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

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.

Hypertonicity during a rapid rise in D-glucose mediates first-phase insulin secretion.

Introduction: Biphasic insulin secretion is an intrinsic characteristic of the pancreatic islet and has clinical relevance due to the loss of first-phase in patients with Type 2 diabetes. As it has long been shown that first-phase insulin secretion only occurs in response to rapid changes in glucose, we tested the hypothesis that islet response to an increase in glucose is a combination of metabolism plus an osmotic effect where hypertonicity is driving first-phase insulin secretion. Methods: Experiments were performed using perifusion analysis of rat, mouse, and human islets. Insulin secretion rate (ISR) and other parameters associated with its regulation were measured in response to combinations of D-glucose and membrane-impermeable carbohydrates (L-glucose or mannitol) designed to dissect the effect of hypertonicity from that of glucose metabolism. Results: Remarkably, the appearance of first-phase responses was wholly dependent on changes in tonicity: no first-phase in NAD(P)H, cytosolic calcium, cAMP secretion rate (cAMP SR), or ISR was observed when increased D-glucose concentration was counterbalanced by decreases in membrane-impermeable carbohydrates. When D-glucose was greater than 8 mM, rapid increases in L-glucose without any change in D-glucose resulted in first-phase responses in all measured parameters that were kinetically similar to D-glucose. First-phase ISR was completely abolished by H89 (a non-specific inhibitor of protein kinases) without affecting first-phase calcium response. Defining first-phase ISR as the difference between glucose-stimulated ISR with and without a change in hypertonicity, the peak of first-phase ISR occurred after second-phase ISR had reached steady state, consistent with the well-established glucose-dependency of mechanisms that potentiate glucose-stimulated ISR. Discussion: The data collected in this study suggests a new model of glucose-stimulated biphasic ISR where first-phase ISR derives from (and after) a transitory amplification of second-phase ISR and driven by hypertonicity-induced rise in H89-inhibitable kinases likely driven by first-phase responses in cAMP, calcium, or a combination of both.

A Compound that Inhibits Glycolysis in Prostate Cancer Controls Growth of Advanced Prostate Cancer.

Purpose: Metastatic castration-resistant prostate cancer remains incurable regardless of recent therapeutic advances. Prostate cancer tumors display highly glycolytic phenotypes as the cancer progresses. Non-specific inhibitors of glycolysis have not been utilized successfully for chemotherapy, because of their penchant to cause systemic toxicity. This study reports the preclinical activity, safety, and pharmacokinetics of a novel small molecule preclinical candidate, BKIDC-1553, with antiglycolytic activity. Experimental design: We tested a large battery of prostate cancer cell lines for inhibition of cell proliferation, in vitro. Cell cycle, metabolic and enzymatic assays were used to demonstrate their mechanism of action. A human PDX model implanted in mice and a human organoid were studied for sensitivity to our BKIDC preclinical candidate. A battery of pharmacokinetic experiments, absorption, distribution, metabolism, and excretion experiments, and in vitro and in vivo toxicology experiments were carried out to assess readiness for clinical trials. Results: We demonstrate a new class of small molecule inhibitors where antiglycolytic activity in prostate cancer cell lines is mediated through inhibition of hexokinase 2. These compounds display selective growth inhibition across multiple prostate cancer models. We describe a lead BKIDC-1553 that demonstrates promising activity in a preclinical xenograft model of advanced prostate cancer, equivalent to that of enzalutamide. BKIDC-1553 demonstrates safety and pharmacologic properties consistent with a compound that can be taken into human studies with expectations of a good safety margin and predicted dosing for efficacy. Conclusion: This work supports testing BKIDC-1553 and its derivatives in clinical trials for patients with advanced prostate cancer.

A Compound That Inhibits Glycolysis in Prostate Cancer Controls Growth of Advanced Prostate Cancer.

Metastatic castration-resistant prostate cancer remains incurable regardless of recent therapeutic advances. Prostate cancer tumors display highly glycolytic phenotypes as the cancer progresses. Nonspecific inhibitors of glycolysis have not been utilized successfully for chemotherapy, because of their penchant to cause systemic toxicity. This study reports the preclinical activity, safety, and pharmacokinetics of a novel small-molecule preclinical candidate, BKIDC-1553, with antiglycolytic activity. We tested a large battery of prostate cancer cell lines for inhibition of cell proliferation, in vitro. Cell-cycle, metabolic, and enzymatic assays were used to demonstrate their mechanism of action. A human patient-derived xenograft model implanted in mice and a human organoid were studied for sensitivity to our BKIDC preclinical candidate. A battery of pharmacokinetic experiments, absorption, distribution, metabolism, and excretion experiments, and in vitro and in vivo toxicology experiments were carried out to assess readiness for clinical trials. We demonstrate a new class of small-molecule inhibitors where antiglycolytic activity in prostate cancer cell lines is mediated through inhibition of hexokinase 2. These compounds display selective growth inhibition across multiple prostate cancer models. We describe a lead BKIDC-1553 that demonstrates promising activity in a preclinical xenograft model of advanced prostate cancer, equivalent to that of enzalutamide. BKIDC-1553 demonstrates safety and pharmacologic properties consistent with a compound that can be taken into human studies with expectations of a good safety margin and predicted dosing for efficacy. This work supports testing BKIDC-1553 and its derivatives in clinical trials for patients with advanced prostate cancer.

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

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, in vivo 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.

The mitochondrially targeted peptide elamipretide (SS-31) improves ADP sensitivity in aged mitochondria by increasing uptake through the adenine nucleotide translocator (ANT).

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, in vivo 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. ELAM binds directly to ANT and ATP synthase and ELAM treatment improves ADP sensitivity, increases ATP production, and improves physiological function in old muscles. ADP (adenosine diphosphate), ATP (adenosine triphosphate), VDAC (voltage-dependent anion channel), ANT (adenine nucleotide translocator), H+ (proton), ROS (reactive oxygen species), NADH (nicotinamide adenine dinucleotide), FADH2 (flavin adenine dinucleotide), O2 (oxygen), ELAM (elamipretide), -SH (free thiol), -SSG (glutathionylated protein).

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 conditions required for the maintenance of function and viability. Toward this end, we have developed a multi-channel flow culture system to maintain tissue and cells in culture and continuously assess function and viability by either in-line sensors and/or collection of outflow fractions. The system combines 8-channel, continuous optical sensing of oxygen consumption rate with a built-in fraction collector to simultaneously measure production rates of metabolites and hormone secretion. Although it is able to maintain and assess a wide range of tissue and cell models, including islets, muscle, and hypothalamus, here we describe its operating principles and the experimental preparations/protocols that we have used to investigate bioenergetic regulation of isolated mouse retina, mouse retinal pigment epithelium (RPE)-choroid-sclera, and cultured human RPE cells. Innovations in the design of the system, such as pumpless fluid flow, have produced a greatly simplified operation of a multi-channel flow system. Videos and images are shown that illustrate how to assemble, prepare the instrument for an experiment, and load the different tissue/cell models into the perifusion chambers. In addition, guidelines for selecting conditions for protocol- and tissue-specific experiments are delineated and discussed, including setting the correct flow rate to tissue ratio to obtain consistent and stable culture conditions and accurate determinations of consumption and production rates. The combination of optimal tissue maintenance and real-time assessment of multiple parameters yields highly informative data sets that will have great utility for research in the physiology of the eye and drug discovery for the treatment of impaired vision.

A peptide triple agonist of GLP-1, neuropeptide Y1, and neuropeptide Y2 receptors promotes glycemic control and weight loss.

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.

A versatile pumpless multi-channel fluidics system for maintenance and real-time functional assessment of tissue and cells.

To address the needs of the life sciences community and the pharmaceutical industry in pre-clinical drug development to both maintain and continuously assess tissue metabolism and function with simple and rapid systems, we improved on the initial BaroFuse to develop it into a fully functional, pumpless, scalable multi-channel fluidics instrument that continuously measures changes in oxygen consumption and other endpoints in response to test compounds. We and several other laboratories assessed it with a wide range of tissue types including retina, pancreatic islets, liver, and hypothalamus with both aqueous and gaseous test compounds. The setup time was less than an hour for all collaborating groups, and there was close agreement between data obtained from the different laboratories. This easy-to-use system reliably generates real-time metabolic and functional data from tissue and cells in response to test compounds that will address a critical need in basic and applied research.

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

Oxygen (O2) and other dissolved gases such as the gasotransmitters H2S, 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 O2 and H2S. Protocols aimed at emulating hypoxia-reperfusion conditions resolved a previously unrecognized transient spike in O2 consumption rate (OCR) following replenishment of O2, and tissue-specific recovery of OCR following hypoxia. The system revealed both inhibitory and stimulatory effects of H2S 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.

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

Corrination is the conjugation of a corrin ring containing molecule, such as vitamin B12 (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 in vivo 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 (22), comparable with Ex4, was successfully screened and subsequently assayed for insulin secretion in rat islets and in vivo in shrews for glucoregulatory and emetic behavior, relative to Ex4.