Epigallocatechin gallate (EGCG) reduces the intensity of pancreatic amyloid fibrils in human islet amyloid polypeptide (hIAPP) transgenic mice

The formation of amyloid fibrils by human islet amyloid polypeptide protein (hIAPP) has been implicated in pancreas dysfunction and diabetes. However, efficient treatment options to reduce amyloid fibrils in vivo are still lacking. Therefore, we tested the effect of epigallocatechin gallate (EGCG) on fibril formation in vitro and in vivo. To determine the binding of hIAPP and EGCG, in vitro interaction studies were performed. To inhibit amyloid plaque formation in vivo, homozygous (tg/tg), hemizygous (wt/tg), and control mice (wt/wt) were treated with EGCG. EGCG bound to hIAPP in vitro and induced formation of amorphous aggregates instead of amyloid fibrils. Amyloid fibrils were detected in the pancreatic islets of tg/tg mice, which was associated with disrupted islet structure and diabetes. Although pancreatic amyloid fibrils could be detected in wt/tg mice, these animals were non-diabetic. EGCG application decreased amyloid fibril intensity in wt/tg mice, however it was ineffective in tg/tg animals. Our data indicate that EGCG inhibits amyloid fibril formation in vitro and reduces fibril intensity in non-diabetic wt/tg mice. These results demonstrate a possible in vivo effectiveness of EGCG on amyloid formation and suggest an early therapeutical application

Identification of the Secreted Proteins Originated from Primary Human Hepatocytes and HepG2 Cells

The liver plays a pivotal role in whole-body carbohydrate, lipid, and protein metabolism. One of the key regulators of glucose and lipid metabolism are hepatokines, which are found among the liver secreted proteins, defined as liver secretome. To elucidate the composition of the human liver secretome and identify hepatokines in primary human hepatocytes (PHH), we conducted comprehensive protein profiling on conditioned medium (CM) of PHH. Secretome profiling using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) identified 691 potential hepatokines in PHH. Subsequently, pathway analysis assigned these proteins to acute phase response, coagulation, and complement system pathways. The secretome of PHH was compared to the secreted proteins of the liver hepatoma cell line HepG2. Although the secretome of PHH and HepG2 cells show a high overlap, the HepG2 secretome rather mirrors the fetal liver with some cancer characteristics. Collectively, our study represents one of the most comprehensive secretome profiling approaches for PHH, allowing new insights into the composition of the secretome derived from primary human material, and points out strength and weakness of using HepG2 cell secretome as a model for the analysis of the human liver secretome

Augmented contractility of murine femoral arteries in a streptozotocin diabetes model is related to increased phosphorylation of MYPT1

Diabetes mellitus (DM) is a metabolic disorder with high prevalence, and a major risk factor for macro- and microvascular abnormalities. This study was undertaken to explore the mechanisms of hypercontractility of murine femoral arteries (FA) obtained from mice with streptozotocin (STZ)-induced diabetes and its relation to the phosphorylation profile of the myosin phosphatase target subunit 1, MYPT1. The immunoreactivity of MYPT1 toward phospho-MYPT1-T696, MYPT1-T853, or MYPT1-S695, used as a read out for MYPT1 phosphorylation, has been studied by Western Blotting. Contractile activity of FA from control and STZ mice has been studied by wire myography. At basal conditions (no treatment), the immunoreactivity of MYPT1-T696/T853 was similar to 2-fold higher in the STZ arteries compared with controls. No changes in MYPT1-T696/853 phosphorylation were observed after stimulation with the Thromboxan-A(2) analog, U46619. Neither basal nor U46619-stimulated phosphorylation of MYPT1 at S695 was affected by STZ treatment. Mechanical distensibility and basal tone of FA obtained from STZ animals were similar to controls. Maximal force after treatment of FA with the contractile agonists phenylephrine (10 mu mol/L) or U46619 (1 mu mol/L) was augmented in the arteries of STZ mice by similar to 2- and similar to 1.5-fold, respectively. In summary, our study suggests that development of a hypercontractile phenotype in murine FA in STZ diabetes is at least partially related to an increase in phosphorylation of MLCP at MYPT1-T696/853. Interestingly, the phosphorylation at S695 site was not altered in STZ-induced diabetes, supporting the view that S695 may serve as a sensor for mechanical activity which is not directly involved in tone regulation

Impaired Insulin Signaling is Associated with Hepatic Mitochondrial Dysfunction in IR+/--IRS-1(+/-) Double Heterozygous (IR-IRS1dh) Mice

Mitochondria play a pivotal role in energy metabolism, but whether insulin signaling per se could regulate mitochondrial function has not been identified yet. To investigate whether mitochondrial function is regulated by insulin signaling, we analyzed muscle and liver of insulin receptor (IR)(+/-)-insulin receptor substrate-1 (IRS-1)(+/-) double heterozygous (IR-IRS1dh) mice, a well described model for insulin resistance. IR-IRS1dh mice were studied at the age of 6 and 12 months and glucose metabolism was determined by glucose and insulin tolerance tests. Mitochondrial enzyme activities, oxygen consumption, and membrane potential were assessed using spectrophotometric, respirometric, and proton motive force analysis, respectively. IR-IRS1dh mice showed elevated serum insulin levels. Hepatic mitochondrial oxygen consumption was reduced in IR-IRS1dh animals at 12 months of age. Furthermore, 6-month-old IR-IRS1dh mice demonstrated enhanced mitochondrial respiration in skeletal muscle, but a tendency of impaired glucose tolerance. On the other hand, 12-month-old IR-IRS1dh mice showed improved glucose tolerance, but normal muscle mitochondrial function. Our data revealed that deficiency in IR/IRS-1 resulted in normal or even elevated skeletal muscle, but impaired hepatic mitochondrial function, suggesting a direct cross-talk between insulin signaling and mitochondria in the liver

CREB-1α Is Recruited to and Mediates Upregulation of the Cytochrome c Promoter during Enhanced Mitochondrial Biogenesis Accompanying Skeletal Muscle Differentiation▿ †

To further understand pathways coordinating the expression of nuclear genes encoding mitochondrial proteins, we studied mitochondrial biogenesis during differentiation of myoblasts to myotubes. This energy-demanding process was accompanied by a fivefold increase of ATP turnover, covered by an eightfold increase of mitochondrial activity. While no change in mitochondrial DNA copy number was observed, mRNAs as well as proteins for nucleus-encoded cytochrome c, cytochrome c oxidase subunit IV, and mitochondrial transcription factor A (TFAM) increased, together with total cellular RNA and protein levels. Detailed analysis of the cytochrome c promoter by luciferase reporter, binding affinity, and electrophoretic mobility shift assays as well as mutagenesis studies revealed a critical role for cyclic AMP responsive element binding protein 1 (CREB-1) for promoter activation. Expression of two CREB-1 isoforms was observed by using specific antibodies and quantitative reverse transcription-PCR, and a shift from phosphorylated CREB-1Δ in myoblasts to phosphorylated CREB-1α protein in myotubes was shown, while mRNA ratios remained unchanged. Chromatin immunoprecipitation assays confirmed preferential binding of CREB-1α in situ to the cytochrome c promoter in myotubes. Overexpression of constitutively active and dominant-negative forms supported the key role of CREB-1 in regulating the expression of genes encoding mitochondrial proteins during myogenesis and probably also in other situations of enhanced mitochondrial biogenesis

Dissociation of Fatty Liver and Insulin Resistance in I148M PNPLA3 Carriers: Differences in Diacylglycerol (DAG) FA18:1 Lipid Species as a Possible Explanation

Fatty liver is tightly associated with insulin resistance and the development of type 2 diabetes. I148M variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene is associated with high liver fat but normal insulin sensitivity. The underlying mechanism of the disassociation between high liver fat but normal insulin sensitivity remains obscure. We investigated the effect of I148M variant on hepatic lipidome of subjects with or without fatty liver, using the Lipidyzer method. Liver samples of four groups of subjects consisting of normal liver fat with wild-type PNPLA3 allele (group 1); normal liver fat with variant PNPLA3 allele (group 2); high liver fat with wild-type PNPLA3 allele (group 3); high liver fat with variant PNPLA3 allele (group 4); were analyzed. When high liver fat to normal liver fat groups were compared, wild-type carriers (group 3 vs. group 1) showed similar lipid changes compared to I148M PNPLA3 carriers (group 4 vs. group 2). On the other hand, in wild-type carriers, increased liver fat significantly elevated the proportion of specific DAGs (diacylglycerols), mostly DAG (FA18:1) which, however, remained unchanged in I148M PNPLA3 carriers. Since DAG (FA18:1) has been implicated in hepatic insulin resistance, the unaltered proportion of DAG (FA18:1) in I148M PNPLA3 carriers with fatty liver may explain the normal insulin sensitivity in these subjects

Inceptor correlates with markers of prostate cancer progression and modulates insulin/IGF1 signaling and cancer cell migration

Objective: The insulin/insulin-like growth factor 1 (IGF1) pathway is emerging as a crucial component of prostate cancer progression. Therefore, we investigated the role of the novel insulin/IGF1 signaling modulator inceptor in prostate cancer. Methods: We analyzed the expression of inceptor in human samples of benign prostate epithelium and prostate cancer. Further, we performed signaling and functional assays using prostate cancer cell lines. Results: We found that inceptor was expressed in human benign and malignant prostate tissue and its expression positively correlated with various genes of interest, including genes involved in androgen signaling. In vitro, total levels of inceptor were increased upon androgen deprivation and correlated with high levels of androgen receptor in the nucleus. Inceptor overexpression was associated with increased cell migration, altered IGF1R trafficking and higher IGF1R activation. Conclusions: Our in vitro results showed that inceptor expression was associated with androgen status, increased migration, and IGF1R signaling. In human samples, inceptor expression was significantly correlated with markers of prostate cancer progression. Taken together, these data provide a basis for investigation of inceptor in the context of prostate cancer

Transcript Levels of Aldo-Keto Reductase Family 1 Subfamily C (AKR1C) Are Increased in Prostate Tissue of Patients with Type 2 Diabetes

Aldo-keto reductase family 1 (AKR1) enzymes play a crucial role in diabetic complications. Since type 2 diabetes (T2D) is associated with cancer progression, we investigated the impact of diabetes on AKR1 gene expression in the context of prostate cancer (PCa) development. In this study, we analyzed benign (BEN) prostate and PCa tissue of patients with and without T2D. Furthermore, to replicate hyperglycemia in vitro, we treated the prostate adenocarcinoma cell line PC3 with increasing glucose concentrations. Gene expression was quantified using real-time qPCR. In the prostate tissue of patients with T2D, AKR1C1 and AKR1C2 transcripts were higher compared to samples of patients without diabetes. In PC3 cells, high glucose treatment induced the gene expression levels of AKR1C1, C2, and C3. Furthermore, both in human tissue and in PC3 cells, the transcript levels of AKR1C1, C2, and C3 showed positive associations with oncogenes, which are involved in proliferation processes and HIF1α and NFκB pathways. These results indicate that in the prostate glands of patients with T2D, hyperglycemia could play a pivotal role by inducing the expression of AKR1C1, C2, and C3. The higher transcript level of AKR1C was furthermore associated with upregulated HIF1α and NFκB pathways, which are major drivers of PCa carcinogenesis

Increased Expressions of Matrix Metalloproteinases (MMPs) in Prostate Cancer Tissues of Men with Type 2 Diabetes

Type 2 diabetes (T2D) is associated with worse prognosis of prostate cancer (PCa). The molecular mechanisms behind this association are still not fully understood. The aim of this study was to identify key factors, which contribute to the more aggressive PCa phenotype in patients with concurrent T2D. Therefore, we investigated benign and PCa tissue of PCa patients with and without diabetes using real time qPCR. Compared to patients without diabetes, patients with T2D showed a decreased E-cadherin/N-cadherin (CDH1/CDH2) ratio in prostate tissue, indicating a switch of epithelial-mesenchymal transition (EMT), which is a pivotal process in carcinogenesis. In addition, the gene expression levels of matrix metalloproteinases (MMPs) and CC chemokine ligands (CCLs) were higher in prostate samples of T2D patients. Next, prostate adenocarcinoma PC3 cells were treated with increasing glucose concentrations to replicate hyperglycemia in vitro. In these cells, high glucose induced expressions of MMPs and CCLs, which showed significant positive associations with the proliferation marker proliferating cell nuclear antigen (PCNA). These results indicate that in prostate tissue of men with T2D, hyperglycemia may induce EMT, increase MMP and CCL gene expressions, which in turn activate invasion and inflammatory processes accelerating the progression of PCa

Efficient Isolation of Pure and Functional Mitochondria from Mouse Tissues Using Automated Tissue Disruption and Enrichment with Anti-TOM22 Magnetic Beads

To better understand molecular mechanisms regulating changes in metabolism, as observed e. g. in diabetes or neuronal disorders, the function of mitochondria needs to be precisely determined. The usual isolation methods such as differential centrifugation result in isolates of highly variable quality and quantity. To fulfill the need of a reproducible isolation method from solid tissues, which is suitable to handle parallel samples simultaneously, we developed a protocol based on anti-TOM22 (translocase of outer mitochondrial membrane 22 homolog) antibody-coupled magnetic beads. To measure oxygen consumption rate in isolated mitochondria from various mouse tissues, a traditional Clark electrode and the high-throughput XF Extracellular Flux Analyzer were used. Furthermore, Western blots, transmission electron microscopic and proteomic studies were performed to analyze the purity and integrity of the mitochondrial preparations. Mitochondrial fractions isolated from liver, brain and skeletal muscle by anti-TOM22 magnetic beads showed oxygen consumption capacities comparable to previously reported values and little contamination with other organelles. The purity and quality of isolated mitochondria using anti-TOM22 magnetic beads was compared to traditional differential centrifugation protocol in liver and the results indicated an obvious advantage of the magnetic beads method compared to the traditional differential centrifugation technique