Refine
Has Fulltext
- yes (104)
Year of publication
Document Type
- Postprint (104) (remove)
Language
- English (104) (remove)
Keywords
- inflammation (6)
- LC-MS/MS (5)
- oxidative stress (5)
- α-amylase/trypsin inhibitors (5)
- cancer (4)
- insulin (4)
- obesity (4)
- sphingolipids (4)
- acid sphingomyelinase (3)
- carotenoids (3)
Institute
- Institut für Ernährungswissenschaft (104) (remove)
Prostaglandins, released from Kupffer cells, have been shown to mediate the increase in hepatic glycogenolysis by various stimuli such as zymosan, endotoxin, immune complexes, and anaphylotoxin C3a involving prostaglandin (PG) receptors coupled to phospholipase C via a G(0) protein. PGs also decreased glucagon-stimulated glycogenolysis in hepatocytes by a different signal chain involving PGE(2) receptors coupled to adenylate cyclase via a G(i) protein (EP(3) receptors). The source of the prostaglandins for this latter glucagon-antagonistic action is so far unknown. This study provides evidence that Kupffer cells may be one source: in Kupffer cells, maintained in primary culture for 72 hours, glucagon (0.1 to 10 nmol/ L) increased PGE(2), PGF(2 alpha), and PGD(2) synthesis rapidly and transiently. Maximal prostaglandin concentrations were reached after 5 minutes. Glucagon (1 nmol/L) elevated the cyclic adenosine monophosphate (cAMP) and inositol triphosphate (InsP(3)) levels in Kupffer cells about fivefold and twofold, respectively. The increase in glyco gen phosphorylase activity elicited by 1 nmol/L glucagon was about twice as large in monocultures of hepatocytes than in cocultures of hepatocytes and Kupffer cells with the same hepatocyte density. Treatment of cocultures with 500 mu mol/L acetylsalicylic acid (ASA) to irreversibly inhibit cyclooxygenase (PGH-synthase) 30 minutes before addition of glucagon abolished this difference. These data support the hypothesis that PGs produced by Kupffer cells in response to glucagon might participate in a feedback loop inhibiting glucagon-stimulated glycogenolysis in hepatocytes.
Macrophages in pathologically expanded dysfunctional white adipose tissue are exposed to a mix of potential modulators of inflammatory response, including fatty acids released from insulin-resistant adipocytes, increased levels of insulin produced to compensate insulin resistance, and prostaglandin E₂ (PGE₂) released from activated macrophages. The current study addressed the question of how palmitate might interact with insulin or PGE₂ to induce the formation of the chemotactic pro-inflammatory cytokine interleukin-8 (IL-8). Human THP-1 cells were differentiated into macrophages. In these macrophages, palmitate induced IL-8 formation. Insulin enhanced the induction of IL-8 formation by palmitate as well as the palmitate-dependent stimulation of PGE₂ synthesis. PGE₂ in turn elicited IL-8 formation on its own and enhanced the induction of IL-8 release by palmitate, most likely by activating the EP4 receptor. Since IL-8 causes insulin resistance and fosters inflammation, the increase in palmitate-induced IL-8 formation that is caused by hyperinsulinemia and locally produced PGE₂ in chronically inflamed adipose tissue might favor disease progression in a vicious feed-forward cycle.
Rat serum, in which the complement sytem had been activated by incubation with zymosan, increased the glucose and lactate output, and reduced and redistributed the flow in isolated perfused rat liver clearly more than the control serum. Heat inactivation of the rat serum prior to zymosan incubation abolished this difference. Metabolic and hemodynamic alterations caused by the activated serum were dose dependent. They were almost completely inhibited by the cyclooxygenase inhibitor indomethacin and by the thromboxane antagonist 4-[2-(4-chlorobenzenesulfonamide)-ethyl]-benzene-acetica cid (BM 13505), but clearly less efficiently by the 5’-lipoxygenase inhibitor nordihydroguaiaretic acid and the leukotriene antagonist N-{3-[3-(4-acetyl-3-hydroxy-2-propyl-phenoxy)-propoxy]-4-chlorine-6-methyl-phenyl}-1H-tetrazole-5-carboxamide sodium salt (CGP 35949 B). Control serum and to a much larger extent complement-activated serum, caused an overflow of thromboxane B₂ and prostaglandin F₂α into the hepatic vein. It is concluded that the activated complement system of rat serum can influence liver metabolism and hemodynamics via release from nonparenchymal liver cells of thromboxane and prostaglandins, the latter of which can in turn act on the parenchymal cells.
Background: Transport of methylmercury (MeHg) across the blood-brain barrier towards the brain side is well discussed in literature, while ethylmercury (EtHg) and inorganic mercury are not adequately characterized regarding their entry into the brain. Studies investigating a possible efflux out of the brain are not described to our knowledge.
Methods: This study compares, for the first time, effects of organic methylmercury chloride (MeHgCl), EtHg-containing thiomersal and inorganic Hg chloride (HgCl2) on as well as their transfer across a primary porcine in vitro model of the blood-brain barrier.
Results: With respect to the barrier integrity, the barrier model exhibited a much higher sensitivity towards HgCl2 following basolateral incubation (brain-facing side) as compared to apical application (blood-facing side). These HgCl2 induced effects on the barrier integrity after brain side incubation are comparable to that of the organic species, although MeHgCl and thiomersal exerted much higher cytotoxic effects in the barrier building cells. Hg transfer rates following exposure to organic species in both directions argue for diffusion as transfer mechanism. Inorganic Hg application surprisingly resulted in a Hg transfer out of the brain-facing compartment.
Conclusions: In case of MeHgCl and thiomersal incubation, mercury crossed the barrier in both directions, with a slight accumulation in the basolateral, brain-facing compartment, after simultaneous incubation in both compartments. For HgCl2, our data provide first evidence that the blood-brain barrier transfers mercury out of the brain.
The protein fraction, important for coffee cup quality, is modified during post-harvest treatment prior to roasting. Proteins may interact with phenolic compounds, which constitute the major metabolites of coffee, where the processing affects these interactions. This allows the hypothesis that the proteins are denatured and modified via enzymatic and/or redox activation steps. The present study was initiated to encompass changes in the protein fraction. The investigations were limited to major storage protein of green coffee beans. Fourteen Coffea arabica samples from various processing methods and countries were used. Different extraction protocols were compared to maintain the status quo of the protein modification. The extracts contained about 4–8 µg of chlorogenic acid derivatives per mg of extracted protein. High-resolution chromatography with multiple reaction monitoring was used to detect lysine modifications in the coffee protein. Marker peptides were allocated for the storage protein of the coffee beans. Among these, the modified peptides K.FFLANGPQQGGK.E and R.LGGK.T of the α-chain and R.ITTVNSQK.I and K.VFDDEVK.Q of β-chain were detected. Results showed a significant increase (p < 0.05) of modified peptides from wet processed green beans as compared to the dry ones. The present study contributes to a better understanding of the influence of the different processing methods on protein quality and its role in the scope of coffee cup quality and aroma. View Full-Text
The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.
The objective of this work was to investigate the potential effect of cereal α-amylase/trypsin inhibitors (ATIs) on growth parameters and selective digestive enzymes of Tenebrio molitor L. larvae. The approach consisted of feeding the larvae with wheat, sorghum and rice meals containing different levels and composition of α-amylase/trypsin inhibitors. The developmental and biochemical characteristics of the larvae were assessed over feeding periods of 5 h, 5 days and 10 days, and the relative abundance of α-amylase and selected proteases in larvae were determined using liquid chromatography tandem mass spectrometry. Overall, weight gains ranged from 21% to 42% after five days of feeding. The larval death rate significantly increased in all groups after 10 days of feeding (p < 0.05), whereas the pupation rate was about 25% among larvae fed with rice (Oryza sativa L.) and Siyazan/Esperya wheat meals, and only 8% and 14% among those fed with Damougari and S35 sorghum meals. As determined using the Lowry method, the protein contents of the sodium phosphate extracts ranged from 7.80 ± 0.09 to 9.42 ± 0.19 mg/mL and those of the ammonium bicarbonate/urea reached 19.78 ± 0.16 to 37.47 ± 1.38 mg/mL. The total protein contents of the larvae according to the Kjeldahl method ranged from 44.0 and 49.9 g/100 g. The relative abundance of α-amylase, CLIP domain-containing serine protease, modular serine protease zymogen and C1 family cathepsin significantly decreased in the larvae, whereas dipeptidylpeptidase I and chymotrypsin increased within the first hours after feeding (p < 0.05). Trypsin content was found to be constant independently of time or feed material. Finally, based on the results we obtained, it was difficult to substantively draw conclusions on the likely effects of meal ATI composition on larval developmental characteristics, but their effects on the digestive enzyme expression remain relevant.
Two decades ago, sphingosine 1-phosphate (S1P) was discovered as a novel bioactive molecule that regulates a variety of cellular functions. The plethora of S1P-mediated effects is due to the fact that the sphingolipid not only modulates intracellular functions but also acts as a ligand of G protein-coupled receptors after secretion into the extracellular environment. In the plasma, S1P is found in high concentrations, modulating immune cell trafficking and vascular endothelial integrity. The liver is engaged in modulating the plasma S1P content, as it produces apolipoprotein M, which is a chaperone for the S1P transport. Moreover, the liver plays a substantial role in glucose and lipid homeostasis. A dysfunction of glucose and lipid metabolism is connected with the development of liver diseases such as hepatic insulin resistance, non-alcoholic fatty liver disease, or liver fibrosis. Recent studies indicate that S1P is involved in liver pathophysiology and contributes to the development of liver diseases. In this review, the current state of knowledge about S1P and its signaling in the liver is summarized with a specific focus on the dysregulation of S1P signaling in obesity-mediated liver diseases. Thus, the modulation of S1P signaling can be considered as a potential therapeutic target for the treatment of hepatic diseases.
Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma–based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data.
Zinc deficiency has a fundamental influence on the immune defense, with multiple effects on different immune cells, resulting in a major impairment of human health. Monocytes and macrophages are among the immune cells that are most fundamentally affected by zinc, but the impact of zinc on these cells is still far from being completely understood. Therefore, this study investigates the influence of zinc deficiency on monocytes of healthy human donors. Peripheral blood mononuclear cells, which include monocytes, were cultured under zinc deficient conditions for 3 days. This was achieved by two different methods: by application of the membrane permeable chelator N,N,N0´,N0´-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) or by removal of zinc from the culture medium using a CHELEX 100 resin. Subsequently, monocyte functions were analyzed in response to Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. Zinc depletion had differential effects. On the one hand, elimination of bacterial pathogens by phagocytosis and oxidative burst was elevated. On the other hand, the production of the inflammatory cytokines tumor necrosis factor (TNF)-a and interleukin (IL)-6 was reduced. This suggests that monocytes shift from intercellular communication to basic innate defensive functions in response to zinc deficiency. These results were obtained regardless of the method by which zinc deficiency was achieved. However, CHELEX-treated medium strongly augmented cytokine production, independently from its capability for zinc removal. This side-effect severely limits the use of CHELEX for investigating the effects of zinc deficiency on innate immunity.
1) During orthograde perfusion of rat liver human anaphylatoxin C3a caused an increase in glucose and lactate output and reduction of flow. These effects could be enhanced nearly twofold by co-infusion of the carboxypeptidase inhibitor MERGETPA, which reduced inactivation of C3a to C3adesArg. 2) During retrograde perfusion C3a caused a two- to threefold larger increase in glucose and lactate output and reduction of flow than in orthograde perfusions. These actions tended to be slightly enhanced by MERGETPA. 3) The elimination of C3a plus C3adesArg immunoreactivity during a single liver passage was around 67%, irrespective of the perfusion direction and the presence of the carboxypeptidase inhibitor MERGETPA; however, less C3adesArg and more intact C3a appeared in the perfusate in the presence of MERGETPA in orthograde and retrogade perfusions It is concluded that rat liver inactivated human anaphylatoxin C3a by conversion to C3adesArg and moreover eliminated it by an additional process. The inactivation to C3adesArg seemed to be located predominantly in the proximal periportal region of the liver sinusoid, since C3a was less effective in orthograde perfusions, when C3a first passed the proximal periportal region before reaching the predominant mass of parenchyma as its site of action, than in retrograde perfusions, when it first passed the perivenous area. These data may be evidence for a periportal scavenger mechanism, by which the liver protects itself from systemically released mediators of inflammation that interfere with the local regulation of liver metabolism and hemodynamics.
Wheat alpha-amylase/trypsin inhibitors remain a subject of interest considering the latest findings showing their implication in wheat-related non-celiac sensitivity (NCWS). Understanding their functions in such a disorder is still unclear and for further study, the need for pure ATI molecules is one of the limiting problems. In this work, a simplified approach based on the successive fractionation of ATI extracts by reverse phase and ion exchange chromatography was developed. ATIs were first extracted from wheat flour using a combination of Tris buffer and chloroform/methanol methods. The separation of the extracts on a C18 column generated two main fractions of interest F1 and F2. The response surface methodology with the Doehlert design allowed optimizing the operating parameters of the strong anion exchange chromatography. Finally, the seven major wheat ATIs namely P01083, P17314, P16850, P01085, P16851, P16159, and P83207 were recovered with purity levels (according to the targeted LC-MS/MS analysis) of 98.2 ± 0.7; 98.1 ± 0.8; 97.9 ± 0.5; 95.1 ± 0.8; 98.3 ± 0.4; 96.9 ± 0.5, and 96.2 ± 0.4%, respectively. MALDI-TOF-MS analysis revealed single peaks in each of the pure fractions and the mass analysis yielded deviations of 0.4, 1.9, 0.1, 0.2, 0.2, 0.9, and 0.1% between the theoretical and the determined masses of P01083, P17314, P16850, P01085, P16851, P16159, and P83207, respectively. Overall, the study allowed establishing an efficient purification process of the most important wheat ATIs. This paves the way for further in-depth investigation of the ATIs to gain more knowledge related to their involvement in NCWS disease and to allow the absolute quantification in wheat samples.
Dermal Delivery of the High-Molecular-Weight Drug Tacrolimus by Means of Polyglycerol-Based Nanogels
(2019)
Polyglycerol-based thermoresponsive nanogels (tNGs) have been shown to have excellent skin hydration properties and to be valuable delivery systems for sustained release of drugs into skin. In this study, we compared the skin penetration of tacrolimus formulated in tNGs with a commercial 0.1% tacrolimus ointment. The penetration of the drug was investigated in ex vivo abdominal and breast skin, while different methods for skin barrier disruption were investigated to improve skin permeability or simulate inflammatory conditions with compromised skin barrier. The amount of penetrated tacrolimus was measured in skin extracts by liquid chromatography tandem-mass spectrometry (LC-MS/MS), whereas the inflammatory markers IL-6 and IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). Higher amounts of tacrolimus penetrated in breast as compared to abdominal skin or in barrier-disrupted as compared to intact skin, confirming that the stratum corneum is the main barrier for tacrolimus skin penetration. The anti-proliferative effect of the penetrated drug was measured in skin tissue/Jurkat cells co-cultures. Interestingly, tNGs exhibited similar anti-proliferative effects as the 0.1% tacrolimus ointment. We conclude that polyglycerol-based nanogels represent an interesting alternative to paraffin-based formulations for the treatment of inflammatory skin conditions.
Trace elements, like Cu, Zn, Fe, or Se, are important for the proper functioning of antioxidant enzymes. However, in excessive amounts, they can also act as pro-oxidants. Accordingly, trace elements influence redox-modulated signaling pathways, such as the Nrf2 pathway. Vice versa, Nrf2 target genes belong to the group of transport and metal binding proteins. In order to investigate whether Nrf2 directly regulates the systemic trace element status, we used mice to study the effect of a constitutive, whole-body Nrf2 knockout on the systemic status of Cu, Zn, Fe, and Se. As the loss of selenoproteins under Se-deprived conditions has been described to further enhance Nrf2 activity, we additionally analyzed the combination of Nrf2 knockout with feeding diets that provide either suboptimal, adequate, or supplemented amounts of Se. Experiments revealed that the Nrf2 knockout partially affected the trace element concentrations of Cu, Zn, Fe, or Se in the intestine, liver, and/or plasma. However, aside from Fe, the other three trace elements were only marginally modulated in an Nrf2-dependent manner. Selenium deficiency mainly resulted in increased plasma Zn levels. One putative mediator could be the metal regulatory transcription factor 1, which was up-regulated with an increasing Se supply and downregulated in Se-supplemented Nrf2 knockout mice.
The α-amylase/trypsin inhibitors (ATIs) are discussed as being responsible for non-celiac wheat sensitivity (NCWS), besides being known as allergenic components for baker’s asthma. Different approaches for characterization and quantification including proteomics-based methods for wheat ATIs have been documented. In these studies generally the major ATIs have been addressed. The challenge of current study was then to develop a more comprehensive workflow encompassing all reviewed wheat-ATI entries in UniProt database. To substantially test proof of concept, 46 German and Turkish wheat samples were used. Two extractions systems based on chloroform/methanol mixture (CM) and under buffered denaturing conditions were evaluated. Three aspects were optimized, tryptic digestion, chromatographic separation, and targeted tandem mass spectrometric analysis (HPLC-MS/MS). Preliminary characterization with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) documented the purity of the extracted ATIs with CM mixture and the amylase (60–80%)/trypsin (10–20%) inhibition demonstrated the bifunctional activity of ATIs. Thirteen (individual/common) biomarkers were established. Major ATIs (7–34%) were differently represented in samples. Finally, to our knowledge, the proposed HPLC-MS/MS method allowed for the first time so far the analysis of all 14 reviewed wheat ATI entries reported.
Many technical challenges still need to be overcome to improve the quality of the green coffee beans. In this work, the wet Arabica coffee processing in batch and continuous modus were investigated. Coffee beans samples as well as by-products and wastewaters collected at different production steps were analyzed in terms of their content in total phenols, antioxidant capacity, caffeine content, organic acids, reducing sugars, free amino group and protein content. The results showed that 40% of caffeine was removed with pulp. Green coffee beans showed highest concentration of organic acids and sucrose (4.96 ± 0.25 and 5.07 ± 0.39 g/100 g DW for the batch and continuous processing). Batch green coffee beans contained higher amount of phenols. 5-caffeoylquinic Acid (5-CQA) was the main constituent (67.1 and 66.0% for the batch and continuous processing, respectively). Protein content was 15 and 13% in the green coffee bean in batch and continuous processing, respectively. A decrease of 50 to 64% for free amino groups during processing was observed resulting in final amounts of 0.8 to 1.4% in the processed beans. Finally, the batch processing still revealed by-products and wastewater with high nutrient content encouraging a better concept for valorization.
Chronic psychosocial stress adversely affects human morbidity and is a risk factor for inflammatory disorders, liver diseases, obesity, metabolic syndrome, and major depressive disorder (MDD). In recent studies, we found an association of MDD with an increase of acid sphingomyelinase (ASM) activity. Thus, we asked whether chronic psychosocial stress as a detrimental factor contributing to the emergence of MDD would also affect ASM activity and sphingolipid (SL) metabolism. To induce chronic psychosocial stress in male mice we employed the chronic subordinate colony housing (CSC) paradigm and compared them to non-stressed single housed control (SHC) mice. We determined Asm activity in liver and serum, hepatic SL concentrations as well as hepatic mRNA expression of genes involved in SL metabolism. We found that hepatic Asm activity was increased by 28% (P = 0.006) and secretory Asm activity by 47% (P = 0.002) in stressed mice. C16:0-Cer was increased by 40% (P = 0.008). Gene expression analysis further revealed an increased expression of tumor necrosis factor (TNF)-alpha (P = 0.009) and of several genes involved in SL metabolism (Cers5, P = 0.028; Cers6, P = 0.045; Gba, P = 0.049; Gba2, P = 0.030; Ormdl2, P = 0.034; Smpdl3B; P = 0.013). Our data thus provides first evidence that chronic psychosocial stress, at least in mice, induces alterations in SL metabolism, which in turn might be involved in mediating the adverse health effects of chronic psychosocial stress and peripheral changes occurring in mood disorders.
Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM's catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.
Prostaglandin (PG)F₂α has previously been shown to increase glucose output from perfused livers and isolated hepatocytes, where it stimulated glycogen phosphorylase via an inositol-trisphosphatedependent signal pathway. In this study, PGF₂α binding sites on hepatocyte plasma membranes, that might represent the putative receptor, were characterized. Binding studies could not be performed with intact hepatocytes, because PGF₂α accumulated within the cells even at 4°C. The intracellular accumulation was an order of magnitude higher than binding to plasma membranes. Purified hepatocyte plasma membranes had a high-affinity/low-capacity and a low-affinity/highcapacity binding'site for PGF₂α. The respective binding constants for the high-affinity site were Kd = 3 nM and Bmax = 6 fmol/mg membrane protein, and for the low-affinity site Kd = 426 nM and Bmax = 245 fmol/mg membrane protein. Specific PGF₂α binding to the low-affinity site, but not to the high-affinity site, could be enhanced most potently by GTP[γS] followed by GDP[ϐS] and GTP, but not by ATP[γS] or GMP. PGF₂α competed most potently with [³H]PGF₂α for specific binding to hepatocyte plasma membranes, followed by PGD₂ and PGE₂. Since the low-affinity PGF₂α-binding site had a Kd in the concentration range in which PG had previously been shown to be half-maximally active, and since this binding site showed a sensitivity to GTP, it is concluded that it might represent the receptor involved in the PGF₂α signal chain in hepatocytes. A biological function of the high-affinity site is currently not known.
Mitochondria are critical for hypothalamic function and regulators of metabolism. Hypothalamic mitochondrial dysfunction with decreased mitochondrial chaperone expression is present in type 2 diabetes (T2D). Recently, we demonstrated that a dysregulated mitochondrial stress response (MSR) with reduced chaperone expression in the hypothalamus is an early event in obesity development due to insufficient insulin signaling. Although insulin activates this response and improves metabolism, the metabolic impact of one of its members, the mitochondrial chaperone heat shock protein 10 (Hsp10), is unknown. Thus, we hypothesized that a reduction of Hsp10 in hypothalamic neurons will impair mitochondrial function and impact brain insulin action. Therefore, we investigated the role of chaperone Hsp10 by introducing a lentiviral-mediated Hsp10 knockdown (KD) in the hypothalamic cell line CLU-183 and in the arcuate nucleus (ARC) of C57BL/6N male mice. We analyzed mitochondrial function and insulin signaling utilizing qPCR, Western blot, XF96 Analyzer, immunohistochemistry, and microscopy techniques. We show that Hsp10 expression is reduced in T2D mice brains and regulated by leptin in vitro. Hsp10 KD in hypothalamic cells induced mitochondrial dysfunction with altered fatty acid metabolism and increased mitochondria-specific oxidative stress resulting in neuronal insulin resistance. Consequently, the reduction of Hsp10 in the ARC of C57BL/6N mice caused hypothalamic insulin resistance with acute liver insulin resistance.