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Inhibition of insulin-degrading enzyme (IDE) has been proposed as a possible therapeutic target for type 2 diabetes treatment. However, many aspects of IDE's role in glucose homeostasis need to be clarified. In light of this, new preclinical models are required to elucidate the specific role of this protease in the main tissues related to insulin handling. To address this, here we generated a novel line of mice with selective deletion of the Ide gene within pancreatic beta-cells, B-IDE-KO mice, which have been characterized in terms of multiple metabolic end points, including blood glucose, plasma C-peptide, and intraperitoneal glucose tolerance tests. In addition, glucose-stimulated insulin secretion was quantified in isolated pancreatic islets and beta-cell differentiation markers and insulin secretion machinery were characterized by RT-PCR. Additionally, IDE was genetically and pharmacologically inhibited in INS-1E cells and rodent and human islets, and insulin secretion was assessed. Our results show that, in vivo, life-long deletion of IDE from beta-cells results in increased plasma C-peptide levels. Corroborating these findings, isolated islets from B-IDE-KO mice showed constitutive insulin secretion, a hallmark of beta-cell functional immaturity. Unexpectedly, we found 60% increase in Glut1 (a high-affinity/low- Km glucose transporter), suggesting increased glucose transport into the beta-cell at low glucose levels, which may be related to constitutive insulin secretion. In parallel, IDE inhibition in INS-1E and islet cells resulted in impaired insulin secretion after glucose challenge. We conclude that IDE is required for glucose-stimulated insulin secretion. When IDE is inhibited, insulin secretion machinery is perturbed, causing either inhibition of insulin release at high glucose concentrations or constitutive secretion.

Producción Científica, Insulin-degrading enzyme (IDE) is a highly conserved and ubiquitously expressed metalloprotease that degrades insulin and several other intermediate-size peptides. For many decades, IDE had been assumed to be involved primarily in hepatic insulin clearance, a key process that regulates availability of circulating insulin levels for peripheral tissues. Emerging evidence, however, suggests that IDE has several other important physiological functions relevant to glucose and insulin homeostasis, including the regulation of insulin secretion from pancreatic β-cells. Investigation of mice with tissue-specific genetic deletion of Ide in the liver and pancreatic β-cells (L-IDE-KO and B-IDE-KO mice, respectively) has revealed additional roles for IDE in the regulation of hepatic insulin action and sensitivity. In this review, we discuss current knowledge about IDE’s function as a regulator of insulin secretion and hepatic insulin sensitivity, both evaluating the classical view of IDE as an insulin protease and also exploring evidence for several non-proteolytic functions. Insulin proteostasis and insulin sensitivity have both been highlighted as targets controlling blood sugar levels in type 2 diabetes, so a clearer understanding the physiological functions of IDE in pancreas and liver could led to the development of novel therapeutics for the treatment of this disease., Ministerio de Economía, Industria y Competitividad - (grant SAF2016-77871-C2-1-R; SAF2016-77871-C2-2-R), Ministerio de Ciencia e Innovación - (grant PID2019-110496RB-C21; PID2019-110496RB-C22), Fundación Europea para el Estudio de la Diabetes (Programa Europeo de Investigación de la Diabetes sobre nuevos objetivos para el tipo 2 Diabetes apoyada por MSD-2017), US National Institutes of Health - (grant GM115617), Fundación “la Caixa" - (grant LCF/PR/PR18/51130007), Junta de Castilla y León y Fondo Social Europeo - ((ORDER EDU/574/2018 and ORDER EDU/556/2019)

The study was partially presented as a poster in the 53rd Annual Meeting of the European Association for the Study of Diabetes, Lisbon 2017., The role of insulin-degrading enzyme (IDE), a metalloprotease with high affinity for insulin, in insulin clearance remains poorly understood. OBJECTIVE: This study aimed to clarify whether IDE is a major mediator of insulin clearance, and to define its role in the etiology of hepatic insulin resistance., [Methods] We generated mice with liver-specific deletion of Ide (L-IDE-KO) and assessed insulin clearance and action., [Results] L-IDE-KO mice exhibited higher (~20%) fasting and non-fasting plasma glucose levels, glucose intolerance and insulin resistance. This phenotype was associated with ~30% lower plasma membrane insulin receptor levels in liver, as well as ~55% reduction in insulin-stimulated phosphorylation of the insulin receptor, and its downstream signaling molecules, AKT1 and AKT2 (reduced by ~40%). In addition, FoxO1 was aberrantly distributed in cellular nuclei, in parallel with up-regulation of the gluconeogenic genes Pck1 and G6pc. Surprisingly, L-IDE-KO mice showed similar plasma insulin levels and hepatic insulin clearance as control mice, despite reduced phosphorylation of the carcinoembryonic antigen-related cell adhesion molecule 1, which upon its insulin-stimulated phosphorylation, promotes receptor-mediated insulin uptake to be degraded., [Conclusion] IDE is not a rate-limiting regulator of plasma insulin levels in vivo., This work was supported by grants from the Ministerio de Economía, Industria y Competitividad: SAF2014-58702-C2-1-R and SAF2016-77871-C2-1-R to ICC; SAF2014-58702-C2-2-R and SAF2016-77871-C2-2-R to GP; supported by the EFSD European Research Programme on New Targets for Type 2 Diabetes supported by an educational research grant from MSD to ICC and GP; the National Institutes of Health: R01-DK054254, R01-DK083850 and RO1-HL-112248 to SMN, and R01-GM115617 to MAL; and the American Diabetes Association: Career Development Award 7-11-CD-13 to MAL.

© 2020 The Authors., The insulin-degrading enzyme (IDE) is a metalloendopeptidase with a high affinity for insulin. Human genetic polymorphisms in Ide have been linked to increased risk for T2DM. In mice, hepatic Ide ablation causes glucose intolerance and insulin resistance when mice are fed a regular diet. [Objective]: These studies were undertaken to further investigate its regulatory role in glucose homeostasis and insulin sensitivity in diet-induced obesity. [Methods]: To this end, we have compared the metabolic effects of loss versus gain of IDE function in mice fed a high-fat diet (HFD). [Results]: We demonstrate that loss of IDE function in liver (L-IDE-KO mouse) exacerbates hyperinsulinemia and insulin resistance without changes in insulin clearance but in parallel to an increase in pancreatic β-cell function. Insulin resistance was associated with increased FoxO1 activation and a ~2-fold increase of GLUT2 protein levels in the liver of HFD-fed mice in response to an intraperitoneal injection of insulin. Conversely, gain of IDE function (adenoviral delivery) improves glucose tolerance and insulin sensitivity, in parallel to a reciprocal ~2-fold reduction in hepatic GLUT2 protein levels. Furthermore, in response to insulin, IDE co-immunoprecipitates with the insulin receptor in liver lysates of mice with adenoviral-mediated liver overexpression of IDE. [Conclusions]: We conclude that IDE regulates hepatic insulin action and whole-body glucose metabolism in diet-induced obesity via insulin receptor levels., This work was supported by grants from the Ministerio de Economía, Industria y Competitividad: SAF2016-77871-C2-1-R to IC; SAF2016-77871-C2-2-R to GP; This work was supported by grants from the Ministerio de Ciencia e Innovación PID2019-110496RB-C21 to IC; PID2019-110496RB-C22 to GP. European Foundation for the Study of Diabetes (European Diabetes Research Programme on New Targets for Type 2 Diabetes supported by MSD-2017) to IC and GP. The project leading to these results has received funding from “la Caixa” Foundation, under agreement LCF/PR/PR18/51130007 to GP. This work was suppoted by grant from NIH GM115617 to ML.

Producción Científica, The worldwide epidemics of obesity and diabetes have been linked to increased sugar consumption in humans. Here, we review fructose and glucose metabolism, as well as potential molecular mechanisms by which excessive sugar consumption is associated to metabolic diseases and insulin resistance in humans. To this end, we focus on understanding molecular and cellular mechanisms of fructose and glucose transport and sensing in the intestine, the intracellular signaling effects of dietary sugar metabolism, and its impact on glucose homeostasis in health and disease. Finally, the peripheral and central effects of dietary sugars on the gut–brain axis will be reviewed., Ministerio de Economía, Industria y Competitividad (grants SAF2016-77871-C2-1-R and SAF2016-77871-C2-2-R), Fundación La-Caixa - Fundación Caja de Burgos (grant CAIXA-UBU001)

Insulin resistance in humans and mice is an important hallmark of metabolic diseases. Therefore, assessment of insulin sensitivity/resistance in animal models provides valuable information in the pathophysiology of diabetes and obesity. Depending on the nature of the information required, we can choose between direct and indirect techniques available for the determination of insulin sensitivity. Thus, the complex hyperinsulinemic-euglycemic glucose clamps and the insulin suppression test assess insulin-mediated glucose utilization under steady-state conditions, whereas less complex methods, such as the insulin tolerance test (ITT), rely on measurements of blood glucose levels in animals subjected to intraperitoneal insulin loading. Finally, surrogated indexes derived from blood glucose and plasma insulin levels are also available for assessment of insulin sensitivity/resistance in vivo. In this chapter, we focus on the intraperitoneal insulin tolerance test (IPITT) protocol for measuring insulin resistance in mice., This work was supported by grants from the Ministerio de Economía, Industria y Competitividad: SAF2014-58702-C2-1-R and SAF2016-77871-C2-1-R to IC; SAF2014-58702-C2-2-R and SAF2016-77871-C2-2-R to GP. Supported by the EFSD European Research Programme on New Targets for Type 2 Diabetes supported by an educational research grant from MSD to ICC and GP.

This article belongs to the Special Issue Progress on Marine Natural Products as Lead Compounds., Two new chloro-furanocembranolides (1, 2) and two new 1,4-diketo cembranolides (3, 4) were isolated from the crude extract of Leptogorgia sp. together with a new seco-furanocembranolide (5) and the known Z-deoxypukalide (6), rubifolide (7), scabrolide D (8) and epoxylophodione (9). Their structures were determined based on spectroscopic evidence. Four compounds: 1, 2, 7 and 8 were found to activate the proliferation of pancreatic insulin-producing (beta) cells., This work was supported by the Ministerio de Ciencia e Innovación (SAF2009-0839 and RTA 2015-00010-C03-02). ARDM acknowledges funding from IMBRAIN project (FP7-REGPOT-2012-CT2012-31637-IMBRAIN) and from Cabildo de Tenerife (Agustín de Betancourt Programme). A.B.G. would like to thank Convenio Universidad de Magallanes (Chile) and CSIC, project 2009CL0031, for financial support.

Hepatocyte growth factor (HGF) is a cytokine that increases glucose transport ex vivo in skeletal muscle. The aim of this work was to decipher the impact of whether conditional overexpression of HGF in vivo could improve glucose homeostasis and insulin sensitivity in mouse skeletal muscle. Following tetracyclin administration, muscle HGF levels were augmented threefold in transgenic mice (SK-HGF) compared to control mice without altering plasma HGF levels. In conditions of normal diet, SK-HGF mice showed no differences in body weight, plasma triglycerides, blood glucose, plasma insulin and glucose tolerance compared to control mice. Importantly, obese SK-HGF mice exhibited improved whole-body glucose tolerance independently of changes in body weight or plasma triglyceride levels compared to control mice. This effect on glucose homeostasis was associated with significantly higher (∼80 %) levels of phosphorylated protein kinase B in muscles from SK-HGF mice compared to control mice. In conclusion, muscle expression of HGF counteracts obesity-mediated muscle insulin resistance and improves glucose tolerance in mice., This study was supported by a grant from ISCIII (CP08/00106), MINECO (SAF2009-11282) and FP7-PEOPLE-2009-RG (PIRG06-GA-2009-25369) to GP; a grant from MINECO (RYC-2011-08101) to IC; and grants from the NIH R-01 (DK067351 and DK077096) to AGO.

This article belongs to the Special Issue Marine Compounds and Inflammation II, 2017., Type 2 diabetes (T2DM) is a complex disease linked to pancreatic beta-cell failure and insulin resistance. Current antidiabetic treatment regimens for T2DM include insulin sensitizers and insulin secretagogues. We have previously demonstrated that leptolide, a member of the furanocembranolides family, promotes pancreatic beta-cell proliferation in mice. Considering the beneficial effects of leptolide in diabetic mice, in this study, we aimed to address the capability of leptolide to improve insulin resistance associated with the pathology of obesity. To this end, we tested the hypothesis that leptolide should protect against fatty acid-induced insulin resistance in hepatocytes. In a time-dependent manner, leptolide (0.1 µM) augmented insulin-stimulated phosphorylation of protein kinase B (PKB) by two-fold above vehicle-treated HepG2 cells. In addition, leptolide (0.1 µM) counteracted palmitate-induced insulin resistance by augmenting by four-fold insulin-stimulated phosphorylation of PKB in HepG2 cells. In vivo, acute intraperitoneal administration of leptolide (0.1 mg/kg and 1 mg/kg) improved glucose tolerance and insulin sensitivity in lean mice. Likewise, prolonged leptolide treatment (0.1 mg/kg) in diet-induced obese mice improved insulin sensitivity. These effects were paralleled with an ~50% increased of insulin-stimulated phosphorylation of PKB in liver and skeletal muscle and reduced circulating pro-inflammatory cytokines in obese mice. We concluded that leptolide significantly improves insulin sensitivity in vitro and in obese mice, suggesting that leptolide may be another potential treatment for T2DM, This research has been funded by Sociedad Española de Diabetes (Ayudas Investigación Básica 2014), Salud Castilla y León (BIO/VA40/15) and Ministerio de Economía y Competitividad-Spain (SAF2014-58702-C2-1-R) to I.C. and Ministerio de Economía y Competitividad-Spain (SAF2014-58702-C2-2-R) to G.P., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

Although age remains the main risk factor to suffer Alzheimer's disease (AD) and vascular dementia (VD), type 2 diabetes (T2D) has turned up as a relevant risk factor for dementia. However, the ultimate underlying mechanisms for this association remain unclear. In the present study we analyzed central nervous system (CNS) morphological and functional consequences of long-term insulin resistance and T2D in db/db mice (leptin receptor KO mice). We also included C57Bl6 mice fed with high fat diet (HFD) and a third group of C57Bl6 streptozotocin (STZ) treated mice. Db/db mice exhibited pathological characteristics that mimic both AD and VD, including age dependent cognitive deterioration, brain atrophy, increased spontaneous hemorrhages and tau phosphorylation, affecting the cortex preferentially. A similar profile was observed in STZ-induced diabetic mice. Moreover metabolic parameters, such as body weight, glucose and insulin levels are good predictors of many of these alterations in db/db mice. In addition, in HFD-induced hyperinsulinemia in C57Bl6 mice, we only observed mild CNS alterations, suggesting that central nervous system dysfunction is associated with well established T2D. Altogether our results suggest that T2D may promote many of the pathological and behavioral alterations observed in dementia, supporting that interventions devoted to control glucose homeostasis could improve dementia progress and prognosis., M.G.-A. was supported by Ramon y Cajal program RYC-2008-02333, ISCIII – Ministerio de Economía y Competitividad, Spain. Junta de Andalucia, Proyectos de Investigación de Excelencia (P11-CTS-7847), Instituto de Salud Carlos III and FEDER (European Union), cofinanced by Fondo Europeo de Desarrollo Regional “Una manera de hacer Europa” PI12/00675 (Monica Garcia-Alloza). I.C.-C. was supported by RYC-2011-08101, ISCIII-Subdirección General de Evaluación y Fomento de la Investigación, Spain (PS09/00671), Europe-FP7 Marie Curie Grant (IRG-247835).

Placentas of women with gestational diabetes mellitus (GDM) exhibit an altered lipid metabolism. The mechanism by which GDM is linked to alterations in placental lipid metabolism remains obscure. We hypothesized that high glucose levels reduce mitochondrial fatty acid oxidation (FAO) and increase triglyceride accumulation in human placenta. To test this hypothesis, we measured FAO, fatty acid esterification, de novo fatty acid synthesis, triglyceride levels, and carnitine palmitoyltransferase activities (CPT) in placental explants of women with GDM or no pregnancy complication. In women with GDM, FAO was reduced by ~30% without change in mitochondrial content, and triglyceride content was threefold higher than in the control group. Likewise, in placental explants of women with no complications, high glucose levels reduced FAO by ~20%, and esterification increased linearly with increasing fatty acid concentrations. However, de novo fatty acid synthesis remained unchanged between high and low glucose levels. In addition, high glucose levels increased triglyceride content approximately twofold compared with low glucose levels. Furthermore, etomoxir-mediated inhibition of FAO enhanced esterification capacity by ~40% and elevated triglyceride content 1.5-fold in placental explants of women, with no complications. Finally, high glucose levels reduced CPT I activity by ~70% and phosphorylation levels of acetyl-CoA carboxylase by ~25% in placental explants of women, with no complications. We reveal an unrecognized regulatory mechanism on placental fatty acid metabolism by which high glucose levels reduce mitochondrial FAO through inhibition of CPT I, shifting flux of fatty acids away from oxidation toward the esterification pathway, leading to accumulation of placental triglycerides. © 2013 the American Physiological Society., This study was supported by a grant from the Carlos III Health Institute (CP08/00106), the Spanish Ministry of Science and Innovation (SAF2009-11282), and the FP7-PEOPLE-2009-RG (PIRG06-GA-2009-25369) to G. Perdomo; a grant from the Consejería de Salud, Junta de Andalucía (N°0269/05.2005) to J. L. Bartha; and grant from the Carlos III Health Institute (PI11/00676) to F. Bugatto.

OBJECTIVE Most knowledge on human β-cell cycle control derives from immunoblots of whole human islets, mixtures of β-cells and non-β-cells. We explored the presence, subcellular localization, and function of five early G1/S phase molecules—cyclins D1–3 and cdk 4 and 6—in the adult human β-cell. RESEARCH DESIGN AND METHODS Immunocytochemistry for the five molecules and their relative abilities to drive human β-cell replication were examined. Human β-cell replication, cell death, and islet function in vivo were studied in the diabetic NOD-SCID mouse. RESULTS Human β-cells contain easily detectable cdks 4 and 6 and cyclin D3 but variable cyclin D1. Cyclin D2 was only marginally detectable. All five were principally cytoplasmic, not nuclear. Overexpression of the five, alone or in combination, led to variable increases in human β-cell replication, with the cdk6/cyclin D3 combination being the most robust (15% versus 0.3% in control β-cells). A single molecule, cdk6, proved to be capable of driving human β-cell replication in vitro and enhancing human islet engraftment/proliferation in vivo, superior to normal islets and as effectively as the combination of cdk6 plus a D-cyclin. CONCLUSIONS Human β-cells contain abundant cdk4, cdk6, and cyclin D3, but variable amounts of cyclin D1. In contrast to rodent β-cells, they contain little or no detectable cyclin D2. They are primarily cytoplasmic and likely ineffective in basal β-cell replication. Unexpectedly, cyclin D3 and cdk6 overexpression drives human β-cell replication most effectively. Most importantly, a single molecule, cdk6, supports robust human β-cell proliferation and function in vivo.

OBJECTIVES To comprehensively inventory the proteins that control the G1/S cell cycle checkpoint in the human islet and compare them with those in the murine islet, to determine whether these might therapeutically enhance human β-cell replication, to determine whether human β-cell replication can be demonstrated in an in vivo model, and to enhance human β-cell function in vivo. RESEARCH DESIGN AND METHODS Thirty-four G1/S regulatory proteins were examined in human islets. Effects of adenoviruses expressing cdk-6, cdk-4, and cyclin D1 on proliferation in human β-cells were studied in both invitro and in vivo models. RESULTS Multiple differences between murine and human islets occur, most strikingly the presence of cdk-6 in human β-cells versus its low abundance in the murine islet. Cdk-6 and cyclin D1 in vitro led to marked activation of retinoblastoma protein phosphorylation and cell cycle progression with no induction of cell death. Human islets transduced with cdk-6 and cyclin D1 were transplanted into diabetic NOD-SCID mice and markedly outperformed native human islets in vivo, maintaining glucose control for the entire 6 weeks of the study. CONCLUSIONS The human G1/S proteome is described for the first time. Human islets are unlike their rodent counterparts in that they contain easily measurable cdk-6. Cdk-6 overexpression, alone or in combination with cyclin D1, strikingly stimulates human β-cell replication, both in vitro as well as in vivo, without inducing cell death or loss of function. Using this model, human β-cell replication can be induced and studied in vivo.

Arterial expression of PTH-related protein is markedly induced by angioplasty. PTH-related protein contains a nuclear localization signal (NLS). PTH-related protein mutants lacking the NLS (ΔNLS-PTH-related protein) are potent inhibitors of arterial vascular smooth muscle cell (VSMC) proliferation in vitro. This is of clinical relevance because adenoviral delivery of ΔNLS-PTH-related protein at angioplasty completely inhibits arterial restenosis in rats. In this study we explored the cellular mechanisms through which ΔNLS-PTH-related protein arrests the cell cycle. In vivo, adenoviral delivery of ΔNLS-PTH-related protein at angioplasty markedly inhibited VSMC proliferation as compared with angioplastied carotids infected with control adenovirus (Ad.LacZ). In vitro, ΔNLS-PTH-related protein overexpression was associated with a decrease in phospho-pRb, and a G0/G1 arrest. This pRb underphosphorylation was associated with stable levels of cdks 2, 4, and 6, the D and E cyclins, p16, p18, p19, and p21, but was associated with a dramatic decrease in cdk-2 and cdk4 kinase activities. Cyclin A was reduced, but restoring cyclin A adenovirally to normal did not promote cell cycle progression in ΔNLS-PTH-related protein VSMC. More importantly, p15INK4 and p27kip1, two critical inhibitors of the G1/S progression, were markedly increased. Normalization of both p15INK4b and p27kip1 by small interfering RNA knockdown normalized cell cycle progression. These data indicate that the changes in p15INK4b and p27kip1 fully account for the marked cell cycle slowing induced by ΔNLS-PTH-related protein in VSMCs. Finally, ΔNLS-PTH-related protein is able to induce p15INK4 and p27kip1 expression when delivered adenovirally to primary murine VSMCs. These studies provide a mechanistic understanding of ΔNLS-PTH-related protein actions, and suggest that ΔNLS-PTH-related protein may have particular efficacy for the prevention of arterial restenosis. This study provides the mechanistic underpinnings for understanding how Δ-NLS-PTHrP functions, and suggests that Δ-NLS-PTHrP may have particular efficacy for the prevention of arterial re-stenosis.

Animal studies show that G(1/S) regulatory molecules (D-cyclins, cdk-4, p18, p21, p27) are critical for normal regulation of beta-cell proliferation, mass, and function. The retinoblastoma protein, pRb, is positioned at the very end of a cascade of these regulatory proteins and is considered the final checkpoint molecule that maintains beta-cell cycle arrest. Logically, removal of pRb from the beta-cell should result in unrestrained beta-cell replication, increased beta-cell mass, and insulin-mediated hypoglycemia. Because global loss of both pRb alleles is embryonic lethal, this hypothesis has not been tested in beta-cells. We developed two types of conditional knockout (CKO) mice in which both alleles of the pRb gene were inactivated specifically in beta-cells. Surprisingly, although the pRb gene was efficiently recombined in beta-cells of both CKO models, changes in beta-cell mass, beta-cell replication rates, insulin concentrations, and blood glucose levels were limited or absent. Other pRb family members, p107 and p130, were not substantially upregulated. In contrast to dogma, the pRb protein is not essential to maintain cell cycle arrest in the pancreatic beta-cell. This may reflect fundamental inaccuracies in models of beta-cell cycle control or complementation for pRb by undefined proteins.

Parathyroid hormone–related protein (PTHrP) is present in vascular smooth muscle (VSM), is markedly upregulated in response to arterial injury, is essential for normal VSM proliferation, and also markedly accentuates neointima formation following rat carotid angioplasty. PTHrP contains a nuclear localization signal (NLS) through which it enters the nucleus and leads to marked increases in retinoblastoma protein (pRb) phosphorylation and cell cycle progression. Our goal was to define key cell cycle molecules upstream of pRb that mediate cell cycle acceleration induced by PTHrP. The cyclin D/cdk-4,-6 system and its upstream regulators, the inhibitory kinases (INKs), are not appreciably influenced by PTHrP. In striking contrast, cyclin E/cdk-2 kinase activity is markedly increased by PTHrP, and this is a result of a specific, marked, PTHrP-induced proteasomal degradation of p27 kip1 . Adenoviral restoration of p27 kip1 fully reverses PTHrP-induced cell cycle progression, indicating that PTHrP mediates its cell cycle acceleration in VSM via p27 kip1 . In confirmation, adenoviral delivery of PTHrP to murine primary vascular smooth muscle cells (VSMCs) significantly decreases p27 kip1 expression and accelerates cell cycle progression. p27 kip1 is well known to be a central cell cycle regulatory molecule involved in both normal and pathological VSM proliferation and is a target of widely used drug-eluting stents. The current observations define a novel “PTHrP/p27 kip1 pathway” in the arterial wall and suggest that this pathway is important in normal arterial biology and a potential target for therapeutic manipulation of the arterial response to injury.

Type 1 and type 2 diabetes both result from inadequate production of insulin by the beta-cells of the pancreatic islet. Accordingly, strategies that lead to increased pancreatic beta-cell mass, as well as retained or enhanced function of islets, would be desirable for the treatment of diabetes. Although pancreatic beta-cells have long been viewed as terminally differentiated and irreversibly arrested, evidence now indicates that beta-cells can and do replicate, that this replication can be enhanced by a variety of maneuvers, and that beta-cell replication plays a quantitatively significant role in maintaining pancreatic beta-cell mass and function. Because beta-cells have been viewed as being unable to proliferate, the science of beta-cell replication is undeveloped. In the past several years, however, this has begun to change at a rapid pace, and many laboratories are now focused on elucidating the molecular details of the control of cell cycle in the beta-cell. In this review, we review the molecular details of cell cycle control as they relate to the pancreatic beta-cell. Our hope is that this review can serve as a common basis and also a roadmap for those interested in developing novel strategies for enhancing beta-cell replication and improving insulin production in animal models as well as in human pancreatic beta-cells.

We hypothesized that combined transgenic overexpression of hepatocyte growth factor (HGF) and placental lactogen in islets would lead to even greater increases in β-cell mass and replication than either growth factor alone. This did not occur, suggesting that β-cell replication is saturable or subject to molecular restraint. We therefore performed the first comprehensive G1/S cell cycle survey in islets, cataloguing the broad range of kinases, cyclins, and kinase inhibitors that control the G1/S transition in islets from normal, HGF, placental lactogen, and doubly transgenic mice. Many of the G1/S checkpoint regulators (E2Fs; pRb; p107; p130; cyclins D1,2,3, A, and E; cdk-2; cdk-4; p15; p16; p18; p19; p21; p27; MDM2; p53; c-Myc; and Egr-1) are present in the murine islet. Most of these proteins were unaltered by overexpression of HGF or placental lactogen, either alone or in combination. In contrast, p21cip was uniquely, dramatically, and reproducibly upregulated in placental lactogen and HGF islets. p21cip was also present in, and upregulated in, proliferating human islets, localizing specifically in β-cells and translocating to the nucleus on mitogenic stimulation. Homozygous p21cip loss releases islets from growth inhibition, markedly enhancing proliferation in response to HGF and placental lactogen.

Islet transplantation for diabetes is limited by the availability of human islet donors. Hepatocyte growth factor (HGF) is a potent beta-cell mitogen and survival factor and improves islet transplant outcomes in a murine model. However, the murine model employs renal subcapsular transplant and immunodeficient mice, features not representative of human islet transplantation protocols. Therefore, we have developed a more rigorous, marginal-mass rat islet transplant model that more closely resembles human islet transplantation protocols: islet donors are allogeneic Lewis islets; recipients are normal Sprague Dawley rats; islets are delivered intraportally; and immunosuppression is accomplished using the same immunosuppressants employed by the Edmonton group. We demonstrate that 1) surprisingly, the Edmonton immunosuppression regimen induces marked insulin resistance and beta-cell toxicity in rats, 2) adenovirus does not adversely affect islet transplant outcomes, 3) the Edmonton immunosuppressants may delay or block rejection of adenovirally transduced islets, and more importantly, 4) pretransplant islet adenoviral gene therapy with HGF markedly improves islet transplant outcomes, 5) this enhanced function persists for months, and 6) HGF enhances islet function and survival even in the setting of immunosuppressant-induced insulin resistance and beta-cell toxicity. This approach may enhance islet transplantation outcomes in humans.

Diabetes is a consequence of a decrease on functional β-cell mass. We have recently demonstrated that epoxypukalide (Epoxy) is a natural compound with beneficial effects on primary cultures of rat islets. In this study, we extend our previous investigations to test the hypothesis that Epoxy protects β-cells and improves glucose metabolism in STZ-induced diabetic mice. We used 3-months old male mice that were treated with Epoxy at 200 μg/kg body weight. Glucose intolerance was induced by multiple intraperitoneal low-doses of streptozotocin (STZ) on 5 consecutive days. Glucose homeostasis was evaluated measuring plasma insulin levels and glucose tolerance. Histomorphometry was used to quantify the number of pancreatic β-cells per islet. β-cell proliferation was assessed by BrdU incorporation, and apoptosis by TUNEL staining. Epoxy treatment significantly improved glucose tolerance and plasma insulin levels. These metabolic changes were associated with increased β-cell numbers, as a result of a two-fold increase in β-cell proliferation and a 50% decrease in β-cell death. Our results demonstrate that Epoxy improves whole-body glucose homeostasis by preventing pancreatic β-cell death due to STZ-induced toxicity in STZ-treated mice., This work was supported by grants from: Programa Ramón y Cajal (RYC-2011-08101) Ministerio de Economía y Competitividad (Spain); Sociedad Española de Diabetes Basic Research Grant (2014) to IC.

Ghrelin is a peptidic hormone, which stimulates cell proliferation and inhibits apoptosis in several tissues, including pancreas. In preclinical stage of type 1 diabetes, proinflammatory cytokines generate a destructive environment for β-cells known as insulitis, which results in loss of β-cell mass and impaired insulin secretion, leading to diabetes. Our aim was to demonstrate that ghrelin could preserve β-cell viability, turnover rate, and insulin secretion acting as a counter balance of cytokines. In the present work we reproduced proinflammatory milieu found in insulitis stage by treating murine cell line INS-1E and rat islets with a cytokine cocktail including IL-1β, IFNγ, and TNFα and/or ghrelin. Several proteins involved in survival pathways (ERK 1/2 and Akt/PKB) and apoptosis (caspases and Bcl-2 protein family and endoplasmic reticulum stress markers) as well as insulin secretion were analyzed. Our results show that ghrelin alone has no remarkable effects on β-cells in basal conditions, but interestingly it activates cell survival pathways, downregulates apoptotic mediators and endoplasmic reticulum stress, and restores insulin secretion in response to glucose when beta-cells are cytokine-exposed. These data suggest a potential role of ghrelin in preventing or slowing down the transition from a preclinical to clinically established diabetes by ameliorating the effects of insulitis on β-cells., This work was funded by a grant by the Andalusian Government (PI 0765-2011).

[Introduction]: To evaluate the impact of the pro-inflammatory cytokine hepatocyte growth factor (HGF) on the regulation of glucose and lipid placental metabolism. [Methods]: HGF levels were quantified in amniotic fluid and placenta from control and obese women. 2-deoxy-glucose (2-DOG) uptake, glycolysis, fatty acid oxidation (FAO), fatty acid esterification, de novo fatty acid synthesis, triglyceride levels and carnitine palmitoyltransferase activities (CPT) were measured in placental explants upon addition of pathophysiological HGF levels. [Results]: In obese women, total- and -activated-HGF levels in amniotic fluid were elevated ∼24%, and placental HGF levels were ∼3-fold higher than in control women. At a similar dose to that present in amniotic fluid of obese women, HGF (30 ng/mL) increased Glut-1 levels and 2-DOG uptake by ∼25-30% in placental explants. HGF-mediated effect on 2-DOG uptake was dependent on the activation of phosphatidylinositol 3-kinase signaling pathway. In addition, HGF decreased ∼20% FAO, whereas esterification and de novo fatty acid synthesis increased ∼15% and ∼25% respectively, leading to 2-fold triglyceride accumulation in placental explants. In parallel, HGF reduced CPT-I activity ∼70%. [Discussion]: HGF is a cytokine elevated in amniotic fluid and placental tissue of obese women, which through its ability to stimulate 2-DOG uptake and metabolism impairs FAO and enhances esterification and de novo fatty acid synthesis, leading to accumulation of placental triglycerides., This study was supported by a grant from the Carlos III Health Institute (CP08/00106), the Spanish Ministry of Science and Innovation (SAF2009-11282) and the FP7-PEOPLE-2009-RG (PIRG06-GA-2009-25369) to GP; grant from the Consejería de Salud, Junta de Andalucía (N°0269/05.2005) to JLB; grant from the Carlos III Health Institute (PI11/00676) and grant from the Consejería de Salud, Junta de Andalucía (PI-0794-2010) to FB; grant from Ministerio de Economía y Competitividad (RYC-2011-08101) to IC.

et al., Aging remains the main risk factor to suffer Alzheimer's disease (AD), though epidemiological studies also support that type 2 diabetes (T2D) is a major contributor. In order to explore the close relationship between both pathologies we have developed an animal model presenting both AD and T2D, by crossing APP/PS1 mice (AD model) with db/db mice (T2D model). We traced metabolic and cognitive evolution before T2D or AD pathology is present (4 weeks of age), when T2D has debuted but no senile plaques are present (14 weeks of age) and when both pathologies are well established (26 weeks of age). APP/PS1xdb/db mice showed an age-dependent synergistic effect between T2D and AD. Significant brain atrophy and tau pathology were detected in the cortex by 14 weeks, that spread to the hippocampus by 26 weeks of age. Severe cognitive impairment was also detected as soon as at 14 weeks of age. Interestingly, in APP/PS1xdb/db mice we observed a shift in Aβ soluble/insoluble levels, and whereas more toxic soluble species were favoured, senile plaques (SP) were reduced. An overall increase of microglia activation was observed in APP/PS1xdb/db mice. We also found exacerbated hemorrhagic burden in APP/PS1xdbd/db mice, suggesting that blood brain barrier alterations may be responsible for the early pathological features observed. Moreover, metabolic parameters can predict many of these alterations, supporting a role for T2D in AD pathology. This new model provides a relevant tool to further explore the relationship between T2D, AD and vascular implications, offering the possibility to assess therapeutic approaches, that by improving T2D metabolic control could delay or prevent AD pathology., Junta de Andalucia, Proyectos de Excelencia, Consejería de Economía, Innovación, Ciencia y Empleo (P11-CTS-7847), Fundación Eugenio Rodríguez Pascual 2015, ISCIII–Subdirección General de Evaluación y Fomento de la Investigación and cofinanced by the European Union (Fondo Europeo de Desarrollo Regional, FEDER) “Una manera de hacer Europa” PI12/00675 (Monica Garcia-Alloza).

Placental metabolism is an important mechanism for the regulation of fetal growth and long-term health of the newborns. In this study, we investigated the effects of maternal metabolic environment on human placental fatty acid and glucose metabolism. We used placental explants from uncomplicated pregnancies or pregnancies complicated with gestational diabetes mellitus (GDM), undergoing vaginal delivery (VD) or cesarean section (CS). Fatty acid oxidation (FAO) and glucose uptake (2-DOG) were similar in both modes of delivery in normal and GDM pregnancies. However, placental explants from GDM exhibited 40% to 50% reduced FAO capacity compared to control placentas in women undergoing VD or CS. In contrast, 2-DOG uptake was 2- to 3-fold higher in placental explants from GDM compared to control placentas in women undergoing VD or CS, respectively. In conclusion, ex vivo placental fuel selection is influenced by maternal GDM, but placental metabolic characteristics are not altered by the mode of delivery.

This is an open-access article distributed under the terms of the Creative Commons Attribution License., Type 2 diabetes (T2D) is an important risk factor to suffer dementia, including Alzheimer's disease (AD), and some neuropathological features observed in dementia could be mediated by T2D metabolic alterations. Since brain atrophy and impaired neurogenesis have been observed both T2D and AD we analyzed central nervous system (CNS) morphological alterations in the db/db mice (leptin receptor KO mice), as a model of long-term insulin resistance and T2D, and in C57Bl6 mice fed with high fat diet (HFD), as a model of diet induced insulin resistance and prediabetes. Db/db mice showed an age-dependent cortical and hippocampal atrophy, whereas in HFD mice cortex and hippocampus were preserved. We also detected increased neurogenesis and cell proliferation rates in young db/db mice when compared with control littermates. Our study shows that metabolic parameters serve as predictors of both atrophy and altered proliferation and neurogenesis in the CNS. Moreover in the cortex, atrophy, cell proliferation and neurogenesis were significantly correlated. Our data suggest that T2D may underline some of the pathological features observed in the dementia process. They also support that blood glucose control in elderly patients could help to slow down dementia evolution and maybe, improve its prognosis. © 2014 Ramos-Rodriguez et al., RYC-2008-02333, Junta de Andalucia, Proyectos de Excelencia (P11-CTS-7847), Instituto de Salud Carlos III and FEDER (European Union), cofinanced by Fondo Europeo de Desarrollo Regional ‘‘Una manera de hacer Europa’’ PI12/00675 (Monica Garcia-Alloza) IC-C: RYC-2011-08101, ISCIII Subdirección General de Evaluación y Fomento de la Investigación, Spain (PS09/00671), Europe-FP7 Marie Curie grant (IRG-247835).

This is an open-access article distributed under the terms of the Creative Commons Attribution License., There is an urgency to find new treatments for the devastating epidemic of diabetes. Pancreatic b-cells viability and function are impaired in the two most common forms of diabetes, type 1 and type 2. Regeneration of pancreatic b-cells has been proposed as a potential therapy for diabetes. In a preliminary study, we screened a collection of marine products for b-cell proliferation. One unique compound (epoxypukalide) showed capability to induce b-cell replication in the cell line INS1 832/ 13 and in primary rat cell cultures. Epoxypukalide was used to study b-cell proliferation by [3H]thymidine incorporation and BrdU incorporation followed by BrdU/insulin staining in primary cultures of rat islets. AKT and ERK1/2 signalling pathways were analyzed. Cell cycle activators, cyclin D2 and cyclin E, were detected by western-blot. Apoptosis was studied by TUNEL and cleaved caspase 3. b-cell function was measured by glucose-stimulated insulin secretion. Epoxypukalide induced 2.5- fold increase in b-cell proliferation; this effect was mediated by activation of ERK1/2 signalling pathway and upregulation of the cell cycle activators, cyclin D2 and cyclin E. Interestingly, epoxypukalide showed protection from basal (40% lower versus control) and cytokine-induced apoptosis (80% lower versus control). Finally, epoxypukalide did not impair b-cell function when measured by glucose-stimulated insulin secretion. In conclusion, epoxypukalide induces b-cell proliferation and protects against basal and cytokine-mediated b-cell death in primary cultures of rat islets. These findings may be translated into new treatments for diabetes. © 2013 López-Acosta et al., This work was supported by ISCIII-Subdireccion Geeneral de Evaluacion y Fomento de la Investigacion, Spain (PS09/00671 and CP08/00094), Europe-FP7 Marie Curie grant (IRG-247835) and Servicio Andaluz de Salud (PI-0256/2009).

Type 2 diabetes (T2D) mellitus and Alzheimer's disease (AD) are two prevalent diseases with comparable pathophysiological features and genetic predisposition. Patients with AD are more susceptible to develop T2D. However, the molecular mechanism linking AD and T2D remains elusive. In this study, we have generated a new mouse model to test the hypothesis that AD would prompt the onset of T2D in mice. To test our hypothesis, we crossed Alzheimer APPswe/PS1dE9 (APP/PS1) transgenic mice with mice partially deficient in leptin signaling (db/+). Body weight, plasma glucose, and insulin levels were monitored. Phenotypic characterization of glucose metabolism was performed using glucose and insulin tolerance tests. β-Cell mass, islet volume, and islet number were analyzed by histomorphometry. APP/PS1 coexpression in mice with intact leptin receptor signaling did not show any metabolic perturbations in glucose metabolism or insulin sensitivity. In contrast, APP/PS1 coexpression in db/+ mice resulted in nonfasting hyperglycemia, hyperinsulinemia, and hypercholesterolemia without changes in body weight. Conversely, fasting blood glucose and cholesterol levels remained unchanged. Coinciding with altered glucose metabolism, APP/PS1 coexpression in db/+ mice resulted in glucose intolerance, insulin resistance, and impaired insulin signaling. In addition, histomorphometric analysis of pancreata revealed augmented β-cell mass. Taken together, these findings provide experimental evidence to support the notion that aberrant Aβ production might be a mechanistic link underlying the pathology of insulin resistance and T2D in AD., This work was supported by grants from Instituto de Salud Carlos III (ISCIII)-Subdirección General de Evaluación y Fomento de la Investigación, Spain (PS09/00671 and CP08/00094), and a Europe-FP7 Marie Curie grant (IRG-247835) to I. Cózar-Castellano and by ISCIII-Subdirección General de Evaluación y Fomento de la Investigación, Spain (PS09/00969), RYC-2008- 02333, Proyecto de Excelencia Junta de Andalucía (P11-CTS-7847), and Fundación Dr. Eugenio Rodríguez Pascual to M. García-Alloza.

Apolipoprotein D (ApoD) is an atypical apolipoprotein with an incompletely understood function in the regulation of triglyceride and glucose metabolism. We have demonstrated that elevated ApoD production in mice results in improved postprandial triglyceride clearance. This work studies the role of ApoD deficiency in the regulation of triglyceride and glucose metabolism and its dependence on aging. We used ApoD knockout (ApoD-KO) mice of 3 and 21 months of age. Body weight and food intake were measured. Hepatic histology, triglyceride content, lipoprotein lipase levels, and plasma metabolites were studied. Phenotypic characterization of glucose metabolism was performed using glucose tolerance test. β-Cell mass, islet volume, and islet number were analyzed by histomorphometry. Apolipoprotein D deficiency results in nonfasting hypertriglyceridemia in young (P =.01) and aged mice (P =.002). In young ApoD-KO mice, hypertriglyceridemia was associated with 30% to 50% increased food intake in nonfasting and fasting conditions, respectively, without changes in body weight. In addition, lipoprotein lipase levels were reduced by 35% in adipose tissue (P =.006). In aged ApoD-KO mice, hypertriglyceridemia was not associated with changes in food intake or body weight, whereas hepatic triglyceride levels were reduced by 35% (P =.02). Furthermore, nonfasting plasma insulin levels were elevated by 2-fold in young (P =.016) and aged (P =.004) ApoD-KO mice, without changes in blood glucose levels, glucose tolerance, β-cell mass, or islet number. These findings underscore the importance of ApoD in the regulation of plasma insulin levels and triglyceride metabolism, suggesting that ApoD plays an important role in the pathogenesis of dyslipidemia. © 2011 Elsevier Inc. All rights reserved., This work was supported by grants from the Carlos III Health Institute (CP08/00106), the Spanish Ministry of Science and Innovation (SAF2009-11282), and the FP7-PEOPLE-2009-RG (PIRG06-GA-2009-256369) to GP; grant from the Spanish Ministry of Science and Innovation (BFU2008-01170) to MDG and DS; and grants from the Carlos III Health Institute (PS09/ 00671) and the FP7-PEOPLE-2009-RG (IRG247835) to IC. MJP was supported by the Carlos III Health Institute (Spain).

Type 1 (formerly “juvenile”) diabetes is believed to be an autoimmune disease in which host immune cells fail to recognize the insulin-producing beta cells contained within pancreatic islets of Langerhans as being “self” and inappropriately elect to destroy these critical insulin-producing cells. The traditional methods of treating type 1 diabetes are good, but not perfect. The studies reported by Sapir et al. (1) in this issue of PNAS suggest that new, more acceptable approaches to beta cell replacement may be feasible. In the current treatment of type 1 diabetes, insulin is given by injection or insulin pump. Patients must frequently check their blood glucose by finger stick to calculate and adjust their insulin doses. Undertreatment (hyperglycemia) and overtreatment (hypoglycemia) are common. The ideal therapy for diabetes would mimic the two essential features of normal beta cells: the ability to sense glucose continually coupled with intelligent and appropriate release of insulin in response to changes in blood glucose. One solution could be an implantable, glucose-sensing insulin delivery system. Attempts to develop such a computer-driven, “closed-loop” insulin delivery system have been aided substantially by the miniaturization of computer chips and insulin pumps. Important hurdles relate to development of reliable, long-term glucose sensors, miniaturization, replacement of batteries, and refillable reservoirs for the necessary insulin pumps. Whole pancreas transplant is another option. Long-term graft function is excellent, but the procedure requires major surgery, is limited by the small numbers of pancreata available for transplant, and is accompanied by morbidity from immunosuppressive drugs used to prevent organ rejection (2). In 2000, a group in Edmonton, Canada, demonstrated the feasibility of pancreatic islet transplantation (3), which requires only trivial outpatient surgery. The Edmonton demonstration that long-term islet graft survival and function are possible has reinvigorated attempts at beta cell replacement therapy. In broad terms, there are …