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.

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.

Exercise training impacts exercise tolerance and bioenergetics in gastrocnemius muscle of non-obese type-2 diabetic Goto-Kakizaki rat in vivo.

The functional and bioenergetics impact of regular physical activity upon type-2 diabetic skeletal muscle independently of confounding factors of overweight remains undocumented. Here, gastrocnemius muscle energy fluxes, mitochondrial capacity and mechanical performance were assessed noninvasively and longitudinally in non-obese diabetic Goto-Kakizaki rats using magnetic resonance (MR) imaging and dynamic 31-phosphorus MR spectroscopy (31P-MRS) throughout a 6-min fatiguing bout of exercise performed before, in the middle (4-week) and at the end of an 8-week training protocol consisting in 60-min daily run on a treadmill. The training protocol reduced plasmatic insulin level (-61%) whereas blood glucose and non-esterified fatty acids levels remained unaffected, thereby indicating an improvement of insulin sensitivity. It also increased muscle mitochondrial citrate synthase activity (+45%) but this increase did not enhance oxidative ATP synthesis capacity in working muscle in vivo while glycolytic ATP production was increased (+33%). On the other hand, the training protocol impaired maximal force-generating capacity (-9%), total amount of force produced (-12%) and increased ATP cost of contraction (+32%) during the fatiguing exercise. Importantly, these deleterious effects were transiently worsened in the middle of the 8-week period, in association with reduced oxidative capacity and increased basal [Pi]/[PCr] ratio (an in vivo biomarker of muscle damage). These data demonstrate that the beneficial effect of regular training on insulin sensitivity in non-obese diabetic rat occurs separately from any improvement in muscle mitochondrial function and might be linked to an increased capacity for metabolizing glucose through anaerobic process in exercising muscle.

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.

Insulin Resistance Is Not Associated with an Impaired Mitochondrial Function in Contracting Gastrocnemius Muscle of Goto-Kakizaki Diabetic Rats In Vivo.

Insulin resistance, altered lipid metabolism and mitochondrial dysfunction in skeletal muscle would play a major role in type 2 diabetes mellitus (T2DM) development, but the causal relationships between these events remain conflicting. To clarify this issue, gastrocnemius muscle function and energetics were investigated throughout a multidisciplinary approach combining in vivo and in vitro measurements in Goto-Kakizaki (GK) rats, a non-obese T2DM model developing peripheral insulin resistant without abnormal level of plasma non-esterified fatty acids (NEFA). Wistar rats were used as controls. Mechanical performance and energy metabolism were assessed strictly non-invasively using magnetic resonance (MR) imaging and 31-phosphorus MR spectroscopy (31P-MRS). Compared with control group, plasma insulin and glucose were respectively lower and higher in GK rats, but plasma NEFA level was normal. In resting GK muscle, phosphocreatine content was reduced whereas glucose content and intracellular pH were both higher. However, there were not differences between both groups for basal oxidative ATP synthesis rate, citrate synthase activity, and intramyocellular contents for lipids, glycogen, ATP and ADP (an important in vivo mitochondrial regulator). During a standardized fatiguing protocol (6 min of maximal repeated isometric contractions electrically induced at a frequency of 1.7 Hz), mechanical performance and glycolytic ATP production rate were reduced in diabetic animals whereas oxidative ATP production rate, maximal mitochondrial capacity and ATP cost of contraction were not changed. These findings provide in vivo evidence that insulin resistance is not caused by an impairment of mitochondrial function in this diabetic model.

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.

Hypothalamic serotonin-insulin signaling cross-talk and alterations in a type 2 diabetic model.

Serotonin and insulin are key regulators of homeostatic mechanisms in the hypothalamus. However, in type 2 diabetes, the hypothalamic responsiveness to serotonin is not clearly established. We used a diabetic model, the Goto Kakizaki (GK) rats, to explore insulin receptor expression, insulin and serotonin efficiency in the hypothalamus and liver by means of Akt phosphorylation. Insulin or dexfenfluramine (stimulator of serotonin) treatment induced Akt phosphorylation in Wistar rats but not in GK rats that exhibit down-regulated insulin receptor. Studies in a neuroblastoma cell line showed that serotonin-induced Akt phosphorylation is PI3-kinase dependent. Finally, in response to food intake, hypothalamic serotonin release was reduced in GK rats, indicating impaired responsiveness of this neurotransmitter. In conclusion, hypothalamic serotonin as insulin efficiency is impaired in diabetic GK rats. The insulin-serotonin cross-talk and impairment observed is one potential key modification in the brain during the onset of diabetes.

Activation of the GLP-1 receptor signalling pathway: a relevant strategy to repair a deficient beta-cell mass.

Recent preclinical studies in rodent models of diabetes suggest that exogenous GLP-1R agonists and DPP-4 inhibitors have the ability to increase islet mass and preserve beta-cell function, by immediate reactivation of beta-cell glucose competence, as well as enhanced beta-cell proliferation and neogenesis and promotion of beta-cell survival. These effects have tremendous implication in the treatment of T2D because they directly address one of the basic defects in T2D, that is, beta-cell failure. In human diabetes, however, evidence that the GLP-1-based drugs alter the course of beta-cell function remains to be found. Several questions surrounding the risks and benefits of GLP-1-based therapy for the diabetic beta-cell mass are discussed in this review and require further investigation.

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.

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.

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.

Islet endothelial activation and oxidative stress gene expression is reduced by IL-1Ra treatment in the type 2 diabetic GK rat.

BACKGROUND: Inflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra). METHODOLOGY/PRINCIPAL FINDINGS: Gene expression was analyzed by quantitative RT-PCR on islets isolated from 10-week-old diabetic GK and control Wistar rats. Furthermore, GK rats were treated s.c twice daily with IL-1Ra (Kineret, Amgen, 100 mg/kg/day) or saline, from 4 weeks of age onwards (onset of diabetes). Four weeks later, islet gene analysis and pancreas immunochemistry were performed. Thirty-two genes were selected encoding molecules involved in endothelial cell activation, particularly fibrinolysis, vascular tone, OS, angiogenesis and also inflammation. All genes except those encoding angiotensinogen and epoxide hydrolase (that were decreased), and 12-lipoxygenase and vascular endothelial growth factor (that showed no change), were significantly up-regulated in GK islets. After IL-1Ra treatment of GK rats in vivo, most selected genes implied in endothelium/OS/immune cells/fibrosis were significantly down-regulated. IL-1Ra also improved islet vascularization, reduced fibrosis and ameliorated glycemia. CONCLUSIONS/SIGNIFICANCE: GK rat islets have increased mRNA expression of markers of early islet endothelial cell activation, possibly triggered by several metabolic factors, and also some defense mechanisms. The beneficial effect of IL-1Ra on most islet endothelial/OS/immune cells/fibrosis parameters analyzed highlights a major endothelial-related role for IL-1 in GK islet alterations. Thus, metabolically-altered islet endothelium might affect the beta-cell microenvironment and contribute to progressive type 2 diabetic beta-cell dysfunction in GK rats. Counteracting islet endothelial cell inflammation might be one way to ameliorate/prevent beta-cell dysfunction in type 2 diabetes.

Mechanisms of KGF mediated signaling in pancreatic duct cell proliferation and differentiation.

BACKGROUND: Keratinocyte growth factor (KGF; palifermin) is a growth factor with a high degree of specificity for epithelial cells. KGF is an important effector of epithelial growth and tissue homeostasis in various organs including the pancreas. Here we investigated the intracellular signaling pathways involved in the mediation of pancreatic ductal cell proliferation and differentiation induced by exogenous KGF during beta-cell regeneration in diabetic rat. METHODOLOGY AND RESULTS: In vitro and in vivo duct cell proliferation was measured by BrdU incorporation assay. The implication of MAPK-ERK1/2 in the mediation of KGF-induced cell proliferation was determined by inactivation of this pathway, using the pharmacological inhibitor or antisense morpholino-oligonucleotides against MEK1. In vivo KGF-induced duct cell differentiation was assessed by the immunolocalization of PDX1 and Glut2 in ductal cells and the implication of PI3K/AKT in this process was investigated. We showed that KGF exerted a potent mitogenic effect on ductal cells. Both in vitro and in vivo, its effect on cell proliferation was mediated through the activation of ERK1/2 as evidenced by the abolition of duct cell proliferation in the context of MEK/ERK inactivation. In vivo, KGF treatment triggered ductal cell differentiation as revealed by the expression of PDX1 and Glut2 in a subpopulation of ductal cells via a PI3K-dependent mechanism. CONCLUSION: Here we show that KGF promotes beta-cell regeneration by stimulating duct cell proliferation in vivo. Moreover, we demonstrated for the first time that KGF directly induces the expression of PDX1 in some ductal cells thus inducing beta-cell neogenesis. We further explored the molecular mechanisms involved in these processes and showed that the effects of KGF on duct cell proliferation are mediated by the MEK-ERK1/2 pathway, while the KGF-induced cell differentiation is mediated by the PI3K/AKT pathway. These findings might have important implications for the in vivo induction of duct-to-beta cell neogenesis in patients with beta-cell deficiency.

Islet inflammation and fibrosis in a spontaneous model of type 2 diabetes, the GK rat.

The molecular pathways leading to islet fibrosis in diabetes are unknown. Therefore, we studied gene expression in islets of 4-month-old Goto-Kakizaki (GK) and Wistar control rats. Of 71 genes found to be overexpressed in GK islets, 24% belong to extracellular matrix (ECM)/cell adhesion and 34% to inflammatory/immune response families. Based on gene data, we selected several antibodies to study fibrosis development during progression of hyperglycemia by immunohistochemistry. One-month-old GK and Wistar islets appeared to be similar. Two-month-old GK islets were strongly heterogenous in terms of ECM accumulation compared with Wistar islets. GK islet vascularization, labeled by von Willebrand factor, was altered after 1 month of mild hyperglycemia. Numerous macrophages (major histocompatibility complex class II(+) and CD68(+)) and granulocytes were found in/around GK islets. These data demonstrate that marked inflammatory reaction accompanies GK islet fibrosis and suggest that islet alterations in this nonobese model of type 2 diabetes develop in a way reminiscent of microangiopathy.

Restitution of defective glucose-stimulated insulin secretion in diabetic GK rat by acetylcholine uncovers paradoxical stimulatory effect of beta-cell muscarinic receptor activation on cAMP production.

Because acetylcholine (ACh) is a recognized potentiator of glucose-stimulated insulin release in the normal beta-cell, we have studied ACh's effect on islets of the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes. We first verified that ACh was able to restore the insulin secretory glucose competence of the GK beta-cell. Then, we demonstrated that in GK islets 1) ACh elicited a first-phase insulin release at low glucose, whereas it had no effect in Wistar; 2) total phospholipase C activity, ACh-induced inositol phosphate production, and intracellular free calcium concentration ([Ca2+]i) elevation were normal; 3) ACh triggered insulin release, even in the presence of thapsigargin, which induced a reduction of the ACh-induced [Ca2+]i response (suggesting that ACh produces amplification signals that augment the efficacy of elevated [Ca2+]i on GK exocytosis); 4) inhibition of protein kinase C did not affect [Ca2+]i nor the insulin release responses to ACh; and 5) inhibition of cAMP-dependent protein kinases (PKAs), adenylyl cyclases, or cAMP generation, while not affecting the [Ca2+]i response, significantly lowered the insulinotropic response to ACh (at low and high glucose). In conclusion, ACh acts mainly through activation of the cAMP/PKA pathway to potently enhance Ca2+-stimulated insulin release in the GK beta-cell and, in doing so, normalizes its defective glucose responsiveness.

Persistent improvement of type 2 diabetes in the Goto-Kakizaki rat model by expansion of the beta-cell mass during the prediabetic period with glucagon-like peptide-1 or exendin-4.

In the Goto-Kakizaki (GK) rat, a genetic model of type 2 diabetes, the neonatal beta-cell mass deficit is considered to be the primary defect leading to basal hyperglycemia, which is detectable for the first time 3 weeks after birth. We investigated in GK females the short- and the long-term effects of a treatment with glucagon-like peptide-1 (GLP-1) or its long-acting analog exendin-4 (Ex-4) during the first postnatal week (during the prediabetic period). GK rats were treated with daily injections of glucagon-like peptide-1 (400 microg x kg(-1) x day(-1)) or Ex-4 (3 microg x kg(-1) x day(-1)) from day 2 to day 6 after birth and were evaluated against Wistar and untreated GK rats. Under these conditions, on day 7 both treatments enhanced pancreatic insulin content and total beta-cell mass by stimulating beta-cell neogenesis and regeneration. Follow-up of biological characteristics from day 7 to adult age (2 months) showed that such a GLP-1 or Ex-4 treatment exerted long-term favorable influences on beta-cell mass and glycemic control at adult age. As compared to untreated GK rats, 2-month-old treated rats exhibited significantly decreased basal plasma glucose. Their glucose-stimulated insulin secretion, in vivo after intravenous glucose load or in vitro using isolated perfused pancreas, was slightly improved. This contributed at least partly to improve the in vivo plasma glucose disappearance rate, which was found to be increased in both treated GK groups compared to the untreated GK group. These findings in the GK model indicated, for the first time, that GLP-1 or Ex-4 treatment limited to the prediabetic period delays the installation and limits the severity of type 2 diabetes. Under these conditions, GLP-1 represents a unique tool because of its beta-cell replenishing effect in spontaneously diabetic rodents. It may prove to be an invaluable agent for the prevention of human type 2 diabetes.