Selective (α)-l-Rhamnosylation and Neuroprotective Activity Exploration of Cardiotonic Steroids.

This work describes the studies on the direct C3-glycosylation of the C19-hydroxylated cardiotonic steroids strophanthidol, anhydro-ouabagenin, and ouabagenin using a strategy based on in situ protection of the C5 and C19 hydroxyl groups with boronic acids. While this strategy resulted in a successful one-pot C3-selective glycosylation of strophanthidol and anhydro-ouabegenin, it failed to provide ouabain from ouabagenin. The neuroprotective activity of the synthetic and natural glycosides against LPS-induced neuroinflammation was explored in neonatal mouse primary glia cells. Co-administration of natural and synthetic C3-glycosides at 200 nM concentrations resulted in the significant reduction of the LPS-induced neuroinflammatory markers IL-6, IL-1, TNFα, and IKBKE, with the anhydro-ouabagenin-3-(α)-l-rhamnoside (anhydro-ouabain) showing the most significant effect. At the same time, unglycosylated anhydro-ouabagenin enhanced rather than suppressed LPS-induced neuroinflammation.

Mast Cell Deficiency in Mice Attenuates Insulin Phenolic Preservative-Induced Inflammation.

One major obstacle that limits the lifespan of insulin infusion pumps is surmounting the tissue site reaction at the device implantation site. All commercial insulin formulations contain insulin phenolic preservatives (IPPs) designed to ensure insulin protein stability and prolong shelf-life. However, our laboratory demonstrated that these preservatives are cytotoxic and induce inflammation. Mature mast cells (MCs) reside in cutaneous tissue and are one of the first responders to an epidermal breach. Upon activation, MCs release proinflammatory and immunomodulatory prepacked mediators that exacerbate these inflammatory reactions. Thus, we hypothesized that once the epidermis is breached, cutaneous MCs are triggered inciting the inflammatory response to IPP-induced inflammation. This hypothesis was pursued utilizing our modified in vivo mouse air pouch model, including a c-kit dependent (C57BL/6J-kitW-sh/W-sh) and a c-kit independent (Cpa3-Cre; Mcl-1fl/fl) MC-deficient mouse model. Leukocytes were quantified in the mouse air pouch lavage fluid following flow cytometry analysis for IPP infusion under three different states, insulin-containing phenolic preservatives (Humalog®), insulin preservatives alone, and normal saline as a control. The air pouch wall was assessed using histopathological evaluations. Flow cytometry analysis demonstrated a statistically significant difference in inflammatory cell recruitment for both MC-deficient mouse models when compared to the control strain including infused control saline. Significantly less inflammation was observed at the site of infusion for the MC-deficient strains compared to the control strain. Overall, concordant results were obtained in both mouse types, C57Bl6-kitW-sh/W-sh and Cpa3-Cre; Mcl-1fl/fl. These findings in multiple model systems support the conclusion that MCs have important or possible unique roles in IPP-induced inflammation.

Intrinsic sexual dimorphism in the placenta determines the differential response to benzene exposure.

Maternal immune activation (MIA) by environmental challenges is linked to severe developmental complications, such as neurocognitive disorders, autism, and even fetal/maternal death. Benzene is a major toxic compound in air pollution that affects the mother as well as the fetus and has been associated with reproductive complications. Our objective was to elucidate whether benzene exposure during gestation triggers MIA and its impact on fetal development. We report that benzene exposure during pregnancy leads MIA associated with increased fetal resorptions, fetal growth, and abnormal placenta development. Furthermore, we demonstrate the existence of a sexual dimorphic response to benzene exposure in male and female placentas. The sexual dimorphic response is a consequence of inherent differences between male and female placenta. These data provide crucial information on the origins or sexual dimorphism and how exposure to environmental factors can have a differential impact on the development of male and female offspring.

Prenatal benzene exposure in mice alters offspring hypothalamic development predisposing to metabolic disease in later life.

Maternal exposure to environmental contaminants during pregnancy poses a significant threat to a developing fetus, as these substances can easily cross the placenta and disrupt the neurodevelopment of offspring. Specifically, the hypothalamus is essential in the regulation of metabolism, notably during critical windows of development. An abnormal hormonal and inflammatory milieu during development can trigger persistent changes in the function of hypothalamic circuits, leading to long-lasting effects on the body's energy homeostasis and metabolism. We recently demonstrated that gestational exposure to clinically relevant levels of benzene induces severe metabolic dysregulation in the offspring. Given the central role of the hypothalamus in metabolic control, we hypothesized that prenatal exposure to benzene impacts hypothalamic development, contributing to the adverse metabolic effects in the offspring. C57BL/6JB dams were exposed to benzene at 50 ppm in the inhalation chambers exclusively during pregnancy (from E0.5 to E19). Transcriptomic analysis of the exposed offspring at postnatal day 21 (P21) revealed hypothalamic changes in genes related to metabolic regulation, inflammation, and neurodevelopment exclusively in males. Moreover, the hypothalamus of prenatally benzene-exposed male offspring displayed alterations in orexigenic and anorexigenic projections, impairments in leptin signaling, and increased microgliosis. Additional exposure to benzene during lactation did not promote further microgliosis or astrogliosis in the offspring, while the high-fat diet (HFD) challenge in adulthood exacerbated glucose metabolism and hypothalamic inflammation in benzene-exposed offspring of both sexes. These findings reveal the persistent adverse effects of prenatal benzene exposure on hypothalamic circuits and neuroinflammation, predisposing the offspring to long-lasting metabolic health conditions.

Efficient SABRE-SHEATH Hyperpolarization of Potent Branched-Chain-Amino-Acid Metabolic Probe [1-13C]ketoisocaproate.

Efficient 13C hyperpolarization of ketoisocaproate is demonstrated in natural isotopic abundance and [1-13C]enriched forms via SABRE-SHEATH (Signal Amplification By Reversible Exchange in SHield Enables Alignment Transfer to Heteronuclei). Parahydrogen, as the source of nuclear spin order, and ketoisocaproate undergo simultaneous chemical exchange with an Ir-IMes-based hexacoordinate complex in CD3OD. SABRE-SHEATH enables spontaneous polarization transfer from parahydrogen-derived hydrides to the 13C nucleus of transiently bound ketoisocaproate. 13C polarization values of up to 18% are achieved at the 1-13C site in 1 min in the liquid state at 30 mM substrate concentration. The efficient polarization build-up becomes possible due to favorable relaxation dynamics. Specifically, the exponential build-up time constant (14.3 ± 0.6 s) is substantially lower than the corresponding polarization decay time constant (22.8 ± 1.2 s) at the optimum polarization transfer field (0.4 microtesla) and temperature (10 °C). The experiments with natural abundance ketoisocaproate revealed polarization level on the 13C-2 site of less than 1%-i.e., one order of magnitude lower than that of the 1-13C site-which is only partially due to more-efficient relaxation dynamics in sub-microtesla fields. We rationalize the overall much lower 13C-2 polarization efficiency in part by less favorable catalyst-binding dynamics of the C-2 site. Pilot SABRE experiments at pH 4.0 (acidified sample) versus pH 6.1 (unaltered sodium [1-13C]ketoisocaproate) reveal substantial modulation of SABRE-SHEATH processes by pH, warranting future systematic pH titration studies of ketoisocaproate, as well as other structurally similar ketocarboxylate motifs including pyruvate and alpha-ketoglutarate, with the overarching goal of maximizing 13C polarization levels in these potent molecular probes. Finally, we also report on the pilot post-mortem use of HP [1-13C]ketoisocaproate in a euthanized mouse, demonstrating that SABRE-hyperpolarized 13C contrast agents hold promise for future metabolic studies.

Growth hormone receptor (GHR) in AgRP neurons regulates thermogenesis in a sex-specific manner.

Evidence for hypothalamic regulation of energy homeostasis and thermoregulation in brown adipose tissue (BAT) during aging has been well recognized, yet the central molecular mediators involved in this process are poorly understood. The arcuate hypothalamus, orexigenic agouti-related peptide (AgRP) neurons control nutrient intake, energy homeostasis, and BAT thermogenesis. To determine the roles of growth hormone receptor (GHR) signaling in the AgRP neurons, we used mice with the AgRP-specific GHR deletion (AgRPΔGHR). We found that female AgRPΔGHR mice were resistant to temperature adaptation, and their body core temperature remained significantly lower when held at 10 °C, 22 °C, or 30 °C, compared to control mice. Low body core temperature in female AgRPΔGHR mice has been associated with significant reductions in Ucp1 and Pgc1α expression in the BAT. Further, neuronal activity in AgRP in response to cold exposure was blunted in AgRPΔGHR female mice, while the number of Fos+ AgRP neurons was increased in female controls exposed to cold. Global transcriptome from BAT identified increased the expression of genes related to immune responses and chemokine activity and decreased the expression of genes involved in triglyceride synthesis and metabolic pathways in AgRPΔGHR female mice. Importantly, these were the same genes that are downregulated by thermoneutrality in control mice but not in the AgRPΔGHR animals. Collectively, these data demonstrate a novel sex-specific role for GHR signaling in AgRP neurons in thermal regulation, which might be particularly relevant during aging.

Prenatal benzene exposure alters offspring hypothalamic development predisposing to metabolic disease in later life.

The hypothalamus is essential in the regulation of metabolism, notably during critical windows of development. An abnormal hormonal and inflammatory milieu during development can trigger persistent changes in the function of hypothalamic circuits, leading to long-lasting effects on the body’s energy homeostasis and metabolism. We recently demonstrated that gestational exposure to benzene at smoking levels induces severe metabolic dysregulation in the offspring. Given the central role of the hypothalamus in metabolic control, we hypothesized that prenatal exposure to benzene impacts hypothalamic development, contributing to the adverse metabolic effects in the offspring. C57BL/6JB dams were exposed to benzene in the inhalation chambers exclusively during pregnancy (from E0.5 to E19). The transcriptome analysis of the offspring hypothalamus at postnatal day 21 (P21) revealed changes in genes related to metabolic regulation, inflammation, and neurodevelopment exclusively in benzene-exposed male offspring. Moreover, the hypothalamus of prenatally benzene-exposed male offspring displayed alterations in orexigenic and anorexigenic projections, impairments in leptin signaling, and increased microgliosis. Additional exposure to benzene during lactation did not promote further microgliosis or astrogliosis in the offspring, while the high-fat diet (HFD) challenge in adulthood exacerbated glucose metabolism and hypothalamic inflammation in benzene-exposed offspring of both sexes. These findings reveal the persistent impact of prenatal benzene exposure on hypothalamic circuits and neuroinflammation, predisposing the offspring to long-lasting metabolic health conditions.

Canagliflozin retards age-related lesions in heart, kidney, liver, and adrenal gland in genetically heterogenous male mice.

Canagliflozin (Cana), a clinically important anti-diabetes drug, leads to a 14% increase in median lifespan and a 9% increase in the 90th percentile age when given to genetically heterogeneous male mice from 7 months of age, but does not increase lifespan in female mice. A histopathological study was conducted on 22-month-old mice to see if Cana retarded diverse forms of age-dependent pathology. This agent was found to diminish incidence or severity, in male mice only, of cardiomyopathy, glomerulonephropathy, arteriosclerosis, hepatic microvesicular cytoplasmic vacuolation (lipidosis), and adrenal cortical neoplasms. Protection against atrophy of the exocrine pancreas was seen in both males and females. Thus, the extension of lifespan in Cana-treated male mice, which is likely to reflect host- or tumor-mediated delay in lethal neoplasms, is accompanied by parallel retardation of lesions, in multiple tissues, that seldom if ever lead to death in these mice. Canagliflozin thus can be considered a drug that acts to slow the aging process and should be evaluated for potential protective effects against many other late-life conditions.

The Novel Peptide Chm-273s Has Therapeutic Potential for Metabolic Disorders: Evidence from In Vitro Studies and High-Sucrose Diet and High-Fat Diet Rodent Models.

The aim of this study was to develop a novel peptide potentially applicable for the treatment of metabolic conditions, such as obesity and type 2 diabetes (T2D). We identified CHM-273S from the list of peptides from milk hydrolysate obtained by HPLC/MS-MS. In vitro analysis of primary murine fibroblasts indicated the potential of CHM-273S to upregulate IRS2 mRNA expression. CHM-273S showed a prominent anorexigenic effect in mice with the induction of a key mechanism of leptin signaling via STAT3 in the hypothalamus as a possible effector. In the animal model of metabolic disease, CHM-273S alleviated glucose intolerance and insulin resistance, and induced phosphorylation of Akt at Ser473 and Thr308 in the hepatocytes of high-sucrose diet-fed rats. In a murine model of T2D, CHM-273S mitigated high-fat diet-induced hyperglycemia and insulin resistance and improved low-grade inflammation by diminishing serum TNFα. Mice treated with chronic CHM-273S had a significant reduction in body weight, with a lower visceral fat pad weight and narrow adipocytes. The effects of the peptide administration were comparable to those of metformin. We show the potential of CHM-273S to alleviate diet-induced metabolic alterations in rodents, substantiating its further development as a therapeutic for obesity, T2D, and other metabolic conditions.

Prenatal Pollutant Exposures and Hypothalamic Development: Early Life Disruption of Metabolic Programming.

Environmental contaminants in ambient air pollution pose a serious risk to long-term metabolic health. Strong evidence shows that prenatal exposure to pollutants can significantly increase the risk of Type II Diabetes (T2DM) in children and all ethnicities, even without the prevalence of obesity. The central nervous system (CNS) is critical in regulating whole-body metabolism. Within the CNS, the hypothalamus lies at the intersection of the neuroendocrine and autonomic systems and is primarily responsible for the regulation of energy homeostasis and satiety signals. The hypothalamus is particularly sensitive to insults during early neurodevelopmental periods and may be susceptible to alterations in the formation of neural metabolic circuitry. Although the precise molecular mechanism is not yet defined, alterations in hypothalamic developmental circuits may represent a leading cause of impaired metabolic programming. In this review, we present the current knowledge on the links between prenatal pollutant exposure and the hypothalamic programming of metabolism.

Neuroprotective effects of Canagliflozin: Lessons from aged genetically diverse UM-HET3 mice.

The aging brain is characterized by progressive increases in neuroinflammation and central insulin resistance, which contribute to neurodegenerative diseases and cognitive impairment. Recently, the Interventions Testing Program demonstrated that the anti-diabetes drug, Canagliflozin (Cana), a sodium-glucose transporter 2 inhibitor, led to lower fasting glucose and improved glucose tolerance in both sexes, but extended median lifespan by 14% in male mice only. Here, we show that Cana treatment significantly improved central insulin sensitivity in the hypothalamus and the hippocampus of 30-month-old male mice. Aged males produce more robust neuroimmune responses than aged females. Remarkably, Cana-treated male and female mice showed significant reductions in age-associated hypothalamic gliosis with a decrease in inflammatory cytokine production by microglia. However, in the hippocampus, Cana reduced microgliosis and astrogliosis in males, but not in female mice. The decrease in microgliosis was partially correlated with reduced phosphorylation of S6 kinase in microglia of Cana-treated aged male, but not female mice. Thus, Cana treatment improved insulin responsiveness in aged male mice. Furthermore, Cana treatment improved exploratory and locomotor activity of 30-month-old male but not female mice. Taken together, we demonstrate the sex-specific neuroprotective effects of Cana treatment, suggesting its application for the potential treatment of neurodegenerative diseases.

17-α-Estradiol Has Sex-Specific Effects on Neuroinflammation That Are Partly Reversed by Gonadectomy.

17-α-Estradiol (17aE2) treatment from 4 months of age extends life span in male mice and can reduce neuroinflammatory responses in the hypothalamus of 12-month-old males. Although 17aE2 improves longevity in males, female mice are unaffected, suggesting a sexually dimorphic pattern of life-span regulation. We tested whether the sex-specific effects of 17aE2 on neuroinflammatory responses are affected by gonadal removal and whether hypothalamic changes extend to other brain regions in old age. We show that sex-specific effects of 17aE2 on age-associated gliosis are brain region specific and are partially dependent on gonadectomy. 17aE2 treatment started at 4 months of age protected 25-month-old males from hypothalamic inflammation. Castration before 17aE2 exposure reduced the effect of 17aE2 on hypothalamic astrogliosis in males. In contrast, sex-specific inhibition of microgliosis generated by 17aE2 was not significantly affected by castration. In the hippocampus, gonadectomy influenced the severity of gliosis and the responsiveness to 17aE2 in a region-dependent manner. The male-specific effects of 17aE2 correlate with increases in hypothalamic estrogen receptor alpha expression, specifically in gonadally intact males, consistent with the idea that 17aE2 might act through this receptor. Our results indicate that neuroinflammatory responses to 17aE2 are partially controlled by the presence of sex-specific gonads. Loss of gonadal function and age-associated neuroinflammation could, therefore, influence late-life health and disease onset, leading to sexual dimorphism in both aging and in response to drugs that modify the pace of aging.

ARCGHR Neurons Regulate Muscle Glucose Uptake.

The growth hormone receptor (GHR) is expressed in brain regions that are known to participate in the regulation of energy homeostasis and glucose metabolism. We generated a novel transgenic mouse line (GHRcre) to characterize GHR-expressing neurons specifically in the arcuate nucleus of the hypothalamus (ARC). Here, we demonstrate that ARCGHR+ neurons are co-localized with agouti-related peptide (AgRP), growth hormone releasing hormone (GHRH), and somatostatin neurons, which are activated by GH stimulation. Using the designer receptors exclusively activated by designer drugs (DREADD) technique to control the ARCGHR+ neuronal activity, we demonstrate that the activation of ARCGHR+ neurons elevates a respiratory exchange ratio (RER) under both fed and fasted conditions. However, while the activation of ARCGHR+ promotes feeding, under fasting conditions, the activation of ARCGHR+ neurons promotes glucose over fat utilization in the body. This effect was accompanied by significant improvements in glucose tolerance, and was specific to GHR+ versus GHRH+ neurons. The activation of ARCGHR+ neurons increased glucose turnover and whole-body glycolysis, as revealed by hyperinsulinemic-euglycemic clamp studies. Remarkably, the increased insulin sensitivity upon the activation of ARCGHR+ neurons was tissue-specific, as the insulin-stimulated glucose uptake was specifically elevated in the skeletal muscle, in parallel with the increased expression of muscle glycolytic genes. Overall, our results identify the GHR-expressing neuronal population in the ARC as a major regulator of glycolysis and muscle insulin sensitivity in vivo.

Hypothalamic GHR-SIRT1 Axis in Fasting.

Many aspects of physiological functions are controlled by the hypothalamus, a brain region that connects the neuroendocrine system to whole-body metabolism. Growth hormone (GH) and the GH receptor (GHR) are expressed in hypothalamic regions known to participate in the regulation of feeding and whole-body energy homeostasis. Sirtuin 1 (SIRT1) is the most conserved mamma-lian nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase that plays a key role in controlling life span and sensing nutrient availability in the hypothalamus in response to caloric restriction. However, the interaction between GHR signaling and SIRT1 in the hypothal-amus is not established. In the arcuate nucleus (ARC) of the hypothalamus, the anorexigenic proopiomelanocortin (POMC)-expressing neurons and the orexigenic agouti-related protein (AgRP)-expressing neurons are the major regulators of feeding and energy expenditure. We show that in the ARC, the majority of GHR-expressing neurons also express SIRT1 and respond to fasting by upregulating SIRT1 expression. Accordingly, hypothalamic upregulation of SIRT1 in response to fasting is blunted in animals with GHR deletion in the AgRP neurons (AgRPEYFPΔGHR). Our data thus reveal a novel interaction between GH and SIRT1 in responses to fasting.

In Utero Maternal Benzene Exposure Predisposes to the Metabolic Imbalance in the Offspring.

Environmental chemicals play a significant role in the development of metabolic disorders, especially when exposure occurs early in life. We have recently demonstrated that benzene exposure, at concentrations relevant to cigarette smoke, induces a severe metabolic imbalance in a sex-specific manner affecting male but not female mice. However, the roles of benzene in the development of aberrant metabolic outcomes following gestational exposure, remain largely unexplored. In this study, we exposed pregnant C57BL/6JB dams to benzene at 50 ppm or filtered air for 6 h/day from gestational day 0.5 (GD0.5) through GD21 and studied male and female offspring metabolic phenotypes in their adult life. While no changes in body weight or body composition were observed between groups, 4-month-old male and female offspring exhibited reduced parameters of energy homeostasis (VO2, VCO2, and heat production). However, only male offspring from benzene-exposed dams were glucose intolerant and insulin resistant at this age. By 6 months of age, both male and female offspring exhibited marked glucose intolerance however, only male offspring developed severe insulin resistance. This effect was accompanied by elevated insulin secretion and increased beta-cell mass only in male offspring. In support, Homeostatic Model Assessment for Insulin Resistance, the index of insulin resistance was elevated only in male but not in female offspring. Regardless, both male and female offspring exhibited a considerable increase in hepatic gene expression associated with inflammation and endoplasmic reticulum stress. Thus, gestational benzene exposure can predispose offspring to increased susceptibility to the metabolic imbalance in adulthood with differential sensitivity between sexes.

Canagliflozin extends life span in genetically heterogeneous male but not female mice.

Canagliflozin (Cana) is an FDA-approved diabetes drug that protects against cardiovascular and kidney diseases. It also inhibits the sodium glucose transporter 2 by blocking renal reuptake and intestinal absorption of glucose. In the context of the mouse Interventions Testing Program, genetically heterogeneous mice were given chow containing Cana at 180 ppm at 7 months of age until their death. Cana extended median survival of male mice by 14%. Cana also increased by 9% the age for 90th percentile survival, with parallel effects seen at each of 3 test sites. Neither the distribution of inferred cause of death nor incidental pathology findings at end-of-life necropsies were altered by Cana. Moreover, although no life span benefits were seen in female mice, Cana led to lower fasting glucose and improved glucose tolerance in both sexes, diminishing fat mass in females only. Therefore, the life span benefit of Cana is likely to reflect blunting of peak glucose levels, because similar longevity effects are seen in male mice given acarbose, a diabetes drug that blocks glucose surges through a distinct mechanism, i.e., slowing breakdown of carbohydrate in the intestine. Interventions that control daily peak glucose levels deserve attention as possible preventive medicines to protect from a wide range of late-life neoplastic and degenerative diseases.

Paraventricular, subparaventricular and periventricular hypothalamic IRS4-expressing neurons are required for normal energy balance.

Understanding the neural components modulating feeding-related behavior and energy expenditure is crucial to combating obesity and its comorbidities. Neurons within the paraventricular nucleus of the hypothalamus (PVH) are a key component of the satiety response; activation of the PVH decreases feeding and increases energy expenditure, thereby promoting negative energy balance. In contrast, PVH ablation or silencing in both rodents and humans leads to substantial obesity. Recent studies have identified genetically-defined PVH subpopulations that control discrete aspects of energy balance (e.g. oxytocin (OXT), neuronal nitric oxide synthase 1 (NOS1), melanocortin 4-receptor (MC4R), prodynorphin (PDYN)). We previously demonstrated that non-OXT NOS1PVH neurons contribute to PVH-mediated feeding suppression. Here, we identify and characterize a non-OXT, non-NOS1 subpopulation of PVH and peri-PVH neurons expressing insulin-receptor substrate 4 (IRS4PVH) involved in energy balance control. Using Cre-dependent viral tools to activate, trace and silence these neurons, we highlight the sufficiency and necessity of IRS4PVH neurons in normal feeding and energy expenditure regulation. Furthermore, we demonstrate that IRS4PVH neurons lie within a complex hypothalamic circuitry that engages distinct hindbrain regions and is innervated by discrete upstream hypothalamic sites. Overall, we reveal a requisite role for IRS4PVH neurons in PVH-mediated energy balance which raises the possibility of developing novel approaches targeting IRS4PVH neurons for anti-obesity therapies.