Early hypermethylation of hepatic Igfbp2 results in its reduced expression preceding fatty liver in mice.

Obesity and ectopic fat disposition are risk factors for metabolic disease. Recent data indicate that IGFBP2 expression in liver is epigenetically inhibited during hepatic steatosis. The aim of this study was to investigate if epigenetic de-regulation of hepatic Igfbp2 occurs already early in life and is associated with increased risk for diet-induced obesity (DIO) during adolescence. Male C57BL/6J mice received a high-fat diet. After 3 weeks on this diet (age of 6 weeks), DIO-susceptible (responder, Resp) and DIO-resistant (non-responder, nResp) mice were identified by early weight gain. At the age of 6 weeks, Resp mice exhibited elevated blood glucose (p < 0.05), plasma insulin (p < 0.01), HOMA-IR and leptin/adiponectin ratio, whereas liver triglycerides were identical but significantly increased (p < 0.01) in Resp mice at 20 weeks of age. Igfbp2 expression was reduced in young Resp compared with nResp mice (p < 0.01), an effect that correlated with elevated DNA methylation of intronic CpG2605 (p < 0.01). The epigenetic inhibition of Igfbp2 was stable over time and preceded DIO and hepatosteatosis in adult mice. In vitro studies demonstrated that selective methylation of CpG2605 significantly reduced reporter activity by ∼85%, indicating that Igfbp2 expression is modulated by methylation. In human whole blood cells, methylation of IGFBP2 at the homologous CpG site was increased in obese men with impaired glucose tolerance. In conclusion, our data show that increased methylation of hepatic Igfbp2 during infancy predicts the development of fatty liver later in life and is linked to deterioration of glucose metabolism.

Caloric restriction and intermittent fasting alter hepatic lipid droplet proteome and diacylglycerol species and prevent diabetes in NZO mice.

Caloric restriction and intermittent fasting are known to improve glucose homeostasis and insulin resistance in several species including humans. The aim of this study was to unravel potential mechanisms by which these interventions improve insulin sensitivity and protect from type 2 diabetes. Diabetes-susceptible New Zealand Obese mice were either 10% calorie restricted (CR) or fasted every other day (IF), and compared to ad libitum (AL) fed control mice. AL mice showed a diabetes prevalence of 43%, whereas mice under CR and IF were completely protected against hyperglycemia. Proteomic analysis of hepatic lipid droplets revealed significantly higher levels of PSMD9 (co-activator Bridge-1), MIF (macrophage migration inhibitor factor), TCEB2 (transcription elongation factor B (SIII), polypeptide 2), ACY1 (aminoacylase 1) and FABP5 (fatty acid binding protein 5), and a marked reduction of GSTA3 (glutathione S-transferase alpha 3) in samples of CR and IF mice. In addition, accumulation of diacylglycerols (DAGs) was significantly reduced in livers of IF mice (P=0.045) while CR mice showed a similar tendency (P=0.062). In particular, 9 DAG species were significantly reduced in response to IF, of which DAG-40:4 and DAG-40:7 also showed significant effects after CR. This was associated with a decreased PKCε activation and might explain the improved insulin sensitivity. In conclusion, our data indicate that protection against diabetes upon caloric restriction and intermittent fasting associates with a modulation of lipid droplet protein composition and reduction of intracellular DAG species.

GLP-1-oestrogen attenuates hyperphagia and protects from beta cell failure in diabetes-prone New Zealand obese (NZO) mice.

AIMS/HYPOTHESIS: Oestrogens have previously been shown to exert beta cell protective, glucose-lowering effects in mouse models. Therefore, the recent development of a glucagon-like peptide-1 (GLP-1)-oestrogen conjugate, which targets oestrogen into cells expressing GLP-1 receptors, offers an opportunity for a cell-specific and enhanced beta cell protection by oestrogen. The purpose of this study was to compare the effects of GLP-1 and GLP-1-oestrogen during beta cell failure under glucolipotoxic conditions. METHODS: Male New Zealand obese (NZO) mice were treated with daily s.c. injections of GLP-1 and GLP-1-oestrogen, respectively. Subsequently, the effects on energy homeostasis and beta cell integrity were measured. In order to clarify the targeting of GLP-1-oestrogen, transcription analyses of oestrogen-responsive genes in distinct tissues as well as microarray analyses in pancreatic islets were performed. RESULTS: In contrast to GLP-1, GLP-1-oestrogen significantly decreased food intake resulting in a substantial weight reduction, preserved normoglycaemia, increased glucose tolerance and enhanced beta cell protection. Analysis of hypothalamic mRNA profiles revealed elevated expression of Pomc and Leprb. In livers from GLP-1-oestrogen-treated mice, expression of lipogenic genes was attenuated and hepatic triacylglycerol levels were decreased. In pancreatic islets, GLP-1-oestrogen altered the mRNA expression to a pattern that was similar to that of diabetes-resistant NZO females. However, conventional oestrogen-responsive genes were not different, indicating rather indirect protection of pancreatic beta cells. CONCLUSIONS/INTERPRETATION: GLP-1-oestrogen efficiently protects NZO mice against carbohydrate-induced beta cell failure by attenuation of hyperphagia. In this regard, targeted delivery of oestrogen to the hypothalamus by far exceeds the anorexigenic capacity of GLP-1 alone.

Exercise training alters DNA methylation patterns in genes related to muscle growth and differentiation in mice.

The adaptive response of skeletal muscle to exercise training is tightly controlled and therefore requires transcriptional regulation. DNA methylation is an epigenetic mechanism known to modulate gene expression, but its contribution to exercise-induced adaptations in skeletal muscle is not well studied. Here, we describe a genome-wide analysis of DNA methylation in muscle of trained mice (n = 3). Compared with sedentary controls, 2,762 genes exhibited differentially methylated CpGs (P < 0.05, meth diff >5%, coverage >10) in their putative promoter regions. Alignment with gene expression data (n = 6) revealed 200 genes with a negative correlation between methylation and expression changes in response to exercise training. The majority of these genes were related to muscle growth and differentiation, and a minor fraction involved in metabolic regulation. Among the candidates were genes that regulate the expression of myogenic regulatory factors (Plexin A2) as well as genes that participate in muscle hypertrophy (Igfbp4) and motor neuron innervation (Dok7). Interestingly, a transcription factor binding site enrichment study discovered significantly enriched occurrence of CpG methylation in the binding sites of the myogenic regulatory factors MyoD and myogenin. These findings suggest that DNA methylation is involved in the regulation of muscle adaptation to regular exercise training.

Protein kinase-D1 overexpression prevents lipid-induced cardiac insulin resistance.

In the insulin resistant heart, energy fuel selection shifts away from glucose utilization towards almost complete dependence on long-chain fatty acids (LCFA). This shift results in excessive cardiac lipid accumulation and eventually heart failure. Lipid-induced cardiomyopathy may be averted by strategies that increase glucose uptake without elevating LCFA uptake. Protein kinase-D1 (PKD1) is involved in contraction-induced glucose, but not LCFA, uptake allowing to hypothesize that this kinase is an attractive target to treat lipid-induced cardiomyopathy. For this, cardiospecific constitutively active PKD1 overexpression (caPKD1)-mice were subjected to an insulin resistance-inducing high fat-diet for 20-weeks. Substrate utilization was assessed by microPET and cardiac function by echocardiography. Cardiomyocytes were isolated for measurement of substrate uptake, lipid accumulation and insulin sensitivity. Wild-type mice on a high fat-diet displayed increased basal myocellular LCFA uptake, increased lipid deposition, greatly impaired insulin signaling, and loss of insulin-stimulated glucose and LCFA uptake, which was associated with concentric hypertrophic remodeling. The caPKD1 mice on high-fat diet showed none of these characteristics, whereas on low-fat diet a shift towards cardiac glucose utilization in combination with hypertrophy and ventricular dilation was observed. In conclusion, these data suggest that PKD pathway activation may be an attractive therapeutic strategy to mitigate lipid accumulation, insulin resistance and maladaptive remodeling in the lipid-overloaded heart, but this requires further investigation.

Calcium signaling recruits substrate transporters GLUT4 and CD36 to the sarcolemma without increasing cardiac substrate uptake.

Activation of AMP-activated protein kinase (AMPK) in cardiomyocytes induces translocation of glucose transporter GLUT4 and long-chain fatty acid (LCFA) transporter CD36 from endosomal stores to the sarcolemma to enhance glucose and LCFA uptake, respectively. Ca(2+)/calmodulin-activated kinase kinase-ß (CaMKKß) has been positioned directly upstream of AMPK. However, it is unknown whether acute increases in [Ca(2+)]i stimulate translocation of GLUT4 and CD36 and uptake of glucose and LCFA or whether Ca(2+) signaling converges with AMPK signaling to exert these actions. Therefore, we studied the interplay between Ca(2+) and AMPK signaling in regulation of cardiomyocyte substrate uptake. Exposure of primary cardiomyocytes to inhibitors or activators of Ca(2+) signaling affected neither AMPK-Thr(172) phosphorylation nor basal and AMPK-mediated glucose and LCFA uptake. Despite their lack of an effect on substrate uptake, Ca(2+) signaling activators induced GLUT4 and CD36 translocation. In contrast, AMPK activators stimulated GLUT4/CD36 translocation as well as glucose/LCFA uptake. When cardiomyocytes were cotreated with Ca(2+) signaling and AMPK activators, Ca(2+) signaling activators further enhanced AMPK-induced glucose/LCFA uptake. In conclusion, Ca(2+) signaling shows no involvement in AMPK-induced GLUT4/CD36 translocation and substrate uptake but elicits transporter translocation via a separate pathway requiring CaMKKß/CaMKs. Ca(2+)-induced transporter translocation by itself appears to be ineffective to increase substrate uptake but requires additional AMPK activation to effectuate transporter translocation into increased substrate uptake. Ca(2+)-induced transporter translocation might be crucial under excessive cardiac stress conditions that require supraphysiological energy demands. Alternatively, Ca(2+) signaling might prepare the heart for substrate uptake during physiological contraction by inducing transporter translocation.

Cardiac troponin in ischemic cardiomyocytes: intracellular decrease before onset of cell death.

AIM: Cardiac troponin I (cTnI) and T (cTnT) are the most important biomarkers in the diagnosis of acute myocardial infarction (AMI). Nevertheless, they can be elevated in the absence of AMI. It is unclear if such elevations represent irreversible cardiomyocyte-damage or leakage from viable cardiomyocytes. Our objective is to evaluate whether cTn is released from viable cardiomyocytes in response to ischemia and to identify differences in the release of cTn and its molecular forms. METHODS AND RESULTS: HL-1 cardiomyocytes (mouse) were subjected to ischemia (modeled by anoxia with glucose deprivation). The total contents and molecular forms of cTn were determined in culture media and cell lysates. Cell viability was assessed from the release of lactate dehydrogenase (LDH). Before the release of LDH, the intracellular cTn content in ischemic cells decreased significantly compared to control (52% for cTnI; 23% for cTnT) and was not matched by a cTn increase in the medium. cTnI decreased more rapidly than cTnT, resulting in an intracellular cTnT/cTnI ratio of 25.5 after 24 h of ischemia. Western blots revealed changes in the relative amounts of fragmented cTnI and cTnT in ischemic cells. CONCLUSIONS: HL-1 cardiomyocytes subjected to simulated ischemia released cTnI and cTnT only in combination with the release of LDH. We find no evidence of cTn release from viable cardiomyocytes, but did observe a significant decrease in cTn content, before the onset of cell death. Intracellular decrease of cTn in viable cardiomyocytes can have important consequences for the interpretation of cTn values in clinical practice.

Overexpression of vesicle-associated membrane protein (VAMP) 3, but not VAMP2, protects glucose transporter (GLUT) 4 protein translocation in an in vitro model of cardiac insulin resistance.

Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.

Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes.

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.

Ex vivo tetramer staining and cell surface phenotyping for early activation markers CD38 and HLA-DR to enumerate and characterize malaria antigen-specific CD8+ T-cells induced in human volunteers immunized with a Plasmodium falciparum adenovirus-vectored malaria vaccine expressing AMA1.

BACKGROUND: Malaria is responsible for up to a 600,000 deaths per year; conveying an urgent need for the development of a malaria vaccine. Studies with whole sporozoite vaccines in mice and non-human primates have shown that sporozoite-induced CD8+ T cells targeting liver stage antigens can mediate sterile protection. There is a need for a direct method to identify and phenotype malaria vaccine-induced CD8+ T cells in humans. METHODS: Fluorochrome-labelled tetramers consisting of appropriate MHC class I molecules in complex with predicted binding peptides derived from Plasmodium falciparum AMA-1 were used to label ex vivo AMA-1 epitope specific CD8+ T cells from research subjects responding strongly to immunization with the NMRC-M3V-Ad-PfCA (adenovirus-vectored) malaria vaccine. The identification of these CD8+ T cells on the basis of their expression of early activation markers was also investigated. RESULTS: Analyses by flow cytometry demonstrated that two of the six tetramers tested: TLDEMRHFY: HLA-A*01:01 and NEVVVKEEY: HLA-B*18:01, labelled tetramer-specific CD8+ T cells from two HLA-A*01:01 volunteers and one HLA-B*18:01 volunteer, respectively. By contrast, post-immune CD8+ T cells from all six of the immunized volunteers exhibited enhanced expression of the CD38 and HLA-DRhi early activation markers. For the three volunteers with positive tetramer staining, the early activation phenotype positive cells included essentially all of the tetramer positive, malaria epitope- specific CD8+ T cells suggesting that the early activation phenotype could identify all malaria vaccine-induced CD8+ T cells without prior knowledge of their exact epitope specificity. CONCLUSIONS: The results demonstrated that class I tetramers can identify ex vivo malaria vaccine antigen-specific CD8+ T cells and could therefore be used to determine their frequency, cell surface phenotype and transcription factor usage. The results also demonstrated that vaccine antigen-specific CD8+ T cells could be identified by activation markers without prior knowledge of their antigen-specificity, using a subunit vaccine for proof-of-concept. Whether, whole parasite or adjuvanted protein vaccines will also induce {CD38 and HLA-DRhi}+ CD8+ T cell populations reflective of the antigen-specific response will the subject of future investigations.

Protein kinase D increases maximal Ca2+-activated tension of cardiomyocyte contraction by phosphorylation of cMyBP-C-Ser315.

Cardiac myosin-binding protein C (cMyBP-C) is involved in the regulation of cardiac myofilament contraction. Recent evidence showed that protein kinase D (PKD) is one of the kinases that phosphorylate cMyBP-C. However, the mechanism by which PKD-induced cMyBP-C phosphorylation affects cardiac contractile responses is not known. Using immunoprecipitation, we showed that, in contracting cardiomyocytes, PKD binds to cMyBP-C and phosphorylates it at Ser(315). The effect of PKD-mediated phosphorylation of cMyBP-C on cardiac myofilament function was investigated in permeabilized ventricular myocytes, isolated from wild-type (WT) and from cMyBP-C knockout (KO) mice, incubated in the presence of full-length active PKD. In WT myocytes, PKD increased both myofilament Ca(2+) sensitivity (pCa(50)) and maximal Ca(2+)-activated tension of contraction (T(max)). In cMyBP-C KO skinned myocytes, PKD increased pCa(50) but did not alter T(max). This suggests that cMyBP-C is not involved in PKD-mediated sensitization of myofilaments to Ca(2+) but is essential for PKD-induced increase in T(max). Furthermore, the phosphorylation of both PKD-Ser(916) and cMyBP-C-Ser(315) was contraction frequency-dependent, suggesting that PKD-mediated cMyBP-C phosphorylation is operational primarily during periods of increased contractile activity. Thus, during high contraction frequency, PKD facilitates contraction of cardiomyocytes by increasing Ca(2+) sensitivity and by an increased T(max) through phosphorylation of cMyBP-C.

Subcellular trafficking of the substrate transporters GLUT4 and CD36 in cardiomyocytes.

Cardiomyocytes use glucose as well as fatty acids for ATP production. These substrates are transported into the cell by glucose transporter 4 (GLUT4) and the fatty acid transporter CD36. Besides being located at the sarcolemma, GLUT4 and CD36 are stored in intracellular compartments. Raised plasma insulin concentrations and increased cardiac work will stimulate GLUT4 as well as CD36 to translocate to the sarcolemma. As so far studied, signaling pathways that regulate GLUT4 translocation similarly affect CD36 translocation. During the development of insulin resistance and type 2 diabetes, CD36 becomes permanently localized at the sarcolemma, whereas GLUT4 internalizes. This juxtaposed positioning of GLUT4 and CD36 is important for aberrant substrate uptake in the diabetic heart: chronically increased fatty acid uptake at the expense of glucose. To explain the differences in subcellular localization of GLUT4 and CD36 in type 2 diabetes, recent research has focused on the role of proteins involved in trafficking of cargo between subcellular compartments. Several of these proteins appear to be similarly involved in both GLUT4 and CD36 translocation. Others, however, have different roles in either GLUT4 or CD36 translocation. These trafficking components, which are differently involved in GLUT4 or CD36 translocation, may be considered novel targets for the development of therapies to restore the imbalanced substrate utilization that occurs in obesity, insulin resistance and diabetic cardiomyopathy.

Differential regulation of cardiac glucose and fatty acid uptake by endosomal pH and actin filaments.

Insulin and contraction stimulate both cardiac glucose and long-chain fatty acid (LCFA) uptake via translocation of the substrate transporters GLUT4 and CD36, respectively, from intracellular compartments to the sarcolemma. Little is known about the role of vesicular trafficking elements in insulin- and contraction-stimulated glucose and LCFA uptake in the heart, especially whether certain trafficking elements are specifically involved in GLUT4 versus CD36 translocation. Therefore, we studied the role of coat proteins, actin- and microtubule-filaments and endosomal pH on glucose and LCFA uptake into primary cardiomyocytes under basal conditions and during stimulation with insulin or oligomycin (contraction-like AMP-activated protein kinase activator). Inhibition of coat protein targeting to Golgi/endosomes decreased insulin/oligomycin-stimulated glucose (-42%/-51%) and LCFA (-39%/-68%) uptake. Actin disruption decreased insulin/oligomycin-stimulated glucose uptake (-41%/-75%), while not affecting LCFA uptake. Microtubule disruption did not affect substrate uptake under any condition. Endosomal alkalinization increased basal sarcolemmal CD36 (2-fold), but not GLUT4, content, and concomitantly decreased basal intracellular membrane GLUT4 and CD36 content (-60% and -62%, respectively), indicating successful CD36 translocation and incomplete GLUT4 translocation. Additionally, endosomal alkalinization elevated basal LCFA uptake (1.4-fold) in a nonadditive manner to insulin/oligomycin, and decreased insulin/oligomycin-stimulated glucose uptake (-32%/-68%). In conclusion, 1) CD36 translocation, just like GLUT4 translocation, is a vesicle-mediated process depending on coat proteins, and 2) GLUT4 and CD36 trafficking are differentially dependent on endosomal pH and actin filaments. The latter conclusion suggests novel strategies to alter cardiac substrate preference as part of metabolic modulation therapy.

Etomoxir-induced partial carnitine palmitoyltransferase-I (CPT-I) inhibition in vivo does not alter cardiac long-chain fatty acid uptake and oxidation rates.

Although CPT-I (carnitine palmitoyltransferase-I) is generally regarded to present a major rate-controlling site in mitochondrial beta-oxidation, it is incompletely understood whether CPT-I is rate-limiting in the overall LCFA (long-chain fatty acid) flux in the heart. Another important site of regulation of the LCFA flux in the heart is trans-sarcolemmal LCFA transport facilitated by CD36 and FABPpm (plasma membrane fatty acid-binding protein). Therefore, we explored to what extent a chronic pharmacological blockade of the LCFA flux at the level of mitochondrial entry of LCFA-CoA would affect sarcolemmal LCFA uptake. Rats were injected daily with saline or etomoxir, a specific CPT-I inhibitor, for 8 days at 20 mg/kg of body mass. Etomoxir-treated rats displayed a 44% reduced cardiac CPT-I activity. Sarcolemmal contents of CD36 and FABPpm, as well as the LCFA transport capacity, were not altered in the hearts of etomoxir-treated versus control rats. Furthermore, rates of LCFA uptake and oxidation, and glucose uptake by cardiac myocytes from etomoxir-treated rats were not different from control rats, neither under basal nor under acutely induced maximal metabolic demands. Finally, hearts from etomoxir-treated rats did not display triacylglycerol accumulation. Therefore CPT-I appears not to present a major rate-controlling site in total cardiac LCFA flux. It is likely that sarcolemmal LCFA entry rather than mitochondrial LCFA-CoA entry is a promising target for normalizing LCFA flux in cardiac metabolic diseases.

Antidiabetic Effects of a Tripeptide That Decreases Abundance of Na+-d-glucose Cotransporter SGLT1 in the Brush-Border Membrane of the Small Intestine.

In enterocytes, protein RS1 (RSC1A1) mediates an increase of glucose absorption after ingestion of glucose-rich food via upregulation of Na+-d-glucose cotransporter SGLT1 in the brush-border membrane (BBM). Whereas RS1 decelerates the exocytotic pathway of vesicles containing SGLT1 at low glucose levels between meals, RS1-mediated deceleration is relieved after ingestion of glucose-rich food. Regulation of SGLT1 is mediated by RS1 domain RS1-Reg, in which Gln-Ser-Pro (QSP) is effective. In contrast to QSP and RS1-Reg, Gln-Glu-Pro (QEP) and RS1-Reg with a serine to glutamate exchange in the QSP motif downregulate the abundance of SGLT1 in the BBM at high intracellular glucose concentrations by about 50%. We investigated whether oral application of QEP improves diabetes in db/db mice and affects the induction of diabetes in New Zealand obese (NZO) mice under glucolipotoxic conditions. After 6-day administration of drinking water containing 5 mM QEP to db/db mice, fasting glucose was decreased, increase of blood glucose in the oral glucose tolerance test was blunted, and insulin sensitivity was increased. When QEP was added for several days to a high fat/high carbohydrate diet that induced diabetes in NZO mice, the increase of random plasma glucose was prevented, accompanied by lower plasma insulin levels. QEP is considered a lead compound for development of new antidiabetic drugs with more rapid cellular uptake. In contrast to SGLT1 inhibitors, QEP-based drugs may be applied in combination with insulin for the treatment of type 1 and type 2 diabetes, decreasing the required insulin amount, and thereby may reduce the risk of hypoglycemia.

Regulation of sarcolemmal glucose and fatty acid transporters in cardiac disease.

Circulating long-chain fatty acids (LCFA) and glucose are the main sources for energy production in the heart. In the healthy heart the ratio of glucose and LCFA oxidation is sensitively balanced and chronic alterations in this substrate mix are closely associated with cardiac dysfunction. While it has been accepted for several years that cardiac glucose uptake is mediated by facilitated transport, i.e. by means of the glucose transport proteins GLUT1 and GLUT4, only in the last few years it has become clear that proteins with high-affinity binding sites to LCFA, referred to as LCFA transporters, are responsible for bulk LCFA uptake. Similar to the GLUTs, the LCFA transporters CD36 and FABP(pm) can be recruited from an intracellular storage compartment to the sarcolemma to increase the rate of substrate uptake. Permanent relocation of LCFA transporters, mainly CD36, from intracellular stores to the sarcolemma is accompanied by accumulation of lipids and lipid metabolites in the heart. As a consequence, insulin signalling and glucose utilization are impaired, leading to decreased contractile activity of the heart. These observations underline the particular role and interplay of substrate carriers for glucose and LCFA in modulating cardiac metabolism, and the development of heart failure. The signalling and trafficking pathways and subcellular machinery regulating translocation of glucose and LCFA transporters are beginning to be unravelled. More knowledge on substrate transporter recycling, especially the similarities and differences between glucose and LCFA transporters, is expected to enable novel therapies aimed at changing the subcellular distribution of glucose and LCFA transporters, thereby manipulating the substrate preference of the diseased heart to help restore cardiac function.

A novel method to monitor insulin-stimulated GTP-loading of Rab11a in cardiomyocytes.

As a member of the Rab small GTPase family, Rab11a has been shown to be involved in different vesicle trafficking processes. In earlier work we identified Rab11a to be present in GLUT4-containing vesicles after insulin stimulation and showed its involvement in insulin-dependent glucose uptake. However, it remained elusive if Rab11a is directly activated by the insulin signalling cascade and at which step a potential activation occurs. To examine the GTP-loading of Rab11a, we introduced a biotinylated GTP-analog into H9c2-hIR cells, transiently overexpressing HA-tagged Rab11a, and measured its binding to the GTPase after insulin stimulation. We observed that Rab11a is transiently GTP-loaded after insulin stimulation with a 2.3 (+/-0.3) fold activation (n=5), reaching its maximum after 4 min and declining back to basal after additional 2 min. The activation of Rab11a is phosphatidylinositol 3-kinase (PI3-kinase) dependent and downstream of Akt, as shown by in vitro knockdown of this kinase. These data show that Rab11a is directly activated by insulin and represents an element of the GLUT4 trafficking machinery.

The dog that did not bark: malaria vaccines without antibodies.

To date, the only pre-blood stage vaccine to confer protection against malaria in field trials elicits both antigen-specific antibody and T-cell responses. Recent clinical trials of new heterologous prime-boost malaria vaccine regimens using DNA, fowlpox or MVA, have chiefly elicited T-cell responses that have promisingly reduced hepatic merozoites in challenge trials, but failed to protect in field trials. These encouraging results suggest further augmentation of T-cell responses to pre-blood stage antigens might one day contribute to a highly protective vaccine. We envision that a highly protective pre-erythrocytic vaccine will likely be based upon a heterologous prime-boost regimen that induces both appropriate T-cell responses as well as robust and protracted antibody production.

Elevated hepatic DPP4 activity promotes insulin resistance and non-alcoholic fatty liver disease.

OBJECTIVE: Increased hepatic expression of dipeptidyl peptidase 4 (DPP4) is associated with non-alcoholic fatty liver disease (NAFLD). Whether this is causative for the development of NAFLD is not yet clarified. Here we investigate the effect of hepatic DPP4 overexpression on the development of liver steatosis in a mouse model of diet-induced obesity. METHODS: Plasma DPP4 activity of subjects with or without NAFLD was analyzed. Wild-type (WT) and liver-specific Dpp4 transgenic mice (Dpp4-Liv-Tg) were fed a high-fat diet and characterized for body weight, body composition, hepatic fat content and insulin sensitivity. In vitro experiments on HepG2 cells and primary mouse hepatocytes were conducted to validate cell autonomous effects of DPP4 on lipid storage and insulin sensitivity. RESULTS: Subjects suffering from insulin resistance and NAFLD show an increased plasma DPP4 activity when compared to healthy controls. Analysis of Dpp4-Liv-Tg mice revealed elevated systemic DPP4 activity and diminished active GLP-1 levels. They furthermore show increased body weight, fat mass, adipose tissue inflammation, hepatic steatosis, liver damage and hypercholesterolemia. These effects were accompanied by increased expression of PPARγ and CD36 as well as severe insulin resistance in the liver. In agreement, treatment of HepG2 cells and primary hepatocytes with physiological concentrations of DPP4 resulted in impaired insulin sensitivity independent of lipid content. CONCLUSIONS: Our results give evidence that elevated expression of DPP4 in the liver promotes NAFLD and insulin resistance. This is linked to reduced levels of active GLP-1, but also to auto- and paracrine effects of DPP4 on hepatic insulin signaling.

Hepatic DPP4 DNA Methylation Associates With Fatty Liver.

Hepatic DPP4 expression is elevated in subjects with ectopic fat accumulation in the liver. However, whether increased dipeptidyl peptidase 4 (DPP4) is involved in the pathogenesis or is rather a consequence of metabolic disease is not known. We therefore studied the transcriptional regulation of hepatic Dpp4 in young mice prone to diet-induced obesity. Already at 6 weeks of age, expression of hepatic Dpp4 was increased in mice with high weight gain, independent of liver fat content. In the same animals, methylation of four intronic CpG sites was decreased, amplifying glucose-induced transcription of hepatic Dpp4 In older mice, hepatic triglyceride content was increased only in animals with elevated Dpp4 expression. Expression and release of DPP4 were markedly higher in the liver compared with adipose depots. Analysis of human liver biopsy specimens revealed a correlation of DPP4 expression and DNA methylation to stages of hepatosteatosis and nonalcoholic steatohepatitis. In summary, our results indicate a crucial role of the liver in participation to systemic DPP4 levels. Furthermore, the data show that glucose-induced expression of Dpp4 in the liver is facilitated by demethylation of the Dpp4 gene early in life. This might contribute to early deteriorations in hepatic function, which in turn result in metabolic disease such as hepatosteatosis later in life.