Rapid cold hardening increases axonal Na+/K+-ATPase activity and enhances performance of a visual motion detection circuit in Locusta migratoria.

Rapid cold hardening (RCH) is a type of phenotypic plasticity that delays the occurrence of chill coma in insects. Chill coma is mediated by a spreading depolarization of neurons and glia in the CNS, triggered by a failure of ion homeostasis. We used biochemical and electrophysiological approaches in the locust, Locusta migratoria, to test the hypothesis that the protection afforded by RCH is mediated by activation of the Na+/K+-ATPase (NKA) in neural tissue. RCH did not affect NKA activity measured in a biochemical assay of homogenized thoracic ganglia. However, RCH hyperpolarized the axon of a visual interneuron (DCMD) and increased the amplitude of an activity-dependent hyperpolarization (ADH) shown previously to be blocked by ouabain. RCH also improved performance of the visual circuitry presynaptic to DCMD to minimize habituation and increase excitability. We conclude that RCH enhances in situ NKA activity in the nervous system but also affects other neuronal properties that promote visual processing in locusts.

Discovery of Insulin Receptor Partial Agonists MK-5160 and MK-1092 as Novel Basal Insulins with Potential to Improve Therapeutic Index.

We have identified a series of novel insulin receptor partial agonists (IRPAs) with a potential to mitigate the risk of hypoglycemia associated with the use of insulin as an antidiabetic treatment. These molecules were designed as dimers of native insulin connected via chemical linkers of variable lengths with optional capping groups at the N-terminals of insulin chains. Depending on the structure, the maximal activation level (%Max) varied in the range of ∼20-70% of native insulin, and EC50 values remained in sub-nM range. Studies in minipig and dog demonstrated that IRPAs had sufficient efficacy to normalize plasma glucose levels in diabetes, while providing reduction of hypoglycemia risk. IRPAs had a prolonged duration of action, potentially making them suitable for once-daily dosing. Two lead compounds with %Max values of 30 and 40% relative to native insulin were selected for follow up studies in the clinic.

Utilizing comparative models in biomedical research.

This review serves as an introduction to a Special Issue of Comparative Biochemistry and Physiology, focused on using non-human models to study biomedical physiology. The concept of a model differs across disciplines. For example, several models are used primarily to gain an understanding of specific human pathologies and disease states, whereas other models may be focused on gaining insight into developmental or evolutionary mechanisms. It is often the case that animals initially used to gain knowledge of some unique biochemical or physiological process finds foothold in the biomedical community and becomes an established model. The choice of a particular model for biomedical research is an ongoing process and model validation must keep pace with existing and emerging technologies. While the importance of non-mammalian models, such as Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Xenopus laevis, is well known, we also seek to bring attention to emerging alternative models of both invertebrates and vertebrates, which are less established but of interest to the comparative biochemistry and physiology community.

WITHDRAWN: Utilizing comparative models in biomedical research.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, Volume 255, 2021, 110593, https://doi.org/10.1016/j.cbpb.2021.110593. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Measuring enzyme activities in crude homogenates: Na+/K+-ATPase as a case study in optimizing assays.

In this review of assays of Na+/K+-ATPase (NKA), we explore the choices made by researchers assaying the enzyme to investigate its role in physiological regulation. We survey NKA structure and function in the context of how it is typically assayed, and how technical choices influence what can be said about the enzyme. In comparing different methods for extraction and assay of NKA, we identified a series of common pitfalls that compromise the veracity of results. We include experimental work to directly demonstrate how choices in detergents, salts and substrates influence NKA activities measured in crude homogenates. Our review of assay approaches integrates what is known from enzymology, biomedical physiology, cell biology and evolutionary biology, offering a more robust method for assaying the enzyme in meaningful ways, identifying caveats and future directions to explore its structure and function. The goal is to provide the sort of background on the enzyme that should be considered in exploring the function of the enzyme in comparative physiology.

Getting the most out of reductionist approaches in comparative biochemistry and physiology.

This review serves as an introduction to a Special Issue of CBP focused on the use of reductionist approaches to explore questions in comparative biochemistry and physiology of animals. An overarching goal for research is to provide new insight and knowledge to advance the field. The significance of the research is dependent upon utilizing the most appropriate approach to get the most reliable data, which requires being knowledgeable about the experimental system and its limitations. It is not a trivial task to decide which level of biological organization is best suited to answer the question of interest, because each choice is a balance between strengths and weaknesses. Reporting caveats and limitations is perceived to detract from the definitiveness and value of a study, and so these are typically avoided, or included begrudgingly to appease a reviewer. Reductionist approaches are most valuable when the results can be translated to other biological levels of organization, providing physiological context for the work. Such extensions must also be accompanied by the appropriate assumptions and caveats arising from both the experimental system or its translation to higher levels of biological organization. In preparing this review, we seek to encourage authors to share the weaknesses and caveats in their approaches, and address the challenges associated with demonstrating the relevance of a reductionist approach to higher levels of organization.

Special issue on aquaculture: New opportunities to address global food supply for comparative biochemistry and physiology.

This article serves as an introduction to a Virtual Special Issue of Comparative Biochemistry and Physiology (CBP) focused on aquaculture. CBP has not traditionally had a focus on aquaculture, and the Editors sought to use this Special Issue to identify opportunities for synergy between traditional comparative physiology and applied physiology, such as aquaculture. Each of the four CBP journals has a dedicated special issue, with manuscripts that span the breadth of vertebrate and invertebrate species cultured around the globe. This overview is intended to identify the major themes of the submissions, as well as articulate a vision for the types of aquaculture-focused research that are well suited for CBP publications.

Superior Glycemic Control With a Glucose-Responsive Insulin Analog: Hepatic and Nonhepatic Impacts.

We evaluated the hepatic and nonhepatic responses to glucose-responsive insulin (GRI). Eight dogs received GRI or regular human insulin (HI) in random order. A primed, continuous intravenous infusion of [3-3H]glucose began at -120 min. Basal sampling (-30 to 0 min) was followed by two study periods (150 min each), clamp period 1 (P1) and clamp period 2 (P2). At 0 min, somatostatin and GRI (36 ± 3 pmol/kg/min) or HI (1.8 pmol/kg/min) were infused intravenously; basal glucagon was replaced intraportally. Glucose was infused intravenously to clamp plasma glucose at 80 mg/dL (P1) and 240 mg/dL (P2). Whole-body insulin clearance and insulin concentrations were not different in P1 versus P2 with HI, but whole-body insulin clearance was 23% higher and arterial insulin 16% lower in P1 versus P2 with GRI. Net hepatic glucose output was similar between treatments in P1. In P2, both treatments induced net hepatic glucose uptake (HGU) (HI mean ± SEM 2.1 ± 0.5 vs. 3.3 ± 0.4 GRI mg/kg/min). Nonhepatic glucose uptake in P1 and P2, respectively, differed between treatments (2.6 ± 0.3 and 7.4 ± 0.6 mg/kg/min with HI vs. 2.0 ± 0.2 and 8.1 ± 0.8 mg/kg/min with GRI). Thus, glycemia affected GRI but not HI clearance, with resultant differential effects on HGU and nonHGU. GRI holds promise for decreasing hypoglycemia risk while enhancing glucose uptake under hyperglycemic conditions.

Sensing and responding to energetic stress: Evolution of the AMPK network.

AMP-activated protein kinase is an enzyme that mediates communication between cellular energy status and diverse effector proteins, particularly those that play roles in determining the metabolic phenotype. By phosphorylating metabolic enzymes, transcriptional regulators and proteins involved in cellular structure, it can modify energy metabolism in both the short term and long term. Its basic features are highly conserved, with homologues in all eukaryotes. Gene and/or genome duplications endowed early vertebrates with paralogs of AMPK subunits, though the nature of their subfunctionalization remains uncertain, even in mammals. While most research focuses on the role of the enzyme in human health, a great deal can be learned from comparative studies targeting non-traditional model animals. Fish, in particular, are interesting models because of the diversity in the metabolic properties and complex relationships between metabolism and environmental challenges. In this review, we examine what is known about AMPK structure and function though the lens of comparative physiology, looking for opportunities to better understand how this vital energy sensor has evolved in animals.

Subfunctionalization of COX4 paralogs in fish.

Cytochrome c oxidase (COX) subunit 4 has two paralogs in most vertebrates. The mammalian COX4-2 gene is hypoxia responsive, and the protein has a disrupted ATP-binding site that confers kinetic properties on COX that distinguish it from COX4-1. The structure-function of COX4-2 orthologs in other vertebrates remains uncertain. Phylogenetic analyses suggest the two paralogs arose in basal vertebrates, but COX4-2 orthologs diverged faster than COX4-1 orthologs. COX4-1/4-2 protein levels in tilapia tracked mRNA levels across tissues, and did not change in hypoxia, arguing against a role for differential post-translational regulation of paralogs. The heart, and to a lesser extent the brain, showed a size-dependent shift from COX4-1 to COX4-2 (transcript and protein). ATP allosterically inhibited both velocity and affinity for oxygen in COX assayed from both muscle (predominantly COX4-2) and gill (predominantly COX4-1). We saw some evidence of cellular and subcellular discrimination of COX4 paralogs in heart. In cardiac ventricle, some non-cardiomyocyte cells were COX positive but lacked detectible COX4-2. Within heart, the two proteins partitioned to different mitochondrial subpopulations. Cardiac subsarcolemmal mitochondria had mostly COX4-1 and intermyofibrillar mitochondria had mostly COX4-2. Collectively, these data argue that, despite common evolutionary origins, COX4-2 orthologs of fish show unique patterns of subfunctionalization with respect to transcriptional and posttranslation regulation relative to the rodents and primates that have been studied to date.

Investigation of piperazine benzamides as human ß3 adrenergic receptor agonists for the treatment of overactive bladder.

The synthesis of a novel class of piperazine benzamide (reverse amides) targeting the human ß3-adrenergic receptor for the treatment of overactive bladder (OAB) is described. The SAR studies directed towards maintaining well established ß3 potency and selectivities while improving the overall pharmacokinetic profile in the reverse amide class will be evaluated. The results and consequences associated with functional activity at the norepinephrine transporter (NET) will also be discussed.

Evaluating the role of NRF-1 in the regulation of the goldfish COX4-1 gene in response to temperature.

Cold acclimation in fish typically increases muscle mitochondrial enzymes. In mammals, stressors that increase mitochondrial content are mediated though transcriptional regulators, including nuclear respiratory factor-1 (NRF-1). Focusing on the goldfish gene for cytochrome c oxidase (COX) subunit 4-1, we analysed the regulatory regions in various contexts to identify a mechanistic link between NRF-1 and cold-induced mitochondrial proliferation. Promoter analysis implicated two putative NRF-1 sites: one in the proximal promoter and a second in exon 1, which encodes the 5' untranslated region (5'-UTR). Transfection into mouse myoblasts showed that deletion of a region that included the proximal NRF-1 site reduced promoter activity by 30%; however, mutagenesis of the specific sequence had no effect. Thermal sensitivity analyses performed in rainbow trout gonadal fibroblasts (RTG-2) showed no effect of temperature (4 vs 19°C) on reporter gene expression. Likewise, reporters injected into muscle of thermally acclimated goldfish (4 vs 26°C) showed no elevation in expression. There was no difference in thermal responses of COX4-1 promoter reporters constructed from homologous regions of eurythermal goldfish and stenothermal zebrafish genes. NRF-1 chromatin immunoprecipitation of thermally acclimated goldfish muscle showed no temperature effect on NRF-1 binding to either the proximal promoter or 5'-UTR. It remains possible that the cold-induced upregulation of COX4-1 expression is a result of NRF-1 binding to distal regulatory regions or through indirect effects on other transcription factors. However, the proximal promoter does not appear to play a role in mediating the thermal response of the COX4-1 gene in fish.

Discovery of Vibegron: A Potent and Selective ß3 Adrenergic Receptor Agonist for the Treatment of Overactive Bladder.

The discovery of vibegron, a potent and selective human ß3-AR agonist for the treatment of overactive bladder (OAB), is described. An early-generation clinical ß3-AR agonist MK-0634 (3) exhibited efficacy in humans for the treatment of OAB, but development was discontinued due to unacceptable structure-based toxicity in preclinical species. Optimization of a series of second-generation pyrrolidine-derived ß3-AR agonists included reducing the risk for phospholipidosis, the risk of formation of disproportionate human metabolites, and the risk of formation of high levels of circulating metabolites in preclinical species. These efforts resulted in the discovery of vibegron, which possesses improved druglike properties and an overall superior preclinical profile compared to MK-0634. Structure-activity relationships leading to the discovery of vibegron and a summary of its preclinical profile are described.

Design of Potent and Orally Active GPR119 Agonists for the Treatment of Type II Diabetes.

We report herein the design and synthesis of a series of potent and selective GPR119 agonists. Our objective was to develop a GPR119 agonist with properties that were suitable for fixed-dose combination with a DPP4 inhibitor. Starting from a phenoxy analogue (1), medicinal chemistry efforts directed toward reducing half-life and increasing solubility led to the synthesis of a series of benzyloxy analogues. Compound 28 was chosen for further profiling because of its favorable physicochemical properties and excellent GPR119 potency across species. This compound exhibited a clean off-target profile in counterscreens and good in vivo efficacy in mouse oGTT.

mRNA degradation: an underestimated factor in steady-state transcript levels of cytochrome c oxidase subunits?

Steady-state mRNA levels are determined by synthesis and degradation; however, changes in mRNA levels are usually attributed to transcription. For cytochrome c oxidase (COX), cold acclimation typically leads to an increase in COX activity while transcript levels for the nuclear-encoded subunits change non-stoichiometrically. Whether those patterns are caused by differences in subunit transcription rates, decay rates or both was not known. We assessed decay rates of transcripts for COX subunits, including representatives that decreased, increased in parallel with COX or increased in excess of COX. Low temperature reduced the decay rate of all transcripts; however, COX subunits displayed higher thermal sensitivity than housekeeping genes. The lower decay rates for COX transcripts might explain some of their increase in response to cold acclimation. The reason for the exaggerated transcript response of two subunits (COX6B-1 and COX7A-2) may be due to decreased decay. However, decay rate differences could not explain the patterns seen with another subunit that did not change in mRNA level with thermal acclimation (COX6A-2). Further, the decay patterns differed between two thermal acclimation experiments, which may explain some of the heterogeneity seen in fish studies. The differences in decay rates suggest that the lack of stoichiometry in mRNA levels is exacerbated by post-transcriptional mechanisms. Collectively, these results suggest that temperature-induced differences in COX subunit mRNA levels and deviations from stoichiometry between them may partially arise from subunit-specific sensitivities to degradation. We suggest that all subunits are controlled by transcription, and that exaggerated responses of some subunits are due to reduced decay rates.

Design, synthesis, and evaluation of conformationally restricted acetanilides as potent and selective ß3 adrenergic receptor agonists for the treatment of overactive bladder.

A series of conformationally restricted acetanilides were synthesized and evaluated as ß3-adrenergic receptor agonists (ß3-AR) for the treatment of overactive bladder (OAB). Optimization studies identified a five-membered ring as the preferred conformational lock of the acetanilide. Further optimization of both the aromatic and thiazole regions led to compounds such as 19 and 29, which have a good balance of potency and selectivity. These compounds have significantly reduced intrinsic clearance compared to our initial series of pyridylethanolamine ß3-AR agonists and thus have improved unbound drug exposures. Both analogues demonstrated dose dependent ß3-AR mediated responses in a rat bladder hyperactivity model.

Metabolism in the age of 'omes'.

Much research in comparative physiology is now performed using 'omics' tools and many results are interpreted in terms of the effects of changes in gene expression on energy metabolism. However, 'metabolism' is a complex phenomenon that spans multiple levels of biological organization. In addition rates and directions of flux change dynamically under various physiological circumstances. Within cells, message level cannot be equated with protein level because multiple mechanisms are at play in the 'regulatory hierarchy' from gene to mRNA to enzyme protein. This results in many documented instances wherein change in mRNA levels and change in enzyme levels are unrelated. It is also known from metabolic control analysis that the influence of single steps in pathways on flux is often small. Flux is a system property and its control tends to be distributed among multiple steps. Consequently, change in enzyme levels cannot be equated with change in flux. Approaches developed by Hans Westerhoff and colleagues, called 'hierarchical regulation analysis', allow quantitative determination of the extent to which 'hierarchical regulation', involving change in enzyme level, and 'metabolic regulation', involving the modulation of the activity of preexisting enzyme, regulate flux. We outline these approaches and provide examples to show their applicability to problems of interest to comparative physiologists.

Transcriptional regulation of temperature-induced remodeling of muscle bioenergetics in goldfish.

Central to mammalian mitochondrial biogenesis is the transcriptional master regulator peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α), and a network of DNA-binding proteins it coactivates. We explored the role of this pathway in muscle mitochondrial biogenesis in response to thermal acclimation in goldfish (Carassius auratus). We investigated the transcriptional response of PGC-1α, PGC-1ß, and their antagonist the nuclear receptor interacting protein 1 (RIP140), as well as the mRNA and protein patterns of DNA-binding proteins that bind PGC-1, including nuclear respiratory factors (NRF) 1 and 2, retinoid X receptor α (RXRα), estrogen-related receptor α (ERRα), thyroid receptor α-1 (TRα-1), PPARα, and PPARß/δ, and the host cell factor 1 (HCF1), which links PGC-1 and NRF-2. Cold-acclimated (4°C) fish had higher COX activities (4.5-fold) and COX4-1 mRNA levels (3.5-fold per total RNA; 6.5-fold per gram tissue) than warm-acclimated (32°C) fish. The transcription factor patterns were profoundly influenced by changes in RNA per gram tissue (2-fold higher in cold fish) and nuclear protein content (2-fold higher in warm fish). In cold-acclimated fish, mRNA per gram tissue was elevated for PGC-1ß, RIP140, NRF-1, HCF1, NRF-2α, NRF-2ß-2, ERRα, PPAR ß/δ, and RXRα, but other transcriptional regulators either did not change (PGC-1α, PPARα) or even decreased (TRα-1). Nuclear protein levels in cold-acclimated fish were higher only for NRF-1; other proteins were either unaffected (NRF-2α, ERRα) or decreased (NRF-2ß1/2, TRα, RXRα). Collectively, these data support the role for NRF-1 in regulating cold-induced mitochondrial biogenesis in goldfish, with effects mediated by PGC-1ß, rather than PGC-1α.

Evolution of mitochondrial-encoded cytochrome oxidase subunits in endothermic fish: the importance of taxon-sampling in codon-based models.

While endothermy is ubiquitous in birds and mammals, it is not exclusive to these most recently arisen vertebrate classes. The ability to warm specific organs and/or tissues above ambient temperature (regional endothermy) has evolved at least three times in phylogentically discrete fish lineages: lamnid sharks (Lamnidae), tunas (Scombridae) and billfishes (Istiophoridae and Xiphidae). Given the links between endothermy and metabolic rate, we looked for evidence of convergent molecular evolution in mtDNA-encoded cytochrome c oxidase (COX) subunits in each of these discrete lineages. We found no evidence that the endothermic phenotype in fishes is driven or accompanied by molecular convergence. Though we found little evidence for positively-selected sites in any of the lineages in any subunit, the conclusions were sensitive to the choice of maximum-likelihood model. Several sites identified by Naïve Empirical Bayes (NEB) were not found when Bayes Empirical Bayes (BEB) was employed. As well, conclusions were profoundly influenced by taxon-sampling. Several of the putative sites of positive selection in COX II were no longer apparent as we augmented taxon sampling. The lack of convergent molecular evolution in these remarkable taxa, combined with the profound influence of model choice and taxon sampling provide a cautionary note on the use of rates of non-synonymous to synonymous mutations (dN/dS) to explore questions of the evolution of physiological function.