Ablation of TSC2 enhances insulin secretion by increasing the number of mitochondria through activation of mTORC1

Aim

We previously found that chronic tuberous sclerosis protein 2 (TSC2) deletion induces activation of mammalian target of rapamycin Complex 1 (mTORC1) and leads to hypertrophy of pancreatic beta cells from pancreatic beta cell-specific TSC2 knockout (βTSC2^−/−) mice. The present study examines the effects of TSC2 ablation on insulin secretion from pancreatic beta cells.

Methods

Isolated islets from βTSC2^−/− mice and TSC2 knockdown insulin 1 (INS-1) insulinoma cells treated with small interfering ribonucleic acid were used to investigate insulin secretion, ATP content and the expression of mitochondrial genes.

Results

Activation of mTORC1 increased mitochondrial DNA expression, mitochondrial density and ATP production in pancreatic beta cells of βTSC2^−/− mice. In TSC2 knockdown INS-1 cells, mitochondrial DNA expression, mitochondrial density and ATP production were increased compared with those in control INS-1 cells, consistent with the phenotype of βTSC2^−/− mice. TSC2 knockdown INS-1 cells also exhibited augmented insulin secretory response to glucose. Rapamycin inhibited mitochondrial DNA expression and ATP production as well as insulin secretion in response to glucose. Thus, βTSC2^−/− mice exhibit hyperinsulinemia due to an increase in the number of mitochondria as well as enlargement of individual beta cells via activation of mTORC1.

Conclusion

Activation of mTORC1 by TSC2 ablation increases mitochondrial biogenesis and enhances insulin secretion from pancreatic beta cells.     

Essential Role of Mitochondrial Ca2+ Uniporter in the Generation of Mitochondrial pH Gradient and Metabolism-Secretion Coupling in Insulin-releasing Cells

In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca²⁺ influx, which triggers insulin exocytosis. The mitochondrial Ca²⁺ uniporter (MCU) mediates Ca²⁺ uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca²⁺ rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca²⁺. Suppression of the putative Ca²⁺/H⁺ antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca²⁺-induced matrix acidification. These results demonstrate that MCU-mediated Ca²⁺ uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.     

Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells

Cannabinoid 1 receptors (CB1Rs) are expressed in peripheral tissues, including islets of Langerhans, where their function(s) is under scrutiny. Using mouse β‐cell lines, human islets and CB1R‐null (CB1R^?/?) mice, we have now investigated the role of CB1Rs in modulating β‐cell function and glucose responsiveness. Synthetic CB1R agonists diminished GLP‐1‐mediated cAMP accumulation and insulin secretion as well as glucose‐stimulated insulin secretion in mouse β‐cell lines and human islets. In addition, silencing CB1R in mouse β cells resulted in an increased expression of pro‐insulin, glucokinase (GCK) and glucose transporter 2 (GLUT2), but this increase was lost in β cells lacking insulin receptor. Furthermore, CB1R^?/? mice had increased pro‐insulin, GCK and GLUT2 expression in β cells. Our results suggest that CB1R signalling in pancreatic islets may be harnessed to improve β‐cell glucose responsiveness and preserve their function. Thus, our findings further support that blocking peripheral CB1Rs would be beneficial to β‐cell function in type 2 diabetes.     

Chemiosmotic lysis and insulin secretion: studies of isolated granules, intact and permeabilised rat islets of Langerhans

The possible involvement of chemiosmotic lysis of secretory granules in the exocytosis of insulin from pancreatic beta cells was investigated by comparing insulin release from isolated secretory granules, from intact islets of Langerhans, and from electrically permeabilised islets. Lysis of isolated granules was stimulated by ATP in the presence of Mg2+. ATP-induced granule lysis was pH and temperature dependent and was inhibited by collapsing the pH gradient across the granule membrane by removal of permeant anions, or by increasing the extragranular osmolarity. However, insulin secretion from intact islets in response to glucose, a phosphodiesterase inhibitor or a Ca2+ ionophore was only partially inhibited by anion replacement, while Ca2+ -induced insulin release from electrically permeabilised islets was not affected by altering the extragranular or intragranular pH. These results suggest that studies of the stability of isolated granules in vitro do not necessarily relate to insulin release from whole cells, and do not support a major role for chemiosmotic lysis of secretory granules in the exocytotic release of insulin.     

Glutamine Stimulates Biosynthesis and Secretion of Insulin-like Growth Factor 2 (IGF2), an Autocrine Regulator of Beta Cell Mass and Function

IGF2 is an autocrine ligand for the beta cell IGF1R receptor and GLP-1 increases the activity of this autocrine loop by enhancing IGF1R expression, a mechanism that mediates the trophic effects of GLP-1 on beta cell mass and function. Here, we investigated the regulation of IGF2 biosynthesis and secretion. We showed that glutamine rapidly and strongly induced IGF2 mRNA translation using reporter constructs transduced in MIN6 cells and primary islet cells. This was followed by rapid secretion of IGF2 via the regulated pathway, as revealed by the presence of mature IGF2 in insulin granule fractions and by inhibition of secretion by nimodipine and diazoxide. When maximally stimulated by glutamine, the amount of secreted IGF2 rapidly exceeded its initial intracellular pool and tolbutamide, and high K⁺ increased IGF2 secretion only marginally. This indicates that the intracellular pool of IGF2 is small and that sustained secretion requires de novo synthesis. The stimulatory effect of glutamine necessitates its metabolism but not mTOR activation. Finally, exposure of insulinomas or beta cells to glutamine induced Akt phosphorylation, an effect that was dependent on IGF2 secretion, and reduced cytokine-induced apoptosis. Thus, glutamine controls the activity of the beta cell IGF2/IGF1R autocrine loop by increasing the biosynthesis and secretion of IGF2. This autocrine loop can thus integrate changes in feeding and metabolic state to adapt beta cell mass and function.     

The Granular Chloride Channel ClC-3 Is Permissive for Insulin Secretion

Insulin secretion from pancreatic β cells is dependent on maturation and acidification of the secretory granule, processes necessary for prohormone convertase cleavage of proinsulin. Previous studies in isolated β cells revealed that acidification may be dependent on the granule membrane chloride channel ClC-3, in a step permissive for a regulated secretory response. In this study, immuno-EM of β cells revealed colocalization of ClC-3 and insulin on secretory granules. Clcn3^−/− mice as well as isolated islets demonstrate impaired insulin secretion; Clcn3^−/− β cells are defective in regulated insulin exocytosis and granular acidification. Increased amounts of proinsulin were found in the majority of secretory granules in the Clcn3^−/− mice, while in Clcn3^+/+ cells, proinsulin was confined to the immature secretory granules. These results demonstrate that in pancreatic β cells, chloride channels, specifically ClC-3, are localized on insulin granules and play a role in insulin processing as well as insulin secretion through regulation of granular acidification.     

Characterization of Stimulus-Secretion Coupling in the Human Pancreatic EndoC-βH1 Beta Cell Line

Aims/Hypothesis

Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.

Methods

Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca²⁺ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.

Results

Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.

Conclusions/Interpretation

Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca²⁺ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.     

Sigma receptor knockdown augments dysfunction and apoptosis of beta cells induced by palmitate

Sigma-1 receptor (Sig-1R) is located in the endoplasmic reticulum (ER) and clustered on the mitochondria related endoplasmic membranes, which are involved in the regulation of nervous system disease. Here, we designed Sig-1R silence MIN6 cells and studied the influence of Sig-1R silence on beta cells. We showed Sig-1R inactivation in MIN6 cells could not only decrease cell proliferation but also inhibit cell cycle, and this inhibitory effect on cell cycle might be achieved by regulating the FoxM1/Plk1/Cenpa pathway. Moreover, Sig-1R deficiency increased MIN6 cells sensitivity to lipotoxicity, exaggerated palmitate (PA)-induced apoptosis, and impaired insulin secretion. On the other hand, ER chaperone GRP78 and ER proapoptotic molecules CHOP increased in Sig-1R knockdown MIN6 cells. The ATP level decreased and reactive oxygen species (ROS) increased in this kind of cells. Furthermore not only GRP78 and CHOP levels, but also ATP and ROS levels changed more in Sig-1R silence cells after cultured with PA. Therefore, Sig-1R deficiency exaggerated PA induced beta cells apoptosis by aggravating ER stress and mitochondrial dysfunction. Together, our study showed that Sig-1R might influence the proliferation, apoptosis, and function of beta cells.     

Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells

Diabetes is a major complication of chronic Glucocorticoids (GCs) treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1) and 2 (Tph2), leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.     

Enhanced GLP-1- and sulfonylurea-induced insulin secretion in islets lacking leptin signaling

We have previously reported that the absence of leptin signaling in β-cells enhances glucose-stimulated insulin secretion and improves glucose tolerance in vivo. To investigate the relevance of β-cell leptin signaling in the context of postprandial or therapeutic insulin secretion, we examined the cross talk between leptin and glucagon-like peptide (GLP)-1 and sulfonylurea actions. Single and size-matched islets isolated from control or pancreas-specific leptin receptor knockout (pancreas-ObR-KO) mice were treated either with GLP-1 or with glibenclamide. Leptin suppressed GLP-1-stimulated intracellular Ca(2+) concentrations ([Ca(2+)](i)) increase that paralleled the decrease in insulin secretion in controls. In contrast, and as expected, the ObR-KO islets were nonresponsive to leptin, and instead, showed a 2.8-fold greater GLP-1-stimulated [Ca(2+)](i) increase and a 1.7-fold greater insulin secretion. Phosphorylation of cAMP-responsive element binding protein was enhanced, and phosphodiesterase enzymatic activity was suppressed in MIN6 β-cells with ObR knockdown compared with controls. The ObR-KO islets also showed significantly higher glibenclamide-induced insulin secretion compared with control islets, whereas [Ca(2+)](i) was similar to the controls. These data support enhanced insulinotropic effects of glucose, GLP-1, and sulfonylureas in the islets lacking leptin signaling with potential therapeutic implications.     

Isolation,Characterization and Potential Role in Beta Cell-Endothelium Cross-Talk of Extracellular Vesicles Released from Human Pancreatic Islets

The cross-talk between beta cells and endothelium plays a key role in islet physiopathology and in the revascularization process after islet transplantation. However, the molecular mechanisms involved in this cross-talk are not fully elucidated. Extracellular vesicles (EVs) are secreted membrane nanoparticles involved in inter-cellular communication through the transfer of proteins and nucleic acids. The aims of this study were: 1) isolation and characterization of EVs from human islets; 2) evaluation of the pro-angiogenic effect of islet-derived EVs on human islet endothelial cells (IECs). EVs were isolated by ultracentrifugation from conditioned medium of human islets and characterized by nanotrack analysis (Nanosight), FACS, western blot, bioanalyzer, mRNA/microRNA RT-PCR array. On IECs, we evaluated EV-induced insulin mRNA transfer, proliferation, resistance to apoptosis, in vitro angiogenesis, migration, gene and protein profiling. EVs sized 236±54 nm, expressed different surface molecules and islet-specific proteins (insulin, C-peptide, GLP1R) and carried several mRNAs (VEGFa, eNOS) and microRNAs (miR-27b, miR-126, miR-130 and miR-296) involved in beta cell function, insulin secretion and angiogenesis. Purified EVs were internalized into IECs inducing insulin mRNA expression, protection from apoptosis and enhancement of angiogenesis. Human islets release biologically active EVs able to shuttle specific mRNAs and microRNAs (miRNAs) into target endothelial cells. These results suggest a putative role for islet-derived EVs in beta cell-endothelium cross-talk and in the neoangiogenesis process which is critical for engraftment of transplanted islets.     

Sphingosine 1-phosphate (S1P) regulates glucose-stimulated insulin secretion in pancreatic beta cells

Recent studies suggest that sphingolipid metabolism is altered during type 2 diabetes. Increased levels of the sphingolipid ceramide are associated with insulin resistance. However, a role for sphingolipids in pancreatic beta cell function, or insulin production, and release remains to be established. Our studies in MIN6 cells and mouse pancreatic islets demonstrate that glucose stimulates an intracellular rise in the sphingolipid, sphingosine 1-phosphate (S1P), whereas the levels of ceramide and sphingomyelin remain unchanged. The increase in S1P levels by glucose is due to activation of sphingosine kinase 2 (SphK2). Interestingly, rises in S1P correlate with increased glucose-stimulated insulin secretion (GSIS). Decreasing S1P levels by treatment of MIN6 cells or primary islets with the sphingosine kinase inhibitor reduces GSIS. Moreover, knockdown of SphK2 alone results in decreased GSIS, whereas knockdown of the S1P phosphatase, Sgpp1, leads to a rise in GSIS. Treatment of mice with the sphingosine kinase inhibitor impairs glucose disposal due to decreased plasma insulin levels. Altogether, our data suggest that glucose activates SphK2 in pancreatic beta cells leading to a rise in S1P levels, which is important for GSIS.     

Taurine supplementation increases KATP channel protein content,improving Ca2+ handling and insulin secretion in islets from malnourished mice fed on a high-fat diet

Pancreatic β-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and K_ATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5 % Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced K_ATP inhibition and Cav activity. RH islets secreted less insulin at high K⁺ concentration and showed enhanced K_ATP activity. Tau supplementation normalized K⁺-induced secretion and enhanced glucose-induced Ca²⁺ influx in RHT islets. R islets presented lower Ca²⁺ influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the K_ATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the K_ATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the K_ATP channels and enhancing Synt-1 islet content.     

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes ^1-3. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass ^4-6. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets ^4,7-15. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function ^{5,6,8,9,16,17}.     

Heat shock restores insulin secretion after injury by nitric oxide by maintaining glucokinase activity in rat islets

Heat shock protein (hsp), including hsp70, has been reported to restore the glucose-induced insulin release suppressed by nitric oxide (NO). However, the mechanism underlying this recovery remains unclear. In the present study, we examine the effects, in rat islets, of heat shock on insulin secretion inhibited by a small amount of NO and also on glucose metabolism, the crucial factor in insulin release. Exposure to a higher dose (15 U/ml) of interleukin-1beta (IL-1beta) abolished the insulin release by stimulation of glucose or KCl in both control and heat shocked islets. In rat islets exposed to a lower dose (1.5 U/ml) of IL-1beta, insulin secretion in response to glucose, but not to glyceraldehydes (GA), ketoisocaproate (KIC), or KCl, was selectively impaired, concomitantly with lower ATP concentrations in the presence of 16.7 mM glucose, while such suppression of insulin secretion and ATP content was not observed in heat shock-treated islets. NO production in islets exposed to 1.5 U/ml IL-1beta was significantly, but only partly, decreased by heat shock treatment. The glucose utilization rate measurement using [5-3H]-glucose and [2-3H]-glucose and the glucokinase activity in vitro were reduced in islets treated with 1.5 U/ml IL-1beta. In heat shock-treated islets, glucose utilization and glucokinase activity were not affected by 1.5 U/ml IL-1beta. These data suggest that heat shock restores glucose-induced insulin release inhibited by NO by maintaining glucokinase activity and the glucose utilization rate in islets in addition to reducing endogenous NO production.     

Slow oscillations persist in pancreatic beta cells lacking phosphofructokinase M

Pulsatile insulin secretion by pancreatic beta cells is necessary for tight glucose control in the body. Glycolytic oscillations have been proposed as the mechanism for generating the electrical oscillations underlying pulsatile insulin secretion. The glycolytic enzyme 6-phosphofructokinase-1 (PFK) synthesizes fructose-1,6-bisphosphate (FBP) from fructose-6-phosphate. It has been proposed that the slow electrical and Ca²⁺ oscillations (periods of 3–5 min) observed in islets result from allosteric feedback activation of PFKM by FBP. Pancreatic beta cells express three PFK isozymes: PFKL, PFKM, and PFKP. A prior study of mice that were engineered to lack PFKM using a gene-trap strategy to delete Pfkm produced a mosaic reduction in global Pfkm expression, but the islets isolated from the mice still exhibited slow Ca²⁺ oscillations. However, these islets still expressed residual PFKM protein. Thus, to more fully test the hypothesis that beta cell PFKM is responsible for slow islet oscillations, we made a beta-cell-specific knockout mouse that completely lacked PFKM. While PFKM deletion resulted in subtle metabolic changes in vivo, islets that were isolated from these mice continued to exhibit slow oscillations in electrical activity, beta cell Ca²⁺ concentrations, and glycolysis, as measured using PKAR, an FBP reporter/biosensor. Furthermore, simulations obtained with a mathematical model of beta cell activity shows that slow oscillations can persist despite PFKM loss provided that one of the other PFK isoforms, such as PFKP, is present, even if its level of expression is unchanged. Thus, while we believe that PFKM may be the main regulator of slow oscillations in wild-type islets, PFKP can provide functional redundancy. Our model also suggests that PFKM likely dominates, in vivo, because it outcompetes PFKP with its higher FBP affinity and lower ATP affinity. We thus propose that isoform redundancy may rescue key physiological processes of the beta cell in the absence of certain critical genes.