 cells, knockdown of MT1 but not MT2 potentiated GSIS, suggesting that
Mt1
rather than
Mt2
affects beta cell function.
Conclusions/interpretation
These findings implicate
Mt1
as a negative regulator of insulin secretion. The downregulation of
Mt1
is associated with beta cell compensation in obesity, whereas increased
Mt1
accompanies beta cell failure and type 2 diabetes.
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.
Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins whose precise biological roles have not been fully characterized. Existing evidence implicated MTs in heavy ...metal detoxification, metal ion homeostasis and antioxidant defense. MTs were thus categorized as protective effectors that contribute to cellular homeostasis and survival. This view has, however, been challenged by emerging evidence in different medical fields revealing novel pathophysiological roles of MTs, including inflammatory bowel disease, neurodegenerative disorders, carcinogenesis and diabetes. In the present focused review, we discuss the evidence for the role of MTs in pancreatic beta-cell biology and insulin secretion. We highlight the pattern of specific isoforms of MT gene expression in rodents and human beta-cells. We then discuss the mechanisms involved in the regulation of MTs in islets under physiological and pathological conditions, particularly type 2 diabetes, and analyze the evidence revealing adaptive and negative roles of MTs in beta-cells and the potential mechanisms involved. Finally, we underscore the unsettled questions in the field and propose some future research directions.
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.
Sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i), or gliflozins, are anti-diabetic drugs that lower glycemia by promoting glucosuria, but they also stimulate endogenous glucose and ketone ...body production. The likely causes of these metabolic responses are increased blood glucagon levels, and decreased blood insulin levels, but the mechanisms involved are hotly debated. This study verified whether or not SGLT2i affect glucagon and insulin secretion by a direct action on islet cells in three species, using multiple approaches.
We tested the in vivo effects of two selective SGLT2i (dapagliflozin, empagliflozin) and a SGLT1/2i (sotagliflozin) on various biological parameters (glucosuria, glycemia, glucagonemia, insulinemia) in mice. mRNA expression of SGLT2 and other glucose transporters was assessed in rat, mouse, and human FACS-purified α- and β-cells, and by analysis of two human islet cell transcriptomic datasets. Immunodetection of SGLT2 in pancreatic tissues was performed with a validated antibody. The effects of dapagliflozin, empagliflozin, and sotagliflozin on glucagon and insulin secretion were assessed using isolated rat, mouse and human islets and the in situ perfused mouse pancreas. Finally, we tested the long-term effect of SGLT2i on glucagon gene expression.
SGLT2 inhibition in mice increased the plasma glucagon/insulin ratio in the fasted state, an effect correlated with a decline in glycemia. Gene expression analyses and immunodetections showed no SGLT2 mRNA or protein expression in rodent and human islet cells, but moderate SGLT1 mRNA expression in human α-cells. However, functional experiments on rat, mouse, and human (29 donors) islets and the in situ perfused mouse pancreas did not identify any direct effect of dapagliflozin, empagliflozin or sotagliflozin on glucagon and insulin secretion. SGLT2i did not affect glucagon gene expression in rat and human islets.
The data indicate that the SGLT2i-induced increase of the plasma glucagon/insulin ratio in vivo does not result from a direct action of the gliflozins on islet cells.
•Gliflozins (SGLT2 and SGLT1/2 inhibitors) increase plasma glucagon/insulin ratio.•SGLT2 is not expressed in rodent and human pancreatic α- and β-cells.•SGLT1 is however expressed in human α-cells.•SGLT2 and SGLT1/2 inhibitors do not directly affect glucagon and insulin secretion.
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