Insulin release from pancreatic β-cells is required to maintain normal glucose homoeostasis in man and many other animals. Defective insulin secretion underlies all forms of diabetes mellitus, a ...disease currently reaching epidemic proportions worldwide. Although the destruction of β-cells is responsible for Type 1 diabetes (T1D), both lowered β-cell mass and loss of secretory function are implicated in Type 2 diabetes (T2D). Emerging results suggest that a functional deficiency, involving de-differentiation of the mature β-cell towards a more progenitor-like state, may be an important driver for impaired secretion in T2D. Conversely, at least in rodents, reprogramming of islet non-β to β-cells appears to occur spontaneously in models of T1D, and may occur in man. In the present paper, we summarize the biochemical properties which define the 'identity' of the mature β-cell as a glucose sensor par excellence. In particular, we discuss the importance of suppressing a group of 11 'disallowed' housekeeping genes, including Ldha and the monocarboxylate transporter Mct1 (Slc16a1), for normal nutrient sensing. We then survey the changes in the expression and/or activity of β-cell-enriched transcription factors, including FOXO1, PDX1, NKX6.1, MAFA and RFX6, as well as non-coding RNAs, which may contribute to β-cell de-differentiation and functional impairment in T2D. The relevance of these observations for the development of new approaches to treat T1D and T2D is considered.
Background
The prognostic value of tumor‐associated dendritic cells (DC) in colon cancer remains poorly understood. This may be in part due to the interchangeable expression of immunostimulatory and ...immunoinhibitory molecules on DC. Here we investigated the prognostic impact of CD11c+ DC co–expressing the immunoinhibitory molecule PD‐L1 and their spatial relationship with CD8+ T‐cells in patients treated for stage III colon cancer.
Methods
Tissue microarrays containing representative cores of central tumor, leading edge, and adjacent normal tissue from 221 patients with stage III colon cancer were immunostained for CD8, CD11c, PD‐L1, and cytokeratin using immunofluorescent probes. Cells were quantified using StrataQuest digital image analysis software, with intratumoral and stromal regions analyzed separately. Kaplan‐Meier estimates and Cox regression were used to assess survival.
Results
Intratumoral CD8+ cell density (HR = .52, 95% confidence interval CI .33‐.83, P = .007), stromal CD11c+ cell density (HR = .52, 95% CI .33‐.83, P = .006), intratumoral CD11c+PD‐L1+ cell density (HR = .57, 95% CI .35‐.92, P = .021), and stromal CD11c+PD‐L1+ cell density (HR = .48, 95% CI .30‐.77, P = .003) on leading‐edge cores were all significantly associated with good survival. CD8+ cell density was positively correlated with both CD11c+ cell density and CD11c+PD‐L1+ cell density in tumor epithelium and stromal compartments.
Conclusion
Here we showed that PD‐L1‐expressing DC in the tumor microenvironment are associated with improved survival in stage III colon cancer and likely reflect an immunologically “hot” tumor microenvironment. Further investigation into the expression of immunomodulatory molecules by tumor‐associated DC may help to further elucidate their prognostic value.
In this study, using immunofluorescence analysis of fixed surgical samples, we demonstrate that PD‐L1 expressing dendritic cells are associated with good prognosis in stage III colon cancer. However, densities of PD‐L1 expressing dendritic cells were correlated to CD8 T cell density, indicating that they may represent an immunologically “hot” tumor microenvironment. Further investigation into the expression of immunomodulatory molecules by DC is required.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
All forms of diabetes mellitus involve the loss or dysfunction of pancreatic beta cells, with the former predominating in type 1 diabetes and the latter in type 2 diabetes. Deeper understanding of ...the coupling mechanisms that link glucose metabolism in these cells to the control of insulin secretion is therefore likely to be essential to develop new therapies. Beta cells display a remarkable metabolic specialisation, expressing high levels of metabolic sensing enzymes, including the glucose transporter GLUT2 (encoded by
SLC2A2
) and glucokinase (encoded by
GCK
). Genetic evidence flowing from both monogenic forms of diabetes and genome-wide association studies for the more common type 2 diabetes, supports the importance for normal glucose-stimulated insulin secretion of metabolic signalling via altered ATP generation, while also highlighting unsuspected roles for Zn
2+
storage, intracellular lipid transfer and other processes. Intriguingly, genes involved in non-oxidative metabolic fates of the sugar, such as those for lactate dehydrogenase (
LDHA
) and monocarboxylate transporter-1 (MCT-1
SLC16A1
), as well as the acyl-CoA thioesterase (
ACOT7
) and others, are selectively repressed (‘disallowed’) in beta cells. Furthermore, mutations in genes critical for mitochondrial oxidative metabolism, such as
TRL-CAG1–7
encoding tRNALeu, are linked to maternally inherited forms of diabetes. Correspondingly, impaired Ca
2+
uptake into mitochondria, or collapse of a normally interconnected mitochondrial network, are associated with defective insulin secretion. Here, we suggest that altered mitochondrial metabolism may also impair beta cell–beta cell communication. Thus, we argue that defective oxidative glucose metabolism is central to beta cell failure in diabetes, acting both at the level of single beta cells and potentially across the whole islet to impair insulin secretion.
Graphical abstract
Full text
Available for:
EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Glucose induces insulin release from pancreatic β-cells by stimulating ATP synthesis, membrane depolarisation and Ca(2+) influx. As well as activating ATP-consuming processes, cytosolic Ca(2+) ...increases may also potentiate mitochondrial ATP synthesis. Until recently, the ability to study the role of mitochondrial Ca(2+) transport in glucose-stimulated insulin secretion has been hindered by the absence of suitable approaches either to suppress Ca(2+) uptake into these organelles, or to examine the impact on β-cell excitability. Here, we have combined patch-clamp electrophysiology with simultaneous real-time imaging of compartmentalised changes in Ca(2+) and ATP/ADP ratio in single primary mouse β-cells, using recombinant targeted (Pericam or Perceval, respectively) as well as entrapped intracellular (Fura-Red), probes. Through shRNA-mediated silencing we show that the recently-identified mitochondrial Ca(2+) uniporter, MCU, is required for depolarisation-induced mitochondrial Ca(2+) increases, and for a sustained increase in cytosolic ATP/ADP ratio. By contrast, silencing of the mitochondrial Na(+)-Ca(2+) exchanger NCLX affected the kinetics of glucose-induced changes in, but not steady state values of, cytosolic ATP/ADP. Exposure to gluco-lipotoxic conditions delayed both mitochondrial Ca(2+) uptake and cytosolic ATP/ADP ratio increases without affecting the expression of either gene. Mitochondrial Ca(2+) accumulation, mediated by MCU and modulated by NCLX, is thus required for normal glucose sensing by pancreatic β-cells, and becomes defective in conditions mimicking the diabetic milieu.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Aims/hypothesis
Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca
2+
uptake into pancreatic beta cell mitochondria potentiates or antagonises ...this process is still a matter of debate. Although the mitochondrial Ca
2+
importer (MCU) complex is thought to represent the main route for Ca
2+
transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo.
Methods
Here, we inactivated the pore-forming subunit of the MCU, encoded by
Mcu
, selectively in mouse beta cells using
Ins1
Cre
-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca
2+
concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance.
Results
Glucose-stimulated mitochondrial Ca
2+
accumulation (
p
< 0.05), ATP production (
p
< 0.05) and insulin secretion (
p
< 0.01) were strongly inhibited in beta cell-specific
Mcu
-null (β
Mcu
-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca
2+
concentrations increased (
p
< 0.001), whereas mitochondrial membrane depolarisation improved in β
Mcu
-KO animals. β
Mcu
-KO mice displayed impaired in vivo insulin secretion at 5 min (
p
< 0.001) but not 15 min post-i.p. injection of glucose, whilst the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (
p
< 0.05) in young β
Mcu
-KO (<12 weeks), but not in older animals vs WT mice.
Conclusions/interpretation
MCU is crucial for mitochondrial Ca
2+
uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in β
Mcu
-KO mice remain to be established.
Full text
Available for:
EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Ca2+ is the key intracellular regulator of insulin secretion, acting in the β‐cell as the ultimate trigger for exocytosis. In response to high glucose, ATP‐sensitive K+ channel closure and plasma ...membrane depolarization engage a sophisticated machinery to drive pulsatile cytosolic Ca2+ changes. Voltage‐gated Ca2+ channels, Ca2+‐activated K+ channels and Na+/Ca2+ exchange all play important roles. The use of targeted Ca2+ probes has revealed that during each cytosolic Ca2+ pulse, uptake of Ca2+ by mitochondria, endoplasmic reticulum (ER), secretory granules and lysosomes fine‐tune cytosolic Ca2+ dynamics and control organellar function. For example, changes in the expression of the Ca2+‐binding protein Sorcin appear to provide a link between ER Ca2+ levels and ER stress, affecting β‐cell function and survival. Across the islet, intercellular communication between highly interconnected “hubs,” which act as pacemaker β‐cells, and subservient “followers,” ensures efficient insulin secretion. Loss of connectivity is seen after the deletion of genes associated with type 2 diabetes (T2D) and follows metabolic and inflammatory insults that characterize this disease. Hubs, which typically comprise ~1%‐10% of total β‐cells, are repurposed for their specialized role by expression of high glucokinase (Gck) but lower Pdx1 and Nkx6.1 levels. Single cell‐omics are poised to provide a deeper understanding of the nature of these cells and of the networks through which they communicate. New insights into the control of both the intra‐ and intercellular Ca2+ dynamics may thus shed light on T2D pathology and provide novel opportunities for therapy.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Heterozygous mutations in the human paired box gene PAX6 lead to impaired glucose tolerance. Although embryonic deletion of the Pax6 gene in mice leads to loss of most pancreatic islet cell types, ...the functional consequences of Pax6 loss in adults are poorly defined. Here we developed a mouse line in which Pax6 was selectively inactivated in β cells by crossing animals with floxed Pax6 alleles to mice expressing the inducible Pdx1CreERT transgene. Pax6 deficiency, achieved by tamoxifen injection, caused progressive hyperglycemia. Although β cell mass was preserved 8 days post-injection, total insulin content and insulin:chromogranin A immunoreactivity were reduced by ∼60%, and glucose-stimulated insulin secretion was eliminated. RNA sequencing and quantitative real-time PCR analyses revealed that, although the expression of key β cell genes, including Ins2, Slc30a8, MafA, Slc2a2, G6pc2, and Glp1r, was reduced after Pax6 deletion, that of several genes that are usually selectively repressed (“disallowed”) in β cells, including Slc16a1, was increased. Assessed in intact islets, glucose-induced ATP:ADP increases were significantly reduced (p < 0.05) in βPax6KO versus control β cells, and the former displayed attenuated increases in cytosolic Ca2+. Unexpectedly, glucose-induced increases in intercellular connectivity were enhanced after Pax6 deletion, consistent with increases in the expression of the glucose sensor glucokinase, but decreases in that of two transcription factors usually expressed in fully differentiated β-cells, Pdx1 and Nkx6.1, were observed in islet “hub” cells. These results indicate that Pax6 is required for the functional identity of adult β cells. Furthermore, deficiencies in β cell glucose sensing are likely to contribute to defective insulin secretion in human carriers of PAX6 mutations.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We and others have previously identified a group of genes, dubbed "disallowed," whose expression is markedly lower in pancreatic islets than in other mammalian cell types. Forced mis-expression of ...several members of this family leads to defective insulin secretion, demonstrating the likely importance of disallowance for normal beta cell function. Up to now, transcriptomic comparisons have been based solely on data from whole islets. This raises the possibilities that (a) there may be important differences in the degree of disallowance of family members between beta and other either neuroendocrine cells; (b) beta (or alpha) cell disallowed genes may have gone undetected. To address this issue, we survey here recent massive parallel sequencing (RNA-Seq) datasets from purified mouse and human islet cells. Our analysis reveals that the most strongly disallowed genes are similar in beta and alpha cells, with 11β-hydroxysteroid dehydrogenase (
mRNA being essentially undetectable in both cell types. The analysis also reveals that several genes involved in cellular proliferation, including
and
4, and previously assumed to be disallowed in both beta and alpha cells, are selectively repressed only in the beta cell. The latter finding supports the view that beta cell growth is selectively restricted in adults, providing a mechanism to avoid excessive insulin production and the risk of hypoglycaemia. Approaches which increase the expression or activity of selected disallowed genes in the beta cell may provide the basis for novel regenerative therapies in type 2 diabetes.
The 2-oxoglutarate dehydrogenase (OGDH) complex is an important control point in vertebrate mitochondrial oxidative metabolism, including in the citrate cycle and catabolism of alternative fuels ...including glutamine. It is subject to allosteric regulation by NADH and the ATP/ADP ratio, and by Ca(2+) through binding to the E1 subunit. The latter involves a unique Ca(2+)-binding site which includes D(114)ADLD (site 1). Here, we describe three splice variants of E1 in which either the exon expressing this site is replaced with another exon (loss of site 1, LS1) or an additional exon is expressed leading to the insertion of 15 amino acids just downstream of site 1 (Insert), or both changes occur together (LS1/Insert). We show that all three variants are essentially Ca(2+)-insensitive. Comparison of massive parallel sequence (RNA-Seq) databases demonstrates predominant expression of the Ca(2+)-sensitive archetype form in heart and skeletal muscle, but substantial expression of the Ca(2+)-insensitive variants in brain, pancreatic islets and other tissues. Detailed proteomic and activity studies comparing OGDH complexes from rat heart and brain confirmed the substantial difference in expression between these tissues. The evolution of OGDH variants was explored using bioinformatics, and this indicated that Ca(2+)-sensitivity arose with the emergence of chordates. In all species examined, this was associated with the co-emergence of Ca(2+)-insensitive variants suggesting a retained requirement for the latter in some settings. Tissue-specific expression of OGDH splice variants may thus provide a mechanism that tunes the control of the enzyme to the specialized metabolic and signalling needs of individual cell types.
PDF
