Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.

Maternal obesity increases hypothalamic miR-505-5p expression in mouse offspring leading to altered fatty acid sensing and increased intake of high-fat food.

In utero exposure to maternal obesity programs increased obesity risk. Animal models show that programmed offspring obesity is preceded by hyperphagia, but the mechanisms that mediate these changes are unknown. Using a mouse model of maternal obesity, we observed increased intake of a high-fat diet (HFD) in offspring of obese mothers that precedes the development of obesity. Through small RNA sequencing, we identified programmed overexpression of hypothalamic miR-505-5p that is established in the fetus, lasts to adulthood and is maintained in hypothalamic neural progenitor cells cultured in vitro. Metabolic hormones and long-chain fatty acids associated with obesity increase miR-505-5p expression in hypothalamic neurons in vitro. We demonstrate that targets of miR-505-5p are enriched in fatty acid metabolism pathways and overexpression of miR-505-5p decreased neuronal fatty acid metabolism in vitro. miR-505-5p targets are associated with increased BMI in human genetic studies. Intra-cerebroventricular injection of miR-505-5p in wild-type mice increased HFD intake, mimicking the phenotype observed in offspring exposed to maternal obesity. Conversely, maternal exercise intervention in an obese mouse pregnancy rescued the programmed increase of hypothalamic miR-505-5p in offspring of obese dams and reduced HFD intake to control offspring levels. This study identifies a novel mechanism by which maternal obesity programs obesity in offspring via increased intake of high-fat foods.

Norepinephrine promotes triglyceride storage in macrophages via beta2-adrenergic receptor activation.

Tissue-resident macrophages are required for homeostasis, but also contribute to tissue dysfunction in pathophysiological states. The sympathetic neurotransmitter norepinephrine (NE) induces an anti-inflammatory and tissue-reparative phenotype in macrophages. As NE has a well-established role in promoting triglyceride lipolysis in adipocytes, and macrophages accumulate triglyceride droplets in various physiological and disease states, we investigated the effect of NE on primary mouse bone marrow-derived macrophage triglyceride metabolism. Surprisingly, our data show that in contrast to the canonical role of NE in stimulating lipolysis, NE acting via beta2-adrenergic receptors (B2ARs) in macrophages promotes extracellular fatty acid uptake and their storage as triglycerides and reduces free fatty acid release from triglyceride-laden macrophages. We demonstrate that these responses are mediated by a B2AR activation-dependent increase in Hilpda and Dgat1 gene expression and activity. We further show that B2AR activation favors the storage of extracellular polyunsaturated fatty acids. Finally, we present evidence that macrophages isolated from hearts after myocardial injury, for which survival critically depends on leukocyte B2ARs, have a transcriptional signature indicative of a transient triglyceride accumulation. Overall, we describe a novel and unexpected role of NE in promoting triglyceride storage in macrophages that could have potential implications in multiple diseases.

Lipotoxicity: a driver of heart failure with preserved ejection fraction?

Heart failure with preserved ejection fraction (HFpEF) is a growing public health concern, with rising incidence alongside high morbidity and mortality. However, the pathophysiology of HFpEF is not yet fully understood. The association between HFpEF and the metabolic syndrome (MetS) suggests that dysregulated lipid metabolism could drive diastolic dysfunction and subsequent HFpEF. Herein we summarise recent advances regarding the pathogenesis of HFpEF in the context of MetS, with a focus on impaired lipid handling, myocardial lipid accumulation and subsequent lipotoxicity.

Lipodystrophy-linked LMNA p.R482W mutation induces clinical early atherosclerosis and in vitro endothelial dysfunction.

OBJECTIVE: Some mutations in LMNA, encoding A-type lamins, are responsible for Dunnigan-type-familial partial lipodystrophy (FPLD2), with altered fat distribution and metabolism. The high prevalence of early and severe cardiovascular outcomes in these patients suggests that, in addition to metabolic risk factors, FPLD2-associated LMNA mutations could have a direct role on the vascular wall cells. APPROACH AND RESULTS: We analyzed the cardiovascular phenotype of 19 FPLD2 patients aged >30 years with LMNA p.R482 heterozygous substitutions, and the effects of p.R482W-prelamin-A overexpression in human coronary artery endothelial cells. In 68% of FPLD2 patients, early atherosclerosis was attested by clinical cardiovascular events, occurring before the age of 45 in most cases. In transduced endothelial cells, exogenous wild-type-prelamin-A was correctly processed and localized, whereas p.R482W-prelamin-A accumulated abnormally at the nuclear envelope. Patients' fibroblasts also showed a predominant nuclear envelope distribution with a decreased rate of prelamin-A maturation. Only p.R482W-prelamin-A induced endothelial dysfunction, with decreased production of NO, increased endothelial adhesion of peripheral blood mononuclear cells, and cellular senescence. p.R482W-prelamin-A also induced oxidative stress, DNA damages, and inflammation. These alterations were prevented by treatment of endothelial cells with pravastatin, which inhibits prelamin-A farnesylation, or with antioxidants. In addition, pravastatin allowed the correct relocalization of p.R482W-prelamin-A within the endothelial cell nucleus. These data suggest that farnesylated p.R482W-prelamin-A accumulation at the nuclear envelope is a toxic event, leading to cellular oxidative stress and endothelial dysfunction. CONCLUSIONS: LMNA p.R482 mutations, responsible for FPLD2, exert a direct proatherogenic effect in endothelial cells, which could contribute to patients' early atherosclerosis.

An adipocentric perspective of pancreatic lipotoxicity in diabetes pathogenesis.

Obesity and diabetes represent two increasing and invalidating public health issues that often coexist. It is acknowledged that fat mass excess predisposes to insulin resistance and type 2 diabetes mellitus (T2D), with the increasing incidence of the two diseases significantly associated. Moreover, emerging evidence suggests that obesity might also accelerate the appearance of type 1 diabetes (T1D), which is now a relatively frequent comorbidity in patients with obesity. It is a common clinical finding that not all patients with obesity will develop diabetes at the same level of adiposity, with gender, genetic, and ethnic factors playing an important role in defining the timing of diabetes appearance. The adipose tissue (AT) expandability hypothesis explains this paradigm, indicating that the individual capacity to appropriately store energy surplus in the form of fat within the AT determines and prevents the toxic deposition of lipids in other organs, such as the pancreas. Thus, we posit that when the maximal storing capacity of AT is exceeded, individuals will develop T2D. In this review, we provide insight into mechanisms by which the AT controls pancreas lipid content and homeostasis in case of obesity to offer an adipocentric perspective of pancreatic lipotoxicity in the pathogenesis of diabetes. Moreover, we suggest that improving AT function is a valid therapeutic approach to fighting obesity-associated complications including diabetes.

SMIM1 absence is associated with reduced energy expenditure and excess weight.

BACKGROUND: Obesity rates have nearly tripled in the past 50 years, and by 2030 more than 1 billion individuals worldwide are projected to be obese. This creates a significant economic strain due to the associated non-communicable diseases. The root cause is an energy expenditure imbalance, owing to an interplay of lifestyle, environmental, and genetic factors. Obesity has a polygenic genetic architecture; however, single genetic variants with large effect size are etiological in a minority of cases. These variants allowed the discovery of novel genes and biology relevant to weight regulation and ultimately led to the development of novel specific treatments. METHODS: We used a case-control approach to determine metabolic differences between individuals homozygous for a loss-of-function genetic variant in the small integral membrane protein 1 (SMIM1) and the general population, leveraging data from five cohorts. Metabolic characterization of SMIM1-/- individuals was performed using plasma biochemistry, calorimetric chamber, and DXA scan. FINDINGS: We found that individuals homozygous for a loss-of-function genetic variant in SMIM1 gene, underlying the blood group Vel, display excess body weight, dyslipidemia, altered leptin to adiponectin ratio, increased liver enzymes, and lower thyroid hormone levels. This was accompanied by a reduction in resting energy expenditure. CONCLUSION: This research identified a novel genetic predisposition to being overweight or obese. It highlights the need to investigate the genetic causes of obesity to select the most appropriate treatment given the large cost disparity between them. FUNDING: This work was funded by the National Institute of Health Research, British Heart Foundation, and NHS Blood and Transplant.

What the genetics of lipodystrophy can teach us about insulin resistance and diabetes.

Genetic lipodystrophic syndromes are rare diseases characterized by generalized or partial fat atrophy (lipoatrophy) associated with severe metabolic complications such as insulin resistance (IR), diabetes, dyslipidemia, nonalcoholic fatty liver disease, and ovarian hyperandrogenism. During the last 15 years, mutations in several genes have been shown to be responsible for monogenic forms of lipodystrophic syndromes, of autosomal dominant or recessive transmission. Although the molecular basis of lipodystrophies is heterogeneous, most mutated genes lead to impaired adipogenesis, adipocyte lipid storage, and/or formation or maintenance of the adipocyte lipid droplet (LD), showing that primary alterations of adipose tissue (AT) can result in severe systemic metabolic and endocrine consequences. The reduced expandability of AT alters its ability to buffer excess caloric intake, leading to ectopic lipid storage that impairs insulin signaling and other cellular functions ("lipotoxicity"). Genetic studies have also pointed out the close relationships between ageing, inflammatory processes, lipodystrophy, and IR.

Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice.

OBJECTIVES: Obesity in humans and mice is associated with elevated levels of two hormones responsive to cellular stress, namely GDF15 and FGF21. Over-expression of each of these is associated with weight loss and beneficial metabolic changes but where they are secreted from and what they are required for physiologically in the context of overfeeding remains unclear. METHODS: Here we used tissue selective knockout mouse models and human transcriptomics to determine the source of circulating GDF15 in obesity. We then generated and characterized the metabolic phenotypes of GDF15/FGF21 double knockout mice. RESULTS: Circulating GDF15 and FGF21 are both largely derived from the liver, rather than adipose tissue or skeletal muscle, in obese states. Combined whole body deletion of FGF21 and GDF15 does not result in any additional weight gain in response to high fat feeding but it does result in significantly greater hepatic steatosis and insulin resistance than that seen in GDF15 single knockout mice. CONCLUSIONS: Collectively the data suggest that overfeeding activates a stress response in the liver which is the major source of systemic rises in GDF15 and FGF21. These hormones then activate pathways which reduce this metabolic stress.