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Although epidemiological studies suggest a lower prostate cancer incidence rate in patients with type 2 diabetes, cancer survival is markedly reduced. Underlying mechanisms that connect the two diseases are still unclear. Potential links between type 2 diabetes and prostate cancer are hallmarks of the metabolic syndrome, such as hyperglycemia and dyslipidemia. Therefore, we explored the systemic metabolism of 103 prostate cancer patients with newly diagnosed and yet untreated prostate cancer compared to 107 healthy controls, who were carefully matched for age and BMI. Here, we report that patients with prostate cancer display higher fasting blood glucose levels and insulin resistance, without changes in insulin secretion. With respect to lipid metabolism, serum triglyceride levels were lower in patients with prostate cancer. In addition, we report increased adrenal steroid biosynthesis in these patients. Our results indicate that higher fasting glucose levels in patients with prostate cancer may be explained at least in part by insulin resistance, due to the enhanced synthesis of adrenal steroids.

Glucotoxic metabolites and pathways play a crucial role in diabetic complications, and new treatment options which improve glucotoxicity are highly warranted. In this study, we analyzed bezafibrate (BEZ) treated, streptozotocin (STZ) injected mice, which showed an improved glucose metabolism compared to untreated STZ animals. In order to identify key molecules and pathways which participate in the beneficial effects of BEZ, we studied plasma, skeletal muscle, white adipose tissue (WAT) and liver samples using non-targeted metabolomics (NMR spectroscopy), targeted metabolomics (mass spectrometry), microarrays and mitochondrial enzyme activity measurements, with a particular focus on the liver. The analysis of muscle and WAT demonstrated that STZ treatment elevated inflammatory pathways and reduced insulin signaling and lipid pathways, whereas BEZ decreased inflammatory pathways and increased insulin signaling and lipid pathways, which can partly explain the beneficial effects of BEZ on glucose metabolism. Furthermore, lysophosphatidylcholine levels were lower in the liver and skeletal muscle of STZ mice, which were reverted in BEZ-treated animals. BEZ also improved circulating and hepatic glucose levels as well as lipid profiles. In the liver, BEZ treatment reduced elevated fumarate levels in STZ mice, which was probably due to a decreased expression of urea cycle genes. Since fumarate has been shown to participate in glucotoxic pathways, our data suggests that BEZ treatment attenuates the urea cycle in the liver, decreases fumarate levels and, in turn, ameliorates glucotoxicity and reduces insulin resistance in STZ mice.

Despite it being the most common incident of cancer among men, the pathophysiological mechanisms contributing to prostate cancer (PCa) are still poorly understood. Altered mitochondrial metabolism is postulated to play a role in the development of PCa. To determine the key metabolites (which included mitochondrial oncometabolites), benign prostatic and cancer tissues of patients with PCa were analyzed using capillary electrophoresis and liquid chromatography coupled with mass spectrometry. Gene expression was studied using real-time PCR. In PCa tissues, we found reduced levels of early tricarboxylic acid cycle metabolites, whereas the contents of urea cycle metabolites including aspartate, argininosuccinate, arginine, proline, and the oncometabolite fumarate were higher than that in benign controls. Fumarate content correlated positively with the gene expression of oncogenic HIF1&alpha, and NF&kappa, B pathways, which were significantly higher in the PCa samples than in the benign controls. Furthermore, data from the TCGA database demonstrated that prostate cancer patients with activated NF&kappa, B pathway had a lower survival rate. In summary, our data showed that fumarate content was positively associated with carcinogenic genes.

Prostate cancer (PCa), the most incident cancer in men, is tightly regulated by endocrine signals. A number of different PCa cell lines are commonly used for in vitro experiments, but these are of diverse origin, and have very different cell-proliferation rates and hormone-response capacities. By analyzing the gene-expression pattern of main hormone pathways, we systematically compared six PCa cell lines and parental primary cells. We compared these cell lines (i) with each other and (ii) with PCa tissue samples from 11 patients. We found major differences in the gene-expression levels of androgen, insulin, estrogen, and oxysterol signaling between PCa tissue and cell lines, and between different cell lines. Our systematic characterization gives researchers a solid basis to choose the appropriate PCa cell model for the hormone pathway of interest.

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.

Low 25-hydroxyvitamin D levels correlate with the prevalence of diabetes; however, the mechanisms remain uncertain. Here, we show that nutritional deprivation–responsive mechanisms regulate vitamin D metabolism. Both fasting and diabetes suppressed hepatic cytochrome P450 (CYP) 2R1, the main vitamin D 25-hydroxylase responsible for the first bioactivation step. Overexpression of coactivator peroxisome proliferator–activated receptor γ coactivator 1-α (PGC-1α), induced physiologically by fasting and pathologically in diabetes, resulted in dramatic downregulation of CYP2R1 in mouse hepatocytes in an estrogen-related receptor α (ERRα)–dependent manner. However, PGC-1α knockout did not prevent fasting-induced suppression of CYP2R1 in the liver, indicating that additional factors contribute to the CYP2R1 repression. Furthermore, glucocorticoid receptor (GR) activation repressed the liver CYP2R1, suggesting GR involvement in the regulation of CYP2R1. GR antagonist mifepristone partially prevented CYP2R1 repression during fasting, suggesting that glucocorticoids and GR contribute to the CYP2R1 repression during fasting. Moreover, fasting upregulated the vitamin D catabolizing CYP24A1 in the kidney through the PGC-1α-ERRα pathway. Our study uncovers a molecular mechanism for vitamin D deficiency in diabetes and reveals a novel negative feedback mechanism that controls crosstalk between energy homeostasis and the vitamin D pathway.

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 ~2‐fold higher in the STZ arteries compared with controls. No changes in MYPT1‐T696/853 phosphorylation were observed after stimulation with the Thromboxan‐A2 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 μmol/L) or U46619 (1 μmol/L) was augmented in the arteries of STZ mice by ~2‐ and ~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.

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.

Background: Nonalcoholic fatty liver disease and hepatic insulin resistance precede diabetes and may further develop into nonalcoholic steatohepatitis and liver fibrosis/cirrhosis. The activation of hepatic stellate cells (HSC), which is characterized by the loss of retinyl-ester stores, plays a crucial role in the pathophysiology of this process. Since cGMP-dependent protein kinase I (cGKI) deficient (cGKI-KO) mice displayed hepatic insulin resistance and disturbed glucose metabolism, as well as, cGKI is only detected in HSC but not in hepatocytes, we hypothesized that cGKI modulates HSC activation and its metabolic consequences. Methods: First, retinol storage and gene expression were studied in cGKI-KO mice. Second, we investigated the effects of cGKI-silencing on gene expression and cellular activation in the human stellate cell line LX2. Finally, cGKI expression was investigated in human liver biopsies covering a wide range of liver fat content. Results: Retinyl-ester level in the liver of cGKI-KO mice was lower compared to wild type animals, which was associated with increased inflammatory gene expression. Activation of LX2 cells showed increased α-smooth muscle actin and matrix metalloproteinase 2 expression, which was augmented by silencing cGKI. On the other hand, activation of LX2 cells by TGFβ suppressed cGKI expression. Furthermore, we detected a negative correlation between cGKI mRNA and liver fat content in human biopsies. Conclusions: The lower mRNA level of cGKI either in TGFβ activated LX2 cells or in liver biopsies with high fat content suggest that cGKI function might be impaired during HSC activation. Furthermore, the higher expression of inflammatory genes and low retinyl-ester levels in cGKI-KO mice as well as the higher levels of activation markers in cGKI-silenced LX2 cells indicate that cGKI inhibits HSC activation. Since cGKI is a downstream target of nitric oxide (NO), it is conceivable that NO may exert protective effects inhibiting liver fibrosis via cGKI. Disclosure A. Franko: None. M. Kovarova: None. R. Wagner: None. C. Weigert: None. R. Feil: None. S. Feil: None. H. Haering: None. S.Z. Lutz: None. A. Peter: None.

In diabetes, hyperamylinemia contributes to cardiac dysfunction. The interplay between hIAPP, blood glucose and other plasma components is, however, not understood. We show that glucose and LDL interact with hIAPP, resulting in β-sheet rich oligomers with increased β-cell toxicity and hemolytic activity, providing mechanistic insights for a direct link between diabetes and cardiovascular diseases.

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.

Background The activation of hepatic stellate cells (HSCs) plays a crucial role in liver fibrosis, however the role of HSCs is less understood in hepatic insulin resistance. Since in the liver cGMP-dependent protein kinase I (cGKI) was detected in HSC but not in hepatocytes, and cGKI-deficient mice that express cGKI selectively in smooth muscle but not in other cell types (cGKI-SM mice) displayed hepatic insulin resistance, we hypothesized that cGKI modulates HSC activation and insulin sensitivity. Materials and Methods To study stellate cell activation in cGKI-SM mice, retinol storage and gene expression were studied. Moreover, in the human stellate cell line LX2, the consequences of cGKI-silencing on gene expression were investigated. Finally, cGKI expression was examined in human liver biopsies covering a wide range of liver fat content. Results Retinyl-ester concentrations in the liver of cGKI-SM mice were lower compared to wild-type animals, which was associated with disturbed expression of genes involved in retinol metabolism and inflammation. cGKI-silenced LX2 cells showed an mRNA expression profile of stellate cell activation, altered matrix degradation and activated chemokine expression. On the other hand, activation of LX2 cells suppressed cGKI expression. In accordance with this finding, in human liver biopsies, we observed a negative correlation between cGKI mRNA and liver fat content. Conclusions These results suggest that the lack of cGKI possibly leads to stellate cell activation, which stimulates chemokine expression and activates inflammatory processes, which could disturb hepatic insulin sensitivity.

Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in β-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.

BACKGROUND Recent data suggest that insulin-like growth factor (IGF)-1 resistance in neurons prolongs longevity. In C. elegans this effect is mediated via DAF-16 the ortholog of the mammalian FoxO transcription factors. 3 different FoxO transcription factors (FoxOs) are expressed in rodent CNS: FoxO1, FoxO3a and FoxO6. METHODS To define whether the different FoxOs are region-, sex- and age-specifically expressed, we analyzed FoxO mRNA levels in different brain regions from 6, 16, 60 and 100 weeks old mice using realtime-PCR. In addition, we fed mice a high fat diet (HFD) to experimentally induce obesity and diabetes and analyzed FoxO mRNA in the different brain regions. RESULTS Interestingly, FoxO1 was predominantly expressed in the hippocampus whereas FoxO3a was quantitatively the most abundant FoxO in the neocortex. During aging, FoxO1 expression peaked in all brain regions at 16 weeks and FoxO6 showed its highest expression at 60 weeks in the parietal and occipital cortex. In 6 weeks old mice FoxO6 expression was higher in male compared to female mice in the hippocampus and all cortical regions. Surprisingly, in HFD animals FoxO3a was significantly less expressed in the cerebellum and all cortical regions compared to control animals. Even more dramatic, FoxO6 expression dropped about 80% in all brain regions in response to HFD. CONCLUSION Thus, FoxOs in the CNS showed a highly distinct expression, which in addition was age- and sex-dependent. In contrast to FoxO1, FoxO3a and FoxO6 were specifically diminished in the CNS of HFD animals possibly contributing to the reduced lifespan observed in these animals.

To identify common regulatory features of nuclear genes encoding mitochondrial proteins we searched for regulatory elements in the Cytochrome c promoter during skeletal muscle differentiation in cell culture. A consensus element with the sequence GCTGCCGCAC-(N4-20)-GGSCGYGGG was found in both rat Cyt c and coxIV promoters. This new sequence element with yet undescribed function, but high abundance in promoters of nuclear genes encoding mitochondrial proteins available from the databases, showed a striking change in protein binding in electromobility shift assays when myoblasts were compared to myotubes. Proteins involved in the observed protein-DNA complexes were isolated from myotubes and identified by MALDI-TOF as BAT1, a DEAD-box protein of yet unknown function, heat shock protein HSP84, and alpha-actinin 4, a non-muscle isoform of the structural protein alpha-actinin. alpha-actinin 4 was found to be preferentially localized in the nucleus upon induction of myogenesis, suggesting a signaling function during muscle differentiation. In conclusion, the analyzed sequence motif may be a new candidate for common regulatory elements specific for nuclear encoded mitochondrial genes, and alpha-actinin 4 may be involved in their regulation.

Background & Aims To determine if diabetic and insulin-resistant states cause mitochondrial dysfunction in liver or if there is long term adaptation of mitochondrial function to these states, mice were (i) fed with a high-fat diet to induce obesity and T2D (HFD), (ii) had a genetic defect in insulin signaling causing whole body insulin resistance, but not full blown T2D ( IR / IRS-1 +/− mice), or (iii) were analyzed after treatment with streptozocin (STZ) to induce a T1D-like state. Methods Hepatic lipid levels were measured by thin layer chromatography. Mitochondrial respiratory chain (RC) levels and function were determined by Western blot, spectrophotometric, oxygen consumption and proton motive force analysis. Gene expression was analyzed by real-time PCR and microarray. Results HFD caused insulin resistance and hepatic lipid accumulation, but RC was largely unchanged. Livers from insulin resistant IR / IRS-1 +/− mice had normal lipid contents and a normal RC, but mitochondria were less well coupled. Livers from severely hyperglycemic and hypoinsulinemic STZ mice had massively depleted lipid levels, but RC abundance was unchanged. However, liver mitochondria isolated from these animals showed increased abundance and activity of the RC, which was better coupled. Conclusions Insulin resistance, induced either by obesity or genetic manipulation and steatosis do not cause mitochondrial dysfunction in mouse liver. Also, mitochondrial dysfunction is not a prerequisite for liver steatosis. However, severe insulin deficiency and high blood glucose levels lead to an enhanced performance and better coupling of the RC. This may represent an adaptation to fuel overload and the high energy-requirement of an unsuppressed gluconeogenesis.

The mouse is widely considered as a toolbox for modeling human diseases: mice are easy to handle and breed, there exist inbred strains, and the mouse genome sequence is available. Mutant mouse lines can be generated by different technologies, and standardized phenotyping of these mutant mouse lines produces a huge amount of valuable data. Useful resources for the scientific community are archives of mutant lines and strains as well as databases delivering information about the mouse lines and their availability. The phenotypic characterization of mutant mouse lines is the bottleneck within the pipeline from the generation via phenotyping to archiving of mutant mouse lines. Mouse clinics generate large data sets by the standardized, comprehensive phenotypic characterization of mutant mouse lines. There is a portfolio of phenotyping protocols available for a broad spectrum of disease areas that is considered as an international standard. For the investigation of human diseases like diabetes, obesity or the metabolic syndrome, metabolic tests to analyze mutant mouse lines become increasingly important. In this respect, challenge experiments have become the major focus to induce disease phenotypes in mutant mice that would remain undiscovered without the environmental challenges. These experimental setups reflect human conditions, where genetic predisposition and the environmental factors originating from different life style act together and enhance each other.

The role of mitochondrial dysfunction in the development of insulin resistance and type 2 diabetes remains controversial. In order to specifically define the relationship between insulin receptor (InsR) signaling, insulin resistance, hyperglycemia, hyperlipidemia and mitochondrial function, we analyzed mitochondrial performance of insulin-sensitive, slow-oxidative muscle in four different mouse models. In obese but normoglycemic ob/ob mice as well as in obese but diabetic mice under high-fat diet, mitochondrial performance remained unchanged even though intramyocellular diacylglycerols (DAGs), triacylglycerols (TAGs), and ceramides accumulated. In contrast, in muscle-specific InsR knockout (MIRKO) and streptozotocin (STZ)-treated hypoinsulinemic, hyperglycemic mice, levels of mitochondrial respiratory chain complexes and mitochondrial function were markedly reduced. In STZ, but not in MIRKO mice, this was caused by reduced transcription of mitochondrial genes mediated via decreased PGC-1α expression. We conclude that mitochondrial dysfunction is not causally involved in the pathogenesis of obesity-associated insulin resistance under normoglycemic conditions. However, obesity-associated type 2 diabetes and accumulation of DAGs or TAGs is not associated with impaired mitochondrial function. In contrast, chronic hypoinsulinemia and hyperglycemia as seen in STZ-treated mice as well as InsR deficiency in muscle of MIRKO mice lead to mitochondrial dysfunction. We postulate that decreased mitochondrial mass and/or performance in skeletal muscle of non-diabetic, obese or type 2 diabetic, obese patients observed in clinical studies must be explained by genetic predisposition, physical inactivity, or other still unknown factors.

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-I) 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. Copyright © 2008, American Society for Microbiology. All Rights Reserved.