High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

Pancreatic ductal adenocarcinoma (PDAC) continues to be a major health problem. A ketogenic diet (KD), characterized by a very low carbohydrate and high fat composition, has gained attention for its anti-tumor potential. We evaluated the effect and mechanisms of feeding a strict KD alone or in combination with gemcitabine in the autochthonous LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx1-Cre (KPC) mouse model. For this purpose, both male and female pancreatic tumor-bearing KPC mice were allocated to a control diet (CD; %kcal: 70% carb, 14% protein, 16% fat), a KD (%kcal: 14% protein, 1% carb, 85% fat), a CD + gemcitabine (CG), or a KD + gemcitabine (KG) group. Mice fed a KD alone or in combination with gemcitabine showed significantly increased blood β-hydroxybutyrate levels compared to mice fed a CD or CG. KPC mice fed a KG had a significant increase in overall median survival compared to KPC mice fed a CD (increased overall median survival by 42%). Interestingly, when the data was disaggregated by sex, the effect of a KG was significant in female KPC mice (60% increase in median overall survival), but not in male KPC mice (28% increase in median overall survival). Mechanistically, the enhanced survival response to a KD combined with gemcitabine was multifactorial, including inhibition of ERK and AKT pathways, regulation of fatty acid metabolism and the modulation of the gut microbiota. In summary, a KD in combination with gemcitabine appears beneficial as a treatment strategy in PDAC in KPC mice, deserving further clinical evaluation.

Prenatal plus postnatal small-quantity lipid-based nutrient supplements (SQ-LNS) improved child growth at 18 months in the International Lipid-Based Nutrient Supplements DYAD trial in Ghana. In this secondary outcome analysis, we determined whether SQ-LNS versus prenatal iron and folic acid (IFA) supplementation improves the cholesterol efflux capacity (CEC) of high-density lipoprotein (HDL) particles and alters their lipidomic, proteomic, or glycoproteomic composition in a subset of 80 children at 18 months of age. HDL CEC was higher among children in the SQ-LNS versus IFA group (20.9 ± 4.1 vs 19.4 ± 3.3%; one-tailed p = 0.038). There were no differences in HDL lipidomic or proteomic composition between groups. Twelve glycopeptides out of the 163 analyzed were significantly altered by SQ-LNS, but none of the group differences remained significant after correction for multiple testing. Exploratory analysis showed that 6 out of the 33 HDL-associated proteins monitored differed in glycopeptide enrichment between intervention groups, and 6 out of the 163 glycopeptides were correlated with CEC. We conclude that prenatal plus postnatal SQ-LNS may modify HDL protein glycoprofiles and improve the CEC of HDL particles in children, which may have implications for subsequent child health outcomes. This trial was registered at as NCT00970866.

Aim: To investigate a novel strategy to target tumor-associated macrophages and reprogram them to an antitumor phenotype in pancreatic adenocarcinoma (PDAC). Methods: M2 peptides were conjugated to HA-PEG/HA-PEI polymer to form self-assembled nanoparticles with miR-125b. The efficacy of HA-PEI/PEG-M2peptide nanoparticles in pancreatic tumors from LSL-KrasG12D/+, LSL-Trp53R172H/+, Pdx1-Cre genetically engineered mice was evaluated. Results: In vitro M2 macrophage-specific delivery of targeted nanoformulations was demonstrated. Intraperitoneal administration of M2-targeted nanoparticles showed preferential accumulation in the pancreas of KPC-PDAC mice and an above fourfold increase in the M1-to-M2 macrophage ratio compared with transfection with scrambled miR. Conclusion: M2-targeted HA-PEI/PEG nanoparticles with miR-125b can transfect tumor-associated macrophages in pancreatic tissues and may have implications for PDAC immunotherapy.

High-density lipoproteins (HDL) play a critical role in cholesterol homeostasis. Apolipoprotein E (APOE), particularly the E4 allele, is a significant risk factor for Alzheimer’s disease but is also a key HDL-associated protein involved in lipid transport in both the periphery and central nervous systems. The objective was to determine the influence of the APOE genotype on HDL function and size in the context of Alzheimer’s disease. HDL from 194 participants (non-demented controls, mild cognitive impairment, and Alzheimer’s disease dementia) were isolated from the plasma. The HDL cholesterol efflux capacity (CEC), lecithin-cholesterol acyltransferase (LCAT) activity, and particle diameter were measured. Neuropsychological test scores, clinical dementia rating, and magnetic resonance imaging scores were used to determine if cognition is associated with HDL function and size. HDL CEC and LCAT activity were reduced in APOE3E4 carriers compared to APOE3E3 carriers, regardless of diagnosis. In APOE3E3 carriers, CEC and LCAT activity were lower in patients. In APOE3E4 patients, the average particle size was lower. HDL LCAT activity and particle size were positively correlated with the neuropsychological scores and negatively correlated with the clinical dementia rating. We provide evidence for the first time of APOE genotype-specific alterations in HDL particles in Alzheimer’s disease and an association between HDL function, size, and cognitive function.

Cytokine signaling in the epidermis has an important role in maintaining barrier function and is perturbed in pathological conditions. Environmental exposures, such as to metal compounds, are of interest for their potential contribution to skin disease. Present work explores the possibility that vanadate is a more effective protein tyrosine phosphatase inhibitor in human keratinocytes than previously observed in fibroblasts. It focuses on the state of phosphorylation of signal transducer and activator of transcription 1 (STAT1) on tyrosine 701 upon treatment of cultured human keratinocytes with the cytokine oncostatin M, a cutaneous inflammatory mediator that is highly effective in suppressing several differentiation markers and in preserving proliferative potential of keratinocytes. Exposure to sodium vanadate in the medium greatly prolonged the phosphorylation of STAT1, but only at high concentration (>30 µM). Inhibitors of protein tyrosine phosphatases known to dephosphorylate STAT1 (SHP2, TCPTP, PTP1B) were ineffective in mimicking the action of vanadate. The irreversible protein tyrosine phosphatase inhibitor phenyl vinyl sulfonate alone induced STAT1 phosphorylation and appeared to induce its limited cleavage. It also inhibited cross-linked envelope formation, a characteristic step of keratinocyte terminal differentiation, likely due to its reaction with the active site cysteine of keratinocyte transglutaminase. Thus, the key protein tyrosine phosphatase responsible for STAT1 dephosphorylation remains to be identified, and an off-target effect of a potential inhibitor was revealed.

Cancer-associated cachexia (CAC) is a critical contributor to pancreatic ductal adenocarcinoma (PDAC) mortality. Thus, there is an urgent need for new strategies to mitigate PDAC-associated cachexia; and the exploration of dietary interventions is a critical component. We previously observed that a ketogenic diet (KD) combined with gemcitabine enhances overall survival in the autochthonous LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx1-Cre (KPC) mouse model. In this study, we investigated the effect and cellular mechanisms of a KD in combination with gemcitabine on the maintenance of skeletal muscle mass in KPC mice. For this purpose, male and female pancreatic tumor-bearing KPC mice were allocated to a control diet (CD), a KD, a CD + gemcitabine (CG), or a KD + gemcitabine (KG) group. We observed that a KD or a KG-mitigated muscle strength declined over time and presented higher gastrocnemius weights compared CD-fed mice. Mechanistically, we observed sex-dependent effects of KG treatment, including the inhibition of autophagy, and increased phosphorylation levels of eIF2α in KG-treated KPC mice when compared to CG-treated mice. Our data suggest that a KG results in preservation of skeletal muscle mass. Additional research is warranted to explore whether this diet-treatment combination can be clinically effective in combating CAC in PDAC patients.

Objectives Cancer cachexia is a multifactorial disorder characterized by involuntary and ongoing wasting of skeletal muscle. More than 80% of patients with pancreatic ductal adenocarcinoma (PDA) suffer from cachexia and up to 20% die directly from it. The ketogenic diet (KD) has been shown to improve health span in old mice. However, its effect in cancer remains elusive. Therefore, our objective was to determine whether a KD mitigates cachexia and/or increases survival in a clinically relevant genetically-engineered LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) model of PDA. Methods After confirming the presence of a pancreatic tumor by high resolution ultrasound imaging, male and female KPC mice were fed either a control diet (CD; %kcal: 14% protein, 70% carb, 16% fat), or a KD (%kcal: 14% protein, < 1% carb, 85% fat) until an endpoint related to PDA was met. Forelimb grip strength, body composition, and non-fasting glucose and ketone levels were evaluated at baseline, monthly and at time of euthanasia. Results After 1 month on the experimental diets, ketone levels were significantly higher in mice fed a KD compared to those fed a CD (p < 0.0001). After two months, KPC mice fed a KD significantly outperformed the KPC mice fed CD in the grip strength test, which assesses skeletal muscle function (p = 0.032). Moreover, the weight of the gastrocnemius muscle was significantly higher in KPC mice fed a KD compared to mice fed a CD (p = 0.024). However, the KD had no significant effect extending KPC survival (p = 0.527), or on the tumor/pancreas weight (p = 0.998). Conclusions A KD maximizes and preserves motor function strength during PDA progression. Additional studies are warranted to evaluate the mechanisms of how KD improves and preserves muscle strength in PDA. Funding Sources Startup funds and a University of California Comprehensive Cancer Center award to GGM. NEC is supported with a fellowship from UC-MEXUS.