Introduction: Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure (HF). Limited insight into underlying mechanisms has culminated in the longstanding absence of evidence-based therapies capable of mitigating the substantial morbidity and mortality associated with the syndrome. Existing clinical and epidemiological evidence suggests that excessive body fat and lipid mishandling contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are unknown. We recently developed a novel, clinically relevant, murine model of HFpEF, uncovering suppression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) signaling pathway as a critical driver of HFpEF pathogenesis. Objectives: To define and manipulate mechanisms downstream of Xbp1s in HFpEF and decipher its cardioprotective actions. Methods and Results: In the myocardium of experimental HFpEF, we detected cardiomyocyte steatosis coupled with increases in the abundance and activity of FoxO1 (Forkhead box protein O1), a conserved transcription factor involved in cell metabolism. FoxO1 depletion, as well as Xbp1s over-expression, in cardiomyocytes each ameliorated the HFpEF phenotype and reduced myocardial lipid accumulation. Strikingly, forced expression of Xbp1s in cardiomyocytes triggered proteasomal degradation of FoxO1. Furthermore, we discovered that FoxO1 is ubiquitinated upon Xbp1s over-expression, and Xbp1s-induced proteasomal degradation of FoxO1 occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-Box-containing protein 1), a protein we identified as a novel and direct transcriptional target of Xbp1s. Conclusions: Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes.

Mitochondrial metabolism plays an integral role in glucose-stimulated insulin secretion (GSIS) in β-cells. In addition, the diabetogenic role of glucagon released from α-cells plays a major role in the etiology of both type 1 and type 2 diabetes because unopposed hyperglucagonemia is a pertinent contributor to diabetic hyperglycemia. Titrating expression levels of the mitochondrial protein mitoNEET is a powerful approach to fine-tune mitochondrial capacity of cells. Mechanistically, β-cell–specific mitoNEET induction causes hyperglycemia and glucose intolerance due to activation of a Parkin-dependent mitophagic pathway, leading to the formation of vacuoles and uniquely structured mitophagosomes. Induction of mitoNEET in α-cells leads to fasting-induced hypoglycemia and hypersecretion of insulin during GSIS. MitoNEET-challenged α-cells exert potent antiapoptotic effects on β-cells and prevent cellular dysfunction associated with mitoNEET overexpression in β-cells. These observations identify that reduced mitochondrial function in α-cells exerts potently protective effects on β-cells, preserving β-cell viability and mass.

The transcriptional regulators Ebf2 and Prdm16 establish and maintain the brown and/or beige fat cell identity. However, the mechanisms operating in white adipocytes to suppress the thermogenic gene program and maintain an energy-storing phenotype are less understood. Here, we report that the transcriptional regulator Zfp423 is critical for maintaining white adipocyte identity through suppression of the thermogenic gene program. Zfp423 expression is enriched in white versus brown adipocytes and suppressed upon cold exposure. Doxycycline-inducible inactivation of Zfp423 in mature adipocytes, combined with β-adrenergic stimulation, triggers a conversion of differentiated adiponectin-expressing inguinal and gonadal adipocytes into beige-like adipocytes; this reprogramming event is sufficient to prevent and reverse diet-induced obesity and insulin resistance. Mechanistically, Zfp423 acts in adipocytes to inhibit the activity of Ebf2 and suppress Prdm16 activation. These data identify Zfp423 as a molecular brake on adipocyte thermogenesis and suggest a therapeutic strategy to unlock the thermogenic potential of white adipocytes in obesity.

Background: Evidence hints at the ability of beta-cells to emerge from non-beta-cells upon genetic or pharmacological interventions. However, their quantitative contributions to the process of autonomous beta-cell regeneration without genetic or pharmacological manipulations remain to be determined.Methods & results: Using PANIC-ATTAC mice, a model of titratable, acute beta-cell apoptosis capable of autonomous, and effective islet mass regeneration, we demonstrate that an extended washout of residual tamoxifen activity is crucial for beta-cell lineage tracing studies using the tamoxifen-inducible Cre/loxP systems. We further establish a doxycycline-inducible system to label different cell types in the mouse pancreas and pursued a highly quantitative assessment to trace adult beta-cells after various metabolic challenges. Beyond proliferation of pre-existing beta-cells, non-beta-cells contribute significantly to the post-challenge regenerated beta-cell pool. alpha-cell trans-differentiation is the predominant mechanism upon post-apoptosis regeneration and multiparity. No contributions from exocrine acinar cells were observed. During diet-induced obesity, about 25% of alpha-cells arise de novo from beta-cells. Ectopic expression of Nkx6.1 promotes alpha-to-beta conversion and insulin production.Conclusions: We identify the origins and fates of adult beta-cells upon post-challenge upon autonomous regeneration of islet mass and establish the quantitative contributions of the different cell types using a lineage tracing system with high temporal resolution. (C) 2016 The Author(s). Published by Elsevier GmbH.

ObjectiveDietary methionine restriction (MR) reduces adiposity and hepatic lipids and increases overall insulin sensitivity in part by reducing lipogenic gene expression in liver, inducing browning of white adipose tissue (WAT), and enhancing the lipogenic and oxidative capacity of the remodeled WAT.MethodsOb/ob mice have compromised β‐adrenergic receptor expression in adipose tissue and were used to test whether MR could ameliorate obesity, insulin resistance, and disordered lipid metabolism.ResultsIn contrast to responses in wild‐type mice, MR failed to slow accumulation of adiposity, increase lipogenic and thermogenic gene expression in adipose tissue, reduce serum insulin, or increase serum adiponectin in ob/ob mice. However, MR produced comparable reductions in hepatic lipids and lipogenic gene expression in both genotypes. In addition, MR was fully effective in increasing insulin sensitivity in adiponectin−/− mice.ConclusionsThese findings show that diet‐induced changes in hepatic lipid metabolism are independent of weight loss and remodeling of WAT and are not required for insulin sensitization. In contrast, the failure of ob/ob mice to mount a normal thermogenic response to MR suggests that the compromised responsiveness of adipose tissue to SNS input is an important component of the inability of the diet to correct their obesity and insulin resistance.

The expansion of white adipose tissue (WAT) in obesity involves de novo differentiation of new adipocytes; however, the cellular origin of these cells remains unclear. Here, we utilize Zfp423(GFP) reporter mice to characterize adipose mural (Pdgfrβ(+)) cells with varying levels of the preadipocyte commitment factor Zfp423. We find that adipose tissue contains distinct mural populations, with levels of Zfp423 distinguishing adipogenic from inflammatory-like mural cells. Using our "MuralChaser" lineage tracking system, we uncover adipose perivascular cells as developmental precursors of adipocytes formed in obesity, with adipogenesis and precursor abundance regulated in a depot-dependent manner. Interestingly, Pdgfrβ(+) cells do not significantly contribute to the initial cold-induced recruitment of beige adipocytes in WAT; it is only after prolonged cold exposure that these cells differentiate into beige adipocytes. These results provide genetic evidence for a mural cell origin of white adipocytes in obesity and suggest that beige adipogenesis may originate from multiple sources.

We recently reported that local overexpression of VEGF-A in white adipose tissue (WAT) protects against diet-induced obesity and metabolic dysfunction. The observation that VEGF-A induces a "brown adipose tissue (BAT)-like" phenotype in WAT prompted us to further explore the direct function of VEGF-A in BAT. We utilized a doxycycline (Dox)-inducible, brown adipocyte-specific VEGF-A transgenic overexpression model to assess direct effects of VEGF-A in BAT in vivo. We observed that BAT-specific VEGF-A expression increases vascularization and up-regulates expression of both UCP1 and PGC-1α in BAT. As a result, the transgenic mice show increased thermogenesis during chronic cold exposure. In diet-induced obese mice, introducing VEGF-A locally in BAT rescues capillary rarefaction, ameliorates brown adipocyte dysfunction, and improves deleterious effects on glucose and lipid metabolism caused by a high-fat diet challenge. These results demonstrate a direct positive role of VEGF-A in the activation and expansion of BAT.

Over the past decade, efforts have focused on the connection between mitochondrial dysfunction and the etiology of obesity, insulin resistance and the progression of type 2 diabetes mellitus (T2DM)1,2. Numerous studies indicate that metabolic disorders are accompanied with reduced mitochondrial content, compromised mitochondrial respiratory capacity, heightened oxidative-stress and consequently, altered whole-body lipid- and glucose metabolism3,4. The mechanisms that prompt compromised mitochondrial activity in obesity-driven T2DM and how targeting these processes will improve metabolic profiles remain largely unknown. Novel preclinical models that elucidate a role of mitochondria in cellular homeostasis have the potential to shed new light on these questions and allow us to define improved therapeutic avenues. Healthy adipose tissue (AT) expansion has potent anti-diabetic effects by providing a safe haven to neutralize and store excess free fatty acids (FFAs). The inability to appropriately expand subcutaneous white adipose tissue (sWAT) may underlie the development of insulin resistance, β-cell failure and T2DM5, by allowing the accumulation of lipid species that promote insulin resistance in cell-types vulnerable to lipotoxic effects. Adipocytes can also secrete adipokines that help buffer these lipotoxic side-effects of excess caloric-intake. A critical player in this area is adiponectin. Secreted exclusively from adipocytes, adiponectin promotes storage of triglycerides (TGs) preferentially in AT5,6 to improve metabolic flexibility. Adiponectin further reduces the accumulation of ceramide species to improve cellular survival and insulin sensitivity. Mice overexpressing adiponectin in an ob/ob background exhibit improved insulin-sensitivity and lipid profiles5; such characteristics are attributed to augmented ceramidase activity7, a redistribution of lipids and increased adipogenesis, concomitant with gross sWAT expansion. However, the underlying mechanisms that initiate this paradoxical phenomenon of lipid-redistribution and chronic AT expansion, to improve metabolic stature, are not fully defined. Mitochondria play a central role in energy homeostasis by partitioning fuels toward β-oxidation or storage as fat. During AT expansion, the oxidation of lipid and carbohydrate fuels requires coordinated regulation of downstream metabolic pathways, such as the tricarboxylic acid cycle and the electron transport chain (ETC). Compromised mitochondrial energy production is a major anomaly in obesity. In particular, obese and type 2 diabetic subjects are known to exhibit lower β-oxidation rates, reduced oxidative enzymatic activities and decreased ETC activity8,9; concomitant with greater glycolytic capacities and increases in cellular fatty acid (FA)-uptake10. While these observations highlight oxidative failure during lipid accumulation, the mechanisms by which diminished β-oxidation and suboptimal mitochondrial function stimulate lipid-uptake and accumulation within obesity, have not been fully established. Here, we take advantage of the unique properties of the mitochondrial membrane protein mitoNEET. Using gain and loss of function models for mitoNEET, we induce chronic and massive AT expansion, at least in part through an upregulation of adiponectin production and release from adipocytes. MitoNEET achieves these effects through a selective modulation of the mitochondrial electron transport activity. This establishes a tight functional connection between mitoNEET, mitochondrial activity and adiponectin release. Originally, mitoNEET was identified as a unique dimeric mitochondrial membrane target crosslinked to the thiazolidinedione (TZD), pioglitazone11,12. Located in the outer mitochondrial membrane, mitoNEET was named according to its C-terminal amino acid sequence, Asn-Glu-Glu-Thr (NEET)11. Furthermore, oriented towards the cytoplasm, the CDGSH domain of mitoNEET can bind redox-active, pH-labile 2Fe-2S clusters13–15; with pioglitazone reported to stabilize the protein against 2Fe-2S cluster release12. MitoNEET achieves its remarkable effects on cellular and systemic metabolic homeostasis on the basis of acting as a powerful regulator of mitochondrial iron content. We have taken advantage of these properties to influence mitochondrial bioenergetics and metabolism in a tissue- and cell-type specific manner; this results in remarkable alterations in whole-body energy homeostasis and further, opens up new avenues for cell-specific manipulation of mitochondrial activity in any cell-type of choice.

Obesity is often associated with mitochondrial dysfunction. What is not clear, however, is whether this is a cause or a consequence of the condition and its detrimental effects on metabolic health. Phil Scherer and colleagues now show that by manipulating a key protein involved in mitochondrial function specifically in adipocytes the mitochondria is crucial in maintaining proper lipid levels and whole-body insulin sensitivity. We examined mouse models with altered adipocyte expression of mitoNEET, a protein residing in the mitochondrial outer membrane, to probe its impact on mitochondrial function and subsequent cellular responses. We found that overexpression of mitoNEET enhances lipid uptake and storage, leading to an expansion of the mass of adipose tissue. Despite the resulting massive obesity, benign aspects of adipose tissue expansion prevail, and insulin sensitivity is preserved. Mechanistically, we also found that mitoNEET inhibits mitochondrial iron transport into the matrix and, because iron is a rate-limiting component for electron transport, lowers the rate of β-oxidation. This effect is associated with a lower mitochondrial membrane potential and lower levels of reactive oxygen species–induced damage, along with increased production of adiponectin. Conversely, a reduction in mitoNEET expression enhances mitochondrial respiratory capacity through enhanced iron content in the matrix, ultimately corresponding to less weight gain on a high-fat diet. However, this reduction in mitoNEET expression also causes heightened oxidative stress and glucose intolerance. Thus, manipulation of mitochondrial function by varying mitoNEET expression markedly affects the dynamics of cellular and whole-body lipid homeostasis.