Mice Deficient in SFRP1 Exhibit Increased Adiposity, Dysregulated Glucose Metabolism

The molecular mechanisms involved in the development of obesity and related complications remain unclear. Wnt signaling plays an important role in preadipocyte differentiation and adipogenesis. The expression of a Wnt antagonist, secreted frizzled related protein 1 (SFRP1), is increased in response to initial weight gain, then levels are reduced under conditions of extreme obesity in both humans and animals. Here we report that loss of Sfrp1 exacerbates weight gain and glucose homeostasis in mice in response to diet induced obesity (DIO). Sfrp1-/- mice fed a high fat diet (HFD) exhibited an increase in body mass accompanied by increases in body fat percentage, visceral WAT mass, and adipocyte size. Fasting glucose levels are elevated, glucose clearance is impaired, hepatic gluconeogenesis regulators are aberrantly upregulated, and glucose transporters are repressed in Sfrp1-/- mice fed a HFD. Additionally, we observed increased steatosis in the livers of Sfrp1-/- mice. Our findings demonstrate that the expression of Sfrp1 is a critical factor required for maintaining appropriate cellular signaling in response to the onset of obesity

Adipocyte lipid synthesis coupled to neuronal control of thermogenic programming

BACKGROUND: The de novo biosynthesis of fatty acids (DNL) through fatty acid synthase (FASN) in adipocytes is exquisitely regulated by nutrients, hormones, fasting, and obesity in mice and humans. However, the functions of DNL in adipocyte biology and in the regulation of systemic glucose homeostasis are not fully understood. METHODS and RESULTS: Here we show adipocyte DNL controls crosstalk to localized sympathetic neurons that mediate expansion of beige/brite adipocytes within inguinal white adipose tissue (iWAT). Induced deletion of FASN in white and brown adipocytes of mature mice (iAdFASNKO mice) enhanced glucose tolerance, UCP1 expression, and cAMP signaling in iWAT. Consistent with induction of adipose sympathetic nerve activity, iAdFASNKO mice displayed markedly increased neuronal tyrosine hydroxylase (TH) and neuropeptide Y (NPY) content in iWAT. In contrast, brown adipose tissue (BAT) of iAdFASNKO mice showed no increase in TH or NPY, nor did FASN deletion selectively in brown adipocytes (UCP1-FASNKO mice) cause these effects in iWAT. CONCLUSIONS: These results demonstrate that downregulation of fatty acid synthesis via FASN depletion in white adipocytes of mature mice can stimulate neuronal signaling to control thermogenic programming in iWAT

Neuronal Modulation of Brown Adipose Activity Through Perturbation of White Adipocyte Lipogenesis [preprint]

White adipose tissue (WAT) secretes factors to communicate with other metabolic organs to maintain energy homeostasis. We previously reported that perturbation of adipocyte de novo lipogenesis (DNL) by deletion of fatty acid synthase (FASN) causes expansion of sympathetic neurons within white adipose tissue (WAT) and the appearance of beige adipocytes. Here we report evidence that white adipocyte DNL activity is also coupled to neuronal regulation and thermogenesis in brown adipose tissue (BAT). Induced deletion of FASN in all adipocytes in mature mice (iAdFASNKO) enhanced sympathetic innervation and neuronal activity as well as UCP1 expression in both WAT and BAT. In contrast, selective ablation of FASN in brown adipocytes of mice (iUCP1FASNKO) failed to modulate sympathetic innervation and the thermogenic program in BAT. Surprisingly, DNL in brown adipocytes was also dispensable in maintaining euthermia when UCP1FASNKO mice were cold-exposed. These results indicate that DNL in white adipocytes influences long distance signaling to BAT, which can modify BAT sympathetic innervation and expression of genes involved in thermogenesis

In Vivo Investigations of Polymer Conjugates as Therapeutics

Polymer conjugates offer a way to introduce materials into the body that would normally be rejected or cause toxicity. Two polymers are investigated in vivo for uses in chemotherapeutic delivery, protein therapeutics, and DNA transfection. A novel polymer, polyMPC, has the ability to increase doxorubicin loading and its solubility, and is conjugated in a way to release its payload in a low pH environment. Through its conjugation, blood clearance time of doxorubicin is increased, and thus tumor exposure to the drug is increased with a single administration. It can be administered at ten times the concentration of free doxorubicin, and three times the concentration of Doxil®, while decreasing the cardio-toxicity normally associated with doxorubicin administration. These results show that polyMPC has the potential to increase treatment efficacy of doxorubicin. With increased circulation time, MPC polymers have additional potential for protein delivery and variations of its design were tested in linear, branched and grafted states, which show limited affect on tissue weight. An additional polymer for use in DNA transfection, NLS2, demonstrated its lack of tissue toxicity when injected intramuscularly. While continued investigation into these polymers is required, this initial data indicates their promising uses as therapeutics

In Vivo Investigations of Polymer Conjugates as Therapeutics

Polymer conjugates offer a way to introduce materials into the body that would normally be rejected or cause toxicity. Two polymers are investigated in vivo for uses in chemotherapeutic delivery, protein therapeutics, and DNA transfection. A novel polymer, polyMPC, has the ability to increase doxorubicin loading and its solubility, and is conjugated in a way to release its payload in a low pH environment. Through its conjugation, blood clearance time of doxorubicin is increased, and thus tumor exposure to the drug is increased with a single administration. It can be administered at ten times the concentration of free doxorubicin, and three times the concentration of Doxil®, while decreasing the cardio-toxicity normally associated with doxorubicin administration. These results show that polyMPC has the potential to increase treatment efficacy of doxorubicin. With increased circulation time, MPC polymers have additional potential for protein delivery and variations of its design were tested in linear, branched and grafted states, which show limited affect on tissue weight. An additional polymer for use in DNA transfection, NLS2, demonstrated its lack of tissue toxicity when injected intramuscularly. While continued investigation into these polymers is required, this initial data indicates their promising uses as therapeutics.Master of Science (M.S.

Rhodiola crenulata induces an early estrogenic response and reduces proliferation and tumorsphere formation over time in MCF7 breast cancer cells

BACKGROUND: Rhodiola crenulata is a Tibetan mountainous plant, commonly used in Eastern alternative medicine. Many phytochemicals possess estrogenic activity, a critical regulator of proliferation in mammary epithelial cells. We have previously characterized anti-cancer properties of R. crenulata in aggressive triple negative breast cancer cells, lacking the expression of estrogen receptor. Currently, it is unknown whether R. crenulata exerts estrogenic effects and as such consumption may be a concern for women with estrogen receptor positive breast cancer that use Rhodiola sp. to relieve mild to moderate depression. PURPOSE: In this study, we wished to determine whether a hydroalcoholic fraction of the R. crenulata root extract exhibits estrogenic activity in estrogen receptor positive (ER+) breast cancer cells in vitro and whether it affects normal mammary epithelial ER target gene expression in vivo. METHODS: ER transcriptional activity was analyzed in MCF7 cells expressing an ERE reporter construct and confirmed via qPCR of endogenous ER target genes. We also monitored cellular proliferation over time. Additionally, to assess stem-like properties in MCF7 cells, we performed a tumorsphere formation assay under anchorage independent conditions. We examined whether R. crenulata treatment reduced β-catenin levels via Western blotting and measured β-catenin transcriptional activity by a reporter assay. To examine the effects of R. crenulata on normal mammary epithelial cells, we performed immunohistochemical staining of ER and PR in the mammary glands of mice fed R. crenulata for 12 weeks. RESULTS: We show an initial activation of ER transcriptional activity by dual reporter assay, qPCR and proliferation of MCF7 ER+ cells in response to 24 h of R. crenulata treatment. However, upon longer treatment basal and R. crenulata induced transcriptional activity was suppressed. There was a decrease in cell doubling times and a decrease in tumorsphere formation. In association with these changes, ERα transcript levels were decreased and active β-catenin levels were reduced in the cells treated for 2 weeks. Finally, we show no change in estrogen targets in normal mammary cells in vivo. CONCLUSION: These data suggest that the R. crenulata extract contains components with estrogenic activity. However, R. crenulata treatment could still be protective in ER+ breast cancer cells, as longer treatment reduced the transcriptional activity of β-catenin and ER responses leading to reduced proliferation and tumorsphere formation. Furthermore, administration of 20 mg/kg/day R. crenulata to mice did not have an observable effect on mammary epithelial ERα target gene expression in vivo

Comparative Analysis of the Mitochondrial Physiology of Pancreatic β Cells

The mitochondrial metabolism of β cells is thought to be highly specialized. Its direct comparison with other cells using isolated mitochondria is limited by the availability of islets/β cells in sufficient quantity. In this study, we have compared mitochondrial metabolism of INS1E/β cells with other cells in intact and permeabilized states. To selectively permeabilize the plasma membrane, we have evaluated the use of perfringolysin-O (PFO) in conjunction with microplate-based respirometry. PFO is a protein that binds membranes based on a threshold level of active cholesterol. Therefore, unless active cholesterol reaches a threshold level in mitochondria, they are expected to remain untouched by PFO. Cytochrome c sensitivity tests showed that in PFO-permeabilized cells, the mitochondrial integrity was completely preserved. Our data show that a time-dependent decline of the oligomycin-insensitive respiration observed in INS1E cells was due to a limitation in substrate supply to the respiratory chain. We predict that it is linked with the β cell-specific metabolism involving metabolites shuttling between the cytoplasm and mitochondria. In permeabilized β cells, the Complex l-dependent respiration was either transient or absent because of the inefficient TCA cycle. The TCA cycle insufficiency was confirmed by analysis of the CO2 evolution. This may be linked with lower levels of NAD+, which is required as a co-factor for CO2 producing reactions of the TCA cycle. β cells showed comparable OxPhos and respiratory capacities that were not affected by the inorganic phosphate (Pi) levels in the respiration medium. They showed lower ADP-stimulation of the respiration on different substrates. We believe that this study will significantly enhance our understanding of the β cell mitochondrial metabolism

Efficacy of PolyMPC–DOX Prodrugs in 4T1 Tumor-Bearing Mice

We report the <i>in vivo</i> efficacy, in tumor-bearing mice, of cancer prodrugs consisting of poly­(methacryloyloxyethyl phosphorylcholine) (polyMPC) conjugated to doxorubicin (DOX). Our synthesis of polyMPC–DOX conjugates established prodrugs with tunable drug loading, pH sensitive release kinetics, and a maximum tolerated dose in the range of 30–50 mg/kg (DOX equivalent) in healthy mice. Here we show prolonged circulation of polyMPC–DOX, with a measured <i>in vivo</i> half-life (<i>t</i>_1/2) 8 times greater than that of the free drug. We observed reduced drug uptake in healthy tissue, and 2–3 times enhanced drug accumulation in tumors for polyMPC–DOX prodrugs compared to free DOX, using BALB/c mice bearing 4T1 tumors. Prolonged survival and reduced tumor growth were observed in mice receiving the polyMPC–DOX prodrug treatment. Moreover, we evaluated immunogenicity of polyMPC–DOX prodrugs by examining complete blood count (CBC) and characteristic cytokine responses, demonstrating no apparent innate or adaptive immune system response

PolyMPC–Doxorubicin Prodrugs

We demonstrate the conjugation of the cancer drug doxorubicin (DOX) to poly­(methacryloyloxyethyl phosphorylcholine) (polyMPC), linked by hydrazone groups, using (1) a one-pot ATRP/click sequence, and (2) a post-polymerization conjugation strategy. While the one-pot method gave polyMPC–DOX conjugates in a facile single step, post-polymerization conjugation gave higher-molecular-weight polymers with very high DOX loadings. DOX release from the polyMPC backbone was pH-dependent (faster at pH 5.0 than at pH 7.4) owing to the hydrazone linkage. Half-life values of DOX release ranged from 2 to 40 h at pH 5.0. Cell culture experiments showed that highly loaded polyMPC–DOX conjugates exhibited higher intracellular drug accumulation and lower half-maximal inhibitory concentration (IC₅₀) values, while a polymer with 30 wt % drug loading showed a maximum tolerated dose in the range of 30–50 mg/kg DOX equivalent weight in healthy mice