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.

New insights in the mechanisms of weight-loss maintenance: Summary from a Pennington symposium.

Obesity is a chronic disease that affects more than 650 million adults worldwide. Obesity not only is a significant health concern on its own, but predisposes to cardiometabolic comorbidities, including coronary heart disease, dyslipidemia, hypertension, type 2 diabetes, and some cancers. Lifestyle interventions effectively promote weight loss of 5% to 10%, and pharmacological and surgical interventions even more, with some novel approved drugs inducing up to an average of 25% weight loss. Yet, maintaining weight loss over the long-term remains extremely challenging, and subsequent weight gain is typical. The mechanisms underlying weight regain remain to be fully elucidated. The purpose of this Pennington Biomedical Scientific Symposium was to review and highlight the complex interplay between the physiological, behavioral, and environmental systems controlling energy intake and expenditure. Each of these contributions were further discussed in the context of weight-loss maintenance, and systems-level viewpoints were highlighted to interpret gaps in current approaches. The invited speakers built upon the science of obesity and weight loss to collectively propose future research directions that will aid in revealing the complicated mechanisms involved in the weight-reduced state.

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.

Weight-independent effects of Roux-en-Y gastric bypass surgery on remission of nonalcoholic fatty liver disease in mice.

OBJECTIVE: Obesity is a driver of non-alcoholic fatty liver disease (NAFLD), and interventions that decrease body weight, such as bariatric surgery and/or calorie restriction (CR), may serve as effective therapies. This study compared the effects of Roux-en-Y gastric bypass surgery (RYGB) and CR on hepatic function in mice with obesity and NAFLD. METHODS: C57BL/6J mice were fed a high-fat diet to promote obesity. At 16 weeks of age, mice were randomized to sham surgery (sham), RYGB, or CR weight matched to RYGB (WM). Body weight/composition, food intake, and energy expenditure (EE) were measured throughout treatment. Liver histopathology was evaluated from H&E-stained sections. Hepatic enzymes and glycogen content were determined by ELISA. Transcriptional signatures were revealed via RNA sequencing. RESULTS: RYGB reduced hepatic lipid content and adiposity while increasing EE and lean body mass relative to WM. Hepatic glycogen and bile acid content were increased after RYGB relative to sham and WM. RYGB activated enterohepatic signaling and genes regulating hepatic lipid homeostasis. CONCLUSIONS: RYGB improved whole-body composition and hepatic lipid homeostasis to a greater extent than CR in mice. RYGB was associated with discrete remodeling of the hepatic transcriptome, suggesting that surgery may be mechanistically additive to CR.

Reduction of Plasma BCAAs following Roux-en-Y Gastric Bypass Surgery Is Primarily Mediated by FGF21.

Type 2 diabetes (T2D) is a challenging health concern worldwide. A lifestyle intervention to treat T2D is difficult to adhere, and the effectiveness of approved medications such as metformin, thiazolidinediones (TZDs), and sulfonylureas are suboptimal. On the other hand, bariatric procedures such as Roux-en-Y gastric bypass (RYGB) are being recognized for their remarkable ability to achieve diabetes remission, although the underlying mechanism is not clear. Recent evidence points to branched-chain amino acids (BCAAs) as a potential contributor to glucose impairment and insulin resistance. RYGB has been shown to effectively lower plasma BCAAs in insulin-resistant or T2D patients that may help improve glycemic control, but the underlying mechanism for BCAA reduction is not understood. Hence, we attempted to explore the mechanism by which RYGB reduces BCAAs. To this end, we randomized diet-induced obese (DIO) mice into three groups that underwent either sham or RYGB surgery or food restriction to match the weight of RYGB mice. We also included regular chow-diet-fed healthy mice as an additional control group. Here, we show that compared to sham surgery, RYGB in DIO mice markedly lowered serum BCAAs most likely by rescuing BCAA breakdown in both liver and white adipose tissues. Importantly, the restored BCAA metabolism following RYGB was independent of caloric intake. Fasting insulin and HOMA-IR were decreased as expected, and serum valine was strongly associated with insulin resistance. While gut hormones such as glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are postulated to mediate various surgery-induced metabolic benefits, mice lacking these hormonal signals (GLP-1R/Y2R double KO) were still able to effectively lower plasma BCAAs and improve glucose tolerance, similar to mice with intact GLP-1 and PYY signaling. On the other hand, mice deficient in fibroblast growth factor 21 (FGF21), another candidate hormone implicated in enhanced glucoregulatory action following RYGB, failed to decrease plasma BCAAs and normalize hepatic BCAA degradation following surgery. This is the first study using an animal model to successfully recapitulate the RYGB-led reduction of circulating BCAAs observed in humans. Our findings unmasked a critical role of FGF21 in mediating the rescue of BCAA metabolism following surgery. It would be interesting to explore the possibility of whether RYGB-induced improvement in glucose homeostasis is partly through decreased BCAAs.

The PYY/Y2R-deficient male mouse is not protected from bone loss due to Roux-en-Y gastric bypass.

BACKGROUND: Peptide YY (PYY) is an anorexigenic gut hormone that also has anti-osteogenic effects, inhibiting osteoblastic activity and inducing catabolic effects. It has been postulated that increases in PYY after Roux-en-Y gastric bypass (RYGB) contribute to declines in bone mineral density (BMD) and increases in bone turnover. The aim of this study is to determine the role of the PYY Y2-receptor in mediating bone loss post-RYGB in mice. METHODS: We compared adult male wildtype (WT) and PYY Y2 receptor-deficient (KO) C57BL/6 mice that received RYGB (WT: n = 8; KO: n = 9), with sham-operated mice (Sham; WT: n = 9; KO: n = 10) and mice that were food-restricted to match the weights of the RYGB-treated group (Weight-Matched, WM; WT: n = 7; KO: n = 5). RYGB or sham surgery was performed at 15-16 weeks of age, and mice sacrificed 21 weeks later. We characterized bone microarchitecture with micro-computed tomography (µCT) at the distal femur (trabecular) and femoral midshaft (cortical). Differences in body weight, bone microarchitecture and biochemical bone markers (parathyroid hormone, PTH; C-telopeptide, CTX; and type 1 procollagen, P1NP) were compared using 2-factor ANOVA with Tukey's adjustments for multiple comparisons. RESULTS: Body weights were similar in the WT-RYGB, WT-WM, KO-RYGB, and KO-WM: 41-44 g; these groups weighed significantly less than the Sham surgery groups: 55-57 g. Trabecular BMD was 31-43 % lower in RYGB mice than either Sham or WM in WT and KO groups. This deficiency in trabecular bone was accompanied by a lower trabecular number (19 %-23 %), thickness (22 %-30 %) and increased trabecular spacing (25 %-34 %) in WT and KO groups (p < 0.001 for all comparisons vs. RYGB). RYGB led to lower cortical thickness, cortical tissue mineral density, and cortical bone area fraction as compared to Sham and WM in WT and KO groups (p ≤ 0.004 for all). There were no interactions between genotype and bone microarchitecture, with patterns of response to RYGB similar in both WT and KO groups. CTX and P1NP were significantly higher in RYGB mice than WM in WT and KO groups. PTH did not differ among groups. CONCLUSIONS: RYGB induced greater trabecular and cortical deficits and high bone turnover than observed in weight-matched mice, with a similar pattern in the WT and Y2RKO mice. Thus, skeletal effects of RYGB are independent of weight loss, and furthermore, PYY signaling through Y2R is not a key mediator of bone loss post-RYGB.

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.

Functional anatomy of the vagus system: How does the polyvagal theory comply?

Due to its pivotal role in autonomic networks and interoception, the vagus attracts continued interest from both basic scientists and therapists of various clinical disciplines. In particular, the widespread use of heart rate variability as an index of autonomic cardiac control and a proposed central role of the vagus in biopsychological concepts, e.g., the polyvagal theory, provide a good opportunity to recall basic features of vagal anatomy. In addition to the "classical" vagal brainstem nuclei, i.e., dorsal motor nucleus, nucleus ambiguus and nucleus tractus solitarii, the spinal trigeminal and paratrigeminal nuclei come into play as targets of vagal afferents. On the other hand, the nucleus of the solitary tract receives and integrates not only visceral but also somatic afferents.

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.

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.

Functional anatomy of the vagus system - Emphasis on the somato-visceral interface.

Due to its pivotal role in autonomic networks, the vagus attracts continuous interest from both basic scientists and clinicians. In particular, recent advances in vagus nerve stimulation strategies and their application to pathological conditions beyond epilepsy provide a good opportunity to recall basic features of vagal peripheral and central anatomy. In addition to the "classical" vagal brainstem nuclei, i.e., dorsal motor nucleus, nucleus ambiguus and nucleus tractus solitarii, the spinal trigeminal and paratrigeminal nuclei come into play as targets of vagal afferents. On the other hand, the nucleus of the solitary tract receives and integrates not only visceral but also somatic afferents. Thus, the vagus system participates significantly in what may be defined as "somato-visceral interface".

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.

Learning of food preferences: mechanisms and implications for obesity & metabolic diseases.

Omnivores, including rodents and humans, compose their diets from a wide variety of potential foods. Beyond the guidance of a few basic orosensory biases such as attraction to sweet and avoidance of bitter, they have limited innate dietary knowledge and must learn to prefer foods based on their flavors and postoral effects. This review focuses on postoral nutrient sensing and signaling as an essential part of the reward system that shapes preferences for the associated flavors of foods. We discuss the extensive array of sensors in the gastrointestinal system and the vagal pathways conveying information about ingested nutrients to the brain. Earlier studies of vagal contributions were limited by nonselective methods that could not easily distinguish the contributions of subsets of vagal afferents. Recent advances in technique have generated substantial new details on sugar- and fat-responsive signaling pathways. We explain methods for conditioning flavor preferences and their use in evaluating gut-brain communication. The SGLT1 intestinal sugar sensor is important in sugar conditioning; the critical sensors for fat are less certain, though GPR40 and 120 fatty acid sensors have been implicated. Ongoing work points to particular vagal pathways to brain reward areas. An implication for obesity treatment is that bariatric surgery may alter vagal function.

Gut-brain communication and obesity: understanding functions of the vagus nerve.

Given the crucial role of the gastrointestinal tract and associated organs in handling nutrient assimilation and metabolism, it has long been known that its communication with the brain is important for the control of ingestive behavior and body weight regulation. It is also clear that gut-brain communication is bidirectional and utilizes both rapid neural and slower humoral mechanisms and pathways. However, progress in understanding these mechanisms and leveraging them for the treatment of obesity and metabolic disease has been hindered by the enormous dimension of the gut mucosa, the complexity of the signaling systems, and lack of specific tools. With the ascent of modern neurobiological technology, our understanding of the role of vagal afferents in gut-brain communication has begun to change. The first function-specific populations of vagal afferents providing nutritional feedback as well as feed-forward signals have been identified with genetics-guided methodology, and it is hoped that extension of the methodology to other neural communication pathways will follow soon. Currently, efficient clinical leveraging of gut-brain communication to treat obesity and metabolic disease is limited to a few gut hormones, but a more complete understanding of function-specific and projection-specific neuronal populations should make it possible to develop selective and more effective neuromodulation approaches.

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.

Physiology of Energy Intake in the Weight-Reduced State.

Physiological adaptations to intentional weight loss can facilitate weight regain. This review summarizes emerging findings on hypothalamic and brainstem circuitry in the regulation of body weight and identifies promising areas for research to improve therapeutic interventions for sustainable weight loss. There is good evidence that body weight is actively regulated in a homeostatic fashion similar to other physiological parameters. However, the defended level of body weight is not fixed but rather depends on environmental conditions and genetic background in an allostatic fashion. In an environment with plenty of easily available energy-dense food and low levels of physical activity, prone individuals develop obesity. In a majority of individuals with obesity, body weight is strongly defended through counterregulatory mechanisms, such as hunger and hypometabolism, making weight loss challenging. Among the options for treatment or prevention of obesity, those directly changing the defended body weight would appear to be the most effective ones. There is strong evidence that the mediobasal hypothalamus is a master sensor of the metabolic state and an integrator of effector actions responsible for the defense of adequate body weight. However, other brain areas, such as the brainstem and limbic system, are also increasingly implicated in body weight defense mechanisms and may thus be additional targets for successful therapies.