The autocrine role of FGF21 in cultured adipocytes.

Exposure to cold alters glucose and lipid metabolism of white and brown adipose tissue via activation of ß-adrenergic receptor (ADRB). Fibroblast growth factor 21 (FGF21) has been shown to be locally released from adipose tissue upon activation of ADRBs and FGF21 increases glucose uptake in adipocytes. Therefore, FGF21 may play an autocrine role in inducing glucose uptake after ß-adrenergic stimulation. To determine the putative autocrine role of FGF21, we stimulated three different types of adipocytes in vitro with Isoprenaline (Iso), an ADRB agonist, in the presence or absence of the FGF receptor (FGFR) inhibitor PD 173074. The three cell lines represent white (3T3-L1), beige (ME3) and brown (WT-1) adipocyte phenotypes, respectively. All three cells systems expressed ß-klotho (KLB) and FGFR1 after differentiation and treatment with recombinant FGF21 increased glucose uptake in 3T3-L1 and WT-1 adipocytes, while no significant effect was observed in ME3. Oppositely, all three cell lines responded to Iso treatment and an increase in glucose uptake and lipolysis were observed. Interestingly, in response to the Iso treatment only the WT-1 adipocytes showed an increase in FGF21 in the medium. This was consistent with the observation that PD 173074 decreased Iso-induced glucose uptake in the WT-1 adipocytes. This suggests that FGF21 plays an autocrine role and increases glucose uptake after ß-adrenergic stimulation of cultured brown WT-1 adipocytes.

Adjuvants Based on Synthetic Mycobacterial Cord Factor Analogues: Biophysical Properties of Neat Glycolipids and Nanoself-Assemblies with DDA.

Synthetic mycobacterial cord factor analogues, e.g., trehalose 6,6'-dibehenate (TDB), are highly promising adjuvants due to their strong immunopotentiating capabilities, but their biophysical properties have remained poorly characterized. Here, we report the synthesis of an array of synthetic TDB analogues varying in acyl chain length, degree of acylation, and headgroup display, which was subjected to biophysical characterization of neat nondispersed self-assembled nanostructures in excess buffer and as aqueous dispersions with cationic dimethyldioctadecylammonium (DDA) bromide. The array comprised trehalose mono- (TMX) and diester (TDX) analogues with symmetrically shortened acyl chains [denoted by X: arachidate (A), stearate (S), palmitate (P), myristate (Myr), and laurate (L)] and an analogue with a short hydrophilic polyethylene glycol (PEG) linker inserted between the trehalose headgroup of TDS and the acyl chains (PEG-TDS). All dispersions were liposomes, but in contrast to the colloidally stable and highly cationic TDX-containing liposomes, the zeta-potential was significantly reduced for DDA/TMX and DDA/PEG-TDS liposomes, suggesting a charge-shielding effect, which compromises the colloidal stability. An increased d-spacing was observed for the lamellar phase of neat TDB analogues in excess buffer (TDS < TMS < PEG-TDS), confirming that the charge shielding is caused by an extended molecular configuration of the more flexible headgroup. Differential scanning calorimetry showed highly cooperative phase transitions for all tested dispersions albeit the monoesters destabilized the lipid bilayers. Langmuir experiments demonstrated that incorporation of TDXs and PEG-TDS stabilized DDA monolayers due to improved hydrogen bonding and reduced intermolecular repulsions. In conclusion, data suggest that the DDA/TDS dispersions exhibit favorable physicochemical properties rendering these DDA/TDS liposomes an attractive vaccine adjuvant, and they emphasize that not only the receptor binding and immune activation but also the biophysical properties of immunopotentiator formulations should be collectively considered when designing adjuvants with optimal safety, efficacy, and storage stability.

Conserved Molecular Superlattices in a Series of Homologous Synthetic Mycobacterial Cell-Wall Lipids Forming Interdigitated Bilayers.

Synthetic analogues of the cell-wall lipid monomycoloyl glycerol (MMG) are promising as next-generation vaccine adjuvants. In the present study, the thermotropic phase behavior of an array of synthetic MMG analogues was examined by using simultaneous small- and wide-angle X-ray scattering under excess water conditions. The MMG analogues differed in the alkyl chain lengths and in the stereochemistry of the polar glycerol headgroup or of the lipid tails (native-like versus alternative compounds). All MMG analogues formed poorly hydrated lamellar phases at low temperatures and inverse hexagonal (H2) phases at higher temperatures prior to melting. MMG analogues with a native-like lipid acid configuration self-assembled into noninterdigitated bilayers whereas the analogues displaying an alternative lipid acid configuration formed interdigitated bilayers in a subgel (Lc') state. This is in contrast to previously described interdigitated phases for other lipids, which are usually in a gel (Lß) state. All investigated MMG analogues displayed an abrupt direct temperature-induced phase transition from Lc' to H2. This transition is ultimately driven by the lipid chain melting and the accompanying change in molecular shape. No intermediate structures were found, but the entire array of MMG analogues displayed phase coexistence during the lamellar to H2 transition. The structural data also showed that the headgroups of the MMG analogues adopting the alternative lipid acid configuration were ordered and formed a two-dimensional molecular superlattice, which was conserved regardless of the lipid tail length. To our knowledge, the MMG analogues with an alternative lipid acid configuration represent the first example of a lipid system showing both interdigitation and superlattice formation, and as such could serve as an interesting model system for future studies. The MMG analogues are also relevant from a subunit vaccine perspective because they are well-tolerated and display promising immunopotentiating activity. The structural characterization described here will serve as a prerequisite for the rational design of nanoparticulate adjuvants with specific and tailored structural features.

Engineering of small interfering RNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles for highly efficient and safe gene silencing: A quality by design-based approach.

Safety and efficacy of therapeutics based on RNA interference, e.g., small interfering RNA (siRNA), are dependent on the optimal engineering of the delivery technology, which is used for intracellular delivery of siRNA to the cytosol of target cells. We investigated the hypothesis that commonly used and poorly tolerated cationic lipids might be replaced with more efficacious and safe lipidoids as the lipid component of siRNA-loaded lipid-polymer hybrid nanoparticles (LPNs) for achieving more efficient gene silencing at lower and safer doses. However, formulation design of such a complex formulation is highly challenging due to a strong interplay between several contributing factors. Hence, critical formulation variables, i.e. the lipidoid content and siRNA:lipidoid ratio, were initially identified, followed by a systematic quality-by-design approach to define the optimal operating space (OOS), eventually resulting in the identification of a robust, highly efficacious and safe formulation. A 17-run design of experiment with an I-optimal approach was performed to systematically assess the effect of selected variables on critical quality attributes (CQAs), i.e. physicochemical properties (hydrodynamic size, zeta potential, siRNA encapsulation/loading) and the biological performance (in vitro gene silencing and cell viability). Model fitting of the obtained data to construct predictive models revealed non-linear relationships for all CQAs, which can be readily overlooked in one-factor-at-a-time optimization approaches. The response surface methodology further enabled the identification of an OOS that met the desired quality target product profile. The optimized lipidoid-modified LPNs revealed more than 50-fold higher in vitro gene silencing at well-tolerated doses and approx. a twofold increase in siRNA loading as compared to reference LPNs modified with the commonly used cationic lipid dioleyltrimethylammonium propane (DOTAP). Thus, lipidoid-modified LPNs show highly promising prospects for efficient and safe intracellular delivery of siRNA.