Translating Animal Models of Obesity and Diabetes to the Clinic

C H A P T E R

1 Translating Animal Models of Obesity and Diabetes to the Clinic B.M. Geiger*, E.N. Pothos† *Department of Biomedical and Nutritional Sciences, Zuckerberg College of Health Sciences, University of Massachusetts, Lowell, Lowell, MA, United States †Program in Pharmacology and Experimental Therapeutics and Pharmacology and Drug Development, Sackler School of Graduate Biomedical Sciences and Department of Immunology, Tufts University School of Medicine, Boston, MA, United States

I INTRODUCTION

A Pathophysiological Similarities to Human Systems

Preclinical models of disease, specifically animal models, are a necessary tool for a robust and innovative drug development process. The use of animal models to identify and validate pharmacological targets has been part of drug development for decades. Experiments in cell culture systems can provide valuable molecular mechanistic information, but they cannot typically model the more complex interactions of multiple cell types that often contribute to the pathophysiology of a disease. Animal models that contribute to our understanding of the CNS are especially important, as acquiring tissue to study from the human brain is not generally feasible or permissible. In this chapter, we discuss the various attributes of valid animal models and illustrate specific examples of animals that we use for translational research in obesity and diabetes.

When considering which animal models to use, one of the first criteria to consider should be how closely does the onset and development of the disease in the animal mimic the corresponding human disease. For example, in humans, an increase in an individual’s body weight and excess blood glucose over time will contribute to decreased sensitivity of their insulin receptors and the eventual development of type 2 diabetes (Reinehr, 2013; Weiss et al., 2004). Therefore, a good model of this disease would also develop insulin resistance over time as body weight, adiposity, and blood glucose levels increase. As an example, C57Bl/6 mouse models of diet-induced obesity have been shown to follow the same metabolic progression of the disease as seen in humans and develop hyperglycemia and insulin resistance over time, similarly to the human disease (Collins, Martin, Surwit, & Robidoux, 2004; Petro et al., 2004; Speakman, Hambly, Mitchell, & Krol, 2007; Wang & Liao, 2012). In studying these models, researchers have been able to elucidate molecular mechanisms involved in the development of insulin resistance over time. We discuss the utility of diet-induced obesity models in greater detail later in this chapter.

II ATTRIBUTES OF VALID ANIMAL MODELS Animal models can be extremely valuable in the translation of our understanding of various diseases to treatments for those diseases in the clinic. Good disease models have some, if not all, of the following attributes: (1) pathophysiological similarities to human disease, (2) a phenotypic match to the disease state, (3) replicability and/or reproducibility, and (4) cost-efficiency (Blass, 2015). The first two criteria directly address the validity of the model (i.e., whether and how it relates to the disease it is supposed to model).

Translational Medicine in CNS Drug Development, Volume 29 ISSN: 1569-7339 https://doi.org/10.1016/B978-0-12-803161-2.00001-1

B Phenotypic Match to Disease State Other times, models may be used, where the exact pathophysiology of a disease is not a match to the human pathophysiology, but the presentation of the disease in the model is similar to the human condition.

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These models are referred to as phenotypic models of disease. The phenotype of a model is the set of characteristics, usually disease-related, that can be observed. If a model is a phenotypic match to a disease, then it has similar observable traits as a human who has that disease. For example, the ob/ob mouse exhibits extreme weight gain in the form of excess adipose tissue, similarly to human obesity. However, the development of obesity in the ob/ob mouse arises from leptin deficiency, while the vast majority of obese individuals have high levels of leptin. Only a very small number of human obese patients are actually leptin-deficient (Farooqi, 2008; Farooqi et al., 2007; Farooqi & O’Rahilly, 2008; Wang, Chandrasekera, & Pippin, 2014). Therefore, this model matches the phenotype of obesity, while the pathophysiology presents differences. Investigators should use caution when using models that are phenotypic models of disease to elucidate the molecular mechanisms of the disease. The altered underlying physiology may not match the human pathophysiology of the disease.

preceding text, if a group is reproducing the results from an MPTP-induced Parkinson’s disease model, but does not have the coordinates or dosage of MPTP used in the original model, reproduction of the original results will be extremely difficult, as different parameters could easily result in ablation of a different set of neurons than the original research. Additionally, many times a study will limit the model to a specific gender. If the reproducing group uses the opposite gender, they may find that gender differences complicate their ability to reproduce the original results. Furthermore, with all animal models, reproducibility is difficult when different litters of animals are used. This is particularly problematic if the animals are from different suppliers as this introduces more variability in the animals’ genetic background and microbiome, which could influence the study results (Masca et al., 2015). Sometimes, animals of the same species, gender, and phenotype may manifest important differences in behavior, physiology, disease state, or microbiome even if they come from different batches of the same or different suppliers housed in different locations and fed different diets (Ussar et al., 2015).

C Replicability and Reproducibility Replicability of a model is the ability to use the same model within a research group and achieve similar results. When conducting research using animal models, a study will often span several months of data collection and use animals that have been born to different dams or in different litters. In this case, replicability of the model is extremely important. If the model is developed using an environmental insult, like 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) injections for the generation of a Parkinson’s disease rodent model, consistent use of the same stereotaxic coordinates is necessary to ensure that the insult is in the same portion of the brain. The results will also replicate better if the same lot and batch of MPTP is used throughout the duration of the study. Numerous other details of the experiment must also be controlled in order to produce results that are replicable ( Jackson-Lewis & Przedborski, 2007; Meredith & Rademacher, 2011). In the case of transgenic animal models, the best design to ensure replicability is to use the wild-type littermates of the knockout animals, supplemented by the appropriate backcrossing to ensure genes neighboring to the targeted ones are not affected, as opposed to a more general wild-type animal like the C57Bl6 mouse (Masca et al., 2015; Wolfer, Crusio, & Lipp, 2002). Reproducibility is the ability of other researchers to reproduce the model in their lab and achieve similar results to those of the original research. Investigators can most easily reproduce results when the original description of the development and use of the model is clear (Masca et al., 2015). Using the same examples as in the

D Cost Efficiency Finally, the cost of husbandry of the various animal models varies widely and should be considered when determining which animal model is best suited for a particular project. The physiology of nonhuman primates is very similar to human physiology. However, the regulations pertinent to a nonhuman primate facility are much more stringent than with other research models, and therefore the cost associated with the establishment, housing and maintenance of these facilities may not be feasible for many researchers. On the other hand, zebra fish are being used more and more as preclinical models of disease because the construction and maintenance of the facilities to house them are much less expensive (Kim, Carlson, Zafreen, Rajpurohit, & Jagadeeswaran, 2009; Paige, Hill, Canterbury, Sweitzer, & RomeroSandoval, 2014). The cost of development, housing, and maintenance of rodent models of disease is somewhere in between the cost of a nonhuman primate colony and a zebra fish facility. For most research organizations, both academic and industrial, protocols and resources for conducting research in rodents are well established.

III TYPES OF ANIMAL MODELS USED FOR TRANSLATIONAL RESEARCH While the vast majority of animal models are rodents, many different animal species are used for translational research in drug discovery and development, ranging

III TYPES OF ANIMAL MODELS USED FOR TRANSLATIONAL RESEARCH

from flies and worms to monkeys and other large animals. In some cases, the species is chosen based on its relative simplicity, while other times, it may be chosen based on the similarity of a particular organ system to the human system. In addition to different animal species that may be used, recent advances in genetics technology now allow us better control of DNA modifications in live animal models. In this section, we discuss some of the more common animal models and the inducible knockout and CRISPR-Cas9 DNA editing technologies that are used in research, including CNS drug development.

A Rodent Models The most common species of animal model used for translational research in the CNS is the rodent model, usually rats or mice. These models have several advantages over other types of models. First, these models are mammalian and have similar physiology to humans for many organ systems, including the brain. While not the least expensive option for this type of research, they are usually more affordable than the other mammalian models that may be used. Finally, researchers are often interested in understanding how learning and memory are affected when developing CNS-related drugs. Robust experimental protocols have been developed to assess changes in these areas in rats and mice that can then be translated to the development of new therapeutics for diseases like Alzheimer’s or Parkinson’s disease. In a later section of the chapter, we apply these principles and discuss the mouse and rat models used for the study of obesity in more detail. We also provide examples of how the information from these models can then be translated to the clinic.

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larval fish, and other brain areas are not as developed in fish as they are in mammals making it difficult to extend the results of the study in zebra fish to mammals (Kalueff, Echevarria, et al., 2014; Kalueff, Stewart, et al., 2014; Stewart et al., 2014). Despite these difficulties, useful zebra fish models related to energy balance and obesity have been developed. We have shown that intestinal melanin-concentrating hormone, which is primarily known for its appetite regulating effects, is upregulated in zebra fish with TNBS-induced enterocolitis constituting a striking molecular similarity to the mouse and human form of the disease (Geiger et al., 2013). A dietinduced obesity model in zebra fish has also been described, which will be useful in understanding more of the pathophysiology associated with the development of obesity (Oka et al., 2010).

C Other Small Animal Models Besides rodents, other small mammalian models may be used in research. These species are typically used very sparingly, but there are instances when they are necessary due to their similarities to specific human organ systems. These species could include dogs, cats, pigs, gerbils, rabbits, and hamsters. For example, if a study protocol is attempting to address administration of a transdermal nociceptive agent, a pig model may be used as the skin of the pig has striking similarities to human skin and delivery of the agent through the skin is an important consideration in the development of transdermal formulations. Cats have been found to be particularly useful in the study of Alzheimer’s disease as they exhibit disease progression very similar to that in humans (Chambers et al., 2015), and dogs are particularly useful in kidney and renal disease research (Norrdin, 1981).

B Zebrafish The use of zebra fish as a model for various diseases, particularly in the CNS, is becoming more common (Kalueff, Echevarria, & Stewart, 2014; Kalueff, Stewart, & Gerlai, 2014; Stewart, Braubach, Spitsbergen, Gerlai, & Kalueff, 2014). In addition to the cost-effectiveness of zebra fish, they can be useful for many other reasons. Many of the same cell types, tissues, and organ systems are similar in the zebra fish and humans, including complex systems like the stress endocrine axis. Additionally, when studying developmental disorders, the transparency of the zebra fish larva allows monitoring and modification of organ systems as they develop in vivo. However, like with any model, there are limitations to the use of zebra fish. In adult zebra fish, drug treatment is often done by solubilizing the drug in tank water, but many drugs are not highly water-soluble. With CNS-specific studies, some behaviors are learned and cannot be studied in

D Nonhuman Primate Models Nonhuman primates are expensive animal models that still have great utility not only in drug pharmacokinetic analysis but also in the drug discovery and development process due to their similarities to human physiology, particularly in the CNS. Their high level of intellectual ability allows researchers to ask and answer questions related to complex social and cognitive behaviors for which other types of animal models cannot be used. For example, the social interactions that surround food intake in humans are extremely complex and difficult to study in the uncontrolled setting of normal everyday life. However, nonhuman primates, including the macaque monkey, exhibit eating behaviors that are extremely similar to humans. Researchers are able to watch and learn from primates in a more controlled social environment than the complex set of parameters under which humans

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operate. Furthermore, many of the same gut and neuropeptide systems control feeding in different situations in these animals as they do in humans, so we can place them in a specific context and evaluate how the eating behavior is affected by the circumstances and which hormones are altered. These correlations can then be used to identify and test specific drug molecules before they are tested in humans (Wilson, Moore, Ethun, & Johnson, 2014).

E Targeted Genetic Modifications of Animal Models Some of the more recent advances in genetic modification have allowed researchers to alter the DNA of traditional animal models with unprecedented precision. Two of these technologies, inducible knockouts using Cre-LoxP systems and DNA editing using CRISPRCas9, are discussed in the succeeding text. 1 Inducible Knockout Animal Models Traditional knockout animal models eliminate the functioning gene from the DNA of the organism at the embryo stage. This approach leads to the elimination of the gene throughout the whole organism for the duration of their life and has been possible to a large extent only in mice. Over the last two decades, new technology has emerged that can alter gene expression in both time- and tissue-specific manner through the use of Cre recombinase fused to a tissue-specific gene and the use of LoxP of the target gene for deletion (Gierut, Jacks, & Haigis, 2014; Lakso et al., 1992; Lobe & Nagy, 1998). The Cre-LoxP system was used to produce a tamoxifen-induced ablation of G protein-coupled receptor kinase 2 (GRK2) in mice with diet-induced obesity. The use of this model validated GRK2 as a potential drug target for the treatment of obesity (Vila-Bedmar et al., 2015). 2 CRISPR-Cas9 DNA Editing One of the most recent major advances in model development was the advent of the CRISPR-Cas9 gene-editing technology that can precisely target and edit specific sequences in genes. By introducing a mutation to study or correct a genetic deficit, this method allows for the study of this editing as a therapy (Barrangou et al., 2007; Cong et al., 2013; Savell & Day, 2017). While research related to the CRISPR-Cas9 technology is still relatively new, this technology provides a unique opportunity for investigators to further identify the effects of genetic manipulations on disease phenotypes and genotypes that have not been previously possible in several species.

IV EXAMPLES OF ANIMALS MODELS USED FOR TRANSLATIONAL OBESITY RESEARCH Because the obesity phenotype is easily identifiable and quantifiable, unlike the phenotype of diseases like schizophrenia, autism, and clinical depression, researchers involved in the discovery and development of obesity therapeutics have used numerous valid and reliable animal models that focus on the CNS pathophysiology of food intake, energy expenditure, and food reward in the translation of disease targets to the clinic. The following section gives an overview of some of these obesityrelated models as examples of the use of preclinical models in CNS translational efforts.

A Behavioral and Environmental Models When some of the contributing factors to the pathogenesis of a disease are environmental and/or behavioral, the preclinical models that best represent the onset and pathophysiology of that disease will often include a simulation of that environment. The rapid increase in obesity in developed countries around the world is in part due to the environment in which we live with increased availability of highly palatable foods and a more sedentary lifestyle. As a result, the most commonly prescribed treatments for obesity are diet and exercise, and some of the most important preclinical models of obesity incorporate diet and/or exercise to investigate the mechanisms by which these factors contribute to the obesity phenotype. In this section, we discuss models that are developed based on behavioral attributes of the animal and specific environmental cues, including diet and exercise. We also include a discussion of the use of changes in the environment within the gut via the gut microbiota and its role in translational obesity research. 1 Animal Models of Dietary Obesity One particularly useful model in obesity research is the diet-induced obesity (DIO) model as it mimics the most common cause of obesity in humans, dietary obesity. In this model, animals, usually rats and mice, although zebra fish DIO models have also been developed, are exposed to a specific diet that has macronutrient and/or caloric differences than their normal chow diet. There are a variety of types of diets that are used that range from high-sucrose and high-fat diets, high-fat only diets, high-saturated fat only diets, to food variety (cafeteria-style) diets (Geiger et al., 2009; Hariri, Gougeon, & Thibault, 2010; Hariri & Thibault, 2010; Hryhorczuk et al., 2016; Oka et al., 2010). Other variations of diets may also be used depending on the research question being addressed. All of these diets are effective at increasing body weight more rapidly in the animals exposed to the diets than control animals in

IV EXAMPLES OF ANIMALS MODELS USED FOR TRANSLATIONAL OBESITY RESEARCH

the experiment that eat a normal lab chow diet. As a result, you can monitor how the increasing adiposity is affecting the various neurochemical pathways that are associated with food intake and energy expenditure. For example, animals that ate a cafeteria-style diet increased weight very rapidly and had excess weight gain over the control animals as early as 2 weeks after the start of the cafeteria diet (Geiger et al., 2009). These animals also showed a preference for a sweetened condensed milk and high-sucrose solutions over other choices that included regular lab chow, chocolate, peanut butter, salami, marshmallow, cookies, and bananas. One major development in obesity preclinical research is the accumulating evidence on the role of central synaptic plasticity on food intake and weight gain. Since the discovery that lesions in the hypothalamus induce obesity (Stricker, 2012; Teitelbaum & Stellar, 1954), there have been numerous studies that focus on the CNS as the regulator of homeostatic mechanisms of energy balance through the activation of serotonin receptors and orexigenic and anorexigenic circuits in the arcuate nucleus and through the melanocortin-4 (MC4) receptor in the paraventricular nucleus of the hypothalamus (PVH) (Garfield & Heisler, 2009). DIO animal models have been extensively employed in this line of research. However, the rapid rise of common dietary obesity in industrialized societies indicates that nonhomeostatic signaling pathways that allow for chronic positive energy intake may be responsible. A crucial question is why laboratory animals and humans keep on eating energy-rich, palatable food to the degree that they become obese. From an evolutionary perspective, it is to be expected that the brain developed a system to respond to natural rewards, such as food. These central mechanisms are conserved across species in order to ensure survival (Kelley & Berridge, 2002) and could interact with or modulate the circuitry that regulates body weight. Therefore, availability of rewarding palatable food may lead to increased caloric intake and weight gain that homeostasis-driven mechanisms, originating primarily in the hypothalamus, may not overcome. This possibility may explain, at least in part, the epidemic proportions of dietary obesity. Prominent among neural systems known to code for the perception of reward are the mesolimbic dopamine pathways, where the release of the neurotransmitter dopamine, particularly in the nucleus accumbens and medial prefrontal cortex terminals originating in the ventral tegmental area (VTA), along with the dorsal striatum (that receives dopaminergic projections from the substantia nigra pars compacta), is known to mediate reward. The activation of these pathways includes elevation of dopamine levels and changes in dopamine turnover after natural rewarding behaviors like feeding (Hernandez & Hoebel, 1988). It is, therefore, reasonable to expect that dietary obesity may be

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linked to the mesolimbic dopamine-releasing ability of palatable high-energy food. The first evidence of linking mesolimbic dopamine release to dietary obesity came through our investigation of chronic exposure (15 weeks) of rats to a highenergy, palatable cafeteria diet along the diet model developed by Anthony Sclafani (Pothos, Sulzer, & Hoebel, 1998). We found that Sprague-Dawley rats took the majority of their daily caloric intake from highcarbohydrate sources and developed diet-induced obesity (Geiger et al., 2009; Pothos et al., 1998). As shown in Fig. 1.1, DIO rats demonstrated depressed basal dopamine release in the nucleus accumbens and an attenuated dopamine response to a standard chow meal or systemic administration of d-amphetamine, a drug that releases the neurotransmitter dopamine into the extracellular space by being a substrate of the plasma membrane and vesicular transporters for dopamine (DAT and VMAT2, respectively) (Pothos & Sulzer, 1998; Sulzer & Rayport, 1990). In further studies on strains of rodents that were selectively bred to develop dietary obesity, we established that the attenuation of basal and stimulated dopamine release in the brain characterized these animal models since early postnatal life (Fig. 1.2) (Geiger et al., 2008). Our findings of decreased dopamine availability in DIO rats were replicated in other studies with obesity animal models (Fulton et al., 2006; Geiger et al., 2008; Geiger et al., 2009; Hryhorczuk et al., 2016; Pothos et al., 1998; Wang et al., 2001) and imaging studies in obese humans (Stice, Spoor, Bohon, & Small, 2008; Stice, Spoor, Bohon, Veldhuizen, & Small, 2008; Wang et al., 2001) and led to the theory of dopamine deficiency in obesity that expanded to the reward deficiency hypothesis. It postulates that hyperphagia, or hedonic food intake in excess of energy needs, is a drive to compensate for the compromised dopamine neurotransmission in the brain of obese animals and patients and their blunting of food reward (Allen et al., 2012). Proof of principle for the importance of reversing dopamine deficits in the brain in order to reduce hyperphagia is the efficacy of the dopamine stimulant D-amphetamine in inducing weight loss (Harris, Ivy, & Searle, 1947). Amphetamine was manufactured as an ephedrine analog in the late 19th century and used as over-the-counter medication until the early 1970s for weight loss. Its withdrawal from the market following its Schedule II classification ended the tenure of amphetamine as weight-loss medication and highlighted concerns over dopaminereleasing drugs, namely, their addictive and neurotoxic potential that can result in severe extrapyramidal symptoms and, in some cases, suicidality, psychosis, and other adverse psychoactive symptoms (Power et al., 2014). The case of amphetamine further strengthens the statement that stimulation of dopamine release in the brain results in inhibition of feeding. Partial dopaminergic

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FIG. 1.1 Basal, amphetamine- and laboratory chow meal-challenged nucleus accumbens dopamine levels are decreased in freely moving dietary obese rats in vivo. (A) Body weight of cafeteria DIO rats during a 14-week period was significantly higher than that of the laboratory chow-fed group beginning at week 2 of the dietary regimen (*P < 0.01 by one-way ANOVA). (B) Basal and amphetamine-challenged extracellular dopamine levels during week 14 in the nucleus accumbens of cafeteria DIO rats (n ¼ 9) was significantly lower than in chow-fed rats (n ¼ 13). (C) The percent increase from baseline after amphetamine was higher in the cafeteria DIO rats than in the chow-fed rats (*P < 0.01 between groups, P < 0.05 within both groups, #P < 0.05 within laboratory chow-fed group only, and ##P < 0.05 within DIO group only relative to baseline prior to the amphetamine injection). (D) A plain chow meal was presented to cafeteria DIO (n ¼ 18) and chow-fed groups (n ¼ 22) after four baseline samples. Only the chow-fed group showed significant increases in dopamine after the meal. The cafeteria meal that was presented to a subset (n ¼ 8) of the cafeteria DIO group 2.5 h after the regular chow meal resulted in a significant increase in dopamine release (**P < 0.05 between groups, #P < 0.05 within the laboratory chow-fed group only, and ##P < 0.05 within the cafeteria DIO group only relative to baseline prior to the laboratory chow or cafeteria meal). From Geiger, B. M., Haburcak, M., Avena, N. M., Moyer, M. C., Hoebel, B. G., & Pothos, E. N. (2009). Deficits of mesolimbic dopamine neurotransmission in rat dietary obesity. Neuroscience, 159(4), 1193–1199.

agonists and reuptake inhibitors with much more moderate effects on body weight continue to be introduced in the market throughout the last decades (Padwal, 2009), although their long-term efficacy and adverse effects remain a concern. Overall, central dopamine deficiency (or inefficiency) in obesity seems to be a consistent foundation of our understanding of the neurobiology of obesity in both animal models and humans, and it remains to be established how this deficit can be translated to any deficiencies in the perception of food reward. More importantly, it is potentially significant to demonstrate novel ways through which dopamine neurotransmission in the brain can be corrected in order to reduce hyperphagia without potential for addiction and other serious adverse effects of weight loss-inducing dopaminergic and other drugs. A number of DIO studies have shown further links between neurochemistry and diet, including showing

that macronutrient composition can directly affect central synaptic plasticity. Recently, a total western diet has been introduced that has not only the high fat content of a typical western diet but also has a modified micronutrient content to better match the dietary pattern of the western diet. Initial studies indicate that animals fed this diet are less susceptible to the excessive weight gain typically seen with high-fat diets. This model provides an opportunity for further investigation into the role of micronutrients in the alteration of CNS pathways involved in feeding behavior and their effect on the development of obesity (Monsanto et al., 2016). As a result of these diet studies, health-care providers may make better dietary and lifestyle recommendations for effective weight loss. Remarkably, weight management interventions in one member of a married couple can have crossover benefits for the other partner even if the latter is not treated (Gorin et al., 2018).

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2 Exercise Models in Obesity Research Along with diet recommendations, exercise is the most commonly recommended treatment for obesity by physicians, as increased exercise will tip the energy balance scales in favor of energy expenditure and weight loss. To study the effects of exercise, rodents are given access to a treadmill, and the amount of time they spend on the treadmill is tracked. In mouse models of dietinduced obesity, exercise has been linked to not only lower body weight but also improvements in other markers of the metabolic syndrome like hyperglycemia and inflammation (Evans et al., 2014). Exercise also influences neurotransmitter release and metabolism that are associated with both energy homeostasis and hedonic feeding behavior, including that of orexin, leptin, norepinephrine, dopamine, and serotonin. These effects are seen in both adult animals and animals immediately post weaning (Greenwood et al., 2011; Meeusen & De Meirleir, 1995; Novak, Kotz, & Levine, 2006; Obici et al., 2015; Patterson, Bouret, DunnMeynell, & Levin, 2009; Patterson, Dunn-Meynell, & Levin, 2008; Patterson & Levin, 2008; Thanos et al., 2010). However, taken to the extreme, excessive exercise can cause addiction-related changes to the opiate pathways in the brain and cause rats to exhibit withdrawal like symptoms (Kanarek, D’Anci, Jurdak, & Mathes, 2009). Taken together, these findings provide more support of the effects of exercise on central synaptic plasticity and the use of exercise, particularly in children, as a safe alternative to pharmacotherapies to combat obesity. In fact, a recent metaanalysis of exercise studies in obese and overweight children indicates that aerobic exercise or a combination of aerobic and strength exercises can decrease body weight and adiposity in this population (Kelley, Kelley, & Pate, 2017).

FIG. 1.2 Reduced dopamine quantal size in VTA-derived neurons from P0-P1 obesity-prone pups. (A) Representative amperometric traces from VTA cultures of obesity-resistant (OR) (top) and obesityprone (OP) (bottom) neonates. Individual events are shown at higher resolution. (B) Quantal size distribution in cultures from neonatal OP and OR animals. Note that the OP distribution is skewed to the left due to the lack of events in the higher quantal size bins. (C) Quantal size (left panel) and amplitude (right panel) of stimulated dopamine release from VTA-derived neuronal cultures from OP pups (gray bars, n ¼ 110 events) is lower than that in cultures derived from OR pups (black bars, n ¼ 182 events). *P < 0.01. From Geiger, B. M., Behr, G. G., Frank, L. E., Caldera-Siu, A. D., Beinfeld, M. C., Kokkotou, E. G., et al. (2008). Evidence for defective mesolimbic dopamine exocytosis in obesity-prone rats. FASEB Journal, 22(8), 2740–2746.

3 Gut Microbiota Models in Obesity Research Recent studies have revealed the gut microbiome as a major contributor to the obesity phenotype in both mice and humans (Boursi et al., 2018; Hildebrandt et al., 2009). However, whether alterations in the microbiome cause obesity or are the result of obesity is still not clear (Moran-Ramos, Lopez-Contreras, & CanizalesQuinteros, 2017; Sanmiguel, Gupta, & Mayer, 2015). In general, obesity is linked to lower diversity of microbes in the gut, particularly in animal models. There are also differences in the by-products produced by these microbes between lean and obese humans and animals. However, the exact significance of these changes is not completely understood (Moran-Ramos, LopezContreras, et al., 2017). Others have also shown that following laparoscopic sleeve gastrectomy, the gut microbiome is altered in patients and these alterations are associated with weight loss, reduced appetite, and decreased hedonic eating (Sanmiguel et al., 2017).

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The molecular mechanisms that contribute to these changes are not well understood. In animal models, changes in the microbiota are induced in a variety of ways. Diet has significant effects on the composition of the gut microbiota, and studies like the one recently published by Moran-Ramos, He, et al. (2017) indicate that even small changes in the diet of an animal can have significant effects on the gut microbiota that lead to improvements in the obesity status of the animal. Another approach that has shown promise as a therapeutic as well is the fecal transfer model. In animals, fecal matter from lean animals is transferred to obese animals, and changes in the gut microbiota and the overall phenotype of the animal have been shown (MoranRamos, Lopez-Contreras, et al., 2017). Based on these results, similar studies are being done in humans that so far have shown similar improvements in obesity (Marotz & Zarrinpar, 2016; Moran-Ramos, LopezContreras, et al., 2017; Vrieze et al., 2012). More work is still necessary in both the preclinical and clinical areas to identify the molecular mechanisms that are altered in these studies, particularly as they relate to the gut-brain axis and changes seen in feeding behavior (Kaczmarek, Musaad, & Holscher, 2017). This work can potentially lead to identification of novel therapeutic targets for obesity.

B Spontaneous and Selectively Bred Animal Models In obesity research, some of the most useful animal models have been the spontaneous and selectively bred models of obesity and diabetes. These types of models exhibit the phenotype of the disease without specific manipulation of a single gene, in contrast to the genetic models discussed in the next section. For obesity and many other complex diseases of the CNS, these spontaneous models are particularly useful, as the disease will develop over time as the result of a complex interaction of multiple genetic factors. The animal models of obesity discussed in this section are summarized in Table 1.1. A spontaneous disease model exhibits the phenotype of the disease without any specific intervention by the breeder. The Zucker rat is a spontaneous model of obesity and hypertension that was first discovered in the 1960s. In contrast, selectively bred models typically arise from exposure to a specific environmental influence, and then the animals are grouped based on their response to the influence. The obesity-prone and obesity-resistant rat model is a good example of a selectively bred model of obesity (Levin et al., 1997). In this model, a colony of Sprague-Dawley rats ate a high-fat diet. The rats that gained the highest and lowest amounts of weight were separated into two groups and selectively bred within

their groups. After several generations of offering the high-fat diet to the animals and selective breeding, the offspring of the high weight gainers would spontaneously weigh about 20% more than the offspring of the low–weight gaining rats, even when all of the rats were fed a normal chow diet (Levin et al., 1997). The rats that gained the excess weight are referred to as diet-induced obese (DIO) or obesity-prone, while the rats that remained leaner are referred to as diet-resistant (DR) or obesity-resistant animals. In this chapter, we use the terms obesity-prone (OP) and obesity-resistant (OR) for selectively bred strains to avoid confusion with our discussion of models that are developed by exposing the animals to specific diets as part of the study, which were discussed earlier in this chapter. In this section the Zucker rat and OP/OR models of obesity as well as others like them are discussed in more detail. 1 Obesity-Prone and Obesity-Resistant Rats in Translational Research The selectively bred OP/OR strains constitute a model with many pathophysiological similarities to the human obese state, which is typically viewed as a polygenic disease. A weight distribution exists between the OP animals and the OR animals, similarly to what is seen in humans. In the presence of a high-fat diet, these weight differences are exacerbated with the OP animals gaining much more weight than the OR animals (Levin et al., 1997). Furthermore, numerous studies have shown that a number of different neuropeptides and other CNS proteins and neurotransmitters are altered in these animals, which also corresponds well to the human condition where multiple neural systems, both homeostatic and hedonic, have been shown to be altered in obese individuals (Berthoud & Morrison, 2008; Levin et al., 1997; Levin & Dunn-Meynell, 2002; Levin & Keesey, 1998; Novak et al., 2006). An analysis of the food reward system in these animals showed an overall depression of dopamine signaling in both males and females (Fig. 1.3). Most importantly, the parent phenotype affected its offspring similarly to human obesity. Specifically, attenuation of dopamine release was evident in neurons that were cultured from the dopamine neurons in newborn pups, indicating that the reduction in dopamine was present from birth and not induced by differences in the animals’ diets once they were born (Fig. 1.2; Geiger et al., 2008). 2 Zucker Rats as a Spontaneous Model of Obesity The Zucker fatty rat is a widely used model of obesity that was discovered in the 1960s when a spontaneous mutation in the recessive fa/fa genotype resulted in obesity and hypertension in these animals (Bray, 1977; Zucker & Zucker, 1967). The fa genotype is a missense mutation on the leptin receptor gene that renders the gene

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IV EXAMPLES OF ANIMALS MODELS USED FOR TRANSLATIONAL OBESITY RESEARCH

TABLE 1.1 Selected Genetic Rodent Models Used in Obesity and Diabetes Research Model

Genotype

Obesity/diabetes phenotype

Reference

Zucker fatty rat (ZFR)

Recessive fa/fa

Obese, hypertension, hyperinsulinemia, hyperlipidemia

Bray (1977), Zucker and Zucker (1967)

Zucker diabetic fatty rat (ZDF)

Recessive fa/fa selectively bred for hyperglycemia

Obese, hypertension, hyperglycemia

Bray (1977)

Obesity-prone rat

Selectively bred polygenic

Obese on lab chow, hyperinsulinemia, hyperglycemia, reduced leptin sensitivity

Levin and Dunn-Meynell (2002), Levin, Dunn-Meynell, Balkan, and Keesey (1997)

Obesity-resistant rat

Selectively bred polygenic

Lean, resistant to diet-induced obesity from high-fat diet

Levin et al. (1997)

Otsuka Long-Evans Tokushima Fatty (OLETF) rats

Cholecystokinin 1 receptor-deficient

Mildly obese, late-onset hyperglycemia, islet hyperplasia, renal disease

Kawano, Hirashima, Mori, and Natori (1994)

ob/ob mouse

Leptin-deficient

Obese, mild hyperglycemia, hyperinsulinemia, altered feeding behavior

Bray and York (1971), Halaas et al. (1995), Pelleymounter et al. (1995)

db/db mouse

Leptin receptor-deficient

Obese, hyperphagia, hyperglycemia, hyperinsulinemia

Coleman and Hummel (1967, 1974)

POMC / mouse

Proopiomelanocortin peptide deficient

Obese, defective adrenal glands, light coat color

Yaswen, Diehl, Brennan, and Hochgeschwender (1999)

Mc4R / mouse

Melanocortin-4 receptordeficient

Obese, hyperphagic, reduced metabolism, hyperinsulinemia, type 2 diabetes

Huszar et al. (1997), Ste Marie, Miura, Marsh, Yagaloff, and Palmiter (2000)

Agrp overexpression mouse

Agouti-related protein overexpression

Obese, hyperinsulinemia, late-onset hyperglycemia, pancreatic-islet hyperplasia

Graham, Shutter, Sarmiento, Sarosi, and Stark (1997)

NMU / mouse

Neuromedin U-deficient

Obese, hyperphagia, reduced activity, hyperinsulinemia, hyperglycemia, hyperleptinemia

Hanada et al. (2004)

MCH / mouse

Melanin-concentrating hormone-deficient

Lean, hyperactive, hypophagic

Shimada, Tritos, Lowell, Flier, and Maratos-Flier (1998)

CB1R / mouse

Cannabinoid 1 receptordeficient

Lean, resistant to diet-induced obesity

Ravinet Trillou, Delgorge, Menet, Arnone, and Soubrie (2004)

200 ms

Stimulus pulse Stimulus pulse OR OP

OR OP

40 30 20 10 0

NAc

DS

PFC

D 150

OR OP

Amplitude (pA)

10 pA

C

# of molecules (×106)

B Amplitude (pA)

A

100 50 0

NAc

DS

PFC

20

10

0

OR

OP

FIG. 1.3 Stimulated dopamine release is attenuated in the nucleus accumbens, dorsal striatum, and prefrontal cortex of adult obesity-prone rats. (A) Representative amperometric traces of stimulated dopamine release from acute accumbens coronal slices. (B and C) Average peak amplitude (pA) (B) and number of molecules released after stimulation of dopamine release (C) were significantly lower in obesity-prone (OP) rats than in obesity-resistant (OR) rats in the nucleus accumbens (NAc) (OP [n ¼ 45] stimulations in 9 slices; OR [n ¼ 53] stimulations in 11 slices), dorsal striatum (DS) (OP [n ¼ 40] stimulations in 8 slices; OR [n ¼ 40] stimulations in 8 slices), and prefrontal cortex (PFC) (OP [n ¼ 32] stimulations in 7 slices; OR [n ¼ 35] stimulations in 9 slices). (D) In 4–5-week-old rats, the average peak amplitude after stimulation of dopamine release was also significantly lower in OP rats than in OR rats (OP [n ¼ 37] stimulations in 10 slices; OR [n ¼ 56] stimulations in 13 slices). *P < 0.01. From Geiger, B. M., Behr, G. G., Frank, L. E., Caldera-Siu, A. D., Beinfeld, M. C., Kokkotou, E. G., et al. (2008). Evidence for defective mesolimbic dopamine exocytosis in obesity-prone rats. FASEB Journal, 22(8), 2740–2746.

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1. TRANSLATING ANIMAL MODELS OF OBESITY AND DIABETES TO THE CLINIC

nonfunctional. In addition to the traditional Zucker fatty rat, it has been further selectively bred for hyperglycemia to produce a diabetic rat model referred to as the Zucker diabetic fatty (ZDF) rat (Bray, 1977; Wang et al., 2014). When investigating the links between obesity, impaired leptin signaling, and dopamine, the Zucker rat was used to show that bromocriptine, a dopamine D2 receptor agonist, could be used to decrease food intake and body fat and increase locomotor activity in these animals (Davis et al., 2009).

In addition to complete deletion or overexpression of specific genes, we are able to target the altered gene expression to specific tissues or during specific times of development. More recently, these techniques have advanced using CRISPR/Cas9 technology so that we now are able to edit a specific gene and introduce specific disease-related mutations to genes of interest. In this section, we describe some of these genetic-based models that relate to our understanding of that pathophysiology of obesity and its translation to the clinic. 1 Genetic Models of Obesity and Diabetes

3 Otsuka Long Evans Tokushima Fatty (OLETF) Rat Obesity Model This model of obesity was first discovered in the 1990s by the spontaneous development of obesity in some of the animals in a colony of Long-Evans rats. These obese animals were then selectively bred to preserve the phenotype (Bi & Moran, 2016; Kawano et al., 1994). The lean control for this model is the Long-Evans Tokushima Otsuka (LETO) rat. The primary deficit in the OLETF rat is the lack of cholecystokinin receptor 1 (CCK1R) in both the GI tract and the brain. CCK is involved in the control of food intake, and disruptions in this system have been shown to cause hyperphagia that leads to increased body weight, reduced insulin sensitivity, and many other characteristics that are consistent with obesity in humans (Anderzhanova, Covasa, & Hajnal, 2007; Hajnal, Margas, & Covasa, 2008). As a result, the neural pathways that are affected by the lack of CCK have been extensively investigated, and the role of brain and gut neuropeptides in the homeostasis of energy balance has been identified (Bi & Moran, 2016; Moran & Bi, 2006). The increased understanding of the neural control of eating that was determined using this model has led to the identification of targets for obesity therapeutics in the clinic. In fact, both CCK1 agonists and antagonists and some of the NPY receptors have been investigated as potential anti-obesity treatments due to their interactions with the homeostatic systems of energy balance. However, in both systems, the single therapy did not result in clinically significant results (Colon-Gonzalez, Kim, Lin, Valentino, & Waldman, 2013; Merlino, Blomain, Aing, & Waldman, 2014).

C The Use of Genetic Models in Translational Obesity Research As our understanding of not only the human genome but also the genome of other species, like mice and rats, has advanced, we have been able to target genes in various animal models for overexpression or deletion to create a modified animal that exhibits a desired disease phenotype (Table 1.1).

Genetically modified animal models are some of the most commonly used models to help understand the obesity and diabetes phenotypes and then translate that knowledge into treatments for these diseases. For example, the leptin-deficient (ob/ob) and leptin receptor– deficient (db/db) mouse models show a distinct obese phenotype, mild hyperglycemia, and altered feeding behavior (Bray & York, 1971; Coleman & Hummel, 1967, 1974; Halaas et al., 1995; Pelleymounter et al., 1995). By providing a disease state that so similarly mimics the human phenotype, we have been able to gain valuable insights into the altered physiology of the obese individual. Such information has in turn helped provide strategies for treatment or prevention of this disease that in this case could be leptin-independent. For example, we found in the ob/ob mouse that there was a significant reduction in central dopamine signaling (Fulton et al., 2006). Because dopamine is a key component of the reward response to feeding, the lack of a proper dopamine response to food and the resulting uncontrolled eating behavior of these animals have contributed to the reward deficiency hypothesis of obesity (Blum et al., 2000). This hypothesis provides a specific target, the dopamine system, for the development of obesity treatments. Another anorexigenic neuropeptide whose role has been elucidated through the use of genetically modified mice is neuromedin U (NMU), a neuropeptide that is associated with a variety of stress-related and feeding behaviors (Hanada et al., 2004; Kaisho et al., 2017). Mice that are lacking either the gene coding for NMU or its receptor that is more common in the CNS, NMUR2, are obese and exhibit hyperinsulinemia and hyperglycemia along with other metabolic disturbances. Kaisho et al. (2017) have recently shown that treatment of dietary obese mice with a specific NMUR2 agonist, NMU-7005, can reduce body weight in these animals, while it has no effect on NMUR2 knockout mice. This study validates the selectivity of NMU-7005 for NMUR2 and provides another possible target for the treatment of dietary obesity (Kaisho et al., 2017). An additional and potentially important target is alpha–melanocyte-stimulating hormone (α-MSH) that shares proopiomelanocortin as a precursor with other MSHs, corticotropin, and endogenous

IV EXAMPLES OF ANIMALS MODELS USED FOR TRANSLATIONAL OBESITY RESEARCH

opiates (Millington & Levell, 2007). Interestingly, aging does not significantly alter hypothalamic mRNA levels of anorexigenic signals such as cocaine- and amphetaminerelated transcript (CART) and α-MSH in mice (Kmiec, 2006), which could allow for interventions to accelerate metabolism in older age. Many other genetically modified models of obesity models have been developed for a wide variety of targets, some of which are proopiomelanocortindeficient, melanocortin-4 receptor–deficient, and agoutirelated peptide overexpressing mice, and have led to several pharmacological interventions under clinical development (Graham et al., 1997; Huszar et al., 1997; Ste Marie et al., 2000; Yaswen et al., 1999). 2 Genetic Models of Leanness Along with the genetically obese animal models, several animal models that exhibit a lean phenotype have also been used to determine what neurobiological pathways and networks may protect from the development of obesity. For example, the melanin-concentrating hormone (MCH /) knockout mouse is lean compared with its wild-type littermates (Shimada et al., 1998). MCH is an appetite-promoting hypothalamic neuropeptide, and the reduced food intake and dysregulation of dopamine neurotransmission in the MCH / mouse contribute to its lean phenotype (Pissios et al., 2008). Based on this phenotype, antagonism of the MCH-1 receptor, which should have a similar effect on feeding behavior as seen in the MCH / mouse, is one approach that researchers are pursuing in the development of obesity therapeutics (Macneil, 2013). Because the MCH system is one of many overlapping hypothalamic systems and because it is well known that obesity is a polygenic disorder, combination therapies including MCH-1 receptor antagonists are also being investigated (ColonGonzalez et al., 2013; Valsamakis, Konstantakou, & Mastorakos, 2017). Interestingly, other knockout mouse models of hypothalamic orexigenic peptides have been less successful in showing the expected lean phenotype. For example, Agouti-related peptide (AgRP) is an orexigenic molecule that has been shown to cause increased food intake and is inhibited by elevated leptin and insulin levels. However, the phenotype of the knockout mouse did not feature significant differences in body weight, response to high-fat diet, or insulin sensitivity compared with its wild-type littermates (Qian et al., 2002). In all likelihood, the use of an AgRP inhibitor alone as a suppressant of food intake in the clinic would not show significant body weight reduction in humans. vTv Therapeutics had an AgRP inhibitor program in which they produced several inhibitors, one of which was evaluated in a phase II clinical trial for safety (Valsamakis et al., 2017). In mice, it was shown that one of the inhibitors, TPP2515, could blunt the effects of AgRP, particularly in situations where AgRP would be

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expected to be elevated. However, there were several off target effects of this compound and limited efficacy in situations where AgRP would not be artificially elevated, and these compounds no longer appear to be in development for the treatment of obesity (Dutia et al., 2013). The endocannabinoid system has also emerged as a potential target for obesity therapeutics due to its actions in both the homeostatic pathways of energy balance and the hedonic system of food reward (Di Marzo, Ligresti, & Cristino, 2009; Melis et al., 2007). Mice lacking the cannabinoid 1 receptor exhibit a lean phenotype and resistance to diet-induced obesity when exposed to a high-fat diet (Ravinet Trillou et al., 2004). Rimonabant is a CB1 receptor antagonist that has been developed for the treatment of obesity. However, it was withdrawn from the market due to its significant effects on patients’ mood that outweighed the clinical benefits of the drug in the treatment of obesity (Richey & Woolcott, 2017). This clinical failure highlights the limitations of murine models in CNS drug development. Specifically, while we have some indications of a mouse’s affective state, it is difficult to assess the extent of mood-related side effects that might occur in humans in a mouse model. 3 Inducible KO Models With Phenotypes Applicable to Food Intake The most effective treatments of disease will target the protein or gene of interest at the target tissue while leaving it unaltered in other areas of the body. Traditional inducible models of disease use a Cre/LoxP systems where gene expression is altered either in specific tissues or following the use of a trigger stimulus that initiates the introduction of the altered DNA into the host’s genome (Gu, Marth, Orban, Mossmann, & Rajewsky, 1994; Heldt & Ressler, 2009). These models have provided invaluable insights into disease mechanisms. Recent advances in Cre/LoxP systems can alter gene expression in the hypothalamus at specific ages or loci in mice and have provided additional insights to the development of obesity. For example, studies have showed that food intake was reduced and energy expenditure increased dramatically by disrupting the synthesis of GABA specifically in agouti-related peptide expressing neurons of the hypothalamus in young adult mice (Meng et al., 2016). These effects were not as significant in older mice, indicating that hypothalamic control of feeding behavior in these neurons is more sensitive to GABA signaling in younger animals. These results demonstrate that targeting GABA signaling might be an effective treatment of obesity in younger populations. Such signaling modulation could be envisioned in human patients, particularly the morbidly obese, through, for example, patterns of localized mild brain stimulation in the hypothalamic region (Palmiter, 2017).

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1. TRANSLATING ANIMAL MODELS OF OBESITY AND DIABETES TO THE CLINIC

4 CRISPR-Cas9 Technology in Translational Research The CRISPR-Cas9 gene-editing technology is proving to be one of the most powerful tools available to alter gene sequences with a high level of accuracy and precision, providing researchers in search of a model to understand the role of specific mutations a way to develop a corresponding animal model more easily than ever before (Cong et al., 2013; Savell & Day, 2017). With respect to obesity and diabetes research, this technology has already been used to alter leptin and ghrelin signaling in both rats and mice. As a result of these studies, hundreds of altered genes have been identified both in the peripheral tissues as well as the CNS that could provide information for new potential targets for obesity pharmacotherapies (Guan et al., 2017; Zallar et al., 2019). As further proof of concept, in mice embryos, both leptin and the leptin receptor have been deleted using CRISPR-Cas9 systems, resulting in phenotypes very similar to the ob/ob and db/db mouse models (Roh et al., 2018). This illustrates the possibility to use the CRISPR-Cas9 technology to produce animal models of obesity in other species. As the difficulties with introducing this technology to neurons specifically are overcome, numerous other applications of CRISPR-Cas9 gene editing may also become critical to biomedical research in the CNS (Savell & Day, 2017).

D Surgical/Pharmacological Models Additional effective approaches to develop a reproducible model of obesity and test novel treatments are through surgical or pharmacological interventions. Bariatric surgery in rats has given us extremely useful insight into the CNS and gut hormone alterations that occur when this surgery is done (Berthoud, Shin, & Zheng, 2011; Hajnal et al., 2010; Tam et al., 2011). In other cases, the disease would be initiated by the surgical or pharmacological treatment. The streptozotocin-induced diabetic rats and mice, which were first used in the 1960s, are an example of this type of model (Szkudelski, 2001). In this section, we discuss examples of each of these types of models and their use in translational research in more detail. 1 Bariatric Surgery In some cases, translational research begins in the clinic, returns to the lab, and then goes back to the clinic. Bariatric surgery is widely considered one of the most successful weight-loss treatments with beneficial effects extending beyond weight reduction. Roux-en-Y gastric bypass surgical techniques were developed in rat and mouse models to investigate what other molecular mechanisms might be altered by this procedure. Interestingly, which type of model is used for the surgery influences the

outcomes of the surgery. For example, DIO mice respond differently to the surgery than ob/ob mice (Hao et al., 2015). It has also been shown that a number of neuropeptides are altered in these animals including CCK, glucagon-like peptide-1, serotonin, and neurotensin (Mumphrey, Patterson, Zheng, & Berthoud, 2013; Shin, Zheng, Townsend, Sigalet, & Berthoud, 2010). Rat models of bariatric surgery have shown alterations to behaviors associated with the hedonic pathways including altered sweet taste and changes in central dopamine (Hajnal et al., 2010; Thanos et al., 2015). Those observations have been confirmed in patients subjected to bariatric surgery through, for instance, functional MRI pilot studies (Wang, Yang, Hajnal, & Rogers, 2016) and may help illustrate changes in alcohol intake observed in human bariatric patients (Smith et al., 2017). More recently, it has been found that the fat-satiety molecule oleoylethanolamide (OEA) is increased in rats that have had Roux-en-Y surgery, while central dopamine signaling through the D1 receptor was also upregulated (Hankir et al., 2017). Taken together, these results indicate that surgery-induced changes in satiety-related gut hormones and a number of contributing neurochemicals may improve weight loss. In the future, as an alternative to surgery, the targeting of these systems or a specific combination of these systems could elicit weight loss in obese individuals. 2 Streptozotocin-Induced Diabetic Rats and Mice The use of pharmacological agents to damage β-cells and produce a diabetic rodent has been utilized for several decades. Streptozotocin is one agent that is often used to create this model. At high doses, it will cause significant damage to the β-cells, similarly to type 1 diabetes. At lower doses, it will cause the damage over time, mimicking the onset of type 2 diabetes (Szkudelski, 2001). In a variation of this model, the animals are also fed a high-fat diet to become obese. This may be one of the most accurate models to link the pathogenesis of diabetes and obesity (Gheibi, Kashfi, & Ghasemi, 2017; Reed et al., 2000). This model has been used to provide valuable insights on the effects of diabetes on central dopamine signaling and reward response. Furthermore, it has been shown that targeting the D1 and D2 receptors can attenuate the hyperphagia that is seen in these animals (Kuo, 2006). Finally, this is a good model of diabetic neuropathy and as the number of diabetic patients increases, the need for safe and effective treatments for pain in diabetics also increases. Tramadol is a commonly prescribed pain medication for diabetic neuropathy. A recent study in the streptozotocin-induced diabetic rat model showed that while tramadol may work in this population, the pathways that it targets are altered when diabetes is present and therefore the patient’s response will also likely be altered (Ezzeldin, Souror, El-Nahhas, Soudi, & Shahat, 2014).

REFERENCES

3 Models of Central Insulin Deficiency Although the role of insulin secretion from pancreatic beta cells is well established in the periphery, an increasingly active area of investigation focuses on the role and function of central insulin receptors in diabetes, obesity, and cognitive function (Arnold et al., 2018). Surprisingly, the targeted deletion of brain insulin receptors does not consistently produce a diabetic phenotype or a phenotype related to metabolic syndrome in mice. Instead, it can produce behavioral indexes of depression and a remarkable decrease in mesolimbic dopamine neurotransmission (Kleinridders, 2016). This is a testament of how animal models can at times offer unexpected contributions to the understanding of disease mechanisms other than the ones originally considered. In this case, the brain insulin animal models may decisively aid in the understanding of the connection between diabetes and neurodegenerative disorders and whether their underlying pathologies are partially overlapping or coincidental and complementary.

V CONCLUSION The use of animal models of disease is crucial to the successful development of new therapeutics to treat diseases of the central nervous system. However, a number of factors should be considered when choosing an appropriate disease model including pathophysiological and phenotypic similarities to the disease in humans, the replicability and reproducibility of the data generated by the model, and the cost of the development and maintenance of the model. With these considerations in mind, models generated using a wide variety of methods ranging from spontaneous development of the disease to complex genetic manipulations that give rise to the disease phenotype have proved to be an invaluable resource in the drug discovery and development process for the treatment of obesity and diabetes.

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