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Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell ...

University of Washington

Corrination is the conjugation of a corrin ring containing molecule, such as vitamin B (B12) or B12 biosynthetic precursor dicyanocobinamide (Cbi), to small molecules, peptides, or proteins with the goal of modifying pharmacology. Recently, a corrinated GLP-1R agonist (GLP-1RA) exendin-4 (Ex4) has been shown  to have reduced penetration into the central nervous system relative to Ex4 alone, producing a glucoregulatory GLP-1RA devoid of anorexia and emesis. The study herein was designed to optimize the lead conjugate for GLP-1R agonism and binding. Two specific conjugation sites were introduced in Ex4, while also utilizing various linkers, so that it was possible to identify Cbi conjugates of Ex4 that exhibit improved binding and agonist activity at the GLP-1R. An optimized conjugate (), comparable with Ex4, was successfully screened and subsequently assayed for insulin secretion in rat islets and  in shrews for glucoregulatory and emetic behavior, relative to Ex4.

Synthesis, Optimization, and Biological Evaluation of Corrinated Conjugates of the GLP-1R Agonist Exendin-4.

Oxygen (O) and other dissolved gases such as the gasotransmitters HS, CO, and NO affect cell metabolism and function. To evaluate effects of dissolved gases on processes in tissue, we developed a fluidics system that controls dissolved gases while simultaneously measuring parameters of electron transport, metabolism, and secretory function. We use pancreatic islets, retina, and liver from rodents to highlight its ability to assess effects of O and HS. Protocols aimed at emulating hypoxia-reperfusion conditions resolved a previously unrecognized transient spike in O consumption rate (OCR) following replenishment of O, and tissue-specific recovery of OCR following hypoxia. The system revealed both inhibitory and stimulatory effects of HS on insulin secretion rate from isolated islets. The unique ability of this new system to quantify metabolic state and cell function in response to precise changes in dissolved gases provides a powerful platform for cell physiologists to study a wide range of disease states.

Fluidics system for resolving concentration-dependent effects of dissolved gases on tissue metabolism.

Islet autoantibodies, including autoantibodies directed against the 65kDa isoform of glutamate decarboxylase (GAD65Ab), are present in the majority of patients with newly diagnosed type 1 diabetes (T1D). Whereas these autoantibodies are historically viewed as an epiphenomenon of the autoimmune response with no significant pathogenic function, we consider in this study the possibility that they impact the major islet function, namely glucose-stimulated insulin secretion. Two human monoclonal GAD65Ab (GAD65 mAb) (b78 and b96.11) were investigated for uptake by live rat beta cells, subcellular localization and their effect on glucose-stimulated insulin secretion. The GAD65 mAbs were internalized by live pancreatic beta cells, where they localized to subcellular structures in an epitope-specific manner. Importantly, GAD65 mAb b78 inhibited, while GAD65 mAb b96.11 enhanced, glucose-stimulated insulin secretion (GSIS). These opposite effects on GSIS rule out non-specific effects of the antibodies and suggest that internalization of the antibody leads to epitope-specific interaction with intracellular machinery regulating insulin granule release. The most likely explanation for the alteration of GSIS by GAD65 Abs is via changes in GABA release due to inhibition or change in GAD65 enzyme activity. This is the first report indicating an active role of GAD65Ab in the pathogenesis of T1D.

Autoantibodies directed against glutamate decarboxylase interfere with glucose-stimulated insulin secretion in dispersed rat islets.

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment,  heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function.

Elamipretide Improves ADP Sensitivity in Aged Mitochondria by Increasing Uptake through the Adenine Nucleotide Translocator (ANT).

Mechanisms underlying long-term sustained weight loss and glycemic normalization after obesity surgery include changes in gut hormone levels, including glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). We demonstrate that two peptide biased agonists (GEP44 and GEP12) of the GLP-1, neuropeptide Y1, and neuropeptide Y2 receptors (GLP-1R, Y1-R, and Y2-R, respectively) elicit Y1-R antagonist-controlled, GLP-1R-dependent stimulation of insulin secretion in both rat and human pancreatic islets, thus revealing the counteracting effects of Y1-R and GLP-1R agonism. These agonists also promote insulin-independent Y1-R-mediated glucose uptake in muscle tissue ex vivo and more profound reductions in food intake and body weight than liraglutide when administered to diet-induced obese rats. Our findings support a role for Y1-R signaling in glucoregulation and highlight the therapeutic potential of simultaneous receptor targeting to achieve long-term benefits for millions of patients.

Varun Kamat

UW Medicine Diabetes Institute

University of Washington

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System