Despite the continuous renewal and turnover of the small intestinal epithelium, the intestinal crypt maintains a ‘soccer ball-like’, alternating pattern of stem and Paneth cells at the base of the crypt. To study the robustness of the alternating pattern, we used intravital two-photon microscopy in mice with fluorescently-labeled Lgr5+ intestinal stem cells and precisely perturbed the mosaic pattern with femtosecond laser ablation. Ablation of one to three cells initiated rapid motion of crypt cells that restored the alternation in the pattern within about two hours with only the rearrangement of pre-existing cells, without any cell division. Crypt cells then performed a coordinated dilation of the crypt lumen, which resulted in peristalsis-like motion that forced damaged cells out of the crypt. Crypt cell motion was reduced with inhibition of the ROCK pathway and attenuated with old age, and both resulted in incomplete pattern recovery. This suggests that in addition to proliferation and self-renewal, motility of stem cells is critical for maintaining homeostasis. Reduction of this newly-identified behavior of stem cells could contribute to disease and age-related changes.

Similar to insulin, central administration of IGF-1 can suppress hepatic glucose production (HGP), but it is unclear whether this effect is mediated via insulin receptors (InsRs) or IGF-1 receptors (IGF-1Rs) in the brain. To this end, we used pharmacologic and genetic approaches in combination with hyperinsulinemic-euglycemic clamps to decipher the role of these receptors in mediating central effects of IGF-1 and insulin on HGP. In rats, we observed that intracerebroventricular (ICV) administration of IGF-1 or insulin markedly increased the glucose infusion rate (GIR) by >50% and suppressed HGP (P < 0.001). However, these effects were completely prevented by preemptive ICV infusion with an IGF-1R and InsR/IGF-1R hybrid (HybridR) blocking antibody. Likewise, ICV infusion of the InsR antagonist, S961, which also can bind HybridRs, interfered with the ability of central insulin, but not IGF-1, to increase the GIR. Furthermore, hyperinsulinemic clamps in mice lacking IGF-1Rs in AgRP neurons revealed ∼30% reduction in the GIR in knockout animals, which was explained by an impaired ability of peripheral insulin to completely suppress HGP (P < 0.05). Signaling studies further revealed an impaired ability of peripheral insulin to trigger ribosomal S6 phosphorylation or phosphatidylinositol (3,4,5)-trisphosphate production in AgRP neurons lacking IGF-1Rs. In summary, these data suggest that attenuation of IGF-1R signaling in the mediobasal hypothalamus, and specifically in AgRP neurons, can phenocopy impaired regulation of HGP as previously demonstrated in mice lacking InsRs in these cells, suggesting a previously unappreciated role for IGF-1Rs and/or HybridRs in the regulation of central insulin/IGF-1 signaling in glucose metabolism.

Surgical parabiosis enables sharing of the circulating milieu between two organisms. This powerful model presents diverse complications based on age, strain, sex, and other experimental parameters. Here, we provide an optimized parabiosis protocol for the surgical union of two mice internally at the elbow and knee joints with continuous external joining of the skin. This protocol incorporates guidance and solutions to complications that can occur, particularly in aging studies, including non-cohesive pairing, variable anesthesia sensitivity, external and internal dehiscence, dehydration, and weight loss. We also offer a straightforward method for validating postoperative blood chimerism and confirming its time course using flow cytometry. Utilization of our optimized protocol can facilitate reproducible parabiosis experimentation to dynamically explore mechanisms of aging and rejuvenation.

Parabiosis is a well-established method to facilitate a shared blood supply between two conjoined animals. In particular, the pairing of mice of dissimilar ages, termed heterochronic parabiosis, has been used extensively for differentiating cell autonomous and non-autonomous mechanisms of aging. Analysis of heterochronic parabionts also has helped to identify individual circulating factors that may act as either pro- or anti-geronics. Heterochronic parabiosis also has proven to be a valuable experimental system to evaluate the effects of specific hallmarks of aging on the process of aging. For example, heterochronic parabiosis was used recently to examine whether cellular senescence was driven via cell autonomous and/or non-autonomous mechanisms. As anticipated, markers of cellular senescence were elevated in old isochronically-paired mice relative to young controls. However, compared to old isochronically paired mice, the senescent cell burden was reduced in multiple tissues of old parabionts joined with young mice. This suggests that the rejuvenation of cells and tissues in old mice by exposure to young blood could be mediated, in part, through suppression or immune clearance of senescent cells. Conversely, young heterochronic parabionts showed increased markers of cellular senescence, demonstrating that exposure to an old circulation is able to drive senescence through a cell non-autonomous mechanism(s), likely contributing to accelerated aging in the young mice. Thus, heterochronic parabiosis is still an important methodology that should continue to be leveraged for evaluating other hallmarks of aging and their mechanisms.