Christiane Bumke-Vogt

Effects of supplemented isoenergetic diets differing in cereal fiber and protein content on insulin sensitivity in overweight humans

BACKGROUND Despite their beneficial effects on weight loss and blood lipids, high-protein (HP) diets have been shown to increase insulin resistance and diabetes risk, whereas high-cereal-fiber (HCF) diets have shown the opposite effects on these outcomes. OBJECTIVE We compared the effects of isoenergetic HP and HCF diets and a diet with moderate increases in both cereal fibers and dietary protein (Mix diet) on insulin sensitivity, as measured by using euglycemic-hyperinsulinemic clamps with infusion of [6,6-(2)H(2)]glucose. DESIGN We randomly assigned 111 overweight adults with features of the metabolic syndrome to 1 of 4 two-phased, 18-wk isoenergetic diets by group-matching. Per 3-d food protocols, the percentages of energy derived from protein and carbohydrates and the intake of cereal fiber per day, respectively, were as follows-after 6 wk: 17%, 52%, and 14 g (control); 17%, 52%, and 43 g (HCF); 28%, 43%, and 13 g (HP); 23%, 44%, and 26 g (Mix); after 18 wk: 17%, 51%, and 15 g (control); 17%, 51%, and 41 g (HCF); 26%, 45%, and 14 g (HP); and 22%, 46%, and 26 g (Mix). Eighty-four participants completed the study successfully and were included in the final analyses. Adherence was supported by the provision of tailored dietary supplements twice daily in all groups. RESULTS Insulin sensitivity expressed as an M value was 25% higher after 6 wk of the HCF diet than after 6 wk of the HP diet (subgroup analysis: 4.61 ± 0.38 compared with 3.71 ± 0.36 mg · kg(-1) · min(-1), P = 0.008; treatment × time interaction: P = 0.005). Effects were attenuated after 18 wk (treatment × time interaction: P = 0.054), which was likely explained by lower adherence to the HP diet. HP intake was associated with a tendency to increased protein expression in adipose tissue of the translation initiation factor serine-kinase-6-1, which is known to mediate amino acid-induced insulin resistance. Biomarkers of protein intake indicated interference of cereal fibers with dietary protein absorption. CONCLUSION Greater changes in insulin sensitivity after intake of an isoenergetic HCF than after intake of an HP diet might help to explain the diverse effects of these diets on diabetes risk. This trial is registered at clinicaltrials.gov as NCT00579657.

Novel Inactivating Mutations of the Calcium-Sensing Receptor: The Calcimimetic NPS R-568 Improves Signal Transduction of Mutant Receptors

CONTEXT AND OBJECTIVE Inactivating mutations in the calcium-sensing receptor (CaSR) gene cause neonatal severe hyperparathyroidism and familial hypocalciuric hypercalcemia (FHH). The aims of the present study were the functional characterization of novel mutations of the CaSR found in FHH patients, the comparison of in vitro receptor function with clinical parameters, and the effect of the allosteric calcimimetic NPS R-568 on the signaling of mutant receptors. METHODS Wild-type and mutant CaSRs (W530G, C568Y, W718X, M734R, L849P, Q926R, and D1005N) were expressed in human embryonic kidney 293 cells. Receptor signaling was studied by measuring intracellular free calcium in response to different concentrations of extracellular calcium ([Ca(2+)](o)). RESULTS Four CaSR mutations (C568Y, W718X, M734R, and L849P) demonstrated a complete lack of a [Ca(2+)](o)-induced cytosolic Ca(2+) response up to 30 mm [Ca(2+)](o), whereas the CaSR mutants W530G, Q926R, and D1005N retained some sensitivity to [Ca(2+)](o). There was no significant relation between the in vitro calcium sensitivity, serum calcium, and intact PTH levels in the patients. Patients with C-terminal CaSR mutations had a calcium to creatine ratio above the established diagnostic threshold of 0.01 for FHH. The calcimimetic NPS R-568 enhanced the responsiveness to [Ca(2+)](o) in CaSR mutants of the extracellular domain (W530G and C568Y) as well as the intracellular C-terminal domain (Q926R and D1005N). CONCLUSION Therefore, calcimimetics might offer medical treatment for symptomatic FHH patients, and more important, for patients with neonatal severe hyperparathyroidism that harbor calcimimetic-sensitive CaSR mutants.

Ghrelin-induced food intake and adiposity depend on central mTORC1/S6K1 signaling.

Signaling through the mammalian target of rapamycin complex 1 (mTORC1) and its effectors the S6-kinases (S6K) in the hypothalamus is thought to be involved in nutrient sensing and control of food intake. Given the anatomical proximity of this pathway to circuits for the hormone ghrelin, we investigated the potential role of the mTORC1/S6K pathway in mediating the metabolic effects of ghrelin. We found that ghrelin promoted phosphorylation of S6K1 in the mouse hypothalamic cell line N-41 and in the rat hypothalamus after intracerebroventricular administration. Rapamycin, an inhibitor of mTORC1, suppressed ghrelin-induced phosphorylation of hypothalamic S6K1 and increased food intake and insulin in rats. Chronic peripheral administration of ghrelin induced a significant increase in body weight, fat mass and food efficiency in wild-type and S6K2-knockout but not in S6K1-knockout mice. We therefore propose that ghrelin-induced hyperphagia, adiposity and insulin secretion are controlled by a central nervous system involving the mTORC1/S6K1 pathway.

The flavones apigenin and luteolin induce FOXO1 translocation but inhibit gluconeogenic and lipogenic gene expression in human cells.

The flavones apigenin (4′,5,7,-trihydroxyflavone) and luteolin (3′,4′,5,7,-tetrahydroxyflavone) are plant secondary metabolites with antioxidant, antiinflammatory, and anticancer activities. We evaluated their impact on cell signaling pathways related to insulin-resistance and type 2 diabetes. Apigenin and luteolin were identified in our U-2 OS (human osteosarcoma) cell screening assay for micronutrients triggering rapid intracellular translocation of the forkhead box transcription factor O1 (FOXO1), an important mediator of insulin signal transduction. Insulin reversed the translocation of FOXO1 as shown by live cell imaging. The impact on the expression of target genes was evaluated in HepG2 (human hepatoma) cells. The mRNA-expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pc), the lipogenic enzymes fatty-acid synthase (FASN) and acetyl-CoA-carboxylase (ACC) were down-regulated by both flavones with smaller effective dosages of apigenin than for luteolin. PKB/AKT-, PRAS40-, p70S6K-, and S6-phosphorylation was reduced by apigenin and luteolin but not that of the insulin-like growth factor receptor IGF-1R by apigenin indicating a direct inhibition of the PKB/AKT-signaling pathway distal to the IGF-1 receptor. N-acetyl-L-cysteine did not prevent FOXO1 nuclear translocation induced by apigenin and luteolin, suggesting that these flavones do not act via oxidative stress. The roles of FOXO1, FOXO3a, AKT, sirtuin1 (SIRT1), and nuclear factor (erythroid-derived2)-like2 (NRF2), investigated by siRNA knockdown, showed differential patterns of signal pathways involved and a role of NRF2 in the inhibition of gluconeogenic enzyme expression. We conclude that these flavones show an antidiabetic potential due to reduction of gluconeogenic and lipogenic capacity despite inhibition of the PKB/AKT pathway which justifies detailed investigation in vivo.

Benzylglucosinolate Derived Isothiocyanate from Tropaeolum majus Reduces Gluconeogenic Gene and Protein Expression in Human Cells

Nasturtium (Tropaeolum majus L.) contains high concentrations of benzylglcosinolate. We found that a hydrolysis product of benzyl glucosinolate—the benzyl isothiocyanate (BITC)—modulates the intracellular localization of the transcription factor Forkhead box O 1 (FOXO1). FoxO transcription factors can antagonize insulin effects and trigger a variety of cellular processes involved in tumor suppression, longevity, development and metabolism. The current study evaluated the ability of BITC—extracted as intact glucosinolate from nasturtium and hydrolyzed with myrosinase—to modulate i) the insulin-signaling pathway, ii) the intracellular localization of FOXO1 and, iii) the expression of proteins involved in gluconeogenesis, antioxidant response and detoxification. Stably transfected human osteosarcoma cells (U-2 OS) with constitutive expression of FOXO1 protein labeled with GFP (green fluorescent protein) were used to evaluate the effect of BITC on FOXO1. Human hepatoma HepG2 cell cultures were selected to evaluate the effect on gluconeogenic, antioxidant and detoxification genes and protein expression. BITC reduced the phosphorylation of protein kinase B (AKT/PKB) and FOXO1; promoted FOXO1 translocation from cytoplasm into the nucleus antagonizing the insulin effect; was able to down-regulate the gene and protein expression of gluconeogenic enzymes; and induced the gene expression of antioxidant and detoxification enzymes. Knockdown analyses with specific siRNAs showed that the expression of gluconeogenic genes was dependent on nuclear factor (erythroid derived)-like2 (NRF2) and independent of FOXO1, AKT and NAD-dependent deacetylase sirtuin-1 (SIRT1). The current study provides evidence that BITC might have a role in type 2 diabetes T2D by reducing hepatic glucose production and increasing antioxidant resistance.

Cell-type specific regulation of the human 11beta-hydroxysteroid dehydrogenase type 1 promoter

Abstract The intracellular enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone into the more active metabolite cortisol. Overexpression of 11beta-HSD1 was associated with features of the metabolic syndrome such as obesity or impaired glucose tolerance. Despite this considerable impact of 11beta-HSD1, the human 11beta-HSD1 promoter has not been described in detail yet. We therefore cloned eight different promoter fragments of the 5′-upstream region of the known transcription/translation-start up to −3034 bp into the luciferase-reporter vector pGL3. A low-cost in-house assay was developed and validated to detect firefly and renilla luciferase activity. Promoter fragments were analysed in human HepG2 and undifferentiated and differentiated murine 3T3-L1 cells. A differential regulation of the human 11beta-HSD1 promoter depending upon the cell type was observed. Specifically, a strong repressor of the basal promoter activity was found between −85 and −172 bp in HepG2 cells only, while an additional repressor appeared to be active between −342 and −823 bp in both, the hepatic and the adipose cell line. The presented data suggest a cell-type specific regulation of the 11beta-HSD1 promoter, which is in agreement with existing expression data from animal and human studies. The described promoter constructs will allow subsequent studies about the role of specific hormonal, metabolic and transcription factors to finally characterise the regulation of the human 11beta-HSD1-promoter in more detail.

Glucagon Decreases IGF-1 Bioactivity in Humans, Independently of Insulin, by Modulating Its Binding Proteins

Context: Depending on its lipolytic activity, glucagon plays a promising role in obesity treatment. Glucagon‐induced growth hormone (GH) release can promote its effect on lipid metabolism, although the underlying mechanisms have not been well‐defined. Objective: The present study highlights the glucagon effect on the GH/insulinlike growth factor 1 (IGF‐1)/IGF‐binding protein (IGFBP) axis in vivo and in vitro, taking into consideration insulin as a confounding factor. Materials and Methods: In a double‐blind, placebo‐controlled study, we investigated changes in GH, IGFBP, and IGF‐1 bioactivity after intramuscular glucagon administration in 13 lean controls, 11 obese participants, and 13 patients with type 1 diabetes mellitus (T1DM). The effect of glucagon on the transcription factor forkhead box protein O1 (FOXO1) translocation, the transcription of GH/IGF‐1 system members, and phosphorylation of protein kinase B (Akt) was further investigated in vitro. Results: Despite unchanged total IGF‐1 and IGFBP‐3 levels, glucagon decreased IGF‐1 bioactivity in all study groups by increasing IGFBP‐1 and IGFBP‐2. The reduction in IGF‐1 bioactivity occurred before the glucagon‐induced surge in GH. In contrast to the transient increase in circulating insulin in obese and lean participants, no change was observed in those with T1DM. In vitro, glucagon dose dependently induced a substantial nuclear translocation of FOXO1 in human osteosarcoma cells and tended to increase IGFBP‐1 and IGFBP‐2 gene expression in mouse primary hepatocytes, despite absent Akt phosphorylation. Conclusions: Our data point to the glucagon‐induced decrease in bioactive IGF‐1 levels as a mechanism through which glucagon induces GH secretion. This insulin‐independent reduction is related to increased IGFBP‐1 and IGFBP‐2 levels, which are most likely mediated via activation of the FOXO/mTOR (mechanistic target of rapamycin) pathway.

Oral administration of nasturtium affects peptide YY secretion in male subjects

SCOPE Nasturtium plants contain the glucosinolate glucotropaeolin and its corresponding breakdown product benzyl isothiocyanate (BITC), the latter being intensively studied with regard to cancer chemoprevention and anti-inflammatory properties. In addition, recent research has shown that isothiocyanates are able to activate the release of several gut hormones in vitro and in rodent studies. Here, we tested the effects of a dietary nasturtium administration on circulating levels of gut hormones in humans. METHODS AND RESULTS Metabolically healthy males (n = 15) received a single oral dose of 10 g freeze-dried nasturtium leaf material suspended in water or only water (control). Blood samples were taken every hour and serum concentrations of insulin, C-peptide, glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and peptide (PYY) were analyzed. Oral nasturtium intake resulted in an increased release of PYY over a time period of 6 h whereas circulating levels of other hormones were not changed. CONCLUSION Given the finding that nasturtium consumption enhances secretion of PYY, a key hormone involved in energy regulation, special diets containing nasturtium, or supplementation with nasturtium or BITC might be considered in the treatment of obesity.