Early-Onset Hypertension and Sex-Specific Residual Risk for Cardiovascular Disease in Type 2 Diabetes.

OBJECTIVE: To investigate whether the sex disparities in type 2 diabetes-associated cardiovascular disease (CVD) risks may be related to early-onset hypertension that could benefit from intensive blood pressure (BP) control. RESEARCH DESIGN AND METHODS: We analyzed intensive versus standard BP control in relation to incident CVD events in women and men with type 2 diabetes, based on their age of hypertension diagnosis. RESULTS: Among 3,792 adults with type 2 diabetes (49% women), multivariable-adjusted CVD risk was increased per decade earlier age at hypertension diagnosis (hazard ratio 1.11 [1.03-1.21], P = 0.006). Excess risk associated with early-diagnosed hypertension was attenuated in the presence of intensive versus standard antihypertensive therapy in women (P = 0.036) but not men (P = 0.76). CONCLUSIONS: Women with type 2 diabetes and early-onset hypertension may represent a higher-risk subpopulation that not only contributes to the female excess in diabetes-related CVD risk but may benefit from intensive BP control.

Gene Regulation and Mitochondrial Activity During White and Brown Adipogenesis Are Modulated by KDM5 Histone Demethylase.

Body fat accumulation differs between males and females and is influenced by both gonadal sex (ovaries vs testes) and chromosomal sex (XX vs XY). We previously showed that an X chromosome gene, Kdm5c, is expressed at higher levels in females compared to males and correlates with adiposity in mice and humans. Kdm5c encodes a KDM5 histone demethylase that regulates gene expression by modulating histone methylation at gene promoters and enhancers. Here, we use chemical inhibition and genetic knockdown to identify a role for KDM5 activity during early stages of white and brown preadipocyte differentiation, with specific effects on white adipocyte clonal expansion, and white and brown adipocyte gene expression and mitochondrial activity. In white adipogenesis, KDM5 activity modulates H3K4 histone methylation at the Dlk1 gene promoter to repress gene expression and promote progression from preadipocytes to mature adipocytes. In brown adipogenesis, KDM5 activity modulates H3K4 methylation and gene expression of Ucp1, which is required for thermogenesis. Unbiased transcriptome analysis revealed that KDM5 activity regulates genes associated with cell cycle regulation and mitochondrial function, and this was confirmed by functional analyses of cell proliferation and cellular bioenergetics. Using genetic knockdown, we demonstrate that KDM5C is the likely KDM5 family member that is responsible for regulation of white and brown preadipocyte programming. Given that KDM5C levels are higher in females compared to males, our findings suggest that sex differences in white and brown preadipocyte gene regulation may contribute to sex differences in adipose tissue function.

The BCKDK inhibitor BT2 is a chemical uncoupler that lowers mitochondrial ROS production and de novo lipogenesis.

Elevated levels of branched chain amino acids (BCAAs) and branched-chain α-ketoacids are associated with cardiovascular and metabolic disease, but the molecular mechanisms underlying a putative causal relationship remain unclear. The branched-chain ketoacid dehydrogenase kinase (BCKDK) inhibitor BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid) is often used in preclinical models to increase BCAA oxidation and restore steady-state BCAA and branched-chain α-ketoacid levels. BT2 administration is protective in various rodent models of heart failure and metabolic disease, but confoundingly, targeted ablation of Bckdk in specific tissues does not reproduce the beneficial effects conferred by pharmacologic inhibition. Here, we demonstrate that BT2, a lipophilic weak acid, can act as a mitochondrial uncoupler. Measurements of oxygen consumption, mitochondrial membrane potential, and patch-clamp electrophysiology show that BT2 increases proton conductance across the mitochondrial inner membrane independently of its inhibitory effect on BCKDK. BT2 is roughly sixfold less potent than the prototypical uncoupler 2,4-dinitrophenol and phenocopies 2,4-dinitrophenol in lowering de novo lipogenesis and mitochondrial superoxide production. The data suggest that the therapeutic efficacy of BT2 may be attributable to the well-documented effects of mitochondrial uncoupling in alleviating cardiovascular and metabolic disease.

Feeding neurons integrate metabolic and reproductive states in mice.

Balance between metabolic and reproductive processes is important for survival, particularly in mammals that gestate their young. How the nervous system coordinates this balance is an active area of study. Herein, we demonstrate that somatostatin (SST) neurons of the tuberal hypothalamus alter feeding in a manner sensitive to metabolic and reproductive states in mice. Whereas chemogenetic activation of SST neurons increased food intake across sexes, ablation decreased food intake only in female mice during proestrus. This ablation effect was only apparent in animals with low body mass. Fat transplantation and bioinformatics analysis of SST neuronal transcriptomes revealed white adipose as a key modulator of these effects. These studies indicate that SST hypothalamic neurons integrate metabolic and reproductive cues by responding to varying levels of circulating estrogens to modulate feeding differentially based on energy stores. Thus, gonadal steroid modulation of neuronal circuits can be context dependent and gated by metabolic status.

The impact of chromosomal sex on cardiometabolic health and disease.

Many aspects of metabolism are sex-biased, from gene expression in metabolic tissues to the prevalence and presentation of cardiometabolic diseases. The influence of hormones produced by male and female gonads has been widely documented, but recent studies have begun to elucidate the impact of genetic sex (XX or XY chromosomes) on cellular and organismal metabolism. XX and XY cells have differential gene dosage conferred by specific genes that escape X chromosome inactivation or the presence of Y chromosome genes that are absent from XX cells. Studies in mouse models that dissociate chromosomal and gonadal sex have uncovered mechanisms for sex-biased epigenetic, transcriptional, and post-transcriptional regulation of gene expression in conditions such as obesity, atherosclerosis, pulmonary hypertension, autoimmune disease, and Alzheimer's disease.

Inclusion of Sex as a Biological Variable in Biomedical Sciences at the Undergraduate Level and Beyond.

To improve research on women's health, and to achieve better understanding of the factors controlling disease across diverse populations of humans, it is imperative to study sex differences in physiology and disease. After the introduction of the "SABV policy" at NIH, which requires investigators using animals or humans to consider sex as a biological factor, it became clear that many investigators were unaware of concepts of sexual differentiation or methods that can be used to study sex as a biological variable (SABV). To remedy this situation, efforts have increased to teach concepts and methods of SABV at all educational levels. The UCLA Scientific Center of Research Excellence (SCORE) grant "Sex differences in the metabolic syndrome" promotes education about SABV through three primary mechanisms: (1) through didactic course content for students at the undergraduate level, (2) by providing pilot funding for early career investigators to study the role of sex in metabolism-related areas, and (3) through curation of a video library, which may be useful for investigators performing research at the graduate, postgraduate, and faculty levels.

The BCKDK inhibitor BT2 is a chemical uncoupler that lowers mitochondrial ROS production and de novo lipogenesis.

Elevated levels of branched chain amino acids (BCAAs) and branched-chain α-ketoacids (BCKAs) are associated with cardiovascular and metabolic disease, but the molecular mechanisms underlying a putative causal relationship remain unclear. The branched-chain ketoacid dehydrogenase kinase (BCKDK) inhibitor BT2 is often used in preclinical models to increase BCAA oxidation and restore steady-state BCAA and BCKA levels. BT2 administration is protective in various rodent models of heart failure and metabolic disease, but confoundingly, targeted ablation of Bckdk in specific tissues does not reproduce the beneficial effects conferred by pharmacologic inhibition. Here we demonstrate that BT2, a lipophilic weak acid, can act as a mitochondrial uncoupler. Measurements of oxygen consumption, mitochondrial membrane potential, and patch-clamp electrophysiology show BT2 increases proton conductance across the mitochondrial inner membrane independently of its inhibitory effect on BCKDK. BT2 is roughly five-fold less potent than the prototypical uncoupler 2,4-dinitrophenol (DNP), and phenocopies DNP in lowering de novo lipogenesis and mitochondrial superoxide production. The data suggest the therapeutic efficacy of BT2 may be attributable to the well-documented effects of mitochondrial uncoupling in alleviating cardiovascular and metabolic disease.

A "Four Core Genotypes" rat model to distinguish mechanisms underlying sex-biased phenotypes and diseases.

Background: We have generated a rat model similar to the Four Core Genotypes mouse model, allowing comparison of XX and XY rats with the same type of gonad. The model detects novel sex chromosome effects (XX vs. XY) that contribute to sex differences in any rat phenotype. Methods: XY rats were produced with an autosomal transgene of Sry , the testis-determining factor gene, which were fathers of XX and XY progeny with testes. In other rats, CRISPR-Cas9 technology was used to remove Y chromosome factors that initiate testis differentiation, producing fertile XY gonadal females that have XX and XY progeny with ovaries. These groups can be compared to detect sex differences caused by sex chromosome complement (XX vs. XY) and/or by gonadal hormones (rats with testes vs. ovaries). Results: We have measured numerous phenotypes to characterize this model, including gonadal histology, breeding performance, anogenital distance, levels of reproductive hormones, body and organ weights, and central nervous system sexual dimorphisms. Serum testosterone levels were comparable in adult XX and XY gonadal males. Numerous phenotypes previously found to be sexually differentiated by the action of gonadal hormones were found to be similar in XX and XY rats with the same type of gonad, suggesting that XX and XY rats with the same type of gonad have comparable levels of gonadal hormones at various stages of development. Conclusion: The results establish a powerful new model to discriminate sex chromosome and gonadal hormone effects that cause sexual differences in rat physiology and disease.

Feeding Neurons Integrate Metabolic and Reproductive States in Mice.

Trade-offs between metabolic and reproductive processes are important for survival, particularly in mammals that gestate their young. Puberty and reproduction, as energetically taxing life stages, are often gated by metabolic availability in animals with ovaries. How the nervous system coordinates these trade-offs is an active area of study. We identify somatostatin neurons of the tuberal nucleus (TNSST) as a node of the feeding circuit that alters feeding in a manner sensitive to metabolic and reproductive states in mice. Whereas chemogenetic activation of TNSST neurons increased food intake across sexes, selective ablation decreased food intake only in female mice during proestrus. Interestingly, this ablation effect was only apparent in animals with a low body mass. Fat transplantation and bioinformatics analysis of TNSST neuronal transcriptomes revealed white adipose as a key modulator of the effects of TNSST neurons on food intake. Together, these studies point to a mechanism whereby TNSST hypothalamic neurons modulate feeding by responding to varying levels of circulating estrogens differentially based on energy stores. This research provides insight into how neural circuits integrate reproductive and metabolic signals, and illustrates how gonadal steroid modulation of neuronal circuits can be context-dependent and gated by metabolic status.

CLSTN3ß enforces adipocyte multilocularity to facilitate lipid utilization.

Multilocular adipocytes are a hallmark of thermogenic adipose tissue1,2, but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3ß) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3ß is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3ß have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3ß is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3ß associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3ß-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.

The role of gonadal hormones and sex chromosomes in sex-dependent effects of early nutrition on metabolic health.

Early-life conditions such as prenatal nutrition can have long-term effects on metabolic health, and these effects may differ between males and females. Understanding the biological mechanisms underlying sex differences in the response to early-life environment will improve interventions, but few such mechanisms have been identified, and there is no overall framework for understanding sex differences. Biological sex differences may be due to chromosomal sex, gonadal sex, or interactions between the two. This review describes approaches to distinguish between the roles of chromosomal and gonadal sex, and summarizes findings regarding sex differences in metabolism. The Four Core Genotypes (FCG) mouse model allows dissociation of the sex chromosome genotype from gonadal type, whereas the XY* mouse model can be used to distinguish effects of X chromosome dosage vs the presence of the Y chromosome. Gonadectomy can be used to distinguish between organizational (permanent) and activational (reversible) effects of sex hormones. Baseline sex differences in a variety of metabolic traits are influenced by both activational and organizational effects of gonadal hormones, as well as sex chromosome complement. Thus far, these approaches have not been widely applied to examine sex-dependent effects of prenatal conditions, although a number of studies have found activational effects of estradiol to be protective against the development of hypertension following early-life adversity. Genes that escape X chromosome inactivation (XCI), such as Kdm5c, contribute to baseline sex-differences in metabolism, while Ogt, another XCI escapee, leads to sex-dependent responses to prenatal maternal stress. Genome-wide approaches to the study of sex differences include mapping genetic loci influencing metabolic traits in a sex-dependent manner. Seeking enrichment for binding sites of hormone receptors among genes showing sexually-dimorphic expression can elucidate the relative roles of hormones. Using the approaches described herein to identify mechanisms underlying sex-dependent effects of early nutrition on metabolic health may enable the identification of fundamental mechanisms and potential interventions.

Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function.

Improving muscle function has great potential to improve the quality of life. To identify novel regulators of skeletal muscle metabolism and function, we performed a proteomic analysis of gastrocnemius muscle from 73 genetically distinct inbred mouse strains, and integrated the data with previously acquired genomics and >300 molecular/phenotypic traits via quantitative trait loci mapping and correlation network analysis. These data identified thousands of associations between protein abundance and phenotypes and can be accessed online (https://muscle.coffeeprot.com/) to identify regulators of muscle function. We used this resource to prioritize targets for a functional genomic screen in human bioengineered skeletal muscle. This identified several negative regulators of muscle function including UFC1, an E2 ligase for protein UFMylation. We show UFMylation is up-regulated in a mouse model of amyotrophic lateral sclerosis, a disease that involves muscle atrophy. Furthermore, in vivo knockdown of UFMylation increased contraction force, implicating its role as a negative regulator of skeletal muscle function.

Ciliary neurotrophic factor-mediated neuroprotection involves enhanced glycolysis and anabolism in degenerating mouse retinas.

Ciliary neurotrophic factor (CNTF) acts as a potent neuroprotective cytokine in multiple models of retinal degeneration. To understand mechanisms underlying its broad neuroprotective effects, we have investigated the influence of CNTF on metabolism in a mouse model of photoreceptor degeneration. CNTF treatment improves the morphology of photoreceptor mitochondria, but also leads to reduced oxygen consumption and suppressed respiratory chain activities. Molecular analyses show elevated glycolytic pathway gene transcripts and active enzymes. Metabolomics analyses detect significantly higher levels of ATP and the energy currency phosphocreatine, elevated glycolytic pathway metabolites, increased TCA cycle metabolites, lipid biosynthetic pathway intermediates, nucleotides, and amino acids. Moreover, CNTF treatment restores the key antioxidant glutathione to the wild type level. Therefore, CNTF significantly impacts the metabolic status of degenerating retinas by promoting aerobic glycolysis and augmenting anabolic activities. These findings reveal cellular mechanisms underlying enhanced neuronal viability and suggest potential therapies for treating retinal degeneration.

Chromosomal and gonadal sex drive sex differences in lipids and hepatic gene expression in response to hypercholesterolemia and statin treatment.

BACKGROUND: Biological sex impacts susceptibility and presentation of cardiovascular disease, which remains the leading cause of death for both sexes. To reduce cardiovascular disease risk, statin drugs are commonly prescribed to reduce circulating cholesterol levels through inhibition of cholesterol synthesis. The effectiveness of statin therapy differs between individuals with a sex bias in the frequency of adverse effects. Limited information is available regarding the mechanisms driving sex-specific responses to hypercholesterolemia or statin treatment. METHODS: Four Core Genotypes mice (XX and XY mice with ovaries and XX and XY mice with testes) on a hypercholesteremic Apoe-/- background were fed a chow diet without or with simvastatin for 8 weeks. Plasma lipid levels were quantified and hepatic differential gene expression was evaluated with RNA-sequencing to identify the independent effects of gonadal and chromosomal sex. RESULTS: In a hypercholesterolemic state, gonadal sex influenced the expression levels of more than 3000 genes, and chromosomal sex impacted expression of nearly 1400 genes, which were distributed across all autosomes as well as the sex chromosomes. Gonadal sex uniquely influenced the expression of ER stress response genes, whereas chromosomal and gonadal sex influenced fatty acid metabolism gene expression in hypercholesterolemic mice. Sex-specific effects on gene regulation in response to statin treatment included a compensatory upregulation of cholesterol biosynthetic gene expression in mice with XY chromosome complement, regardless of presence of ovaries or testes. CONCLUSION: Gonadal and chromosomal sex have independent effects on the hepatic transcriptome to influence different cellular pathways in a hypercholesterolemic environment. Furthermore, chromosomal sex in particular impacted the cellular response to statin treatment. An improved understanding of how gonadal and chromosomal sex influence cellular response to disease conditions and in response to drug treatment is critical to optimize disease management for all individuals.

Simultaneous monitoring of mouse grip strength, force profile, and cumulative force profile distinguishes muscle physiology following surgical, pharmacologic and diet interventions.

Grip strength is a valuable preclinical assay to study muscle physiology in disease and aging by directly determining changes in muscle force generation in active laboratory mice. Existing methods to statistically evaluate grip strength, however, have limitations in the power and scope of the physiological features that are assessed. We therefore designed a microcontroller whose serial measure of resistance-based force enables the simultaneous readout of (1) peak grip strength, (2) force profile (the non-linear progress of force exerted throughout a standard grip strength trial), and (3) cumulative force profile (the integral of force with respect to time of a single grip strength trial). We hypothesized that muscle pathologies of different etiologies have distinct effects on these parameters. To test this, we used our apparatus to assess the three muscle parameters in mice with impaired muscle function resulting from surgically induced peripheral pain, genetic peripheral neuropathy, adverse muscle effects induced by statin drug, and metabolic alterations induced by a high-fat diet. Both surgically induced peripheral nerve injury and statin-associated muscle damage diminished grip strength and force profile, without affecting cumulative force profile. Conversely, genetic peripheral neuropathy resulting from lipin 1 deficiency led to a marked reduction to all three parameters. A chronic high-fat diet led to reduced grip strength and force profile when normalized to body weight. In high-fat fed mice that were exerted aerobically and allowed to recover for 30 min, male mice exhibited impaired force profile parameters, which female mice were more resilient. Thus, simultaneous analysis of peak grip strength, force profile and cumulative force profile distinguishes the muscle impairments that result from distinct perturbations and may reflect distinct motor unit recruitment strategies.

Sex differences in heart mitochondria regulate diastolic dysfunction.

Heart failure with preserved ejection fraction (HFpEF) exhibits a sex bias, being more common in women than men, and we hypothesize that mitochondrial sex differences might underlie this bias. As part of genetic studies of heart failure in mice, we observe that heart mitochondrial DNA levels and function tend to be reduced in females as compared to males. We also observe that expression of genes encoding mitochondrial proteins are higher in males than females in human cohorts. We test our hypothesis in a panel of genetically diverse inbred strains of mice, termed the Hybrid Mouse Diversity Panel (HMDP). Indeed, we find that mitochondrial gene expression is highly correlated with diastolic function, a key trait in HFpEF. Consistent with this, studies of a "two-hit" mouse model of HFpEF confirm that mitochondrial function differs between sexes and is strongly associated with a number of HFpEF traits. By integrating data from human heart failure and the mouse HMDP cohort, we identify the mitochondrial gene Acsl6 as a genetic determinant of diastolic function. We validate its role in HFpEF using adenoviral over-expression in the heart. We conclude that sex differences in mitochondrial function underlie, in part, the sex bias in diastolic function.

Illuminating the Mechanisms Underlying Sex Differences in Cardiovascular Disease.

Sex is a key risk factor for many types of cardiovascular disease. It is imperative to understand the mechanisms underlying sex differences to devise optimal preventive and therapeutic approaches for all individuals. Both biological sex (determined by sex chromosomes and gonadal hormones) and gender (social and cultural behaviors associated with femininity or masculinity) influence differences between men and women in disease susceptibility and pathology. Here, we focus on the application of experimental mouse models that elucidate the influence of 2 components of biological sex-sex chromosome complement (XX or XY) and gonad type (ovaries or testes). These models have revealed that in addition to well-known effects of gonadal hormones, sex chromosome complement influences cardiovascular risk factors, such as plasma cholesterol levels and adiposity, as well as the development of atherosclerosis and pulmonary hypertension. One mechanism by which sex chromosome dosage influences cardiometabolic traits is through sex-biased expression of X chromosome genes that escape X inactivation. These include chromatin-modifying enzymes that regulate gene expression throughout the genome. The identification of factors that determine sex-biased gene expression and cardiometabolic traits will expand our mechanistic understanding of cardiovascular disease processes and provide insight into sex differences that remain throughout the lifespan as gonadal hormone levels alter with age.

Relative contributions of sex hormones, sex chromosomes, and gonads to sex differences in tissue gene regulation.

Sex differences in physiology and disease in mammals result from the effects of three classes of factors that are inherently unequal in males and females: reversible (activational) effects of gonadal hormones, permanent (organizational) effects of gonadal hormones, and cell-autonomous effects of sex chromosomes, as well as genes driven by these classes of factors. Often, these factors act together to cause sex differences in specific phenotypes, but the relative contribution of each and the interactions among them remain unclear. Here, we used the four core genotypes (FCG) mouse model with or without hormone replacement to distinguish the effects of each class of sex-biasing factors on transcriptome regulation in liver and adipose tissues. We found that the activational hormone levels have the strongest influence on gene expression, followed by the organizational gonadal sex effect, and last, sex chromosomal effect, along with interactions among the three factors. Tissue specificity was prominent, with a major impact of estradiol on adipose tissue gene regulation and of testosterone on the liver transcriptome. The networks affected by the three sex-biasing factors include development, immunity and metabolism, and tissue-specific regulators were identified for these networks. Furthermore, the genes affected by individual sex-biasing factors and interactions among factors are associated with human disease traits such as coronary artery disease, diabetes, and inflammatory bowel disease. Our study offers a tissue-specific account of the individual and interactive contributions of major sex-biasing factors to gene regulation that have broad impact on systemic metabolic, endocrine, and immune functions.