History and future of leptin: Discovery, regulation and signaling.

The cloning of leptin 30 years ago in 1994 was an important milestone in obesity research. Prior to the discovery of leptin, obesity was stigmatized as a condition caused by lack of character and self-control. Mutations in either leptin or its receptor were the first single gene mutations found to cause severe obesity, and it is now recognized that obesity is caused mostly by a dysregulation of central neuronal circuits. Since the discovery of the leptin-deficient obese mouse (ob/ob) the cloning of leptin (ob aka lep) and leptin receptor (db aka lepr) genes, we have learned much about leptin and its action in the central nervous system. The first hope that leptin would cure obesity was quickly dampened because humans with obesity have increased leptin levels and develop leptin resistance. Nevertheless, leptin target sites in the brain represent an excellent blueprint to understand how neuronal circuits control energy homeostasis. Our expanding understanding of leptin function, interconnection of leptin signaling with other systems and impact on distinct physiological functions continues to guide and improve the development of safe and effective interventions to treat metabolic illnesses. This review highlights past concepts and current emerging concepts of the hormone leptin, leptin receptor signaling pathways and central targets to mediate distinct physiological functions.

FGF21 as a mediator of adaptive changes in food intake and macronutrient preference in response to protein restriction.

Free-feeding animals navigate complex nutritional landscapes in which food availability, cost, and nutritional value can vary markedly. Animals have thus developed neural mechanisms that enable the detection of nutrient restriction, and these mechanisms engage adaptive physiological and behavioral responses that limit or reverse this nutrient restriction. This review focuses specifically on dietary protein as an essential and independently defended nutrient. Adequate protein intake is required for life, and ample evidence exists to support an active defense of protein that involves behavioral changes in food intake, food preference, and food motivation, likely mediated by neural changes that increase the reward value of protein foods. Available evidence also suggests that the circulating hormone fibroblast growth factor 21 (FGF21) acts in the brain to coordinate these adaptive changes in food intake, making it a unique endocrine signal that drives changes in macronutrient preference in the context of protein restriction. This article is part of the Special Issue on "Food intake and feeding states".

FGF21 acts in the brain to drive macronutrient-specific changes in behavioral motivation and brain reward signaling.

Dietary protein restriction induces adaptive changes in food preference, increasing protein consumption over carbohydrates or fat. We investigated whether motivation and reward signaling underpin these preferences. In an operant task, protein-restricted male mice responded more for liquid protein rewards, but not carbohydrate, fat, or sweet rewards compared to non-restricted mice. The protein restriction-induced increase in operant responding for protein was absent in Fgf21-KO mice and mice with neuron-specific deletion of the FGF21 co-receptor beta-Klotho (Klb Cam2ka ) mice. Fiber photometry recording of VTA dopamine neurons revealed that oral delivery of maltodextrin triggered a larger activation as compared to casein in control-fed mice, whereas casein triggered a larger activation in protein-restricted mice. This restriction-induced shift in nutrient-specific VTA dopamine signaling was lost in Fgf21-KO mice. These data strongly suggest that the increased FGF21 during protein restriction acts in the brain to induce a protein-specific appetite by specifically enhancing the reward value of protein-containing foods and the motivation to consume them.

Hepatic interoception in health and disease.

The liver is a large organ with crucial functions in metabolism and immune defense, as well as blood homeostasis and detoxification, and it is clearly in bidirectional communication with the brain and rest of the body via both neural and humoral pathways. A host of neural sensory mechanisms have been proposed, but in contrast to the gut-brain axis, details for both the exact site and molecular signaling steps of their peripheral transduction mechanisms are generally lacking. Similarly, knowledge about function-specific sensory and motor components of both vagal and spinal access pathways to the hepatic parenchyma is missing. Lack of progress largely owes to controversies regarding selectivity of vagal access pathways and extent of hepatocyte innervation. In contrast, there is considerable evidence for glucose sensors in the wall of the hepatic portal vein and their importance for glucose handling by the liver and the brain and the systemic response to hypoglycemia. As liver diseases are on the rise globally, and there are intriguing associations between liver diseases and mental illnesses, it will be important to further dissect and identify both neural and humoral pathways that mediate hepatocyte-specific signals to relevant brain areas. The question of whether and how sensations from the liver contribute to interoceptive self-awareness has not yet been explored.

Sensory spinal interoceptive pathways and energy balance regulation.

Interoception plays an important role in homeostatic regulation of energy intake and metabolism. Major interoceptive pathways include gut-to-brain and adipose tissue-to brain signaling via vagal sensory nerves and hormones, such as leptin. However, signaling via spinal sensory neurons is rapidly emerging as an additional important signaling pathway. Here we provide an in-depth review of the known anatomy and functions of spinal sensory pathways and discuss potential mechanisms relevant for energy balance homeostasis in health and disease. Because sensory innervation by dorsal root ganglia (DRG) neurons goes far beyond vagally innervated viscera and includes adipose tissue, skeletal muscle, and skin, it is in a position to provide much more complete metabolic information to the brain. Molecular and anatomical identification of function specific DRG neurons will be important steps in designing pharmacological and neuromodulation approaches to affect energy balance regulation in disease states such as obesity, diabetes, and cancer.

Regulation of body weight: Lessons learned from bariatric surgery.

BACKGROUND: Bariatric or weight loss surgery is currently the most effective treatment for obesity and metabolic disease. Unlike dieting and pharmacology, its beneficial effects are sustained over decades in most patients, and mortality is among the lowest for major surgery. Because there are not nearly enough surgeons to implement bariatric surgery on a global scale, intensive research efforts have begun to identify its mechanisms of action on a molecular level in order to replace surgery with targeted behavioral or pharmacological treatments. To date, however, there is no consensus as to the critical mechanisms involved. SCOPE OF REVIEW: The purpose of this non-systematic review is to evaluate the existing evidence for specific molecular and inter-organ signaling pathways that play major roles in bariatric surgery-induced weight loss and metabolic benefits, with a focus on Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG), in both humans and rodents. MAJOR CONCLUSIONS: Gut-brain communication and its brain targets of food intake control and energy balance regulation are complex and redundant. Although the relatively young science of bariatric surgery has generated a number of hypotheses, no clear and unique mechanism has yet emerged. It seems increasingly likely that the broad physiological and behavioral effects produced by bariatric surgery do not involve a single mechanism, but rather multiple signaling pathways. Besides a need to improve and better validate surgeries in animals, advanced techniques, including inducible, tissue-specific knockout models, and the use of humanized physiological traits will be necessary. State-of-the-art genetically-guided neural identification techniques should be used to more selectively manipulate function-specific pathways.

Galanin Regulates Myocardial Mitochondrial ROS Homeostasis and Hypertrophic Remodeling Through GalR2.

The regulatory peptide galanin is broadly distributed in the central nervous systems and peripheral tissues where it modulates numerous physiological and pathological processes through binding to its three G-protein-coupled receptors, GalR1-3. However, the function and identity of the galaninergic system in the heart remain unclear. Therefore, we investigated the expression of the galanin receptors in cardiac cells and tissues and found that GalR2 is the dominant receptor subtype in adult mouse hearts, cardiomyocytes and H9C2 cardiomyoblasts. In vivo, genetic suppression of GalR2 promotes cardiac hypertrophy, fibrosis and mitochondrial oxidative stress in the heart. In vitro, GalR2 silencing by siRNA abolished the beneficial effects of galanin on cell hypertrophy and mitochondrial reactive oxygen species (ROS) production. These findings unravel new insights into the role of galaninergic system in the heart and suggest novel therapeutic strategies in heart disease.

FGF21 is required for protein restriction to extend lifespan and improve metabolic health in male mice.

Dietary protein restriction is increasingly recognized as a unique approach to improve metabolic health, and there is increasing interest in the mechanisms underlying this beneficial effect. Recent work indicates that the hormone FGF21 mediates the metabolic effects of protein restriction in young mice. Here we demonstrate that protein restriction increases lifespan, reduces frailty, lowers body weight and adiposity, improves physical performance, improves glucose tolerance, and alters various metabolic markers within the serum, liver, and adipose tissue of wildtype male mice. Conversely, mice lacking FGF21 fail to exhibit metabolic responses to protein restriction in early life, and in later life exhibit early onset of age-related weight loss, reduced physical performance, increased frailty, and reduced lifespan. These data demonstrate that protein restriction in aging male mice exerts marked beneficial effects on lifespan and metabolic health and that a single metabolic hormone, FGF21, is essential for the anti-aging effect of this dietary intervention.

Lateral hypothalamic galanin neurons are activated by stress and blunt anxiety-like behavior in mice.

Despite the prevalence of anxiety disorders, the molecular identity of neural circuits underlying anxiety remains unclear. The lateral hypothalamus (LH) is one brain region implicated in the regulation of anxiety, and our recent data found that chemogenetic activation of LH galanin neurons attenuated the stress response to a novel environment as measured by the marble burying test. Thus, we hypothesize that LH galanin neurons may contribute to anxiety-related behavior. We used chemogenetics and fiber photometry to test the ability of LH galanin neurons to influence anxiety and stress-related behavior. Chemogenetic activation of LH galanin neurons significantly decreased anxiety-like behavior in the elevated plus maze, open field test, and light dark test. However, LH galanin activation did not alter restraint stress induced HPA activation or freezing behavior in the fear conditioning paradigm. In vivo calcium monitoring by fiber photometry indicated that LH galanin neurons were activated by anxiogenic and/or stressful stimuli including tail suspension, novel mouse interaction, and predator odor. Further, in a fear conditioning task, calcium transients strongly increased during foot shock, but were not affected by the unconditioned stimulus tone. These data indicate that LH galanin neurons both respond to and modulate anxiety, with no influence on stress induced HPA activation or fear behaviors. Further investigation of LH galanin circuitry and functional mediators of behavioral output may offer a more refined pharmacological target as an alternative to first-line broad pharmacotherapies such as benzodiazepines.

Organization of sympathetic innervation of interscapular brown adipose tissue in the mouse.

The interscapular brown adipose tissue (iBAT) is under sympathetic control, and recent studies emphasized the importance of efferent sympathetic and afferent sensory or humoral feedback systems to regulate adipose tissue function and overall metabolic health. However, functional studies of the sympathetic nervous system in the mouse are limited, because details of anatomy and fine structure are lacking. Here, we used reporter mice for tyrosine hydroxylase expressing neurons (TH:tomato mice), iDISCO tissue clearance, confocal, lightsheet, and electron microscopy to clarify that (a) iBAT receives sympathetic input via dorsal rami (instead of often cited intercostal nerves); (b) dorsal rami T1-T5 correspond to the postganglionic input from sympathetic chain ganglia (stellate/T1-T5); (c) dorsal rami serve as conduits for sympathetic axons that branch off in finer nerve bundles to enter iBAT; (d) axonal varicosities show strong differential innervation of brown (dense innervation) versus white (sparse innervation) adipocytes, that surround the core iBAT in the mouse and are intermingled in human adipose tissues, (e) axonal varicosities can form neuro-adipocyte junctions with brown adipocytes. Taken together, we demonstrate that sympathetic iBAT innervation is organized by specific nerves and terminal structures that can be surgically and genetically accessed for neuromodulatory purposes.

Protein Appetite at the Interface between Nutrient Sensing and Physiological Homeostasis.

Feeding behavior is guided by multiple competing physiological needs, as animals must sense their internal nutritional state and then identify and consume foods that meet nutritional needs. Dietary protein intake is necessary to provide essential amino acids and represents a specific, distinct nutritional need. Consistent with this importance, there is a relatively strong body of literature indicating that protein intake is defended, such that animals sense the restriction of protein and adaptively alter feeding behavior to increase protein intake. Here, we argue that this matching of food consumption with physiological need requires at least two concurrent mechanisms: the first being the detection of internal nutritional need (a protein need state) and the second being the discrimination between foods with differing nutritional compositions. In this review, we outline various mechanisms that could mediate the sensing of need state and the discrimination between protein-rich and protein-poor foods. Finally, we briefly describe how the interaction of these mechanisms might allow an animal to self-select between a complex array of foods to meet nutritional needs and adaptively respond to changes in either the external environment or internal physiological state.

Targeting the T-type calcium channel Cav3.2 in GABAergic arcuate nucleus neurons to treat obesity.

OBJECTIVE: Cav3.2, a T-type low voltage-activated calcium channel widely expressed throughout the central nervous system, plays a vital role in neuronal excitability and various physiological functions. However, the effects of Cav3.2 on energy homeostasis remain unclear. Here, we examined the role of Cav3.2 expressed by hypothalamic GABAergic neurons in the regulation of food intake and body weight in mice and explored the underlying mechanisms. METHODS: Male congenital Cana1h (the gene coding for Cav3.2) global knockout (Cav3.2KO) mice and their wild type (WT) littermates were first used for metabolic phenotyping studies. By using the CRISPR-Cas9 technique, Cav3.2 was selectively deleted from GABAergic neurons in the arcuate nucleus of the hypothalamus (ARH) by specifically overexpressing Cas9 protein and Cav3.2-targeting sgRNAs in ARH Vgat (VgatARH) neurons. These male mutants (Cav3.2KO-VgatARH) were used to determine whether Cav3.2 expressed by VgatARH neurons is required for the proper regulation of energy balance. Subsequently, we used an electrophysiological patch-clamp recording in ex vivo brain slices to explore the impact of Cav3.2KO on the cellular excitability of VgatARH neurons. RESULTS: Male Cav3.2KO mice had significantly lower food intake than their WT littermate controls when fed with either a normal chow diet (NCD) or a high-fat diet (HFD). This hypophagia phenotype was associated with increased energy expenditure and decreased fat mass, lean mass, and total body weight. Selective deletion of Cav3.2 in VgatARH neurons resulted in similar feeding inhibition and lean phenotype without changing energy expenditure. These data provides an intrinsic mechanism to support the previous finding on ARH non-AgRP GABA neurons in regulating diet-induced obesity. Lastly, we found that naringenin extract, a predominant flavanone found in various fruits and herbs and known to act on Cav3.2, decreased the firing activity of VgatARH neurons and reduced food intake and body weight. These naringenin-induced inhibitions were fully blocked in Cav3.2KO-VgatARH mice. CONCLUSION: Our results identified Cav3.2 expressed by VgatARH neurons as an essential intrinsic modulator for food intake and energy homeostasis, which is a potential therapeutic target in the treatment of obesity.

Sympathetic innervation of the mouse kidney and liver arising from prevertebral ganglia.

The sympathetic nervous system (SNS) plays a crucial role in the regulation of renal and hepatic functions. Although sympathetic nerves to the kidney and liver have been identified in many species, specific details are lacking in the mouse. In the absence of detailed information of sympathetic prevertebral innervation of specific organs, selective manipulation of a specific function will remain challenging. Despite providing major postganglionic inputs to abdominal organs, limited data are available about the mouse celiac-superior mesenteric complex. We used tyrosine hydroxylase (TH) and dopamine ß-hydroxylase (DbH) reporter mice to visualize abdominal prevertebral ganglia. We found that both the TH and DbH reporter mice are useful models for identification of ganglia and nerve bundles. We further tested if the celiac-superior mesenteric complex provides differential inputs to the mouse kidney and liver. The retrograde viral tracer, pseudorabies virus (PRV)-152 was injected into the cortex of the left kidney or the main lobe of the liver to identify kidney-projecting and liver-projecting neurons in the celiac-superior mesenteric complex. iDISCO immunostaining and tissue clearing were used to visualize unprecedented anatomical detail of kidney-related and liver-related postganglionic neurons in the celiac-superior mesenteric complex and aorticorenal and suprarenal ganglia compared with TH-positive neurons. Kidney-projecting neurons were restricted to the suprarenal and aorticorenal ganglia, whereas only sparse labeling was observed in the celiac-superior mesenteric complex. In contrast, liver-projecting postganglionic neurons were observed in the celiac-superior mesenteric complex and aorticorenal and suprarenal ganglia, suggesting spatial separation between the sympathetic innervation of the mouse kidney and liver.

Maternal cold exposure induces distinct transcriptome changes in the placenta and fetal brown adipose tissue in mice.

BACKGROUND: Brown adipose tissue (BAT) is specialized to dissipate energy in the form of heat. BAT-mediated heat production in rodents and humans is critical for effective temperature adaptation of newborns to the extrauterine environment immediately after birth. However, very little is known about whether and how fetal BAT development is modulated in-utero in response to changes in maternal thermal environment during pregnancy. Using BL6 mice, we evaluated the impact of different maternal environmental temperatures (28 °C and 18 °C) on the transcriptome of the placenta and fetal BAT to test if maternal cold exposure influences fetal BAT development via placental remodeling. RESULTS: Maternal weight gain during pregnancy, the average number of fetuses per pregnancy, and placental weight did not differ between the groups at 28 °C and 18 °C. However, the average fetal weight at E18.5 was 6% lower in the 18 °C-group compared to the 28 °C-group. In fetal BATs, cold exposure during pregnancy induced increased expression of genes involved in de novo lipogenesis and lipid metabolism while decreasing the expression of genes associated with muscle cell differentiation, thus suggesting that maternal cold exposure may promote fetal brown adipogenesis by suppressing the myogenic lineage in bidirectional progenitors. In placental tissues, maternal cold exposure was associated with upregulation of genes involved in complement activation and downregulation of genes related to muscle contraction and actin-myosin filament sliding. These changes may coordinate placental adaptation to maternal cold exposure, potentially by protecting against cold stress-induced inflammatory damage and modulating the vascular and extravascular contractile system in the placenta. CONCLUSIONS: These findings provide evidence that environmental cold temperature sensed by the mother can modulate the transcriptome of placental and fetal BAT tissues. The ramifications of the observed gene expression changes warrant future investigation.

Sympathetic Innervation of White Adipose Tissue: to Beige or Not to Beige?

Obesity research progresses in understanding neuronal circuits and adipocyte biology to regulate metabolism. However, the interface of neuro-adipocyte interaction is less studied. We summarize the current knowledge of adipose tissue innervation and interaction with adipocytes and emphasize adipocyte transitions from white to brown adipocytes and vice versa. We further highlight emerging concepts for the differential neuronal regulation of brown/beige versus white adipocyte and the interdependence of both for metabolic regulation.

IGFBP-2 partly mediates the early metabolic improvements caused by bariatric surgery.

Insulin-like growth factor-binding protein (IGFBP)-2 is a circulating biomarker of cardiometabolic health. Here, we report that circulating IGFBP-2 concentrations robustly increase after different bariatric procedures in humans, reaching higher levels after biliopancreatic diversion with duodenal switch (BPD-DS) than after Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG). This increase is closely associated with insulin sensitization. In mice and rats, BPD-DS and RYGB operations also increase circulating IGFBP-2 levels, which are not affected by SG or caloric restriction. In mice, Igfbp2 deficiency significantly impairs surgery-induced loss in adiposity and early improvement in insulin sensitivity but does not affect long-term enhancement in glucose homeostasis. This study demonstrates that the modulation of circulating IGFBP-2 may play a role in the early improvement of insulin sensitivity and loss of adiposity brought about by bariatric surgery.

Sympathetic innervation of inguinal white adipose tissue in the mouse.

Adipose tissue plays an important role in metabolic homeostasis and its prominent role as endocrine organ is now well recognized. Adipose tissue is controlled via the sympathetic nervous system (SNS). New viral, molecular-genetic tools will soon allow a more detailed study of adipose tissue innervation in metabolic function, yet, the precise anatomical extent of preganglionic and postganglionic inputs to the inguinal white adipose tissue (iWAT) is limited. Furthermore, several viral, molecular-genetic tools will require the use of cre/loxP mouse models, while the available studies on sympathetic iWAT innervation were established in larger species. In this study, we generated a detailed map for the sympathetic innervation of iWAT in male and female mice. We adapted iDISCO tissue clearing to process large, whole-body specimens for an unprecedented view of the natural abdominal SNS. Combined with pseudorabies virus retrograde tracing from the iWAT, we defined the preganglionic and postganglionic sympathetic input to iWAT. We used fluorescence-guided anatomical dissections of sympathetic nerves in reporter mice to further clarify that postganglionic axons connect to iWAT via lateral cutaneous rami (dorsolumbar iWAT portion) and the lumbar plexus (inguinal iWAT portion). Importantly, these rami carry axons that branch to iWAT, as well as axons that travel further to innervate the skin and vasculature, and their functional impact will require consideration in denervation studies. Our study may serve as a comprehensive map for future experiments that employ virally driven neuromodulation techniques to predict anatomy-based viral labeling.

Recent advances in understanding the role of leptin in energy homeostasis.

The hormone leptin plays a critical role in energy homeostasis, although our overall understanding of acutely changing leptin levels still needs improvement. Several developments allow a fresh look at recent and early data on leptin action. This review highlights select recent publications that are relevant for understanding the role played by dynamic changes in circulating leptin levels. We further discuss the relevance for our current understanding of leptin signaling in central neuronal feeding and energy expenditure circuits and highlight cohesive and discrepant findings that need to be addressed in future studies to understand how leptin couples with physiological adaptations of food intake and energy expenditure.

The obesity epidemic in the face of homeostatic body weight regulation: What went wrong and how can it be fixed?

Ever since the pioneering discoveries in the mid nineteen hundreds, the hypothalamus was recognized as a crucial component of the neural system controlling appetite and energy balance. The new wave of neuron-specific research tools has confirmed this key role of the hypothalamus and has delineated many other brain areas to be part of an expanded neural system sub serving these crucial functions. However, despite significant progress in defining this complex neural circuitry, many questions remain. One of the key questions is why the sophisticated body weight regulatory system is unable to prevent the rampant obesity epidemic we are experiencing. Why are pathologically obese body weight levels defended, and what can we do about it? Here we try to find answers to these questions by 1) reminding the reader that the neural controls of ingestive behavior have evolved in a demanding, restrictive environment and encompass much of the brain's major functions, far beyond the hypothalamus and brainstem, 2) hypothesizing that the current obesogenic environment impinges mainly on a critical pathway linking hypothalamic areas with the motivational and reward systems to produce uncompensated hyperphagia, and 3) proposing adequate strategies for prevention and treatment.

FGF21 and the Physiological Regulation of Macronutrient Preference.

The ability to respond to variations in nutritional status depends on regulatory systems that monitor nutrient intake and adaptively alter metabolism and feeding behavior during nutrient restriction. There is ample evidence that the restriction of water, sodium, or energy intake triggers adaptive responses that conserve existing nutrient stores and promote the ingestion of the missing nutrient, and that these homeostatic responses are mediated, at least in part, by nutritionally regulated hormones acting within the brain. This review highlights recent research that suggests that the metabolic hormone fibroblast growth factor 21 (FGF21) acts on the brain to homeostatically alter macronutrient preference. Circulating FGF21 levels are robustly increased by diets that are high in carbohydrate but low in protein, and exogenous FGF21 treatment reduces the consumption of sweet foods and alcohol while alternatively increasing the consumption of protein. In addition, while control mice adaptively shift macronutrient preference and increase protein intake in response to dietary protein restriction, mice that lack either FGF21 or FGF21 signaling in the brain fail to exhibit this homeostatic response. FGF21 therefore mediates a unique physiological niche, coordinating adaptive shifts in macronutrient preference that serve to maintain protein intake in the face of dietary protein restriction.