High fat diet is associated with gut microbiota dysbiosis and decreased gut microbial derived metabolites related to metabolic health in young Göttingen Minipigs.

The objectives were 1) to characterize a Göttingen Minipig model of metabolic syndrome regarding its colon microbiota and circulating microbial products, and 2) to assess whether ovariectomized female and castrated male minipigs show similar phenotypes. Twenty-four nine-week-old Göttingen Minipigs were allocated to four groups based on sex and diet: ovariectomized females and castrated males fed either chow or high-fat diet (HFD) for 12 weeks. At study end, body composition and plasma biomarkers were measured, and a mixed meal tolerance test (MMT) and an intravenous glucose tolerance test (IVGTT) were performed. The HFD groups had significantly higher weight gain, fat percentage, fasting plasma insulin and glucagon compared to the chow groups. Homeostatic model assessment of insulin resistance index (HOMA-IR) was increased and glucose effectiveness derived from the IVGTT and Matsuda´s insulin sensitivity index from the MMT were decreased in the HFD groups. The HFD groups displayed dyslipidemia, with significantly increased total-, LDL- and HDL-cholesterol, and decreased HDL/non-HDL cholesterol ratio. The colon microbiota of HFD minipigs clearly differed from the lean controls (GuniFrac distance matrix). The main bacteria families driving this separation were Clostridiaceae, Fibrobacteraceae, Flavobacteriaceae and Porphyromonadaceae. Moreover, the species richness was significantly decreased by HFD. In addition, HFD decreased the circulating level of short chain fatty acids and beneficial microbial metabolites hippuric acid, xanthine and trigonelline, while increasing the level of branched chain amino acids. Six and nine metabolically relevant genes were differentially expressed between chow-fed and HFD-fed animals in liver and omental adipose tissue, respectively. The HFD-fed pigs presented with metabolic syndrome, gut microbial dysbiosis and a marked decrease in healthy gut microbial products and thus displayed marked parallels to human obesity and insulin resistance. HFD-fed Göttingen Minipig therefore represents a relevant animal model for studying host-microbiota interactions. No significant differences between the castrated and ovariectomized minipigs were observed.

Marked gut microbiota dysbiosis and increased imidazole propionate are associated with a NASH Göttingen Minipig model.

BACKGROUND: Gut microbiota dysbiosis is associated with the development of non-alcoholic steatohepatitis (NASH) through modulation of gut barrier, inflammation, lipid metabolism, bile acid signaling and short-chain fatty acid production. The aim of this study was to describe the impact of a choline-deficient amino acid defined high fat diet (CDAHFD) on the gut microbiota in a male Göttingen Minipig model and on selected pathways implicated in the development of NASH. RESULTS: Eight weeks of CDAHFD resulted in a significantly altered colon microbiota mainly driven by the bacterial families Lachnospiraceae and Enterobacteriaceae, being decreased and increased in relative abundance, respectively. Metabolomics analysis revealed that CDAHFD decreased colon content of short-chain fatty acid and increased colonic pH. In addition, serum levels of the microbially produced metabolite imidazole propionate were significantly elevated as a consequence of CDAHFD feeding. Hepatic gene expression analysis showed upregulation of mechanistic target of rapamycin (mTOR) and Ras Homolog, MTORC1 binding in addition to downregulation of insulin receptor substrate 1, insulin receptor substrate 2 and the glucagon receptor in CDAHFD fed minipigs. Further, the consequences of CDAHFD feeding were associated with increased levels of circulating cholesterol, bile acids, and glucagon but not total amino acids. CONCLUSIONS: Our results indicate imidazole propionate as a new potentially relevant factor in relation to NASH and discuss the possible implication of gut microbiota dysbiosis in the development of NASH. In addition, the study emphasizes the need for considering the gut microbiota and its products when developing translational animal models for NASH.

Molecular Engineering of Efficacious Mono-Valent Ultra-Long Acting Two-Chain Insulin-Fc Conjugates.

Here, we describe molecular engineering of monovalent ultra-long acting two-chain insulin-Fc conjugates. Insulin-Fc conjugates were synthesized using trifunctional linkers with one amino reactive group for reaction with a lysine residue of insulin and two thiol reactive groups used for re-bridging of a disulfide bond within the Fc molecule. The ultra-long pharmacokinetic profile of the insulin-Fc conjugates was the result of concertedly slowing insulin receptor-mediated clearance by (1) introduction of amino acid substitutions that lowered the insulin receptor affinity and (2) conjugating insulin to the Fc element. Fc conjugation leads to recycling by the neonatal Fc receptor and increase in the molecular size, both contributing to the ultra-long pharmacokinetic and pharmacodynamic profiles.

Optimization of pig models for translation of subcutaneous pharmacokinetics of therapeutic proteins: Liraglutide, insulin aspart and insulin detemir.

Prediction of human pharmacokinetics (PK) from data obtained in animal studies is essential in drug development. Here, we present a thorough examination of how to achieve good pharmacokinetic data from the pig model for translational purposes by using single-species allometric scaling for selected therapeutic proteins: liraglutide, insulin aspart and insulin detemir. The predictions were based on non-compartmental analysis of intravenous and subcutaneous PK data obtained from two injection regions (neck, thigh) in two pig breeds, domestic pig and Göttingen Minipig, that were compared with PK parameters reported in humans. The effects of pig breed, injection site and injection depth (insulin aspart only) on the PK of these proteins were also assessed. Results show that the prediction error for human PK was within two-fold for most PK parameters in both pig breeds. Furthermore, pig breed significantly influenced the plasma half-life and mean absorption time (MAT), both being longer in Göttingen Minipigs compared to domestic pigs (P <0.01). In both breeds, thigh vs neck dosing was associated with a higher dose-normalized maximum plasma concentration and area under the curve as well as shorter MAT and plasma half-life (P <0.01). Finally, more superficial injections resulted in faster absorption, higher Cmax/dose and bioavailability of insulin aspart (P <0.05, 3.0 vs 5.0 mm injection depth). In conclusion, pig breed and injection region affected the PK of liraglutide, insulin aspart and insulin detemir and reliable predictions of human PK were demonstrated when applying single-species allometric scaling with the pig as a pre-clinical animal model.

Spatial distribution of soluble insulin in pig subcutaneous tissue: Effect of needle length, injection speed and injected volume.

The spatial distribution of a soluble insulin formulation was visualized and quantified in 3-dimensions using X-ray computed tomography. The drug distribution was visualized for ex vivo injections in pig subcutaneous tissue. Pig subcutaneous tissue has very distinct layers, which could be separated in the tomographic reconstructions and the amount of drug in each tissue class was quantified. With a scan time of about 45min per sample, and a robust segmentation it was possible to analyze differences in the spatial drug distribution between several similar injections. It was studied how the drug distribution was effected by needle length, injection speed and injected volume. For an injected volume of 0.1ml and injection depth of 8mm about 50% of the injections were partly intramuscular. Using a 5mm needle resulted in purely subcutaneous injections with minor differences in the spatial drug distribution between injections. Increasing the injected volume from 0.1ml to 1ml did not increase the intramuscular volume fraction, but gave a significantly higher volume fraction placed in the fascia separating the deep and superficial subcutaneous fat layers. Varying the injection speed from 25l/s up to 300l/s gave no changes in the drug concentration distribution. The method presented gives novel insight into subcutaneous injections of soluble insulin drugs and can be used to optimize the injection technique for subcutaneous drug administration in preclinical studies of rodents.