Emerging role for kynurenines in metabolic pathologies.

PURPOSE OF REVIEW: So far, the tryptophan catabolites generated in the kynurenine pathway have been mainly studied in relation to oncologic and mental health disorders. The current review provides an update on the emerging biomedical interest for kynurenine pathway activity in the field of energy homeostasis and metabolic diseases. RECENT FINDINGS: Kynurenine pathway enzymes are expressed in tissues relevant for energy homeostasis such as fat, skeletal muscle, liver and endocrine pancreas, blood vessel and heart, and are regulated by nutritional and inflammatory signals. Kynurenine pathway metabolites have been proposed as biomarkers for initiation and progression of atherosclerosis and diabetes. Exercise training activation of kynurenine pathway in skeletal muscles increases lipid metabolism and thermogenesis, and it limits weight gain, inflammation, insulin resistance, and glucose intolerance in rodents fed a high-fat diet. Manipulation of kynurenine pathway metabolism through administration of enzyme inhibitors or kynurenine pathway metabolites can serve as novel therapeutic strategy for atherosclerosis, obesity, glucose intolerance, or impaired insulin secretion. SUMMARY: Although we are far from a complete understanding of the role of kynurenine pathway in the modulation of energy homeostasis, targeting kynurenine pathway harbors high potential to expand the range of therapies to prevent and treat metabolic diseases.

HSF1 acetylation decreases its transcriptional activity and enhances glucolipotoxicity-induced apoptosis in rat and human beta cells.

AIMS/HYPOTHESIS: Heat shock factor protein 1 (HSF1) is a transcription factor that regulates the expression of key molecular chaperones, thereby orchestrating the cellular response to stress. This system was recently implicated in the control of insulin sensitivity and is therefore being scrutinised as a novel therapeutic avenue for type 2 diabetes. However, the regulation and biological actions of HSF1 in beta cells remain elusive. Herein, we sought to investigate the regulation of HSF1 in pancreatic beta cells and to study its potential role in cell survival. METHODS: We exposed human islets and beta cell lines to glucolipotoxicity and thapsigargin. HSF1 activity was evaluated by gel shift assay. HSF1 acetylation and interaction with the protein acetylase cAMP response element binding protein (CBP) were investigated by western blot. We measured the expression of HSF1 and its canonical targets in islets from Goto-Kakizaki (GK) rat models of diabetes and delineated the effects of HSF1 acetylation using mutants mimicking constitutive acetylation and deacetylation of the protein. RESULTS: Glucolipotoxicity promoted HSF1 acetylation and interaction with CBP. Glucolipotoxicity-induced HSF1 acetylation inhibited HSF1 DNA binding activity and decreased the expression of its target genes. Restoration of HSF1 activity in beta cells prevented glucolipotoxicity-induced endoplasmic reticulum stress and apoptosis. However, overexpression of a mutant protein (K80Q) mimicking constitutive acetylation of HSF1 failed to confer protection against glucolipotoxicity. Finally, we showed that expression of HSF1 and its target genes were altered in islets from diabetic GK rats, suggesting that this pathway could participate in the pathophysiology of diabetes and constitutes a potential site for therapeutic intervention. CONCLUSIONS/INTERPRETATION: Our results unravel a new mechanism by which HSF1 inhibition is required for glucolipotoxicity-induced beta cell apoptosis. Restoring HSF1 activity may represent a novel strategy for the maintenance of a functional beta cell mass. Our study supports the therapeutic potential of HSF1/heat shock protein-targeting agents in diabetes treatment.

Disrupted dynamics of F-actin and insulin granule fusion in INS-1 832/13 beta-cells exposed to glucotoxicity: partial restoration by glucagon-like peptide 1.

Actin dynamics in pancreatic ß-cells is involved in insulin exocytosis but the molecular mechanisms of this dynamics and its role in biphasic insulin secretion in pancreatic ß-cells is largely unknown. Moreover, the impact of a glucotoxic environment on the sub-cortical actin network dynamics is poorly studied. In this study, we investigate the behavior of insulin granules and the subcortical actin network dynamics in INS-1 832/13 ß-cells submitted to a normal or glucotoxic environment. Our results show that glucose stimulation leads to a reorganization of the subcortical actin network with a rupture of its interactions with t-SNARE proteins (Syntaxin 1A and SNAP-25), promoting insulin secretion in INS-1 832/13 ß-cells. Prolonged exposure of INS-1 832/13 ß-cells to high-glucose levels (glucotoxicity) leads to the densification of the cortical actin network, which prevents its reorganization under acute glucose, and diminishes the glucose-stimulated insulin secretion, as shown by the decreased number of fusion events. The most interesting in our results is the partial restoration by GLP-1 of the insulin secretion ability from high-glucose treated INS-1 832/13 cells. This improved insulin exocytosis is associated with partial restored actin dynamics and fusion events during the two phases of the secretion, with a preferential involvement of Epac2 signaling in the first phase and a rather involvement of PKA signaling in the second phase of insulin exocytosis. All these data provide some new insights into the mechanism by which current therapeutics may be improving insulin secretion.

Role of palmitate-induced sphingoid base-1-phosphate biosynthesis in INS-1 ß-cell survival.

Sphingoid base-1-phosphates represent a very low portion of the sphingolipid pool but are potent bioactive lipids in mammals. This study was undertaken to determine whether these lipids are produced in palmitate-treated pancreatic ß cells and what role they play in palmitate-induced ß cell apoptosis. Our lipidomic analysis revealed that palmitate at low and high glucose supplementation increased (dihydro)sphingosine-1-phosphate levels in INS-1 ß cells. This increase was associated with an increase in sphingosine kinase 1 (SphK1) mRNA and protein levels. Over-expression of SphK1 in INS-1 cells potentiated palmitate-induced accumulation of dihydrosphingosine-1-phosphate. N,N-dimethyl-sphingosine, a potent inhibitor of SphK, potentiated ß-cell apoptosis induced by palmitate whereas over-expression of SphK1 significantly reduced apoptosis induced by palmitate with high glucose. Endoplasmic reticulum (ER)-targeted SphK1 also partially inhibited apoptosis induced by palmitate. Inhibition of INS-1 apoptosis by over-expressed SphK1 was independent of sphingosine-1-phosphate receptors but was associated with a decreased formation of pro-apoptotic ceramides induced by gluco-lipotoxicity. Moreover, over-expression of SphK1 counteracted the defect in the ER-to-Golgi transport of proteins that contribute to the ceramide-dependent ER stress observed during gluco-lipotoxicity. In conclusion, our results suggest that activation of palmitate-induced SphK1-mediated sphingoid base-1-phosphate formation in the ER of ß cells plays a protective role against palmitate-induced ceramide-dependent apoptotic ß cell death.

Role of Sphingosine Kinase 1 in Glucolipotoxicity-Induced Early Activation of Autophagy in INS-1 Pancreatic ß Cells.

Insulin-producing pancreatic ß cells play a crucial role in the regulation of glucose homeostasis, and their failure is a key event for diabetes development. Prolonged exposure to palmitate in the presence of elevated glucose levels, termed gluco-lipotoxicity, is known to induce ß cell apoptosis. Autophagy has been proposed to be regulated by gluco-lipotoxicity in order to favor ß cell survival. However, the role of palmitate metabolism in gluco-lipotoxcity-induced autophagy is presently unknown. We therefore treated INS-1 cells for 6 and 24 h with palmitate in the presence of low and high glucose concentrations and then monitored autophagy. Gluco-lipotoxicity induces accumulation of LC3-II levels in INS-1 at 6 h which returns to basal levels at 24 h. Using the RFP-GFP-LC3 probe, gluco-lipotoxicity increased both autophagosomes and autolysosmes structures, reflecting early stimulation of an autophagy flux. Triacsin C, a potent inhibitor of the long fatty acid acetyl-coA synthase, completely prevents LC3-II formation and recruitment to autophagosomes, suggesting that autophagic response requires palmitate metabolism. In contrast, etomoxir and bromo-palmitate, inhibitors of fatty acid mitochondrial ß-oxidation, are unable to prevent gluco-lipotoxicity-induced LC3-II accumulation and recruitment to autophagosomes. Moreover, bromo-palmitate and etomoxir potentiate palmitate autophagic response. Even if gluco-lipotoxicity raised ceramide levels in INS-1 cells, ceramide synthase 4 overexpression does not potentiate LC3-II accumulation. Gluco-lipotoxicity also still stimulates an autophagic flux in the presence of an ER stress repressor. Finally, selective inhibition of sphingosine kinase 1 (SphK1) activity precludes gluco-lipotoxicity to induce LC3-II accumulation. Moreover, SphK1 overexpression potentiates autophagic flux induced by gluco-lipotxicity. Altogether, our results indicate that early activation of autophagy by gluco-lipotoxicity is mediated by SphK1, which plays a protective role in ß cells.

Local in vivo GSK3ß knockdown promotes pancreatic ß cell and acinar cell regeneration in 90% pancreatectomized rat.

Endocrine and exocrine insufficiencies are associated with serious diseases such as diabetes and pancreatitis, respectively. Pancreatic cells retain the capacity to regenerate in the context of cell deficiency. The remnant pancreas after pancreatectomy (Px) is a valuable target for testing the efficiency of pharmacological interventions to stimulate cell regeneration. Here, we tested the ability of GSK3ß downregulation on the stimulation of ß- and acinar cell regeneration after 90% Px in adult rats. We developed an in vivo approach based on local silencing of GSK3ß, by delivering antisense morpholino-oligonucleotides within the remnant pancreas of 90% pancreatectomized rats, and evaluated its impact on the regenerative potential of pancreatic ß and exocrine cells. ß-Cell (BC) mass was evaluated by morphometry. Cell proliferation and apoptosis were assessed by 5'bromo 2'deoxyuridine (BrdU) incorporation method and TUNEL assay, respectively. The expression of Sox9, Neurogenin-3 (Ngn3), and PDX1 was evaluated by immunohistochemistry. We show that intrapancreatic GSK3ß knockdown leads to increased BC mass (BCM) in 90% pancreatectomized rats by promoting both BC proliferation and differentiation. Moreover, downregulation of GSK3ß significantly improves exocrine growth and prevents acinar cell apoptosis in vivo. Our study designates GSK3ß as a viable drug target for therapeutic intervention on diseases of endocrine and exocrine pancreas associated with cell deficiency.

Oral administration of kynurenic acid delays the onset of type 2 diabetes in Goto-Kakizaki rats.

Kynurenic acid (KYNA) is an endogenous catabolite of tryptophan that has been found to demonstrate neuroprotective properties in psychiatric disorders. Recently, accumulating data have suggested that KYNA may also play a significant role in various metabolic diseases by stimulating energy metabolism in adipose tissue and muscle. However, whether KYNA can serves as an anti-diabetes agent has yet to be studied. In this study, we investigated the potential anti-diabetic effects of administering KYNA orally through drinking water in pre-diabetic Goto-Kakizaki rats and examined how this treatment may influence energy metabolism regulation within the liver. We found that hyperglycemic Goto-Kakizaki rats showed lower plasmatic KYNA levels compared to normal rats. Oral administration of KYNA significantly delayed the onset of diabetes in Goto-Kakizaki rats compared to untreated animals. Moreover, we found that KYNA treatment significantly increased respiration exchange ratio and promoted the energy expenditure by stimulating the expression of uncoupling protein (UCP). We confirmed that KYNA stimulated the UCP expression in HepG2 cells and mouse hepatocytes at mRNA and protein levels. Our study reveals that KYNA could potentially act as an anti-diabetic agent and KYNA-induced UCP upregulation is closely associated with the regulation of energy metabolism. These results provide further evidence for the therapeutic potential of KYNA in diabetes.

Early reduction of circulating homocysteine levels in Goto-Kakizaki rat, a spontaneous nonobese model of type 2 diabetes.

Diabetes mellitus is associated with increased risk for cardiovascular disorders, which are major causes of mortality in this disease. Hyperhomocysteinemia, defined by high plasma homocysteine levels, is an independent risk factor for the development of cardiovascular diseases. Type 2 diabetic patients have higher circulating homocysteine levels than healthy subjects and these levels are even higher in plasma of obese than nonobese diabetic patients. Homocysteine metabolism that has been studied in 2 animal models of type 2 diabetes with obesity led to conflicting data. The aim of the present study was to analyze homocysteine metabolism in a spontaneous nonobese model of type 2 diabetes, the Goto-Kakizaki rats at various successive and well characterized stages of the disease: during early postnatal normoglycemia, at the onset of hyperglycemia (around weaning), and during chronic mild hyperglycemia with progressive insulin resistance. Compared to age-matched Wistar controls, Goto-Kakizaki rats showed lower plasma levels of homocysteine and a falling trend in its major byproduct antioxidant, glutathione, from the prediabetic stage onwards. Concomitantly, Goto-Kakizaki rats exhibited increased liver activity of cystathionine beta synthase, which catalyzes the condensation of homocysteine with serine in the first step of the transsulfuration pathway. These results emphasize a strong association between homocysteine metabolism and insulin via the first step of the hepatic transsulfuration pathway in Goto-Kakizaki rats.

Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in glucolipotoxicity-induced apoptosis of INS-1 ß-cells.

Pancreatic ß-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of glucolipotoxicity-induced ß-cell apoptosis. In INS-1 ß-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)ceramides. Of note, glucolipotoxicity specifically induced the formation of C(18:0), C(22:0) and C(24:1) (dihydro)ceramide molecular species, which was associated with the up-regulation of CerS4 (ceramide synthase 4) levels. Fumonisin-B1, a ceramide synthase inhibitor, partially blocked apoptosis induced by glucolipotoxicity. In contrast, apoptosis was potentiated in the presence of D,L-threo-1-phenyl-2-palmitoylamino-3-morpholinopropan-1-ol, an inhibitor of glucosylceramide synthase. Moreover, overexpression of CerS4 amplified ceramide production and apoptosis induced by palmitate with 30 mM glucose, whereas down-regulation of CerS4 by siRNA (short interfering RNA) reduced apoptosis. CerS4 also potentiates ceramide accumulation and apoptosis induced by another saturated fatty acid: stearate. Collectively, our results suggest that glucolipotoxicity induces ß-cell apoptosis through a dual mechanism involving de novo ceramide biosynthesis and the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content.

Glucocorticoid-Induced Hyperinsulinism in a Preterm Neonate with Inherited ABCC8 Variant.

Glucose homeostasis is a real challenge for extremely preterm infants (EPIs) who have both limited substrate availability and immature glucose metabolism regulation. In the first days of life, EPIs frequently develop transient glucose intolerance, which has a complex pathophysiology that associates unregulated gluconeogenesis, immature insulin secretion, and peripheral insulin resistance. In this population, glucocorticoid therapy is frequently administrated to prevent severe bronchopulmonary dysplasia. During this treatment, glucose intolerance classically increases and may lead to hyperglycemia. We report a case of neonatal hypoglycemia that was concomitant to a glucocorticoids administration, and that led to a congenital hyperinsulinism diagnosis in an EPI with a heterozygous ABCC8 variant. The variant was inherited from his mother, who had developed monogenic onset diabetes of the youth (MODY) at the age of 23. ABCC8 encodes a beta-cell potassium channel unit and causes congenital hyperinsulinism or MODY depending on the mutation location. Moreover, some mutations have been observed in the same patient to cause both hyperinsulinism in infancy and MODY in adulthood. In our case, the baby showed repeated and severe hypoglycemias, which were undoubtedly time-associated with the betamethasone intravenous administration. This hyperinsulinism was transient, and the infant has not yet developed diabetes at three years of age. We take the opportunity presented by this unusual clinical presentation to provide a review of the literature, suggesting new insights regarding the pathophysiology of the beta-pancreatic cells' insulin secretion: glucocorticoids may potentiate basal insulin secretion in patients with ABCC8 mutation.

Kynurenine-3-monooxygenase expression is activated in the pancreatic endocrine cells by diabetes and its blockade improves glucose-stimulated insulin secretion.

Type 2 diabetes is associated with an inflammatory phenotype in the pancreatic islets. We previously demonstrated that proinflammatory cytokines potently activate the tryptophan/kynurenine pathway (TKP) in INS-1 cells and in normal rat islets. Here we examined: (1) the TKP enzymes expression in the diabetic GK islets; (2) the TKP enzymes expression profiles in the GK islets before and after the onset of diabetes; (3) The glucose-stimulated insulin secretion (GSIS) in vitro in GK islets after KMO knockdown using specific morpholino-oligonucleotides against KMO or KMO blockade using the specific inhibitor Ro618048; (4) The glucose tolerance and GSIS after acute in vivo exposure to Ro618048 in GK rats. We report a remarkable induction of the kmo gene in GK islets and in human islets exposed to proinflammatory conditions. It occurred prominently in beta cells. The increased expression and activity of KMO reflected an acquired adaptation. Both KMO knockdown and specific inhibitor Ro618048 enhanced GSIS in vitro in GK islets. Moreover, acute administration of Ro618048 in vivo improved glucose tolerance, GSIS and basal blood glucose levels in GK rats. These results demonstrate that targeting islet TKP is able to correct defective GSIS. KMO inhibition could represent a potential therapeutic strategy for type 2 diabetes.

cAMP-secretion coupling is impaired in diabetic GK/Par rat ß-cells: a defect counteracted by GLP-1.

cAMP-raising agents with glucagon-like peptide-1 (GLP-1) as the first in class, exhibit multiple actions that are beneficial for the treatment of type 2 diabetic (T2D) patients, including improvement of glucose-induced insulin secretion (GIIS). To gain additional insight into the role of cAMP in the disturbed stimulus-secretion coupling within the diabetic ß-cell, we examined more thoroughly the relationship between changes in islet cAMP concentration and insulin release in the GK/Par rat model of T2D. Basal cAMP content in GK/Par islets was significantly higher, whereas their basal insulin release was not significantly different from that of Wistar (W) islets. Even in the presence of IBMX or GLP-1, their insulin release did not significantly change despite further enhanced cAMP accumulation in both cases. The high basal cAMP level most likely reflects an increased cAMP generation in GK/Par compared with W islets since 1) forskolin dose-dependently induced an exaggerated cAMP accumulation; 2) adenylyl cyclase (AC)2, AC3, and G(s)α proteins were overexpressed; 3) IBMX-activated cAMP accumulation was less efficient and PDE-3B and PDE-1C mRNA were decreased. Moreover, the GK/Par insulin release apparatus appears less sensitive to cAMP, since GK/Par islets released less insulin at submaximal cAMP levels and required five times more cAMP to reach a maximal secretion rate no longer different from W. GLP-1 was able to reactivate GK/Par insulin secretion so that GIIS became indistinguishable from that of W. The exaggerated cAMP production is instrumental, since GLP-1-induced GIIS reactivation was lost in the presence the AC blocker 2',5'-dideoxyadenosine. This GLP-1 effect takes place in the absence of any improvement of the [Ca(2+)](i) response and correlates with activation of the cAMP-dependent PKA-dependent pathway.

Effect of Postnatal Nutritional Environment Due to Maternal Diabetes on Beta Cell Mass Programming and Glucose Intolerance Risk in Male and Female Offspring.

Besides the fetal period, the suckling period is a critical time window in determining long-term metabolic health. We undertook the present study to elucidate the impact of a diabetic suckling environment alone or associated with an in utero diabetic environment on beta cell mass development and the risk of diabetes in the offspring in the long term. To that end, we have compared two experimental settings. In setting 1, we used Wistar (W) rat newborns resulting from W ovocytes (oW) transferred into diabetic GK rat mothers (pGK). These oW/pGK neonates were then suckled by diabetic GK foster mothers (oW/pGK/sGK model) and compared to oW/pW neonates suckled by normal W foster mothers (oW/pW/sW model). In setting 2, normal W rat newborns were suckled by diabetic GK rat foster mothers (nW/sGK model) or normal W foster mothers (nW/sW model). Our data revealed that the extent of metabolic disorders in term of glucose intolerance and beta cell mass are similar between rats which have been exposed to maternal diabetes both pre- and postnatally (oW/pGK/sGK model) and those which have been exposed only during postnatal life (nW/sW model). In other words, being nurtured by diabetic GK mothers from birth to weaning was sufficient to significantly alter the beta cell mass, glucose-induced insulin secretion and glucose homeostasis of offspring. No synergistic deleterious effects of pre-and postnatal exposure was observed in our setting.

Paternal High-Protein Diet Programs Offspring Insulin Sensitivity in a Sex-Specific Manner.

The impact of maternal nutrition on offspring is well documented. However, the implication of pre-conceptional paternal nutrition on the metabolic health of the progeny remains underexplored. Here, we investigated the impact of paternal high-protein diet (HPD, 43.2% protein) consumption on the endocrine pancreas and the metabolic phenotype of offspring. Male Wistar rats were given HPD or standard diet (SD, 18.9% protein) for two months. The progenies (F1) were studied at fetal stage and in adulthood. Body weight, glycemia, glucose tolerance (GT), glucose-induced insulin secretion in vivo (GIIS) and whole-body insulin sensitivity were assessed in male and female F1 offspring. Insulin sensitivity, GT and GIIS were similar between F1 females from HPD (HPD/F1) and SD fathers (SD/F1). Conversely, male HPD/F1 exhibited increased insulin sensitivity (p < 0.05) and decreased GIIS (p < 0.05) compared to male SD/F1. The improvement of insulin sensitivity in HPD/F1 was sustained even after 2 months of high-fat feeding. In male HPD/F1, the ß cell mass was preserved and the ß cell plasticity, following metabolic challenge, was enhanced compared to SD/F1. In conclusion, we provide the first evidence of a sex-specific impact of paternal HPD on the insulin sensitivity and GIIS of their descendants, demonstrating that changes in paternal nutrition alter the metabolic status of their progeny in adulthood.

Diabetic GK/Par rat beta-cells are spontaneously protected against H2O2-triggered apoptosis. A cAMP-dependent adaptive response.

The alteration of the beta-cell population in the Goto-Kakizaki rat (GK/Par line), a model of spontaneous type 2 diabetes, has been ascribed to significantly decreased beta-cell replication and neogenesis, while beta-cell apoptosis is surprisingly not enhanced and remains in the normal range. To gain insight into the mechanisms by which those beta-cells are protected from death, we studied ex vivo the apoptotic activity and the expression of a large set of pro/antiapoptotic and pro/antioxidant genes in GK/Par islet cells. This was done in vitro in freshly isolated islets as well as in response to culture conditions and calibrated reactive oxygen species (ROS) exposure (i.e., H2O2). We also investigated the intracellular mechanisms of the diabetic beta-cell response to ROS, the role if any of the intracellular cAMP metabolism, and finally the kinetic of ROS response, taking advantage of the GK/Par rat normoglycemia until weaning. Our results show that the peculiar GK/Par beta-cell phenotype was correlated with an increased expression of a large panel of antioxidant genes as well as pro/antiapoptotic genes. We demonstrate that such combination confers resistance to cytotoxic H2O2 exposure in vitro, raising the possibility that at least some of the activated stress/defense genes have protective effects against H2O2-triggered beta-cell death. We also present some evidence that the GK/Par beta-cell resistance to H2O2 is at least partly cAMP dependent. Finally, we show that such a phenotype is not innate but is spontaneously acquired after diabetes onset as the result of an adaptive response to the diabetic environment.

Neonatal growth and regeneration of beta-cells are regulated by the Wnt/beta-catenin signaling in normal and diabetic rats.

Wnt/beta-catenin signaling is critical for a variety of fundamental cellular processes. Here, we investigated the implication of the Wnt/beta-catenin signaling in the in vivo regulation of beta-cell growth and regeneration in normal and diabetic rats. To this aim, TCF7L2, the distal effector of the canonical Wnt pathway, was knocked down in groups of normal and diabetic rats by the use of specific antisense morpholino-oligonucleotides. In other groups of diabetic rats, the Wnt/beta-catenin pathway was activated by the inhibition of its negative regulator GSK-3beta. GSK-3beta was inactivated by either LiCl or anti-GSK-3beta oligonucleotides. The beta-cell mass was evaluated by morphometry. beta-cell proliferation was assessed in vivo and in vitro by BrdU incorporation method. In vivo beta-cell neogenesis was estimated by the evaluation of PDX1-positive ductal cells and GLUT2-positive ductal cells and the number of beta cells budding from the ducts. We showed that the in vivo disruption of the canonical Wnt pathway resulted in the alteration of normal and compensatory growth of beta-cells mainly through the inhibition of beta-cell proliferation. Conversely, activation of the Wnt pathway through the inhibition of GSK-3beta had a significant stimulatory effect on beta-cell regeneration in diabetic rats. In vitro, GSK-3beta inactivation resulted in the stimulation of beta-cell proliferation. This was mediated by the stabilization of beta-catenin and the induction of cyclin D. Taken together, our results demonstrate the involvement of the canonical Wnt signaling in the neonatal regulation of normal and regenerative growth of pancreatic beta-cells. Moreover, we provide evidence that activation of this pathway by pharmacological maneuvers can efficiently improve beta-cell regeneration in diabetic rats. These findings might have potential clinical applications in the regenerative therapy of diabetes.

Upregulation of eNOS and unchanged energy metabolism in increased susceptibility of the aging type 2 diabetic GK rat heart to ischemic injury.

We investigated the tolerance of the insulin-resistant diabetic heart to ischemic injury in the male Goto-Kakizaki (GK) rat, a model of type 2 diabetes. Changes in energy metabolism, nitric oxide (NO) pathway, and cardiac function were assessed in the presence of physiological substrates. Age-matched control Wistar (n = 19) and GK (n = 18) isolated rat hearts were perfused with 0.4 mM palmitate, 3% albumin, 11 mM glucose, 3 U/l insulin, 0.2 mM pyruvate, and 0.8 mM lactate for 24 min before switching to 1.2 mM palmitate (11 rats/group) during 32 min low-flow (0.5 ml·min(-1)·g wet wt(-1)) ischemia. Next, flow was restored with 0.4 mM palmitate buffer for 32 min. A subset of hearts from each group (n = 8 for control and n = 7 for GK groups) were freeze-clamped for determining baseline values after the initial perfusion of 24 min. ATP, phosphocreatine (PCr), and intracellular pH (pH(i)) were followed using (31)P magnetic resonance spectroscopy with simultaneous measurement of contractile function. The NO pathway was determined by nitric oxide synthase (NOS) isoform expression and total nitrate concentration (NOx) in hearts. We found that coronary flow was 26% lower (P < 0.05) during baseline conditions and 61% lower (P < 0.05) during reperfusion in GK vs. control rat hearts. Rate pressure product was lower during reperfusion in GK vs. control rat hearts (P < 0.05). ATP, PCr, and pH(i) during ischemia-reperfusion were similar in both groups. Endothelial NOS expression was increased in GK rat hearts during baseline conditions (P < 0.05). NOx was increased during baseline conditions (P < 0.05) and after reperfusion (P < 0.05) in GK rat hearts. We report increased susceptibility of type 2 diabetic GK rat heart to ischemic injury that is not associated with impaired energy metabolism. Reduced coronary flow, upregulation of eNOS expression, and increased total NOx levels confirm NO pathway modifications in this model, presumably related to increased oxidative stress. Modifications in the NO pathway may play a major role in ischemia-reperfusion injury of the type 2 diabetic GK rat heart.

Islet structure and function in the GK rat.

Type 2 diabetes mellitus (T2D) arises when the endocrine pancreas fails to secrete sufficient insulin to cope with the metabolic demand because of beta-cell secretory dysfunction and/or decreased beta-cell mass. Defining the nature of the pancreatic islet defects present in T2D has been difficult, in part because human islets are inaccessible for direct study. This review is aimed to illustrate to what extent the Goto-Kakizaki rat, one of the best characterized animal models of spontaneous T2D, has proved to be a valuable tool offering sufficient commonalities to study this aspect. A comprehensive compendium of the multiple functional GK islet abnormalities so far identified is proposed in this perspective. The pathogenesis of defective beta-cell number and function in the GK model is also discussed. It is proposed that the development of T2D in the GK model results from the complex interaction of multiple events: (i) several susceptibility loci containing genes responsible for some diabetic traits (distinct loci encoding impairment of beta-cell metabolism and insulin exocytosis, but no quantitative trait locus for decreased beta-cell mass); (ii) gestational metabolic impairment inducing an epigenetic programming of the offspring pancreas (decreased beta-cell neogenesis and proliferation) transmitted over generations; and (iii) loss of beta-cell differentiation related to chronic exposure to hyperglycaemia/hyperlipidaemia, islet inflammation, islet oxidative stress, islet fibrosis and perturbed islet vasculature.

Implication of glycogen synthase kinase 3 in diabetes-associated islet inflammation.

Islet inflammation is associated with defective ß cell function and mass in type 2 diabetes (T2D). Glycogen synthase kinase 3 (GSK3) has been identified as an important regulator of inflammation in different diseased conditions. However, the role of GSK3 in islet inflammation in the context of diabetes remains unexplored. In this study, we investigated the direct implication of GSK3 in islet inflammation in vitro and tested the impact of GSK3 inhibition in vivo, on the reduction of islet inflammation, and the improvement of glucose metabolism in the Goto-Kakizaki (GK) rat, a spontaneous model of T2D. GK rats were chronically treated with infra-therapeutic doses of lithium, a widely used inhibitor of GSK3. We analyzed parameters of glucose homeostasis as well as islet inflammation and fibrosis in the endocrine pancreas. Ex vivo, we tested the impact of GSK3 inhibition on the autonomous inflammatory response of non-diabetic rat and human islets, exposed to a mix of pro-inflammatory cytokines to mimic an inflammatory environment. Treatment of young GK rats with lithium prevented the development of overt diabetes. Lithium treatment resulted in reduced expression of pro-inflammatory cytokines in the islets. It decreased islet fibrosis and partially restored the glucose-induced insulin secretion in GK rats. Studies in non-diabetic human and rat islets exposed to inflammatory environment revealed the direct implication of GSK3 in the islet autonomous inflammatory response. We show for the first time, the implication of GSK3 in islet inflammation and suggest this enzyme as a viable target to treat diabetes-associated inflammation.

Undernutrition of the GK rat during gestation improves pancreatic IGF-2 and beta-cell mass in the fetuses.

The Goto-Kakizaki (GK) rat is a type 2 diabetes model with a defective beta-cell mass detectable in late fetal development. Diminished IGF-2 production seems to be involved in this effect. Herein, we analyzed the effect of maternal food-restriction on the beta-cell mass of GK fetuses and the involvement of the IGF system, highly responsive to nutritional status in this process. To this end, in undernourished GK fetuses (U-GK), we measured serum GH/IGF levels, beta-cell mass, replication and differentiation, and IGF-1/-2 protein content in liver and pancreas tissue. Pregnant GK females were food restricted (65% restriction) during the last week of gestation. Our results show that maternal malnutrition ameliorates beta-cell mass in U-GK fetuses and a specific pancreatic IGF-2 increase may be instrumental in this effect. Further studies are needed to determine whether maternal undernutrition is sufficient to delay or decrease the risk of the GK rat for developing diabetes.