Glucose-stimulated insulin secretion depends on FFA1 and Gq in neonatal mouse islets.

AIMS/HYPOTHESIS: After birth, the neonatal islets gradually acquire glucose-responsive insulin secretion, a process that is subjected to maternal imprinting. Although NEFA are major components of breastmilk and insulin secretagogues, their role for functional maturation of neonatal beta cells is still unclear. NEFA are the endogenous ligands of fatty acid receptor 1 (FFA1, encoded by Ffar1 in mice), a Gq-coupled receptor with stimulatory effect on insulin secretion. This study investigates the role of FFA1 in neonatal beta cell function and in the adaptation of offspring beta cells to parental high-fat feeding. METHODS: Wild-type (WT) and Ffar1-/- mice were fed high-fat (HFD) or chow diet (CD) for 8 weeks before mating, and during gestation and lactation. Blood variables, pancreas weight and insulin content were assessed in 1-, 6-, 11- and 26-day old (P1-P26) offspring. Beta cell mass and proliferation were determined in P1-P26 pancreatic tissue sections. FFA1/Gq dependence of insulin secretion was evaluated in isolated islets and INS-1E cells using pharmacological inhibitors and siRNA strategy. Transcriptome analysis was conducted in isolated islets. RESULTS: Blood glucose levels were higher in CD-fed Ffar1-/- P6-offspring compared with CD-fed WT P6-offspring. Accordingly, glucose-stimulated insulin secretion (GSIS) and its potentiation by palmitate were impaired in CD Ffar1-/- P6-islets. In CD WT P6-islets, insulin secretion was stimulated four- to fivefold by glucose and five- and sixfold over GSIS by palmitate and exendin-4, respectively. Although parental HFD increased blood glucose in WT P6-offspring, it did not change insulin secretion from WT P6-islets. In contrast, parental HFD abolished glucose responsiveness (i.e. GSIS) in Ffar1-/- P6-islets. Inhibition of Gq by FR900359 or YM-254890 in WT P6-islets mimicked the effect of Ffar1 deletion, i.e. suppression of GSIS and of palmitate-augmented GSIS. The blockage of Gi/o by pertussis toxin (PTX) enhanced (100-fold) GSIS in WT P6-islets and rendered Ffar1-/- P6-islets glucose responsive, suggesting constitutive activation of Gi/o. In WT P6-islets, FR900359 cancelled 90% of PTX-mediated stimulation, while in Ffar1-/- P6-islets it completely abolished PTX-elevated GSIS. The secretory defect of Ffar1-/- P6-islets did not originate from insufficient beta cells, since beta cell mass increased with the offspring's age irrespective of genotype and diet. In spite of that, in the breastfed offspring (i.e. P1-P11) beta cell proliferation and pancreatic insulin content had a genotype- and diet-driven dynamic. Under CD, the highest proliferation rate was reached by the Ffar1-/- P6 offspring (3.95% vs 1.88% in WT P6), whose islets also showed increased mRNA levels of genes (e.g. Fos, Egr1, Jun) typically high in immature beta cells. Although parental HFD increased beta cell proliferation in both WT (4.48%) and Ffar1-/- (5.19%) P11 offspring, only the WT offspring significantly increased their pancreatic insulin content upon parental HFD (5.18 µg under CD to 16.93 µg under HFD). CONCLUSIONS/INTERPRETATION: FFA1 promotes glucose-responsive insulin secretion and functional maturation of newborn islets and is required for adaptive offspring insulin secretion in the face of metabolic challenge, such as parental HFD.

Pancreatic fat cells of humans with type 2 diabetes display reduced adipogenic and lipolytic activity.

Obesity, especially visceral fat accumulation, increases the risk of type 2 diabetes (T2D). The purpose of this study was to investigate the impact of T2D on the pancreatic fat depot. Pancreatic fat pads from 17 partial pancreatectomized patients (PPP) were collected, pancreatic preadipocytes isolated, and in vitro differentiated. Patients were grouped using HbA1c into normal glucose tolerant (NGT), prediabetic (PD), and T2D. Transcriptome profiles of preadipocytes and adipocytes were assessed by RNAseq. Insulin sensitivity was estimated by quantifying AKT phosphorylation on Western blots. Lipogenic capacity was assessed with oil red O staining, lipolytic activity via fatty acid release. Secreted factors were measured using ELISA. Comparative transcriptome analysis of preadipocytes and adipocytes indicates defective upregulation of genes governing adipogenesis (NR1H3), lipogenesis (FASN, SCD, ELOVL6, and FADS1), and lipolysis (LIPE) during differentiation of cells from T2D-PPP. In addition, the ratio of leptin/adiponectin mRNA was higher in T2D than in NGT-PPP. Preadipocytes and adipocytes of NGT-PPP were more insulin sensitive than T2D-PPP cells in regard to AKT phosphorylation. Triglyceride accumulation was similar in NGT and T2D adipocytes. Despite a high expression of the receptors NPR1 and NPR2 in NGT and T2D adipocytes, lipolysis was stimulated by ANP 1.74-fold in NGT cells only. This stimulation was further increased by the PDE5 inhibitor dipyridamole (3.09-fold). Dipyridamole and forskolin increased lipolysis receptor independently 1.88-fold and 1.48-fold, respectively, solely in NGT cells. In conclusion, the metabolic status persistently affects differentiation and lipolysis of pancreatic adipocytes. These alterations could aggravate the development of T2D.

The hepatokine fetuin-A disrupts functional maturation of pancreatic beta cells.

AIMS/HYPOTHESIS: Neonatal beta cells carry out a programme of postnatal functional maturation to achieve full glucose responsiveness. A partial loss of the mature phenotype of adult beta cells may contribute to a reduction of functional beta cell mass and accelerate the onset of type 2 diabetes. We previously found that fetuin-A, a hepatokine increasingly secreted by the fatty liver and a determinant of type 2 diabetes, inhibits glucose-stimulated insulin secretion (GSIS) of human islets. Since fetuin-A is a ubiquitous fetal glycoprotein that declines peripartum, we examined here whether fetuin-A interferes with the functional maturity of beta cells. METHODS: The effects of fetuin-A were assessed during in vitro maturation of porcine neonatal islet cell clusters (NICCs) and in adult human islets. Expression alterations were examined via microarray, RNA sequencing and reverse transcription quantitative real-time PCR (qRT-PCR), proteins were analysed by western blotting and immunostaining, and insulin secretion was quantified in static incubations. RESULTS: NICC maturation was accompanied by the gain of glucose-responsive insulin secretion (twofold stimulation), backed up by mRNA upregulation of genes governing beta cell identity and function, such as NEUROD1, UCN3, ABCC8 and CASR (Log2 fold change [Log2FC] > 1.6). An active TGFß receptor (TGFBR)-SMAD2/3 pathway facilitates NICC maturation, since the TGFBR inhibitor SB431542 counteracted the upregulation of aforementioned genes and de-repressed ALDOB, a gene disallowed in mature beta cells. In fetuin-A-treated NICCs, upregulation of beta cell markers and the onset of glucose responsiveness were suppressed. Concomitantly, SMAD2/3 phosphorylation was inhibited. Transcriptome analysis confirmed inhibitory effects of fetuin-A and SB431542 on TGFß-1- and SMAD2/3-regulated transcription. However, contrary to SB431542 and regardless of cMYC upregulation, fetuin-A inhibited beta cell proliferation (0.27 ± 0.08% vs 1.0 ± 0.1% Ki67-positive cells in control NICCs). This effect was sustained by reduced expression (Log2FC ≤ -2.4) of FOXM1, CENPA, CDK1 or TOP2A. In agreement, the number of insulin-positive cells was lower in fetuin-A-treated NICCs than in control NICCs (14.4 ± 1.2% and 22.3 ± 1.1%, respectively). In adult human islets fetuin-A abolished glucose responsiveness, i.e. 1.7- and 1.1-fold change over 2.8 mmol/l glucose in control- and fetuin-A-cultured islets, respectively. In addition, fetuin-A reduced SMAD2/3 phosphorylation and suppressed expression of proliferative genes. Of note, in non-diabetic humans, plasma fetuin-A was negatively correlated (p = 0.013) with islet beta cell area. CONCLUSIONS/INTERPRETATION: Our results suggest that the perinatal decline of fetuin-A relieves TGFBR signalling in islets, a process that facilitates functional maturation of neonatal beta cells. Functional maturity remains revocable in later life, and the occurrence of a metabolically unhealthy milieu, such as liver steatosis and elevated plasma fetuin-A, can impair both function and adaptive proliferation of beta cells. DATA AVAILABILITY: The RNAseq datasets and computer code produced in this study are available in the Gene Expression Omnibus (GEO): GSE144950; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144950.

Lack of mitochondrial NADP(H)-transhydrogenase expression in macrophages exacerbates atherosclerosis in hypercholesterolemic mice.

The atherosclerosis prone LDL receptor knockout mice (Ldlr-/-, C57BL/6J background) carry a deletion of the NADP(H)-transhydrogenase gene (Nnt) encoding the mitochondrial enzyme that catalyzes NADPH synthesis. Here we hypothesize that both increased NADPH consumption (due to increased steroidogenesis) and decreased NADPH generation (due to Nnt deficiency) in Ldlr-/- mice contribute to establish a macrophage oxidative stress and increase atherosclerosis development. Thus, we compared peritoneal macrophages and liver mitochondria from three C57BL/6J mice lines: Ldlr and Nnt double mutant, single Nnt mutant and wild-type. We found increased oxidants production in both mitochondria and macrophages according to a gradient: double mutant > single mutant > wild-type. We also observed a parallel up-regulation of mitochondrial biogenesis (PGC1a, TFAM and respiratory complexes levels) and inflammatory (iNOS, IL6 and IL1b) markers in single and double mutant macrophages. When exposed to modified LDL, the single and double mutant cells exhibited significant increases in lipid accumulation leading to foam cell formation, the hallmark of atherosclerosis. Nnt deficiency cells showed up-regulation of CD36 and down-regulation of ABCA1 transporters what may explain lipid accumulation in macrophages. Finally, Nnt wild-type bone marrow transplantation into LDLr-/- mice resulted in reduced diet-induced atherosclerosis. Therefore, Nnt plays a critical role in the maintenance of macrophage redox, inflammatory and cholesterol homeostasis, which is relevant for delaying the atherogenesis process.

Coenzyme Q10 protects against ß-cell toxicity induced by pravastatin treatment of hypercholesterolemia.

New onset of diabetes is associated with the use of statins. We have recently demonstrated that pravastatin-treated hypercholesterolemic LDL receptor knockout (LDLr-/- ) mice exhibit reductions in insulin secretion and increased islet cell death and oxidative stress. Here, we hypothesized that these diabetogenic effects of pravastatin could be counteracted by treatment with the antioxidant coenzyme Q 10 (CoQ 10 ), an intermediate generated in the cholesterol synthesis pathway. LDLr -/- mice were treated with pravastatin and/or CoQ 10 for 2 months. Pravastatin treatment resulted in a 75% decrease of liver CoQ 10 content. Dietary CoQ 10 supplementation of pravastatin-treated mice reversed fasting hyperglycemia, improved glucose tolerance (20%) and insulin sensitivity (>2-fold), and fully restored islet glucose-stimulated insulin secretion impaired by pravastatin (40%). Pravastatin had no effect on insulin secretion of wild-type mice. In vitro, insulin-secreting INS1E cells cotreated with CoQ 10 were protected from cell death and oxidative stress induced by pravastatin. Simvastatin and atorvastatin were more potent in inducing dose-dependent INS1E cell death (10-15-fold), which were also attenuated by CoQ 10 cotreatment. Together, these results demonstrate that statins impair ß-cell redox balance, function and viability. However, CoQ 10 supplementation can protect the statins detrimental effects on the endocrine pancreas.

Diabetogenic effect of pravastatin is associated with insulin resistance and myotoxicity in hypercholesterolemic mice.

BACKGROUND: HMG-CoA reductase inhibitors (statins) are cholesterol-lowering drugs widely used to treat hypercholesterolemia and prevent cardiovascular disease. Statins are generally well tolerated, but adverse reactions may occur, particularly myopathy and new onset of diabetes. The exact mechanism of statin-induced myopathy and diabetes has not been fully elucidated. We have previously shown that treatment of hypercholesterolemic (LDLr-/-) mice with pravastatin for 2 months decreased pancreatic islet insulin secretion and increased oxidative stress and cell death, but no glucose intolerance was observed. The purpose of the current work was to study long-term pravastatin effects on glucose homeostasis, insulin sensitivity, muscle protein turnover and cell viability. METHODS: LDLr-/- mice were treated with pravastatin for 3, 6 and 10 months. Glucose tolerance, insulin resistance and glucose-stimulated insulin secretion were evaluated. The rates of protein synthesis and degradation were determined in gastrocnemius muscle after 10 months of treatment. Insulin signalling, oxidative stress and cell death were analysed in vitro using C2C12 myotubes. RESULTS: After 6 and 10 months of treatment, these mice became glucose intolerant, and after 10 months, they exhibited marked insulin resistance. Reduced islet glucose-stimulated insulin secretion was observed after the 3rd month of treatment. Mice treated for 10 months showed significantly decreased body weight and increased muscle protein degradation. In addition, muscle chymotrypsin-like proteasomal activity and lysosomal cathepsin were markedly elevated. C2C12 myotubes exposed to increasing concentrations of pravastatin presented dose-dependent impairment of insulin-induced Akt phosphorylation, increased apoptotic markers (Bax protein and cleaved caspase-3) and augmented superoxide anion production. CONCLUSIONS: In addition to reduced insulin secretion, long-term pravastatin treatment induces insulin resistance and muscle wasting. These results suggest that the diabetogenic effect of statins is linked to the appearance of myotoxicity induced by oxidative stress, impaired insulin signalling, proteolysis and apoptosis.

Effects of adrenergic-stimulated lipolysis and cytokine production on in vitro mouse adipose tissue-islet interactions.

Inflammatory cytokines and non-esterified fatty acids (NEFAs) are obesity-linked factors that disturb insulin secretion. The aim of this study was to investigate whether pancreatic adipose tissue (pWAT) is able to generate a NEFA/cytokine overload within the pancreatic environment and as consequence to impact on insulin secretion. Pancreatic fat is a minor fat depot, therefore we used high-fat diet (HFD) feeding to induce pancreatic steatosis in mice. Relative Adipoq and Lep mRNA levels were higher in pWAT of HFD compared to chow diet mice. Regardless of HFD, Adipoq and Lep mRNA levels of pWAT were at least 10-times lower than those of epididymal fat (eWAT). Lipolysis stimulating receptors Adrb3 and Npr1 were expressed in pWAT and eWAT, and HFD reduced their expression in eWAT only. In accordance, HFD impaired lipolysis in eWAT but not in pWAT. Despite expression of Npr mRNA, lipolysis was stimulated solely by the adrenergic agonists, isoproterenol and adrenaline. Short term co-incubation of islets with CD/HFD pWAT did not alter insulin secretion. In the presence of CD/HFD eWAT, glucose stimulated insulin secretion only upon isoproterenol-induced lipolysis, i.e. in the presence of elevated NEFA. Isoproterenol augmented Il1b and Il6 mRNA levels both in pWAT and eWAT. These results suggest that an increased sympathetic activity enhances NEFA and cytokine load of the adipose microenvironment, including that of pancreatic fat, and by doing so it may alter beta-cell function.

The Presence of Cholesteryl Ester Transfer Protein (CETP) in Endothelial Cells Generates Vascular Oxidative Stress and Endothelial Dysfunction.

Endothelial dysfunction precedes atherosclerosis and is an independent predictor of cardiovascular events. Cholesterol levels and oxidative stress are key contributors to endothelial damage, whereas high levels of plasma high-density lipoproteins (HDL) could prevent it. Cholesteryl ester transfer protein (CETP) is one of the most potent endogenous negative regulators of HDL-cholesterol. However, whether and to what degree CETP expression impacts endothelial function, and the molecular mechanisms underlying the vascular effects of CETP on endothelial cells, have not been addressed. Acetylcholine-induced endothelium-dependent relaxation of aortic rings was impaired in human CETP-expressing transgenic mice, compared to their non-transgenic littermates. However, endothelial nitric oxide synthase (eNOS) activation was enhanced. The generation of superoxide and hydrogen peroxide was increased in aortas from CETP transgenic mice, while silencing CETP in cultured human aortic endothelial cells effectively decreased oxidative stress promoted by all major sources of ROS: mitochondria and NOX2. The endoplasmic reticulum stress markers, known as GADD153, PERK, and ARF6, and unfolded protein response effectors, were also diminished. Silencing CETP reduced endothelial tumor necrosis factor (TNF) α levels, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expression, diminishing monocyte adhesion. These results support the notion that CETP expression negatively impacts endothelial cell function, revealing a new mechanism that might contribute to atherosclerosis.

Ventricular Fibrosis and Coronary Remodeling Following Short-Term Exposure of Healthy and Malnourished Mice to Bisphenol A.

Bisphenol-A (BPA) is an endocrine disruptor associated with higher risk of insulin resistance, type 2 diabetes, and cardiovascular diseases especially in susceptible populations. Because malnutrition is a nutritional disorder associated with high cardiovascular risk, we sought to compare the effects of short-term BPA exposure on cardiovascular parameters of healthy and protein-malnourished mice. Postweaned male mice were fed a normo- (control) or low-protein (LP) diet for 8 weeks and then exposed or not to BPA (50 µg kg-1 day-1) for the last 9 days. Systolic blood pressure was higher in BPA or LP groups compared with the control group. However, diastolic blood pressure was enhanced by BPA only in malnourished mice. Left ventricle (LV) end diastolic pressure (EDP), collagen deposition, and CTGF mRNA expression were higher in the control or malnourished mice exposed to BPA than in the respective nonexposed groups. Nevertheless, mice fed LP diet exposed to BPA exhibited higher angiotensinogen and cardiac TGF-ß1 mRNA expression than mice treated with LP or BPA alone. Wall:lumen ratio and cross-sectional area of intramyocardial arteries were higher either in the LP or BPA group compared with the control mice. Taken together, our data suggest that short-term BPA exposure results in LV diastolic dysfunction and fibrosis, and intramyocardial arteries inward remodeling, besides potentiate protein malnutrition-induced hypertension and cardiovascular risk.

FFA2-, but not FFA3-agonists inhibit GSIS of human pseudoislets: a comparative study with mouse islets and rat INS-1E cells.

The expression of short chain fatty acid receptors FFA2 and FFA3 in pancreatic islets raised interest in using them as drug targets for treating hyperglycemia in humans. This study aims to examine the efficacy of synthetic FFA2- and FFA3-ligands to modulate glucose-stimulated insulin secretion (GSIS) in human pseudoislets which display intact glucose responsiveness. The FFA2-agonists 4-CMTB and TUG-1375 inhibited GSIS, an effect reversed by the FFA2-antagonist CATPB. GSIS itself was not augmented by CATPB. The FFA3-agonists FHQC and 1-MCPC did not affect GSIS in human pseudoislets. For further drug evaluation we used mouse islets. The CATPB-sensitive inhibitory effect of 100 µM 4-CMTB on GSIS was recapitulated. The inhibition was partially sensitive to the Gi/o-protein inhibitor pertussis toxin. A previously described FFA2-dependent increase of GSIS was observed with lower concentrations of 4-CMTB (10 and 30 µM). The stimulatory effect of 4-CMTB on secretion was prevented by the Gq-protein inhibitor FR900359. As in human pseudoislets, in mouse islets relative mRNA levels were FFAR2 > FFAR3 and FFA3-agonists did not affect GSIS. The FFA3-agonists, however, inhibited GSIS in a pertussis toxin-sensitive manner in INS-1E cells and this correlated with relative mRNA levels of Ffar3 > > Ffar2. Thus, in humans, when FFA2-activation impedes GSIS, FFA2-antagonism may reduce glycemia.

The Anti-atherogenic Role of Exercise Is Associated With the Attenuation of Bone Marrow-Derived Macrophage Activation and Migration in Hypercholesterolemic Mice.

An early event in atherogenesis is the recruitment and infiltration of circulating monocytes and macrophage activation in the subendothelial space. Atherosclerosis subsequently progresses as a unresolved inflammatory disease, particularly in hypercholesterolemic conditions. Although physical exercise training has been a widely accepted strategy to inhibit atherosclerosis, its impact on arterial wall inflammation and macrophage phenotype and function has not yet been directly evaluated. Thus, the aim of this study was to investigate the effects of aerobic exercise training on the inflammatory state of atherosclerotic lesions with a focus on macrophages. Hypercholesterolemic LDL-receptor-deficient male mice were subjected to treadmill training for 8 weeks and fed a high-fat diet. Analyses included plasma lipoprotein and cytokine levels; aortic root staining for lipids (oil red O); macrophages (CD68, MCP1 and IL1ß); oxidative (nitrotyrosine and, DHE) and endoplasmic reticulum (GADD) stress markers. Primary bone marrow-derived macrophages (BMDM) were assayed for migration activity, motility phenotype (Rac1 and F-actin) and inflammation-related gene expression. Plasma levels of HDL cholesterol were increased, while levels of proinflammatory cytokines (TNFa, IL1b, and IL6) were markedly reduced in the exercised mice. The exercised mice developed lower levels of lipid content and inflammation in atherosclerotic plaques. Additionally, lesions in the exercised mice had lower levels of oxidative and ER stress markers. BMDM isolated from the exercised mice showed a marked reduction in proinflammatory cytokine gene expression and migratory activity and a disrupted motility phenotype. More importantly, bone marrow from exercised mice transplanted into sedentary mice led to reduced atherosclerosis in the recipient sedentary mice, thus suggesting that epigenetic mechanisms are associated with exercise. Collectively, the presented data indicate that exercise training prevents atherosclerosis by inhibiting bone marrow-derived macrophage recruitment and activation.

Glucose, adrenaline and palmitate antagonistically regulate insulin and glucagon secretion in human pseudoislets.

Isolated human islets do not always meet the quality standards required for transplant survival and reliable functional in vitro studies. The formation of pseudoislets, i.e. the reaggregation of a defined number of islet cells after dissociation, improves insulin secretion. We present a simple method of pseudoislet formation from human islet cells and assess the transcriptome and function of isolated human islets and pseudoislets from the same organ donors. Following pseudoislet formation, insulin content/DNA and mRNA/RPS13 resembled that of islets. In pseudoislets, glucose-stimulated insulin secretion (GSIS) was significantly higher (8-13-fold) than in islets (2-4-fold). GSIS of pseudoislets was partly inhibited by the glucagon-like peptide-1 receptor (GLP-1R) antagonist exendin-9. The stimulatory effects of palmitate and forskolin at 12 mM glucose were also significantly higher in pseudoislets than in islets. Further analysis of pseudoislets revealed that regulation of secretion and insulin and glucagon content was maintained over a longer culture period (6-14 d). While adrenaline inhibited GSIS, adrenaline together with palmitate stimulated glucagon secretion 2-fold at low glucose, an effect suppressed by high glucose. Transcriptome analysis revealed that, unlike islets, pseudoislets were deprived of exocrine and endothelial cells. In conclusion, pseudoislet formation restores functional integrity of human islet cells and allows long-term in vitro testing.

Palmitate and insulin counteract glucose-induced thioredoxin interacting protein (TXNIP) expression in insulin secreting cells via distinct mechanisms.

Glucose and palmitate synergistically stimulate insulin secretion, but chronically elevated they induce apoptotic ß-cell death. The glucotoxic effect has been attributed, at least partly, to the upregulation of the oxidative stress marker thioredoxin interacting protein (TXNIP). Palmitate downregulates TXNIP expression, the functional significance of which is still under debate. This study examines the mechanism and consequence of palmitate-mediated TXNIP regulation in insulin secreting cells. Palmitate (600 µM) reduced TXNIP mRNA levels in isolated human and mouse islets independently of FFAR1/GPR40. Similar effects of palmitate were observed in INS-1E cells and mimicked by other long chain fatty acids. The lowering of TXNIP mRNA was significant already 1 h after addition of palmitate, persisted for 24 h and was directly translated to changes in TXNIP protein. The pharmacological inhibition of palmitate-induced phosphorylation of AMPK, ERK1/2, JNK and PKCα/ß by BML-275, PD98059, SP600125 and Gö6976, respectively, did not abolish palmitate-mediated TXNIP downregulation. The effect of palmitate was superimposed by a time-dependent (8 h and 24 h) decline of TXNIP mRNA and protein. This decline correlated with accumulation of secreted insulin into the medium. Accordingly, exogenously added insulin reduced TXNIP mRNA and protein levels, an effect counteracted by the insulin/IGF-1 receptor antagonist linsitinib. The inhibition of PI3K and Akt/PKB increased TXNIP mRNA levels. The histone deacetylase (HDAC1/2/3) inhibitor MS-275 completely abrogated the time-dependent, insulin-mediated reduction of TXNIP, leaving the effect of palmitate unaltered. Acute stimulation of insulin secretion and chronic accentuation of cell death by palmitate occurred independently of TXNIP regulation. On the contrary, palmitate antagonized glucose-augmented ROS production. In conclusion, glucose-induced TXNIP expression is efficiently antagonized by two independent mechanisms, namely via an autocrine activation of insulin/IGF-1 receptors involving HDAC and by palmitate attenuating oxidative stress of ß-cells.

The Expression of Aldolase B in Islets Is Negatively Associated With Insulin Secretion in Humans.

Context: Reduced ß-cell mass, impaired islet function, and dedifferentiation are considered causal to development of hyperglycemia and type 2 diabetes. In human cohort studies, changes of islet cell-specific expression patterns have been associated with diabetes but not directly with in vivo insulin secretion. Objective: This study investigates alterations of islet gene expression and corresponding gene variants in the context of in vivo glycemic traits from the same patients. Methods: Fasting blood was collected before surgery, and pancreatic tissue was frozen after resection from 18 patients undergoing pancreatectomy. Islet tissue was isolated by laser capture microdissection. Islet transcriptome was analyzed using microarray and quantitative RT-PCR. Proteins were examined by immunohistochemistry and western blotting. The association of gene variants with insulin secretion was investigated with oral glucose tolerance test (OGTT)-derived insulin secretion measured in a large cohort of subjects at increased risk of type 2 diabetes and with hyperglycemic clamp in a subset. Results: Differential gene expression between islets from normoglycemic and hyperglycemic patients was prominent for the glycolytic enzyme ALDOB and the obesity-associated gene FAIM2. The mRNA levels of both genes correlated negatively with insulin secretion and positively with HbA1c. Islets of hyperglycemic patients displayed increased ALDOB immunoreactivity in insulin-positive cells, whereas α- and δ-cells were negative. Exposure of isolated islets to hyperglycemia augmented ALDOB expression. The minor allele of the ALDOB variant rs550915 associated with significantly higher levels of C-peptide and insulin during OGTT and hyperglycemic clamp, respectively. Conclusion: Our analyses suggest that increased ALDOB expression in human islets is associated with lower insulin secretion.

Point mutation of Ffar1 abrogates fatty acid-dependent insulin secretion, but protects against HFD-induced glucose intolerance.

OBJECTIVE: The fatty acid receptor 1 (FFAR1/GPR40) mediates fatty acid-dependent augmentation of glucose-induced insulin secretion (GIIS) in pancreatic ß-cells. Genetically engineered Ffar1-knockout/congenic mice univocally displayed impaired fatty acid-mediated insulin secretion, but in vivo experiments delivered controversial results regarding the function of FFAR1 in glucose homeostasis and liver steatosis. This study presents a new coisogenic mouse model carrying a point mutation in Ffar1 with functional consequence. These mice reflect the situations in humans in which point mutations can lead to protein malfunction and disease development. METHODS: The Munich N-ethyl-N-nitrosourea (ENU) mutagenesis-derived F1 archive containing over 16,800 sperms and corresponding DNA samples was screened for mutations in the coding region of Ffar1. Two missense mutations (R258W and T146S) in the extracellular domain of the protein were chosen and homozygote mice were generated. The functional consequence of these mutations was examined in vitro in isolated islets and in vivo in chow diet and high fat diet fed mice. RESULTS: Palmitate, 50 µM, and the FFAR1 agonist TUG-469, 3 µM, stimulated insulin secretion in islets of Ffar1T146S/T146S mutant mice and of wild-type littermates, while in islets of Ffar1R258W/R258W mutant mice, these stimulatory effects were abolished. Insulin content and mRNA levels of Ffar1, Glp1r, Ins2, Slc2a2, Ppara, and Ppard were not significantly different between wild-type and Ffar1R258W/R258W mouse islets. Palmitate exposure, 600 µM, significantly increased Ppara mRNA levels in wild-type but not in Ffar1R258W/R258W mouse islets. On the contrary, Slc2a2 mRNA levels were significantly reduced in both wild-type and Ffar1R258W/R258W mouse islets after palmitate treatment. HFD feeding induced glucose intolerance in wild-type mice. Ffar1R258W/R258W mutant mice remained glucose tolerant although their body weight gain, liver steatosis, insulin resistance, and plasma insulin levels were not different from those of wild-type littermates. Worth mentioning, fasting plasma insulin levels were lower in Ffar1R258W/R258W mice. CONCLUSION: A point mutation in Ffar1 abrogates the stimulatory effect of palmitate on GIIS, an effect that does not necessarily translate to HFD-induced glucose intolerance.

Chronic use of pravastatin reduces insulin exocytosis and increases ß-cell death in hypercholesterolemic mice.

We have previously demonstrated that hypercholesterolemic LDL receptor knockout (LDLr(-/-)) mice secrete less insulin than wild-type mice. Removing cholesterol from isolated islets using methyl-beta-cyclodextrin reversed this defect. In this study, we hypothesized that in vivo treatment of LDLr(-/-) mice with the HMGCoA reductase inhibitor pravastatin would improve glucose-stimulated insulin secretion. Female LDLr(-/-) mice were treated with pravastatin (400mg/L) for 1-3 months. Isolated pancreatic islets were assayed for insulin secretion rates, intracellular calcium oscillations, cholesterol levels, NAD(P)H and SNARE protein levels, apoptosis indicators and lipidomic profile. Two months pravastatin treatment reduced cholesterol levels in plasma, liver and islets by 35%, 25% and 50%, respectively. Contrary to our hypothesis, pravastatin treatment increased fasting and fed plasma levels of glucose and decreased markedly (40%) fed plasma levels of insulin. In addition, ex vivo glucose stimulated insulin secretion was significantly reduced after two and three months (36-48%, p<0.05) of pravastatin treatment. Although reducing insulin secretion and insulinemia, two months pravastatin treatment did not affect glucose tolerance because it improved global insulin sensitivity. Pravastatin induced islet dysfunction was associated with marked reductions of exocytosis-related SNARE proteins (SNAP25, Syntaxin 1A, VAMP2) and increased apoptosis markers (Bax/Bcl2 protein ratio, cleaved caspase-3 and lower NAD(P)H production rates) observed in pancreatic islets from treated mice. In addition, several oxidized phospholipids, tri- and diacylglycerols and the proapoptotic lipid molecule ceramide were identified as markers of pravastatin-treated islets. Cell death and oxidative stress (H2O2 production) were confirmed in insulin secreting INS-1E cells treated with pravastatin. These results indicate that chronic treatment with pravastatin impairs the insulin exocytosis machinery and increases ß-cell death. These findings suggest that prolonged use of statins may have a diabetogenic effect.