Sara Larsson

Green tea powder and Lactobacillus plantarum affect gut microbiota, lipid metabolism and inflammation in high-fat fed C57BL/6J mice

BackgroundType 2 diabetes is associated with obesity, ectopic lipid accumulation and low-grade inflammation. A dysfunctional gut microbiota has been suggested to participate in the pathogenesis of the disease. Green tea is rich in polyphenols and has previously been shown to exert beneficial metabolic effects. Lactobacillus plantarum has the ability to metabolize phenolic acids. The health promoting effect of whole green tea powder as a prebiotic compound has not been thoroughly investigated previously.MethodsC57BL/6J mice were fed a high-fat diet with or without a supplement of 4% green tea powder (GT), and offered drinking water supplemented with Lactobacillus plantarum DSM 15313 (Lp) or the combination of both (Lp + GT) for 22 weeks. Parameters related to obesity, glucose tolerance, lipid metabolism, hepatic steatosis and inflammation were examined. Small intestinal tissue and caecal content were collected for bacterial analysis.ResultsMice in the Lp + GT group had significantly more Lactobacillus and higher diversity of bacteria in the intestine compared to both mice in the control and the GT group. Green tea strongly reduced the body fat content and hepatic triacylglycerol and cholesterol accumulation. The reduction was negatively correlated to the amount of Akkermansia and/or the total amount of bacteria in the small intestine. Markers of inflammation were reduced in the Lp + GT group compared to control. PLS analysis of correlations between the microbiota and the metabolic variables of the individual mice showed that relatively few components of the microbiota had high impact on the correlation model.ConclusionsGreen tea powder in combination with a single strain of Lactobacillus plantarum was able to promote growth of Lactobacillus in the intestine and to attenuate high fat diet-induced inflammation. In addition, a component of the microbiota, Akkermansia, correlated negatively with several metabolic parameters known to be risk factors for the development of type 2 diabetes.

Characterization of the Lipid Droplet Proteome of a Clonal Insulin-producing β-Cell Line (INS-1 832/13)

Lipids are known to play a crucial role both in the normal control of insulin release and in the deterioration of β-cell function, as observed in type 2 diabetes. Despite this established dual role of lipids, little is known about lipid storage and handling in β-cells. Here, we isolated lipid droplets from oleate-incubated INS-1 832/13 cells and characterized the lipid droplet proteome. In a total of four rounds of droplet isolation and proteomic analysis by HPLC-MS/MS, we identified 96 proteins that were specific to droplets. The proteins fall into six categories based on function or previously observed localization: metabolism, endoplasmic reticulum/ribosomes, mitochondria, vesicle formation and transport, signaling, and miscellaneous. The protein profile reinforces the emerging picture of the lipid droplet as an active and dynamic organelle involved in lipid homeostasis and intracellular trafficking. Proteins belonging to the category mitochondria were highly represented, suggesting that the β-cell mitochondria and lipid droplets form a metabolic unit of potential relevance for insulin secretion.

A rapid method for the separation of vitamin D and its metabolites by ultra-high performance supercritical fluid chromatography–mass spectrometry

In this study, a new supercritical fluid chromatography-mass spectrometry (SFC-MS) method has been developed for the separation of nine vitamin D metabolites within less than eight minutes. This is the first study of analysis of vitamin D and its metabolites carried out by SFC-MS. Six columns of orthogonal selectivity were examined, and the best separation was obtained by using a 1-aminoanthracene (1-AA) column. The number and the position of hydroxyl groups in the structure of the studied compounds as well as the number of unsaturated bonds determine the physiochemical properties and, thus the separation of vitamin D metabolites that is achieved on this column. All D2 and the D3 forms were baseline separated with resolution values>1.5. The effects of pressure, temperature, flow rate and different gradient modes were studied. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were compared in positive mode, both by direct infusion and after SFC separation. The results showed that the sensitivity in APCI(+) was higher than in ESI(+) using direct infusion. In contrast, the sensitivity in APCI(+) was 6-fold lower than in ESI(+) after SFC separation. The SFC-MS method was validated between 10 and 500ng/mL for all analytes with coefficient of determination (R(2))≥0.999 for all calibration curves. The limits of detection (LOD) were found to range between 0.39 and 5.98ng/mL for 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1-hydroxyvitamin D2 (1OHD2), respectively. To show its potential, the method was applied to human plasma samples from healthy individuals. Vitamin D3 (D3), 25-hydroxyvitamin D3 (25OHD3) and 24,25(OH)2D3 were determined in plasma samples and the concentrations were 6.6±3.0ng/mL, 23.8±9.2ng/mL and 5.4±2.7ng/mL, respectively.

Perilipin is present in islets of Langerhans and protects against lipotoxicity when overexpressed in the beta-cell line INS-1.

Lipids have been shown to play a dual role in pancreatic beta-cells: a lipid-derived signal appears to be necessary for glucose-stimulated insulin secretion, whereas lipid accumulation causes impaired insulin secretion and apoptosis. The ability of the protein perilipin to regulate lipolysis prompted an investigation of the presence of perilipin in the islets of Langerhans. In this study evidence is presented for perilipin expression in rat, mouse, and human islets of Langerhans as well as the rat clonal beta-cell line INS-1. In rat and mouse islets, perilipin was verified to be present in beta-cells. To examine whether the development of lipotoxicity could be prevented by manipulating the conditions for lipid storage in the beta-cell, INS-1 cells with adenoviral-mediated overexpression of perilipin were exposed to lipotoxic conditions for 72 h. In cells exposed to palmitate, perilipin overexpression caused increased accumulation of triacylglycerols and decreased lipolysis compared with control cells. Whereas glucose-stimulated insulin secretion was retained after palmitate exposure in cells overexpressing perilipin, it was completely abolished in control beta-cells. Thus, overexpression of perilipin appears to confer protection against the development of beta-cell dysfunction after prolonged exposure to palmitate by promoting lipid storage and limiting lipolysis.

Disturbed cholesterol homeostasis in hormone-sensitive lipase-null mice

Transcriptomics analysis revealed that genes involved in hepatic de novo cholesterol synthesis were downregulated in fed HSL-null mice that had been on a high-fat diet (HFD) for 6 mo. This finding prompted a further analysis of cholesterol metabolism in HSL-null mice, which was performed in fed and 16-h-fasted mice on a normal chow diet (ND) or HFD regimen. Plasma cholesterol was elevated in HSL-null mice, in all tested conditions, as a result of cholesterol enrichment of HDL and VLDL. Hepatic esterified cholesterol content and ATP-binding cassette transporter A1 (ABCA1) mRNA and protein levels were increased in HSL-null mice regardless of the dietary regimen. Unsaturated fatty acid composition of hepatic triglycerides was modified in fasted HSL-null mice on ND and HFD. The increased ABCA1 expression had no major effect on cholesterol efflux from HSL-null mouse hepatocytes. Taken together, the results of this study suggest that HSL plays a critical role in the hydrolysis of cytosolic cholesteryl esters and that increased levels of hepatic cholesteryl esters, due to lack of action of HSL in the liver, are the main mechanism underlying the imbalance in cholesterol metabolism in HSL-null mice.

Different cell cycle kinetic effects of N1,N11-diethylnorspermine-induced polyamine depletion in four human breast cancer cell lines.

Polyamine analogues are presently undergoing clinical evaluation in the treatment of cancer. To better understand under what circumstances treatment with a polyamine analogue will yield beneficial results, we have investigated the effect of N1,N11-diethylnorspermine (DENSPM) on cell cycle kinetics of the human breast cancer cell lines SK-BR-3, MCF-7, HCC1937, and L56Br-C1. A bromodeoxyuridine–DNA flow cytometry method was used to evaluate the treatment with 10 μmol/l DENSPM on cell cycle kinetics. A correlation between polyamine pool size after DENSPM treatment and cell cycle kinetic effects was found. The most sensitive cell cycle phase was the S phase, followed by an effect on the G2+M phase and then the G1/S transition. The levels of a number of cell cycle regulatory proteins such as cyclin E1, cyclin A2, and cyclin B1 were lowered by DENSPM treatment, which may explain the effects on cell cycle kinetics. The two cell lines that were most sensitive to DENSPM treatment belong to the basal-like subtype of breast cancer and they were deficient with respect to p53, BRCA1, and RB1.

Parathyroid hormone induces adipocyte lipolysis via PKA-mediated phosphorylation of hormone-sensitive lipase.

Parathyroid hormone (PTH) is secreted from the parathyroid glands in response to low plasma calcium levels. Besides its classical actions on bone and kidney, PTH may have other important effects, including metabolic effects, as suggested for instance by increased prevalence of insulin resistance and type 2 diabetes in patients with primary hyperparathyroidism. Moreover, secondary hyperparathyroidism may contribute to the metabolic derangements that characterize states of vitamin D deficiency. PTH has been shown to induce adipose tissue lipolysis, but the details of the lipolytic action of PTH have not been described. Here we used primary mouse adipocytes to show that intact PTH (1-84) as well as the N-terminal fragment (1-37) acutely stimulated lipolysis in a dose-dependent manner, whereas the C-terminal fragment (38-84) was without lipolytic effect. The lipolytic action of PTH was paralleled by phosphorylation of known protein kinase A (PKA) substrates, i.e. hormone-sensitive lipase (HSL) and perilipin. The phosphorylation of HSL in response to PTH occurred at the known PKA sites S563 and S660, but not at the non-PKA site S565. PTH-induced lipolysis, as well as phosphorylation of HSL at S563 and S660, was blocked by both the PKA-inhibitor H89 and the adenylate cyclase inhibitor MDL-12330A, whereas inhibitors of extracellular-regulated kinase (ERK), protein kinase B (PKB), AMP-activated protein kinase (AMPK) and Ca(2+)/calmodulin-dependent protein kinase (CaMK) had little or no effect. Inhibition of phosphodiesterase 4 (PDE4) strongly potentiated the lipolytic action of PTH, whereas inhibition of PDE3 had no effect. Our results show that the lipolytic action of PTH is mediated by the PKA signaling pathway with no or minor contribution of other signaling pathways and, furthermore, that the lipolytic action of PTH is limited by simultaneous activation of PDE4. Knowledge of the signaling pathways involved in the lipolytic action of PTH is important for our understanding of how metabolic derangements develop in states of hyperparathyroidism, including vitamin D deficiency.

Lack of cholesterol mobilization in islets of hormone-sensitive lipase deficient mice impairs insulin secretion.

The observations that hormone-sensitive lipase (HSL) is located in close association to insulin granules in beta-cells and that cholesterol ester hydrolase activity is completely blunted in islets of HSL null mice made us hypothesize that the role of HSL in beta-cells is to provide cholesterol for the exocytosis of insulin. To test this hypothesis, wild type (wt) and HSL null islets were depleted of plasma membrane cholesterol using methyl-beta-cyclodextrin (mbetacd). A significant reduction in insulin secretion from HSL null islets was observed whereas wt islets were unaffected. Using synaptosomal protein of 25 kDa (SNAP-25) as indicator of cholesterol-rich microdomains, confocal microscopy was used to show that HSL null beta-cells treated with mbetacd contained fewer clusters than wt beta-cells. These results indicate that HSL plays an important role in insulin secretion by providing free cholesterol for the formation and maintenance of cholesterol-rich patches for docking of SNARE-proteins to the plasma membrane.

Estimating the Total Rate of DNA Replication Using Branching Processes

Increasing the knowledge of various cell cycle kinetic parameters, such as the length of the cell cycle and its different phases, is of considerable importance for several purposes including tumor diagnostics and treatment in clinical health care and a deepened understanding of tumor growth mechanisms. Of particular interest as a prognostic factor in different cancer forms is the S phase, during which DNA is replicated. In the present paper, we estimate the DNA replication rate and the S phase length from bromodeoxyuridine-DNA flow cytometry data. The mathematical analysis is based on a branching process model, paired with an assumed gamma distribution for the S phase duration, with which the DNA distribution of S phase cells can be expressed in terms of the DNA replication rate. Flow cytometry data typically contains rather large measurement variations, however, and we employ nonparametric deconvolution to estimate the underlying DNA distribution of S phase cells; an estimate of the DNA replication rate is then provided by this distribution and the mathematical model.

A Markov model approach shows a large variation in the length of S phase in MCF-7 breast cancer cells.

The potential doubling time of a tumor has been suggested to be a measurement of tumor aggressiveness; therefore, it is of interest to find reliable methods to estimate this time. Because of variability in length of the various cell cycle phases, stochastic modeling of the cell cycle might be a suitable approach.