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.

Lessons from Cre-Mice and Indicator Mice.

The adult human adipose tissue is predominantly composed of white adipocytes. However, within certain depots, adipose tissue contains thermogenically active brown-like adipocytes, which have been evolutionarily conserved in mammals. This chapter will give a brief overview on the methods used to genetically target and trace both white and brown adipocytes using techniques such as bacterial artificial chromosome (BAC) cloning to create transgenic mouse models and the tools with which genetic recombination is mediated in vivo (e.g., Cre-loxP, CreERT, and Tet-On). The chapter furthermore critically discusses the strength and limitation of the various systems used to target mature white and brown adipocytes (ap2-Cre, Adipoq-Cre, and Ucp1-Cre). Based on these systems, it is evident that our knowledge of mature adipocyte categorization into brown, white, brite, or beige adipocytes is strongly influenced by the use of the various genetic mouse models described in this chapter. Our evaluation of different studies using the aforementioned systems focuses on key genes, which have been reported to maintain adipocyte's function (insulin receptor, Raptor, or Atgl).

Short-term feeding of a ketogenic diet induces more severe hepatic insulin resistance than an obesogenic high-fat diet.

KEY POINTS: A ketogenic diet is known to lead to weight loss and is considered metabolically healthy; however there are conflicting reports on its effect on hepatic insulin sensitivity. KD fed animals appear metabolically healthy in the fasted state after 3 days of dietary challenge, whereas obesogenic high-fat diet (HFD) fed animals show elevated insulin levels. A glucose challenge reveals that both KD and HFD fed animals are glucose intolerant. Glucose intolerance correlates with increased lipid oxidation and lower respiratory exchange ratio (RER); however, all animals respond to glucose injection with an increase in RER. Hyperinsulinaemic-euglycaemic clamps with double tracer show that the effect of KD is a result of hepatic insulin resistance and increased glucose output but not impaired glucose clearance or tissue glucose uptake in other tissues. ABSTRACT: Despite being a relevant healthcare issue and heavily investigated, the aetiology of type 2 diabetes (T2D) is still incompletely understood. It is well established that increased endogenous glucose production (EGP) leads to a progressive increase in glucose levels, causing insulin resistance and eventual loss of glucose homeostasis. The consumption of high carbohydrate, high-fat, western style diet (HFD) is linked to the development of T2D and obesity, whereas the consumption of a low carbohydrate, high-fat, ketogenic diet (KD) is considered healthy. However, several days of carbohydrate restriction are known to cause selective hepatic insulin resistance. In the present study, we compare the effects of short-term HFD and KD feeding on glucose homeostasis in mice. We show that, even though KD fed animals appear to be healthy in the fasted state, they exhibit decreased glucose tolerance to a greater extent than HFD fed animals. Furthermore, we show that this effect originates from blunted suppression of hepatic glucose production by insulin, rather than impaired glucose clearance and tissue glucose uptake. These data suggest that the early effects of HFD consumption on EGP may be part of a normal physiological response to increased lipid intake and oxidation, and that systemic insulin resistance results from the addition of dietary glucose to EGP-derived glucose.

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.

Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays.

Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specific detection of compounds with alkyne or azido groups. Here we describe the use of alkyne lipids as substrates for in vitro enzymatic assays of lipid modifying enzymes. The small alkyne moiety is introduced synthetically at the terminus of the hydrocarbon chain of various substrate lipids. After the assay, the label is click-reacted with the azide-bearing fluorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thin-layer chromatography and fluorescence detection, resulting in high sensitivity and wide-range linearity. Kinetic analyses using alkyne-labeled substrates for lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases, and ceramide synthases resulted in Michaelis-Menten constants similar to those for radiolabeled or natural substrates. We tested additional alkyne substrates for several hydrolases and acyltransferases in lipid metabolism. In this pilot study we establish alkyne lipids as a new class of convenient substrates for in vitro enzymatic assays.

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.

Protective roles of adiponectin and molecular signatures of HNF4α and PPARα as downstream targets of adiponectin in pancreatic ß cells.

The disease progression of the metabolic syndrome is associated with prolonged hyperlipidemia and insulin resistance, eventually giving rise to impaired insulin secretion, often concomitant with hypoadiponectinemia. As an adipose tissue derived hormone, adiponectin is beneficial for insulin secretion and ß cell health and differentiation. However, the down-stream pathway of adiponectin in the pancreatic islets has not been studied extensively. Here, along with the overall reduction of endocrine pancreatic function in islets from adiponectin KO mice, we examine PPARα and HNF4α as additional down-regulated transcription factors during a prolonged metabolic challenge. To elucidate the function of ß cell-specific PPARα and HNF4α expression, we developed doxycycline inducible pancreatic ß cell-specific PPARα (ß-PPARα) and HNF4α (ß-HNF4α) overexpression mice. ß-PPARα mice exhibited improved protection from lipotoxicity, but elevated ß-oxidative damage in the islets, and also displayed lowered phospholipid levels and impaired glucose-stimulated insulin secretion. ß-HNF4α mice showed a more severe phenotype when compared to ß-PPARα mice, characterized by lower body weight, small islet mass and impaired insulin secretion. RNA-sequencing of the islets of these models highlights overlapping yet unique roles of ß-PPARα and ß-HNF4α. Given that ß-HNF4α potently induces PPARα expression, we define a novel adiponectin-HNF4α-PPARα cascade. We further analyzed downstream genes consistently regulated by this axis. Among them, the islet amyloid polypeptide (IAPP) gene is an important target and accumulates in adiponectin KO mice. We propose a new mechanism of IAPP aggregation in type 2 diabetes through reduced adiponectin action.

Hyperleptinemia contributes to antipsychotic drug-associated obesity and metabolic disorders.

Despite their high degree of effectiveness in the management of psychiatric conditions, exposure to antipsychotic drugs, including olanzapine and risperidone, is frequently associated with substantial weight gain and the development of diabetes. Even before weight gain, a rapid rise in circulating leptin concentrations can be observed in most patients taking antipsychotic drugs. To date, the contribution of this hyperleptinemia to weight gain and metabolic deterioration has not been defined. Here, with an established mouse model that recapitulates antipsychotic drug-induced obesity and insulin resistance, we not only confirm that hyperleptinemia occurs before weight gain but also demonstrate that hyperleptinemia contributes directly to the development of obesity and associated metabolic disorders. By suppressing the rise in leptin through the use of a monoclonal leptin-neutralizing antibody, we effectively prevented weight gain, restored glucose tolerance, and preserved adipose tissue and liver function in antipsychotic drug-treated mice. Mechanistically, suppressing excess leptin resolved local tissue and systemic inflammation typically associated with antipsychotic drug treatment. We conclude that hyperleptinemia is a key contributor to antipsychotic drug-associated weight gain and metabolic deterioration. Leptin suppression may be an effective approach to reducing the undesirable side effects of antipsychotic drugs.

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.

Adipose tissue hyaluronan production improves systemic glucose homeostasis and primes adipocytes for CL 316,243-stimulated lipolysis.

Plasma hyaluronan (HA) increases systemically in type 2 diabetes (T2D) and the HA synthesis inhibitor, 4-Methylumbelliferone, has been proposed to treat the disease. However, HA is also implicated in normal physiology. Therefore, we generated a Hyaluronan Synthase 2 transgenic mouse line, driven by a tet-response element promoter to understand the role of HA in systemic metabolism. To our surprise, adipocyte-specific overproduction of HA leads to smaller adipocytes and protects mice from high-fat-high-sucrose-diet-induced obesity and glucose intolerance. Adipocytes also have more free glycerol that can be released upon beta3 adrenergic stimulation. Improvements in glucose tolerance were not linked to increased plasma HA. Instead, an HA-driven systemic substrate redistribution and adipose tissue-liver crosstalk contributes to the systemic glucose improvements. In summary, we demonstrate an unexpected improvement in glucose metabolism as a consequence of HA overproduction in adipose tissue, which argues against the use of systemic HA synthesis inhibitors to treat obesity and T2D.

A Genetic Model to Study the Contribution of Brown and Brite Adipocytes to Metabolism.

UCP1-dependent thermogenesis is studied to define new strategies to ameliorate obesity and type 2 diabetes; however, animal models are mostly limited to germline mutations of UCP1, which can effect adaptive changes in UCP1-independent pathways. We develop an inducible mouse model for the sequential ablation of UCP1+ brown and brite/beige adipocytes in adult mice. We demonstrate that activated brown adipocytes can increase systemic energy expenditure (EE) by 30%, while the contribution of brite/beige UCP1+ cells is <5%. Notably, UCP1+ adipocytes do not contribute to circulating FGF21 levels, either at room temperature or after cold exposure. We demonstrate that the FGF21-mediated effects on EE and glucose homeostasis are partially dependent on the presence of UCP1+ cells, while the effect on weight loss is not. In conclusion, acute UCP1+ cell deletion may be a useful model to study the impact of brown and brite/beige adipocytes on metabolism.

Partial leptin deficiency confers resistance to diet-induced obesity in mice.

OBJECTIVE: Hyperleptinemia per se is sufficient to promote leptin resistance in the obese state. Leptin sensitivity can be restored by reducing circulating leptin levels within a physiologically healthy range and is a viable antiobesity and antidiabetic strategy. However, a previous study suggests that partial leptin deficiency favors diet-induced obesity and related metabolic disorders in mice, arguing that a lower leptin level may indeed promote diet-induced obesity and its associated metabolic disorders. Here, we aim to elucidate what the impact of partial leptin deficiency is on fat mass and insulin sensitivity. METHODS: We used two different mouse models of partial leptin deficiency: an adipocyte-specific congenital heterozygous leptin knockout mouse line (LepHZ) and the well-established whole body heterozygous leptin knockout mouse (OBHZ). The metabolic studies of OBHZ and LepHZ mice were performed both on normal carbohydrate-rich chow diet and on a high-fat diet (HFD). Male and female mice were included in the study to account for sex-specific differences. Body weight, food intake, glucose tolerance, and insulin tolerance were tested. Histology of adipose tissue and liver tissue allowed insights into adipose tissue inflammation and hepatic triglyceride content. Immunohistochemistry was paired with RT-PCR analysis for expression levels of inflammatory markers. RESULTS: Both OBHZ and LepHZ mice displayed reduced circulating leptin levels on the chow diet and HFD. On chow diet, male OBHZ and LepHZ mice showed elevated fat mass and body weight, while their glucose tolerance and insulin sensitivity remained unchanged. However, the inability in partially leptin-deficient mice to fully induce circulating leptin during the development of diet-induced obesity results in reduced food intake and leaner mice with lower body weight compared to their littermate controls. Importantly, a strong reduction of adipose tissue inflammation is observed along with improvements in insulin sensitivity and enhanced glucose tolerance. Additionally, partial leptin deficiency protects the mice from fatty liver and liver fibrosis. Chronically HFD-fed OBHZ and LepHZ mice remain more sensitive to exogenous leptin injection, as reflected by their reduced food intake upon an acute leptin treatment. CONCLUSION: In response to HFD feeding, the inability to upregulate leptin levels due to partial leptin deficiency protects mice from diet-induced obesity and metabolic dysregulation. Thus, in an obesogenic environment, maintaining lower leptin levels is highly beneficial for both obesity and diabetes management. Chronic leptin reduction represents a viable preventive strategy whose efficacy awaits clinical testing.

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.

Dysregulation of Amyloid Precursor Protein Impairs Adipose Tissue Mitochondrial Function and Promotes Obesity.

Mitochondrial function in white adipose tissue (WAT) is an important yet understudied aspect in adipocyte biology. Here, we report a role for amyloid precursor protein (APP) in compromising WAT mitochondrial function through a high-fat diet (HFD)-induced, unconventional mis-localization to mitochondria that further promotes obesity. In humans and mice, obese conditions significantly induce APP production in WAT and its enrichment in mitochondria. Mechanistically, a HFD-induced dysregulation of signal recognition particle subunit 54c is responsible for the mis-targeting of APP to adipocyte mitochondria. Mis-localized APP blocks the protein import machinery, leading to mitochondrial dysfunction in WAT. Adipocyte-specific and mitochondria-targeted APP overexpressing mice display increased body mass and reduced insulin sensitivity, along with dysfunctional WAT due to a dramatic hypertrophic program in adipocytes. Elimination of adipocyte APP rescues HFD-impaired mitochondrial function with significant protection from weight gain and systemic metabolic deficiency. Our data highlights an important role of APP in modulating WAT mitochondrial function and obesity-associated metabolic dysfunction.

Inhibition of Mevalonate Pathway Prevents Adipocyte Browning in Mice and Men by Affecting Protein Prenylation.

Recent research focusing on brown adipose tissue (BAT) function emphasizes its importance in systemic metabolic homeostasis. We show here that genetic and pharmacological inhibition of the mevalonate pathway leads to reduced human and mouse brown adipocyte function in vitro and impaired adipose tissue browning in vivo. A retrospective analysis of a large patient cohort suggests an inverse correlation between statin use and active BAT in humans, while we show in a prospective clinical trial that fluvastatin reduces thermogenic gene expression in human BAT. We identify geranylgeranyl pyrophosphate as the key mevalonate pathway intermediate driving adipocyte browning in vitro and in vivo, whose effects are mediated by geranylgeranyltransferases (GGTases), enzymes catalyzing geranylgeranylation of small GTP-binding proteins, thereby regulating YAP1/TAZ signaling through F-actin modulation. Conversely, adipocyte-specific ablation of GGTase I leads to impaired adipocyte browning, reduced energy expenditure, and glucose intolerance under obesogenic conditions, highlighting the importance of this pathway in modulating brown adipocyte functionality and systemic metabolism.

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.