Evaluating the Occurrence of Age-related Peripheral Neuropathy in HET3 Mice and Development of a Whole Tissue Imaging Technique for Analyzing Total Innervation in the Subcutaneous Adipose Depot

Adipose tissue can be characterized as either being a white (energy storing) depot or a brown (energy expending) depot and both have been found to contain dense networks of neural innervation. This adipose nerve supply regulates numerous metabolic functions and likely plays an important role in the function of adipose blood vessels. Recently our lab has shown in the C57BL/6 mouse model that peripheral neuropathy, or the dying back and dysfunction of the nerves in the superficial tissues such as the skin, can extend into the subcutaneous adipose tissue in conditions commonly associated with the neuropathic phenotype (i.e. diabetes, obesity, and aging.) We have collaborated with David Harrison’s research group at the Jackson Laboratory to further investigate the onset and penetrance of this nerve die-back due to age in the genetically heterozygous HET3 mouse model. To do this effectively we had to develop a whole depot immunostaining and imaging technique that would allow us to characterize the nerves within subcutaneous adipose tissue and to quantify the total innervation. With our new method of tissue processing and imaging combined with gene and protein expression measurements we have found that the neuropathic phenotype brought on by aging is present in the skin and muscles of HET3 mice but does not appear to be present in subcutaneous adipose. The differences in age-related neuropathy between HET3 and C57BL/6 mice are likely genetic and may represent the diversity of neuropathy outcomes in the human population

The involvement of neuroimmune cells in adipose innervation.

BACKGROUND: Innervation of adipose tissue is essential for the proper function of this critical metabolic organ. Numerous surgical and chemical denervation studies have demonstrated how maintenance of brain-adipose communication through both sympathetic efferent and sensory afferent nerves helps regulate adipocyte size, cell number, lipolysis, and \u27browning\u27 of white adipose tissue. Neurotrophic factors are growth factors that promote neuron survival, regeneration, and plasticity, including neurite outgrowth and synapse formation. Peripheral immune cells have been shown to be a source of neurotrophic factors in humans and mice. Although a number of immune cells reside in the adipose stromal vascular fraction (SVF), it has remained unclear what roles they play in adipose innervation. We previously demonstrated that adipose SVF secretes brain derived neurotrophic factor (BDNF). METHODS: We now show that deletion of this neurotrophic factor from the myeloid lineage of immune cells led to a \u27genetic denervation\u27 of inguinal subcutaneous white adipose tissue (scWAT), thereby causing decreased energy expenditure, increased adipose mass, and a blunted UCP1 response to cold stimulation. RESULTS: We and others have previously shown that noradrenergic stimulation via cold exposure increases adipose innervation in the inguinal depot. Here we have identified a subset of myeloid cells that home to scWAT upon cold exposure and are Ly6C CONCLUSIONS: We propose that these myeloid lineage, cold induced neuroimmune cells (CINCs) are key players in maintaining adipose innervation as well as promoting adipose nerve remodeling under noradrenergic stimulation, such as cold exposure

The Importance of Peripheral Nerves in Adipose Tissue for the Regulation of Energy Balance

Brown and white adipose tissues are essential for maintenance of proper energy balance and metabolic health. In order to function efficiently, these tissues require both endocrine and neural communication with the brain. Brown adipose tissue (BAT), as well as the inducible brown adipocytes that appear in white adipose tissue (WAT) after simulation, are thermogenic and energy expending. This uncoupling protein 1 (UCP1)-mediated process requires input from sympathetic nerves releasing norepinephrine. In addition to sympathetic noradrenergic signaling, adipose tissue contains sensory nerves that may be important for relaying fuel status to the brain. Chemical and surgical denervation studies of both WAT and BAT have clearly demonstrated the role of peripheral nerves in browning, thermogenesis, lipolysis, and adipogenesis. However, much is still unknown about which subtypes of nerves are present in BAT versus WAT, what nerve products are released from adipose nerves and how they act to mediate metabolic homeostasis, as well as which cell types in adipose are receiving synaptic input. Recent advances in whole-depot imaging and quantification of adipose nerve fibers, as well as other new research findings, have reinvigorated this field of research. This review summarizes the history of research into adipose innervation and brain⁻adipose communication, and also covers landmark and recent research on this topic to outline what we currently know and do not know about adipose tissue nerve supply and communication with the brain

Neuropathy and neural plasticity in the subcutaneous white adipose depot.

The difficulty in obtaining as well as maintaining weight loss, together with the impairment of metabolic control in conditions like diabetes and cardiovascular disease, may represent pathological situations of inadequate neural communication between the brain and peripheral organs and tissues. Innervation of adipose tissues by peripheral nerves provides a means of communication between the master metabolic regulator in the brain (chiefly the hypothalamus), and energy-expending and energy-storing cells in the body (primarily adipocytes). Although chemical and surgical denervation studies have clearly demonstrated how crucial adipose tissue neural innervation is for maintaining proper metabolic health, we have uncovered that adipose tissue becomes neuropathic (ie: reduction in neurites) in various conditions of metabolic dysregulation. Here, utilizing both human and mouse adipose tissues, we present evidence of adipose tissue neuropathy, or loss of proper innervation, under pathophysiological conditions such as obesity, diabetes, and aging, all of which are concomitant with insult to the adipose organ as well as metabolic dysfunction. Neuropathy is indicated by loss of nerve fiber protein expression, reduction in synaptic markers, and lower neurotrophic factor expression in adipose tissue. Aging-related adipose neuropathy particularly results in loss of innervation around the tissue vasculature, which cannot be reversed by exercise. Together with indications of neuropathy in muscle and bone, these findings underscore that peripheral neuropathy is not restricted to classic tissues like the skin of distal extremities, and that loss of innervation to adipose may trigger or exacerbate metabolic diseases. In addition, we have demonstrated stimulation of adipose tissue neural plasticity with cold exposure, which may ameliorate adipose neuropathy and be a potential therapeutic option to re-innervate adipose and restore metabolic health

Neuropathy and neural plasticity in the subcutaneous white adipose depot.

The difficulty in obtaining as well as maintaining weight loss, together with the impairment of metabolic control in conditions like diabetes and cardiovascular disease, may represent pathological situations of inadequate neural communication between the brain and peripheral organs and tissues. Innervation of adipose tissues by peripheral nerves provides a means of communication between the master metabolic regulator in the brain (chiefly the hypothalamus), and energy-expending and energy-storing cells in the body (primarily adipocytes). Although chemical and surgical denervation studies have clearly demonstrated how crucial adipose tissue neural innervation is for maintaining proper metabolic health, we have uncovered that adipose tissue becomes neuropathic (ie: reduction in neurites) in various conditions of metabolic dysregulation. Here, utilizing both human and mouse adipose tissues, we present evidence of adipose tissue neuropathy, or loss of proper innervation, under pathophysiological conditions such as obesity, diabetes, and aging, all of which are concomitant with insult to the adipose organ as well as metabolic dysfunction. Neuropathy is indicated by loss of nerve fiber protein expression, reduction in synaptic markers, and lower neurotrophic factor expression in adipose tissue. Aging-related adipose neuropathy particularly results in loss of innervation around the tissue vasculature, which cannot be reversed by exercise. Together with indications of neuropathy in muscle and bone, these findings underscore that peripheral neuropathy is not restricted to classic tissues like the skin of distal extremities, and that loss of innervation to adipose may trigger or exacerbate metabolic diseases. In addition, we have demonstrated stimulation of adipose tissue neural plasticity with cold exposure, which may ameliorate adipose neuropathy and be a potential therapeutic option to re-innervate adipose and restore metabolic health

Age-related changes to adipose tissue and peripheral neuropathy in genetically diverse HET3 mice differ by sex and are not mitigated by rapamycin longevity treatment

Neural communication between the brain and adipose tissues regulates energy expenditure and metabolism through modulation of adipose tissue functions. We have recently demonstrated that under pathophysiological conditions (obesity, diabetes, and aging), total subcutaneous white adipose tissue (scWAT) innervation is decreased (\u27adipose neuropathy\u27). With advanced age in the C57BL/6J mouse, small fiber peripheral nerve endings in adipose tissue die back, resulting in reduced contact with adipose-resident blood vessels and other cells. This vascular neuropathy and parenchymal neuropathy together likely pose a physiological challenge for tissue function. In the current work, we used the genetically diverse HET3 mouse model to investigate the incidence of peripheral neuropathy and adipose tissue dysregulation across several ages in both male and female mice. We also investigated the anti-aging treatment rapamycin, an mTOR inhibitor, as a means to prevent or reduce adipose neuropathy. We found that HET3 mice displayed a reduced neuropathy phenotype compared to inbred C56BL/6 J mice, indicating genetic contributions to this aging phenotype. Compared to female HET3 mice, male HET3 mice had worse neuropathic phenotypes by 62 weeks of age. Female HET3 mice appeared to have increased protection from neuropathy until advanced age (126 weeks), after reproductive senescence. We found that rapamycin overall had little impact on neuropathy measures, and actually worsened adipose tissue inflammation and fibrosis. Despite its success as a longevity treatment in mice, higher doses and longer delivery paradigms for rapamycin may lead to a disconnect between life span and beneficial health outcomes