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.

CRISPR-Cas9 base editing of pathogenic CaMKIIδ improves cardiac function in a humanized mouse model.

Cardiovascular diseases are the most common cause of worldwide morbidity and mortality, highlighting the necessity for advanced therapeutic strategies. Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is a prominent inducer of various cardiac disorders, which is mediated by 2 oxidation-sensitive methionine residues within the regulatory domain. We have previously shown that ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing enables the heart to recover function from otherwise severe damage following ischemia/reperfusion (IR) injury. Here, we extended this therapeutic concept toward potential clinical translation. We generated a humanized CAMK2D knockin mouse model in which the genomic sequence encoding the entire regulatory domain was replaced with the human sequence. This enabled comparison and optimization of two different editing strategies for the human genome in mice. To edit CAMK2D in vivo, we packaged the optimized editing components into an engineered myotropic adeno-associated virus (MyoAAV 2A), which enabled efficient delivery at a very low AAV dose into the humanized mice at the time of IR injury. CAMK2D-edited mice recovered cardiac function, showed improved exercise performance, and were protected from myocardial fibrosis, which was otherwise observed in injured control mice after IR. Our findings identify a potentially effective strategy for cardioprotection in response to oxidative damage.

PAQR4 regulates adipocyte function and systemic metabolic health by mediating ceramide levels.

PAQR4 is an orphan receptor in the PAQR family with an unknown function in metabolism. Here, we identify a critical role of PAQR4 in maintaining adipose tissue function and whole-body metabolic health. We demonstrate that expression of Paqr4 specifically in adipocytes, in an inducible and reversible fashion, leads to partial lipodystrophy, hyperglycaemia and hyperinsulinaemia, which is ameliorated by wild-type adipose tissue transplants or leptin treatment. By contrast, deletion of Paqr4 in adipocytes improves healthy adipose remodelling and glucose homoeostasis in diet-induced obesity. Mechanistically, PAQR4 regulates ceramide levels by mediating the stability of ceramide synthases (CERS2 and CERS5) and, thus, their activities. Overactivation of the PQAR4-CERS axis causes ceramide accumulation and impairs adipose tissue function through suppressing adipogenesis and triggering adipocyte de-differentiation. Blocking de novo ceramide biosynthesis rescues PAQR4-induced metabolic defects. Collectively, our findings suggest a critical function of PAQR4 in regulating cellular ceramide homoeostasis and targeting PAQR4 offers an approach for the treatment of metabolic disorders.

Inhibition of AXL receptor tyrosine kinase enhances brown adipose tissue functionality in mice.

The current obesity epidemic and high prevalence of metabolic diseases necessitate efficacious and safe treatments. Brown adipose tissue in this context is a promising target with the potential to increase energy expenditure, however no pharmacological treatments activating brown adipose tissue are currently available. Here, we identify AXL receptor tyrosine kinase as a regulator of adipose function. Pharmacological and genetic inhibition of AXL enhance thermogenic capacity of brown and white adipocytes, in vitro and in vivo. Mechanistically, these effects are mediated through inhibition of PI3K/AKT/PDE signaling pathway, resulting in induction of nuclear FOXO1 localization and increased intracellular cAMP levels via PDE3/4 inhibition and subsequent stimulation of the PKA-ATF2 pathway. In line with this, both constitutive Axl deletion as well as inducible adipocyte-specific Axl deletion protect animals from diet-induced obesity concomitant with increases in energy expenditure. Based on these data, we propose AXL receptor as a target for the treatment of obesity.

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).

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.

Adipocyte iron levels impinge on a fat-gut crosstalk to regulate intestinal lipid absorption and mediate protection from obesity.

Iron overload is positively associated with diabetes risk. However, the role of iron in adipose tissue remains incompletely understood. Here, we report that transferrin-receptor-1-mediated iron uptake is differentially required for distinct subtypes of adipocytes. Notably, adipocyte-specific transferrin receptor 1 deficiency substantially protects mice from high-fat-diet-induced metabolic disorders. Mechanistically, low cellular iron levels have a positive impact on the health of the white adipose tissue and can restrict lipid absorption from the intestine through modulation of vesicular transport in enterocytes following high-fat diet feeding. Specific reduction of adipocyte iron by AAV-mediated overexpression of the iron exporter Ferroportin1 in adult mice effectively mimics these protective effects. In summary, our studies highlight an important role of adipocyte iron in the maintenance of systemic metabolism through an adipocyte-enterocyte axis, offering an additional level of control over caloric influx into the system after feeding by regulating intestinal lipid absorption.

Metabolic Messengers: Adiponectin.

Adiponectin is one of the most widely studied adipokines to date. First described in the mid-1990's, studying its regulation, biogenesis and physiological effects has proven to be extremely insightful and improved our understanding of the mechanisms that ensure systemic metabolic homeostasis. Here, we provide a brief overview of the current state of the field with respect to adiponectin, its history, sites and mechanisms of action, and the critical questions that will need to be addressed in the future.

Lipolysis Triggers a Systemic Insulin Response Essential for Efficient Energy Replenishment of Activated Brown Adipose Tissue in Mice.

The coordination of the organ-specific responses regulating systemic energy distribution to replenish lipid stores in acutely activated brown adipose tissue (BAT) remains elusive. Here, we show that short-term cold exposure or acute ß3-adrenergic receptor (ß3AR) stimulation results in secretion of the anabolic hormone insulin. This process is diminished in adipocyte-specific Atgl-/- mice, indicating that lipolysis in white adipose tissue (WAT) promotes insulin secretion. Inhibition of pancreatic ß cells abolished uptake of lipids delivered by triglyceride-rich lipoproteins into activated BAT. Both increased lipid uptake into BAT and whole-body energy expenditure in response to ß3AR stimulation were blunted in mice treated with the insulin receptor antagonist S961 or lacking the insulin receptor in brown adipocytes. In conclusion, we introduce the concept that acute cold and ß3AR stimulation trigger a systemic response involving WAT, ß cells, and BAT, which is essential for insulin-dependent fuel uptake and adaptive thermogenesis.

Author Correction: Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

In the version of this article originally published, the months on the axis labeled projected month of conception in Fig. 1a were out of order. April and March should have been the first and last months listed, respectively. The error has been corrected in the print, PDF and HTML versions of this article.

Publisher Correction: Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

In the version of this article originally published, the bars in the mean temperature graph in Fig. 1a were incorrectly aligned. The left-most bar should have been aligned with the Apr label on the projected month of conception axis. The error has been corrected in the print, PDF and HTML versions of this article.

Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

Recent research has focused on environmental effects that control tissue functionality and systemic metabolism. However, whether such stimuli affect human thermogenesis and body mass index (BMI) has not been explored. Here we show retrospectively that the presence of brown adipose tissue (BAT) and the season of conception are linked to BMI in humans. In mice, we demonstrate that cold exposure (CE) of males, but not females, before mating results in improved systemic metabolism and protection from diet-induced obesity of the male offspring. Integrated analyses of the DNA methylome and RNA sequencing of the sperm from male mice revealed several clusters of co-regulated differentially methylated regions (DMRs) and differentially expressed genes (DEGs), suggesting that the improved metabolic health of the offspring was due to enhanced BAT formation and increased neurogenesis. The conclusions are supported by cell-autonomous studies in the offspring that demonstrate an enhanced capacity to form mature active brown adipocytes, improved neuronal density and more norepinephrine release in BAT in response to cold stimulation. Taken together, our results indicate that in humans and in mice, seasonal or experimental CE induces an epigenetic programming of the sperm such that the offspring harbor hyperactive BAT and an improved adaptation to overnutrition and hypothermia.

LSD1 Makes Fat Colorful.

The histone lysine-specific demethylase 1 (LSD1) is a new and important player in the regulation of brown fat identity and function. In a recent Cell Reports article, Duteil et al. show that LSD1 exerts its effects via regulation of specific histone marks as well as through association with co-repressor complexes.

Bmp4 Promotes a Brown to White-like Adipocyte Shift.

While Bmp4 has a well-established role in the commitment of mesenchymal stem cells into the adipogenic lineage, its role in brown adipocyte formation and activity is not well defined. Here, we show that Bmp4 has a dual function in adipogenesis by inducing adipocyte commitment while inhibiting the acquisition of a brown phenotype during terminal differentiation. Selective brown adipose tissue overexpression of Bmp4 in mice induces a shift from a brown to a white-like adipocyte phenotype. This effect is mediated by Smad signaling and might be in part due to suppression of lipolysis, via regulation of hormone sensitive lipase expression linked to reduced Ppar activity. Given that we observed a strong correlation between BMP4 levels and adipocyte size, as well as insulin sensitivity in humans, we propose that Bmp4 is an important factor in the context of obesity and type 2 diabetes.

Regulation of De Novo Adipocyte Differentiation Through Cross Talk Between Adipocytes and Preadipocytes.

There are many known adipokines differentially secreted from the different adipose depots; however, their paracrine and autocrine effects on de novo adipocyte formation are not fully understood. By developing a coculture method of preadipocytes with primary subcutaneous and visceral adipocytes or tissue explants, we could show that the total secretome inhibited preadipocyte differentiation. Using a proteomics approach with fractionated secretome samples, we were able to identify a spectrum of factors that either positively or negatively affected adipocyte formation. Among the secreted factors, Slc27a1, Vim, Cp, and Ecm1 promoted adipocyte differentiation, whereas Got2, Cpq, interleukin-1 receptor-like 1/ST2-IL-33, Sparc, and Lgals3bp decreased adipocyte differentiation. In human subcutaneous adipocytes of lean subjects, obese subjects, and obese subjects with type 2 diabetes, Vim and Slc27a1 expression was negatively correlated with adipocyte size and BMI and positively correlated with insulin sensitivity, while Sparc and Got2 showed the opposite trend. Furthermore, we demonstrate that Slc27a1 was increased upon weight loss in morbidly obese patients, while Sparc expression was reduced. Taken together, our findings identify adipokines that regulate adipocyte differentiation through positive or negative paracrine and autocrine feedback loop mechanisms, which could potentially affect whole-body energy metabolism.