Acute retinol mobilization by retinol-binding protein 4 in mouse liver induces fibroblast growth factor 21 expression.

Hepatocytes secrete retinol-binding protein 4 (RBP4) into circulation, thereby mobilizing vitamin A from the liver to provide retinol for extrahepatic tissues. Obesity and insulin resistance are associated with elevated RBP4 levels in the blood. However, in a previous study, we observed that chronically increased RBP4 by forced Rbp4 expression in the liver does not impair glucose homeostasis in mice. Here, we investigated the effects of an acute mobilization of hepatic vitamin A stores by hepatic overexpression of RBP4 in mice. We show that hepatic retinol mobilization decreases body fat content and enhances fat turnover. Mechanistically, we found that acute retinol mobilization increases hepatic expression and serum levels of fibroblast growth factor 21 (FGF21), which is regulated by retinol mobilization and retinoic acid in primary hepatocytes. Moreover, we provide evidence that the insulin-sensitizing effect of FGF21 is associated with organ-specific adaptations in retinoid homeostasis. Taken together, our findings identify a novel crosstalk between retinoid homeostasis and FGF21 in mice with acute RBP4-mediated retinol mobilization from the liver.

Tissue-specific signatures of metabolites and proteins in asparagus roots and exudates.

Comprehensive untargeted and targeted analysis of root exudate composition has advanced our understanding of rhizosphere processes. However, little is known about exudate spatial distribution and regulation. We studied the specific metabolite signatures of asparagus root exudates, root outer (epidermis and exodermis), and root inner tissues (cortex and vasculature). The greatest differences were found between exudates and root tissues. In total, 263 non-redundant metabolites were identified as significantly differentially abundant between the three root fractions, with the majority being enriched in the root exudate and/or outer tissue and annotated as 'lipids and lipid-like molecules' or 'phenylpropanoids and polyketides'. Spatial distribution was verified for three selected compounds using MALDI-TOF mass spectrometry imaging. Tissue-specific proteome analysis related root tissue-specific metabolite distributions and rhizodeposition with underlying biosynthetic pathways and transport mechanisms. The proteomes of root outer and inner tissues were spatially very distinct, in agreement with the fundamental differences between their functions and structures. According to KEGG pathway analysis, the outer tissue proteome was characterized by a high abundance of proteins related to 'lipid metabolism', 'biosynthesis of other secondary metabolites' and 'transport and catabolism', reflecting its main functions of providing a hydrophobic barrier, secreting secondary metabolites, and mediating water and nutrient uptake. Proteins more abundant in the inner tissue related to 'transcription', 'translation' and 'folding, sorting and degradation', in accord with the high activity of cortical and vasculature cell layers in growth- and development-related processes. In summary, asparagus root fractions accumulate specific metabolites. This expands our knowledge of tissue-specific plant cell function.

SNP rs6564851 in the BCO1 Gene Is Associated with Varying Provitamin a Plasma Concentrations but Not with Retinol Concentrations among Adolescents from Rural Ghana.

In sub-Saharan Africa, vitamin A deficiency constitutes a severe health problem despite various supplementation and food fortification programs. Given that the intake of preformed vitamin A from animal products remains low in these countries, an efficient metabolization of plant-based provitamin A carotenoids is essential. Previously, adolescents in rural Ghana have shown high total plasma carotenoid concentrations, while 36% had a vitamin A deficiency (defined as plasma retinol < 0.7 µmol/L). Hence, the aim of this cross-sectional study was to identify the relationships between variants in the ß-carotene 15,15'-oxygenase (BCO1) gene and plasma carotenoid concentrations among 189 15-year-old girls and boys in rural Ghana. BCO1 rs6564851, rs7500996, rs10048138 and PKD1L2 rs6420424, and rs8044334 were typed, and carotenoid concentrations were compared among the different genotypes. G allele carriers of rs6564851 (53%) showed higher plasma carotenoid concentrations than T allele carriers (median (interquartile range): 3.07 (2.17-4.02) vs. 2.59 (2.21-3.50) µmol/L, p-value = 0.0424). This was not explained by differences in socio-demographic or dietary factors. In contrast, no differences in plasma retinol concentrations were observed between these genotypes. Pending verification in independent populations, the low conversion efficiency of provitamin A carotenoids among rs6564851 G allele carriers may undermine existing fortification and supplementation programs to improve the vitamin A status in sub-Saharan Africa.

Metabolic footprint and intestinal microbial changes in response to dietary proteins in a pig model.

Epidemiological studies revealed that dietary proteins can contribute to the modulation of the cardiovascular disease risk. Still, direct effects of dietary proteins on serum metabolites and other health-modulating factors have not been fully explored. Here, we compared the effects of dietary lupin protein with the effects of beef protein and casein on the serum metabolite profile, cardiovascular risk markers and the fecal microbiome. Pigs were fed diets containing 15% of the respective proteins for 4 weeks. A classification analysis of the serum metabolites revealed six biomarker sets of two metabolites each that discriminated between the intake of lupin protein, lean beef or casein. These biomarker sets included 1- and 3-methylhistidine, betaine, carnitine, homoarginine and methionine. The study revealed differences in the serum levels of the metabolites 1- and 3- methylhistidine, homoarginine, methionine and homocysteine, which are involved in the one-carbon cycle. However, these changes were not associated with differences in the methylation capacity or the histone methylation pattern. With the exception of serum homocysteine and homoarginine levels, other cardiovascular risk markers, such as the homeostatic model assessment index, trimethylamine-N-oxide and lipids, were not influenced by the dietary protein source. However, the composition of the fecal microorganisms was markedly changed by the dietary protein source. Lupin-protein-fed pigs exhibited more species from the phyla Bacteroidetes and Firmicutes than the other two groups. In conclusion, different dietary protein sources induce distinct serum metabolic fingerprints, have an impact on the cardiovascular risk and modulate the composition of the fecal microbiome.

Liver-secreted RBP4 does not impair glucose homeostasis in mice.

Retinol-binding protein 4 (RBP4) is the major transport protein for retinol in blood. Recent evidence from genetic mouse models shows that circulating RBP4 derives exclusively from hepatocytes. Because RBP4 is elevated in obesity and associates with the development of glucose intolerance and insulin resistance, we tested whether a liver-specific overexpression of RBP4 in mice impairs glucose homeostasis. We used adeno-associated viruses (AAV) that contain a highly liver-specific promoter to drive expression of murine RBP4 in livers of adult mice. The resulting increase in serum RBP4 levels in these mice was comparable with elevated levels that were reported in obesity. Surprisingly, we found that increasing circulating RBP4 had no effect on glucose homeostasis. Also during a high-fat diet challenge, elevated levels of RBP4 in the circulation failed to aggravate the worsening of systemic parameters of glucose and energy homeostasis. These findings show that liver-secreted RBP4 does not impair glucose homeostasis. We conclude that a modest increase of its circulating levels in mice, as observed in the obese, insulin-resistant state, is unlikely to be a causative factor for impaired glucose homeostasis.

Determination of lipid profiles in serum of obese ponies before and after weight reduction by using multi-one-dimensional thin-layer chromatography.

Obesity is a key component of equine metabolic syndrome, which is highly associated with laminitis. Feed restriction and/or exercise are known to alleviate the detrimental effects of insulin resistance in obese ponies. However, little is known about changes in the serum lipid patterns due to weight reduction and its association with disease outcomes. Therefore, the lipid patterns in the serum of 14 mature ponies before and after a 14-week body weight reduction program (BWRP) were investigated by multi-one-dimensional thin-layer chromatography (MOD-TLC). Additionally, sensitivity to insulin (SI), body condition scores (BCS) and cresty neck scores (CNS) were measured. A BWRP resulted in a significant loss of body weight (P<0.001), which was associated with beneficial decreases in BCS and CNS (both, P<0.001). Serum lipid compositions revealed significantly increased free fatty acid (FFA), sphingomyelin (SM; both P<0.001), total cholesterol (C) and cholesterol ester (CE) (both P<0.01) and triacylglycerol (TG; P<0.05) densities. Improvement of SI after the BWRP was associated with increases in neutral lipids (C, CE and TG, all P<0.01), FFA and the phospholipid SM (both, P<0.001). The results show that a BWRP in obese ponies was effective and associated with changes in the concentrations of neutral lipids and the phospholipid SM, indicating that SM may play a role in insulin signaling pathways and thus in the pathogenesis of insulin resistance and the progression of metabolic syndrome in obese ponies.

Adolescent health in rural Ghana: A cross-sectional study on the co-occurrence of infectious diseases, malnutrition and cardio-metabolic risk factors.

In sub-Saharan Africa, infectious diseases and malnutrition constitute the main health problems in children, while adolescents and adults are increasingly facing cardio-metabolic conditions. Among adolescents as the largest population group in this region, we investigated the co-occurrence of infectious diseases, malnutrition and cardio-metabolic risk factors (CRFs), and evaluated demographic, socio-economic and medical risk factors for these entities. In a cross-sectional study among 188 adolescents in rural Ghana, malarial infection, common infectious diseases and Body Mass Index were assessed. We measured ferritin, C-reactive protein, retinol, fasting glucose and blood pressure. Socio-demographic data were documented. We analyzed the proportions (95% confidence interval, CI) and the co-occurrence of infectious diseases (malaria, other common diseases), malnutrition (underweight, stunting, iron deficiency, vitamin A deficiency [VAD]), and CRFs (overweight, obesity, impaired fasting glucose, hypertension). In logistic regression, odds ratios (OR) and 95% CIs were calculated for the associations with socio-demographic factors. In this Ghanaian population (age range, 14.4-15.5 years; males, 50%), the proportions were for infectious diseases 45% (95% CI: 38-52%), for malnutrition 50% (43-57%) and for CRFs 16% (11-21%). Infectious diseases and malnutrition frequently co-existed (28%; 21-34%). Specifically, VAD increased the odds of non-malarial infectious diseases 3-fold (95% CI: 1.03, 10.19). Overlap of CRFs with infectious diseases (6%; 2-9%) or with malnutrition (7%; 3-11%) was also present. Male gender and low socio-economic status increased the odds of infectious diseases and malnutrition, respectively. Malarial infection, chronic malnutrition and VAD remain the predominant health problems among these Ghanaian adolescents. Investigating the relationships with evolving CRFs is warranted.

First trimester TTR-RBP4-ROH complex and angiogenic factors in the prediction of small for gestational age infant's outcome.

PURPOSE: To study the role of the TTR-RBP4-ROH complex components (transthyretin, serum retinol binding protein, retinol) and of angiogenic factors PlGF (placental growth factor) and sFlt-1 (soluble fms-like tyrosine kinase-1) in pregnancies complicated by small for gestational age infants (SGA). METHODS: Case control study conducted on maternal serum collected between 11 + 0 to 13 + 6 weeks of gestation. TTR, RBP4, ROH, PlGF and sFlt-1 were measured in SGA patients (birth weight <10%) who delivered at term (n = 37) and before 37 weeks of gestation (n = 17) and in a matched control group with uneventful pregnancies (n = 37). RESULTS: We found decreased RBP4 in SGA patients that delivered fetuses <3% and in fetuses delivered after the 37 weeks of gestation compared to controls [1.50 (95% CI 1.40-1.75) vs 1.62 (95% CI 1.47-1.98), p < 0.05]. Further, we found lower PlGF and sFlt-1 concentrations in SGA that delivered before 37 weeks of gestation compared to controls (respectively, PIGF and sFlt-1: 39.7 pg/ml (95% CI 32.3-66.3) vs 62.9 pg/ml (95% CI 45.2-78.4) and 906 pg/ml (95% CI 727-1626) vs 1610 pg/ml (95% CI 1088-212), p < 0.05). CONCLUSIONS: First trimester maternal serum RBP4 and angiogenic factors PlGF and sFlt-1 can differently predict the timing of delivery of pregnancies complicated by SGA fetuses.

Caenorhabditis elegans as a model system to study post-translational modifications of human transthyretin.

The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.

The molecular chaperone Hsp70 promotes the proteolytic removal of oxidatively damaged proteins by the proteasome.

One hallmark of aging is the accumulation of protein aggregates, promoted by the unfolding of oxidized proteins. Unraveling the mechanism by which oxidized proteins are degraded may provide a basis to delay the early onset of features, such as protein aggregate formation, that contribute to the aging phenotype. In order to prevent aggregation of oxidized proteins, cells recur to the 20S proteasome, an efficient turnover proteolysis complex. It has previously been shown that upon oxidative stress the 26S proteasome, another form, dissociates into the 20S form. A critical player implicated in its dissociation is the Heat Shock Protein 70 (Hsp70), which promotes an increase in free 20S proteasome and, therefore, an increased capability to degrade oxidized proteins. The aim of this study was to test whether or not Hsp70 is involved in cooperating with the 20S proteasome for a selective degradation of oxidatively damaged proteins. Our results demonstrate that Hsp70 expression is induced in HT22 cells as a result of mild oxidative stress conditions. Furthermore, Hsp70 prevents the accumulation of oxidized proteins and directly promotes their degradation by the 20S proteasome. In contrast the expression of the Heat shock cognate protein 70 (Hsc70) was not changed in recovery after oxidative stress and Hsc70 has no influence on the removal of oxidatively damaged proteins. We were able to demonstrate in HT22 cells, in brain homogenates from 129/SV mice and in vitro, that there is an increased interaction of Hsp70 with oxidized proteins, but also with the 20S proteasome, indicating a role of Hsp70 in mediating the interaction of oxidized proteins with the 20S proteasome. Thus, our data clearly implicate an involvement of Hsp70 oxidatively damaged protein degradation by the 20S proteasome.

Vitamin A: potential misclassification of vitamin A status among patients with type 2 diabetes and hypertension in urban Ghana.

BACKGROUND: Sub-Saharan Africa is facing a double burden of malnutrition: vitamin A deficiency (VAD) prevails, whereas the nutrition-related chronic conditions type 2 diabetes (T2D) and hypertension are emerging. Serum retinol­a VAD marker­increases in kidney disease and decreases in inflammation, which can partly be attributed to alterations in the vitamin A-transport proteins retinol-binding protein 4 (RBP4) and prealbumin. Kidney dysfunction and inflammation commonly accompany T2D and hypertension. OBJECTIVE: Among urban Ghanaians, we investigated the associations of T2D and hypertension with serum retinol as well as the importance of kidney function and inflammation in this regard. DESIGN: A hospital-based, case-control study in individuals for risk factors of T2D, hypertension, or both was conducted in Kumasi, Ghana (328 controls, 197 with T2D, 354 with hypertension, and 340 with T2D plus hypertension). In 1219 blood samples, serum retinol, RBP4, and prealbumin were measured. Urinary albumin and estimated glomerular filtration rate (eGFR) defined kidney function. C-reactive protein (CRP) >5 mg/L indicated inflammation. We identified associations of T2D and hypertension with retinol by linear regression and calculated the contribution of RBP4, prealbumin, urinary albumin, eGFR, and CRP to these associations as the percentages of the explained variance of retinol. RESULTS: VAD (retinol <1.05 µmol/L) was present in 10% of this predominantly female, middle-aged, overweight, and deprived population. Hypertension, but not T2D, was positively associated with retinol (ß: 0.12; 95% CI: 0.08, 0.17), adjusted for age, sex, socioeconomic factors, anthropometric measurements, and lifestyle. In addition to RBP4 (72%) and prealbumin (22%), the effect of increased retinol on individuals with hypertension was mainly attributed to impaired kidney function (eGFR: 30%; urinary albumin: 5%) but not to inflammation. CONCLUSIONS: In patients with hypertension, VAD might be underestimated because of increased serum retinol in the context of kidney dysfunction. Thus, the interpretation of serum retinol in sub-Saharan Africa should account for hypertension status.

First trimester concentrations of the TTR-RBP4-retinol complex components as early markers of insulin-treated gestational diabetes mellitus.

BACKGROUND: The objective of the study was to investigate the relationship between first trimester maternal serum levels of the TTR-RBP4-ROH complex components and the later insurgence of an altered glucose metabolism during pregnancy. METHODS: Retrospective case control study including 96 patients between the 12th and 14th week of gestation, 32 that developed gestational diabetes mellitus (GDM), respectively, 21 non-insulin-treated (dGDM) and 11 insulin-treated (iGDM), 20 large for gestational age fetuses (LGA) without GDM and 44 patients with normal outcome as control. Serum concentrations of RBP4 and TTR were assessed by ELISA; serum concentration of ROH by reverse-phase high performance liquid chromatography (rpHPLC). The molecular heterogeneity of TTR and RBP4 was analyzed after immunoprecipitation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: iGDM patients were characterized by reduced TTR, RBP4 and ROH compared to controls (respectively, iGDM vs. controls, mean±SD: TTR 3.96±0.89 µmol/L vs. 4.68±1.21 µmol/L, RBP4 1.13±0.25 µmol/L vs. 1.33±0.38 µmol/L and ROH 1.33±0.17 µmol/L vs. 1.62±0.29 µmol/L, p<0.05). TTR containing Gly10 in place of Cys10 was lower in the iGDM group (p<0.05) compared to controls. In the final logistic regression model ROH significantly predicted the diagnosis of iGDM (OR 0.93, 95% CI 0.87-0.98, p<0.05). CONCLUSIONS: First trimester maternal serum ROH, RBP4 and TTR represent potential biomarkers associated with the development of iGDM.

Post-translational modifications of transthyretin affect the triiodonine-binding potential.

Transthyretin (TTR) is a visceral protein, which facilitates the transport of thyroid hormones in blood and cerebrospinal fluid. The homotetrameric structure of TTR enables the simultaneous binding of two thyroid hormones per molecule. Each TTR subunit provides a single cysteine residue (Cys10 ), which is frequently affected by oxidative post-translational modifications. As Cys10 is part of the thyroid hormone-binding channel within the TTR molecule, PTM of Cys10 may influence the binding of thyroid hormones. Therefore, we analysed the effects of Cys10 modification with sulphonic acid, cysteine, cysteinylglycine and glutathione on binding of triiodothyronine (T3) by molecular modelling. Furthermore, we determined the PTM pattern of TTR in serum of patients with thyroid disease by immunoprecipitation and mass spectrometry to evaluate this association in vivo. The in silico assays demonstrated that oxidative PTM of TTR resulted in substantial reorganization of the intramolecular interactions and also affected the binding of T3 in a chemotype- and site-specific manner with S-glutathionylation as the most potent modulator of T3 binding. These findings were supported by the in vivo results, which indicated thyroid function-specific patterns of TTR with a substantial decrease in S-sulphonated, S-cysteinylglycinated and S-glutathionylated TTR in hypothyroid patients. In conclusion, this study provides evidence that oxidative modifications of Cys10 seem to affect binding of T3 to TTR probably because of the introduction of a sterical hindrance and induction of conformational changes. As oxidative modifications can be dynamically regulated, this may represent a sensitive mechanism to adjust thyroid hormone availability.

Retinol-binding protein 4 and its membrane receptor STRA6 control adipogenesis by regulating cellular retinoid homeostasis and retinoic acid receptor α activity.

Retinoids are vitamin A (retinol) derivatives and complex regulators of adipogenesis by activating specific nuclear receptors, including the retinoic acid receptor (RAR) and retinoid X receptor (RXR). Circulating retinol-binding protein 4 (RBP4) and its membrane receptor STRA6 coordinate cellular retinol uptake. It is unknown whether retinol levels and the activity of RAR and RXR in adipocyte precursors are linked via RBP4/STRA6. Here, we show that STRA6 is expressed in precursor cells and, dictated by the apo- and holo-RBP4 isoforms, mediates bidirectional retinol transport that controls RARα activity and subsequent adipocyte differentiation. Mobilization of retinoid stores in mice by inducing RBP4 secretion from the liver activated RARα signaling in the precursor cell containing the stromal-vascular fraction of adipose tissue. Retinol-loaded holo-RBP4 blocked adipocyte differentiation of cultured precursors by activating RARα. Remarkably, retinol-free apo-RBP4 triggered retinol efflux that reduced cellular retinoids, RARα activity, and target gene expression and enhanced adipogenesis synergistically with ectopic STRA6. Thus, STRA6 in adipocyte precursor cells links nuclear RARα activity to the circulating RBP4 isoforms, whose ratio in obese mice was shifted toward limiting the adipogenic potential of their precursors. This novel cross talk identifies a retinol-dependent metabolic function of RBP4 that may have important implications for the treatment of obesity.

Low plasma α-tocopherol concentrations and adverse clinical outcomes in diabetic hemodialysis patients.

BACKGROUND AND OBJECTIVES: Trials with the antioxidant vitamin E have failed to show benefit in the general population. Considering the different causes of death in ESRD, this study investigated the association between plasma concentrations of α-tocopherol and specific clinical outcomes in diabetic hemodialysis patients. DESIGN, SETTINGS, PARTICIPANTS, & MEASUREMENTS: In 1046 diabetic hemodialysis patients (participants of the German Diabetes and Dialysis Study), α-tocopherol was measured in plasma by reversed-phase HPLC. By Cox regression analyses, hazard ratios were determined for prespecified end points according to baseline plasma α-tocopherol levels: sudden death (n=134), myocardial infarction (n=172), stroke (n=89), combined cardiovascular events (n=398), fatal infection (n=107), and all-cause mortality (n=508). RESULTS: Patients had a mean age of 66±8 years, and mean plasma α-tocopherol level was 22.8±9.6 µmol/L. Levels of α-tocopherol were highly correlated to triglycerides (r=0.63, P<0.001). Patients in the lowest α-tocopherol quartile had (in unadjusted analyses) a 79% higher risk of stroke and a 31% higher risk of all-cause mortality compared with patients in the highest quartile. The associations were attenuated after adjustment for confounders (hazard ratiostroke=1.56, 95% confidence interval=0.75-3.25; hazard ratiomortality=1.22, 95% confidence interval=0.89-1.69, respectively). There was no association between α-tocopherol and myocardial infarction, sudden death, or infectious death. CONCLUSIONS: Plasma α-tocopherol concentrations were not independently associated with cardiovascular outcomes, infectious deaths, or all-cause mortality in diabetic hemodialysis patients. The lack of association can partly be explained by a confounding influence of malnutrition, which should be considered in the planning of trials to reduce cardiovascular risk in dialysis patients.

Does N-acetylcysteine modulate post-translational modifications of transthyretin in hemodialysis patients?

It is assumed that effects of the thiol antioxidant N-acetylcysteine (NAC) are mediated by interaction with protein-associated cysteine residues, however, information on protein level in vivo are missing. Therefore, we analyzed NAC-induced modifications of the protein transthyretin (TTR) in plasma of hemodialysis patients in a randomized, placebo-controlled study. TTR was selected due to its low molecular weight and the free cysteine residue in the polypeptide chain, which is known to be extensively modified by formation of mixed disulfides. The intravenous application of NAC during a hemodialysis session resulted in a substantial increase of native TTR from median 15% (range 8.8%-30%) to median 40% (37-50) and reduction of S-cysteinylated TTR [51% (44-60) vs. 6.6% (2.4-10)]. Additionally the pronounced formation of a TTR-NAC adduct was detected. However, all these modifications seemed to be reversible. Additionally, in vitro incubation of plasma with NAC confirmed the in vivo results and indicated that changes in post-translational modification pattern of TTR were a function of NAC concentration. Based on these observations and the essential metabolic and biochemical role of protein-associated cysteine residues we hypothesize that the interaction of NAC with proteins may explain altered protein functions due to modification of cysteine residues.

DPP-4 inhibition on top of angiotensin receptor blockade offers a new therapeutic approach for diabetic nephropathy.

BACKGROUND: The need for an improved treatment for diabetic nephropathy is greatest in patients who do not adequately respond to angiotensin II receptor blockers (ARBs). This study investigated the effect of the novel dipeptidyl peptidase-4 inhibitor linagliptin alone and in combination with the ARB telmisartan on the progression of diabetic nephropathy in diabetic endothelial nitric oxide synthase (eNOS) knockout mice. METHODS: Sixty male eNOS knockout C57BL/6J mice were divided into four groups after receiving intraperitoneal highdose streptozotocin: telmisartan (1 mg/kg), linagliptin (3 mg/kg), linagliptin + telmisartan (3 mg/kg + 1 mg/kg) and vehicle. Fourteen mice were used as non-diabetic controls. RESULTS: After 12 weeks, urine and blood were obtained and blood pressure measured. Glucose concentrations were increased and similar in all diabetic groups. Telmisartan alone reduced systolic blood pressure by 5.9 mmHg versus diabetic controls (111.2 ± 2.3 mmHg vs 117.1 ± 2.2 mmHg; mean ± SEM; P=0.071). Combined treatment significantly reduced albuminuria compared with diabetic controls (71.7 ± 15.3 µg/24 h vs. 170.8 ± 34.2 µg/24 h; P=0.017), whereas the effects of single treatment with either telmisartan (97.8 ± 26.4 µg/24 h) or linagliptin (120.8 ± 37.7 µg/24 h) were not statistically significant. DPP-4 inhibition, alone and in combination, led to significantly lower plasma osteopontin levels compared with telmisartan alone. Histological analysis revealed reduced glomerulosclerosis after Linagliptin alone and in combination with telmisartan in comparison to non treated diabetic animals (p<0.01 and p<0.05). Kidney malonaldehyde immune-reactivity, a marker of oxidative stress, was significantly lower in animals treated with linagliptin. CONCLUSIONS: DPP-4 inhibition on top of ARB treatment significantly reduced urinary albumin excretion and oxidative stress in diabetic eNOS knockout mice. Linagliptin on top of an angiotensin II receptor blocker may offer a new therapeutic approach for patients with diabetic nephropathy.

Transthyretin predicts cardiovascular outcome in hemodialysis patients with type 2 diabetes.

OBJECTIVE: BMI and albumin are commonly accepted parameters to recognize wasting in dialysis patients and are powerful predictors of morbidity and mortality. However, both parameters reveal limitations and may not cover the entire range of patients with wasting. The visceral protein transthyretin (TTR) may be helpful in overcoming the diagnostic and prognostic gap. Therefore, the aim of this study was to assess the association of TTR with morbidity and mortality in hemodialysis patients. RESEARCH DESIGN AND METHODS: The TTR concentration was determined in plasma samples of 1,177 hemodialysis patients with type 2 diabetes. Cox regression analyses were used to determine hazard ratios (HRs) for the risk of cardiovascular end points (CVEs) and mortality according to quartiles of TTR concentration for the total study cohort and the subgroups BMI ≥23 kg/m(2), albumin concentration ≥3.8 g/dL, and a combination of both. RESULTS: A low TTR concentration was associated with an increased risk for CVE for the total study cohort (HR 1.65 [95% CI 1.27-2.14]), patients with BMI ≥23 kg/m(2) (1.70 [1.22-2.37]), albumin ≥3.8 g/dL (1.68 [1.17-2.42]), and the combination of both (1.69 [1.13-2.53]). Additionally, a low TTR concentration predicted mortality for the total study cohort (1.79 [1.43-2.24]) and patients with BMI ≥23 kg/m(2) (1.46 [1.09-1.95]). CONCLUSIONS: The current study demonstrated that TTR is a useful predictor for cardiovascular outcome and mortality in diabetic hemodialysis patients. TTR was particularly useful in patients who were not identified to be at risk by BMI or albumin status.

Effects of stimulation of soluble guanylate cyclase on diabetic nephropathy in diabetic eNOS knockout mice on top of angiotensin II receptor blockade.

The prevalence of diabetes mellitus and its complications, such as diabetic nephropathy (DN), is rising worldwide and prevention and treatment are therefore becoming increasingly important. Therapy of DN is particularly important for patients who do not adequately respond to angiotensin receptor blocker (ARB) treatment. Novel approaches include the stimulation of soluble guanylate cyclase (sGC) as it is reported to have beneficial effects on cardiac and renal damage. We aimed to investigate the effects of the sGC stimulator riociguat and ARB telmisartan on kidney function and structure in a hypertensive model of diabetic nephropathy. Seventy-six diabetic male eNOS knockout C57BL/6J mice were randomly divided after having received streptozotocin: telmisartan (1 mg/kg/d), riociguat (3 mg/kg/d), riociguat+telmisartan (3+1 mg/kg/d), and vehicle. Fourteen mice were used as non-diabetic controls. Treatment duration was 11 weeks. Glucose concentrations were increased and similar in all diabetic groups. Telmisartan insignificantly reduced blood pressure by 5.9 mmHg compared with diabetic controls (111.2±2.3 mmHg vs. 117.1±2.2 mmHg; p = 0.071). Treatment with riociguat both alone and in combination with telmisartan led to a significant reduction of blood pressure towards diabetic vehicle (105.2±2.5 mmHg and 105.0±3.2 mmHg, respectively, vs. 117.1±2.2 mmHg). Combined treatment also significantly decreased albuminuria compared with diabetic controls (47.3±9.6 µg/24 h vs. 170.8±34.2 µg/24 h; p = 0.002) reaching levels similar to those of non-diabetic controls (34.4±10.6 µg/24 h), whereas the reduction by single treatment with either telmisartan (97.8±26.4 µg/24 h) or riociguat (97.1±15.7 µg/24 h) was not statistically significant. The combination treatment led to a significant (p<0.01) decrease of tissue immunoreactivity of malondialdehyde, as consequence of reduced oxidative stress. In conclusion, stimulation of sGC significantly reduced urinary albumin excretion in diabetic eNOS knockout mice treated already with ARB. Thus, this new drug class on top of standard ARBs administration may offer a new therapeutic approach for patients resistant to ARB treatment.

Structural and sensory characterization of key pungent and tingling compounds from black pepper (Piper nigrum L.).

To gain a more comprehensive knowledge on whether, besides the well-known piperine, other compounds are responsible for the pungent and tingling oral impression imparted by black pepper, an ethanol extract prepared from black pepper (Piper nigrum L.) was screened for its key sensory-active nonvolatiles by application of taste dilution analysis (TDA). Purification of the compounds perceived with the highest sensory impact, followed by LC-MS and 1D/2D NMR experiments as well as synthesis, led to the structure determination of 25 key pungent and tingling phytochemicals, among which the eight amides 1-(octadeca-2E,4E,13Z-trienyl)piperidine, 1-(octadeca-2E,4E,13Z-trienyl)pyrrolidine, (2E,4E,13Z)-N-isobutyl-octadeca-2,4,13-trienamide, 1-(octadeca-2E,4E,12Z-trienoyl)-pyrrolidine, 1-(eicosa-2E,4E,15Z-trienyl)piperidine, 1-(eicosa-2E,4E,15Z-trienyl)pyrrolidine, (2E,4E,15Z)-N-isobutyl-eicosa-2,4,15-trienamide, and 1-(eicosa-2E,4E,14Z-trienoyl)-pyrrolidine were not yet reported in literature. Sensory studies by means of a modified half-tongue test revealed recognition thresholds ranging from 3.0 to 1150.2 nmol/cm² for pungency and from 520.6 to 2162.1 nmol/cm² for the tingling orosensation depending on their chemical structure.