Quantification of adipocyte numbers in transgenic mice via the Cre-LoxP recombination sites

This protocol describes a method to assess adipocyte numbers within a specific depot based on their inducible genomic label. By extracting DNA from a complete adipose tissue depot stemming from two transgenic mouse lines (Adipoq-CreERT2 x ROSA26-tdRFP and Ucp1-CreERT2 x ROSA26-tdRFP), the number of adipocytes can be determined based on the quantification of the recombined LoxPRed sites. This highly sensitive system allows for the quantification of white, brown, and brite/beige adipocytes in a spatially unbiased and size-independent manner. For complete details on the use and execution of this protocol, please refer to Moser et al. (2021).ISSN:2666-166

Quantification of adipocyte numbers following adipose tissue remodeling

To analyze the capacity of white and brown adipose tissue remodeling, we developed two mouse lines to label, quantitatively trace, and ablate white, brown, and brite/beige adipocytes at different ambient temperatures. We show here that the brown adipocytes are recruited first and reach a peak after 1 week of cold stimulation followed by a decline during prolonged cold exposure. On the contrary, brite/beige cell numbers plateau after 3 weeks of cold exposure. At thermoneutrality, brown adipose tissue, in spite of being masked by a white-like morphology, retains its brown-like physiology, as Ucp1+ cells can be recovered immediately upon beta3-adrenergic stimulation. We further demonstrate that the recruitment of Ucp1+ cells in response to cold is driven by existing adipocytes. In contrast, the regeneration of the interscapular brown adipose tissue following ablation of Ucp1+ cells is driven by de novo differentiation.ISSN:2666-3864ISSN:2211-124

Antioxidants protect against diabetes by improving glucose homeostasis in mouse models of inducible insulin resistance and obesity

Aims/hypothesis In the context of diabetes, the health benefit of antioxidant treatment has been widely debated. In this study, we investigated the effect of antioxidant treatment during the development of insulin resistance and hyperphagia in obesity and partial lipodystrophy. Methods We studied the role of antioxidants in the regulation of insulin resistance using the tamoxifen-inducible fat-specific insulin receptor knockout (iFIRKO) mouse model, which allowed us to analyse the antioxidant’s effect in a time-resolved manner. In addition, leptin-deficient ob/ob mice were used as a hyperphagic, chronically obese and diabetic mouse model to validate the beneficial effect of antioxidants on metabolism. Results Acute induction of insulin receptor knockout in adipocytes changed the substrate preference to fat before induction of a diabetic phenotype including hyperinsulinaemia and hyperglycaemia. In healthy chow-fed animals as well as in morbidly obese mice, this diabetic phase could be reversed within a few weeks. Furthermore, after the induction of insulin receptor knockout in mature adipocytes, iFIRKO mice were protected from subsequent obesity development through high-fat diet feeding. By genetic tracing we show that the persistent fat mass loss in mice after insulin receptor knockout in adipocytes is not caused by the depletion of adipocytes. Treatment of iFIRKO mice with antioxidants postponed and reduced hyperglycaemia by increasing insulin sensitivity. In ob/ob mice, antioxidants rescued both hyperglycaemia and hyperphagia. Conclusions/interpretation We conclude that fat mass reduction through insulin resistance in adipocytes is not reversible. Furthermore, it seems unlikely that adipocytes undergo apoptosis during the process of extreme lipolysis, as a consequence of insulin resistance. Antioxidants have a beneficial health effect not only during the acute phase of diabetes development, but also in a temporary fashion once chronic obesity and diabetes have been established