Acetate Does Not Affect Palmitate Oxidation and AMPK Phosphorylation in Human Primary Skeletal Muscle Cells.

Our recent in vivo human studies showed that colonic administration of sodium acetate (SA) resulted in increased circulating acetate levels, which was accompanied by increments in whole-body fat oxidation in overweight-obese men. Since skeletal muscle has a major role in whole-body fat oxidation, we aimed to investigate effects of SA on fat oxidation and underlying mechanisms in human primary skeletal muscle cells (HSkMC). We investigated the dose (0-5 mmol/L) and time (1, 4, 20, and 24 h) effect of SA on complete and incomplete endogenous and exogenous oxidation of 14C-labeled palmitate in HSkMC derived from a lean insulin sensitive male donor. Both physiological (0.1 and 0.25 mmol/L) and supraphysiological (0.5, 1 and 5 mmol/L) concentrations of SA neither increased endogenous nor exogenous fat oxidation over time in HSkMC. In addition, no effect of SA was observed on Thr172-AMPKα phosphorylation. In conclusion, our previously observed in vivo effects of SA on whole-body fat oxidation in men may not be explained via direct effects on HSkMC fat oxidation. Nevertheless, SA-mediated effects on whole-body fat oxidation may be triggered by other mechanisms including gut-derived hormones or may occur in other metabolically active tissues.

Vitamin D and Tissue-Specific Insulin Sensitivity in Humans With Overweight/Obesity.

Context: Vitamin D deficiency in obesity has been linked to insulin resistance. However, studies that examined the association between plasma 25-hydroxyvitamin D3 [25(OH)D3] as well as plasma 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and tissue-specific insulin sensitivity are scarce. Furthermore, vitamin D receptor (VDR) and vitamin D-metabolizing enzymes [cytochrome 450 (CYP)] expression in adipose tissue (AT) might affect AT insulin sensitivity. Objective: To investigate the association between body mass index (BMI) and plasma 25(OH)D3 and 1,25(OH)2D3, AT VDR; between plasma 25(OH)D3, 1,25(OH)2D3, AT VDR, and tissue-specific insulin sensitivity in individuals with overweight/obesity. Design and Patients: This analysis included 92 adult individuals (BMI, >25 kg/m2). A two-step hyperinsulinemic-euglycemic clamp with a [6,6-2H2]-glucose tracer was performed to assess tissue-specific insulin sensitivity. Abdominal subcutaneous AT (SAT) mRNA expression of VDR and CYP was determined by using quantitative RT-PCR. Setting: University medical center. Main Outcome Measures: Plasma 25(OH)D3, 1,25(OH)2D3, 1,25(OH)2D3/25(OH)D3 ratio, SAT VDR and CYPs mRNA, and tissue-specific insulin sensitivity. Results: BMI was inversely associated with plasma 25(OH)D3 (ß = -0.274; P = 0.011) but not with plasma 1,25(OH)2D3. Plasma 25(OH)D3 was not related to CYPs or VDR expression in SAT. Plasma 1,25(OH)2D3 and 25(OH)D3 were not related to tissue-specific insulin sensitivity. Interestingly, SAT VDR mRNA was negatively associated with AT insulin sensitivity (ß = -0.207; P = 0.025). Conclusions: BMI was inversely associated with 25(OH)D3 concentrations, which could not be explained by alterations in SAT VDR and CYP enzymes. Plasma vitamin D metabolites were not related to tissue-specific insulin sensitivity. However, VDR expression in SAT was negatively associated with AT insulin sensitivity.

Short-Chain Fatty Acids Differentially Affect Intracellular Lipolysis in a Human White Adipocyte Model.

BACKGROUND AND AIMS: Gut-derived short-chain fatty acids (SCFA), formed by microbial fermentation of dietary fibers, are believed to be involved in the etiology of obesity and diabetes. Previous data from our group showed that colonic infusions of physiologically relevant SCFA mixtures attenuated whole-body lipolysis in overweight men. To further study potential mechanisms involved in the antilipolytic properties of SCFA, we aimed to investigate the in vitro effects of SCFA incubations on intracellular lipolysis and signaling using a human white adipocyte model, the human multipotent adipose tissue-derived stem (hMADS) cells. METHODS: hMADS adipocytes were incubated with mixtures of acetate, propionate, and butyrate or single SCFA (acetate, propionate and butyrate) in concentrations ranging between 1 µmol/L and 1 mmol/L. Glycerol release and lipase activation was investigated during basal conditions and following ß-adrenergic stimulation. RESULTS: SCFA mixtures high in acetate and propionate decreased basal glycerol release, when compared to control (P < 0.05), while mixtures high in butyrate had no effect. Also, ß-adrenergic receptor mediated glycerol release was not significantly altered following incubation with SCFA mixtures. Incubation with only acetate decreased basal (1 µmol/L) and ß-adrenergically (1 µmol/L and 1 mmol/L) mediated glycerol release when compared with control (P < 0.05). In contrast, butyrate (1 µmol/L) slightly increased basal and ß-adrenergically mediated glycerol release compared with control (P < 0.05), while propionate had no effect on lipolysis. The antilipolytic effect of acetate was accompanied by a reduced phosphorylation of hormone-sensitive lipase (HSL) at serine residue 650. In addition, inhibition of Gi G proteins following pertussis toxin treatment prevented the antilipolytic effect of acetate. CONCLUSION: The present data demonstrated that acetate was mainly responsible for the antilipolytic effects of SCFA and acts via attenuation of HSL phosphorylation in a Gi-coupled manner in hMADS adipocytes. Therefore, the modulation of colonic and circulating acetate may be an important target to modulate human adipose tissue lipid metabolism.

Postpartum reversal of the pregnancy-induced uterine artery remodeling in young, aging, and eNOS-deficient mice.

OBJECTIVES: The progressive rise in uterine blood flow (UBF) during pregnancy is accompanied by outward hypertrophic remodeling of the uterine artery (UA). After birth, UBF falls in concert with the sudden decline in uterine metabolic demands. Arterial remodeling associated with the reversal of increased blood flow has been described in large arteries. It is unclear whether this situation applies to small-sized resistance arteries such as the UA. We investigated the pattern of UA remodeling postpartum in relation to age and endothelial nitric oxide synthase (eNOS) deficiency. METHODS: Uterine artery of 2 and 10 days postpartum young (age 12 weeks), aged (age 40 weeks), and eNOS-deficient (eNOS( -/-), age 12 weeks) mice were dissected and processed for either morphometric analysis (lumen, wall mass) or immunohistochemistry (cellular differentiation, proliferation, and apoptosis). We used data of previously studied control (nonpregnant) and late-pregnant (17 days gestation) mice as reference. RESULTS: By 2 days postpartum, morphometric and cellular characteristics of the UA did not differ from those of late-pregnant UA. By 10 days postpartum, the UA was wider with wall mass being decreased by approximately 30%. Cytological parameters indicated a stable smooth muscle media. Apoptosis was only present in UA of 2 and 10 days pregnant mice. In eNOS(- /-) and aged mice, changes were smaller or absent, respectively. CONCLUSIONS: The outward hypertrophic response of the UA induced by pregnancy regresses gradually postpartum. We speculate that persisting UA widening facilitates UA remodeling in a next pregnancy thereby favoring placentation and with it, allowing for a higher birth weight as usually observed in a second mammalian pregnancy.

Adipose triglyceride lipase (ATGL) expression in human skeletal muscle is type I (oxidative) fiber specific.

Accumulation of triacylglycerol (TAG) and lipid intermediates in skeletal muscle plays an important role in the etiology of insulin resistance and type 2 diabetes mellitus. Disturbances in skeletal muscle lipid turnover and lipolysis may contribute significantly to this. So far, knowledge on the regulation of muscle lipolysis is limited. Recently the identification of a new lipase was reported: adipose triglyceride lipase (ATGL). ATGL deficient animals show significant lipid accumulation in skeletal muscle, which may indicate that ATGL plays a pivotal role in skeletal muscle lipolysis. However, until now, it is still unknown whether ATGL protein is expressed in human skeletal muscle. Therefore, the aim of the present study was to investigate whether ATGL is expressed at the protein level in human skeletal muscle, and to examine whether its expression is fiber-type specific. To accomplish this, we established an imunohistochemical and immunofluorescent staining procedure to study ATGL protein expression in relation to fiber type in human vastus lateralis muscle of eight male subjects (BMI range: 21.0-34.5 kg/m2 and age: 38-59 years). In the present paper we report for the first time that ATGL protein is indeed expressed in human skeletal muscle. Moreover, ATGL is exclusively expressed in type I (oxidative) muscle fibers, suggesting a pivotal role for ATGL in intramuscular fatty acid handling, lipid storage and breakdown.

Vascular expression of adrenomedullin is increased in Wistar rats during early pregnancy.

OBJECTIVE: Circulating levels of adrenomedullin (ADM)--a vasodilator peptide with long-lasting effects--increase in the course of pregnancy. Neither the site nor the concomitant rate of ADM synthesis in pregnancy is known. The aim of this study was to test the hypothesis that the rise in plasma levels of ADM during pregnancy is paralleled by increased gene expression and protein levels in the vascular bed. STUDY DESIGN: We determined in cardiovascular and reproductive tissues of non-pregnant (n=10) and 10-days pregnant (n=10) Wistar rats ADM gene expression by semi-quantitative RT-PCR (normalized to GAPDH). As a support for the mRNA data, protein concentrations were measured by both ELISA and Western blot analysis. Finally, ADM in these tissues was localized by immunohistochemical staining. Statistical analysis was carried out by applying Mann-Whitney U-test. RESULTS: ADM mRNA levels in the abdominal aorta, renal artery and the kidney were increased during pregnancy. In addition, immunohistochemical staining in the kidney, uterus, abdominal aorta, renal, uterine and superior mesenteric artery was more intense as compared to non-pregnant rats. However, we observed lower concentrations of tissue ADM protein in pregnant rats, indicating an increased release of the hormone by the producing cells. CONCLUSION: Vascular ADM gene expression is increased in the first half of rat pregnancy. This coincides and may be functionally related to the institution of a high flow/low resistance circulation in pregnancy.

Uterine artery remodeling in pseudopregnancy is comparable to that in early pregnancy.

During pregnancy, the lumenal diameter and wall mass of the uterine artery (UA) increase, most likely in response to the increased hemodynamic strain resulting from the chronically elevated uterine blood flow (UBF). In this remodeling process, the phenotype of vascular smooth-muscle cells (VSMC) is transiently altered to enable VSMC proliferation. These phenomena are already seen during early pregnancy, when the rise in UBF is still modest. This raises the question whether the newly instituted endocrine environment of pregnancy is involved in the onset of the pregnancy-related UA remodeling. We tested the hypothesis that the conceptus is not essential for the onset of UA remodeling of pregnancy. Six control and 18 pseudopregnant (Postcopulation Days 5, 11, and 17; n = 6 per subgroup) C57Bl/6 mice were killed and UAs were dissected and processed for either morphometric analysis or immunohistochemistry. The latter consisted of staining UA cross sections for the differentiation markers smooth muscle alpha-actin and smoothelin, and for the proliferation marker MKI67. We analyzed the UA changes in response to pseudopregnancy by ANOVA. Data are presented as mean +/- SD. By Day 11 of pseudopregnancy, the UA lumen was 25% wider and the media cross-sectional area 71% larger than in control mice. These differences were accompanied by reduced smoothelin expression and increased proliferation of UA medial VSMC. All UA morphological differences had returned or were in the process of returning to baseline values by Day 17 of pseudopregnancy. The structural and cellular aspects of UA remodeling as seen at midpregnancy are also seen in pseudopregnancy. These results support the concept that the conceptus does not contribute to the initiation of UA remodeling. We suggest that ovarian hormones trigger the onset of UA remodeling.

Uterine artery remodeling and reproductive performance are impaired in endothelial nitric oxide synthase-deficient mice.

The progressive rise in uterine blood flow during pregnancy is accompanied by outward hypertrophic remodeling of the uterine artery (UA). This process involves changes of the arterial smooth muscle cells and extracellular matrix. Acute increases in blood flow stimulate endothelial production of nitric oxide (NO). It remains to be established whether endothelial NO synthase (eNOS) is involved in pregnancy-related arterial remodeling. We tested the hypothesis that absence of eNOS results in a reduced remodeling capacity of the UA during pregnancy leading to a decline in neonatal outcome. UA of nonpregnant and pregnant wild-type (Nos3+/+) and eNOS-deficient (Nos3-/-) mice were collected and processed for standard morphometrical analyses. In addition, cross sections of UA were processed for cytological (smoothelin, smooth muscle alpha-actin) and proliferation (Ki-67) immunostaining. We compared the pregnancy-related changes longitudinally and, together with the data on pregnancy outcome, transversally by analysis of variance with Bonferroni correction. During pregnancy, the increases in radius and medial cross sectional area of Nos3-/- UA was significantly less than those of Nos3+/+ UA. Smooth muscle cell dedifferentiation and proliferation were impaired in gravid Nos3-/- mice as deduced from the lack of change in the expression of smoothelin and smooth muscle alpha-actin, and the reduced Ki-67 expression. Until 17 days of gestation, litter size did not differ between both genotypes, but at birth the number of viable newborn pups and their weights were smaller in Nos3-/- than in Nos3+/+ mice. We conclude that absence of eNOS adversely affects UA remodeling in pregnancy, which may explain the impaired pregnancy outcome observed in these mice.

Aging blunts remodeling of the uterine artery during murine pregnancy.

OBJECTIVE: The progressive increase in uterine blood flow (UBF) during pregnancy is accommodated by morphologic changes in the uterine artery (UA) in a process defined as arterial remodeling. This process is accompanied by changes in cytoskeletal architecture of the arterial smooth muscle cells (SMCs) and surrounding extracellular matrix (ECM). Aging reduces flow-induced arterial remodeling. We studied changes in the murine UA during pregnancy and on the effects of aging on the capacity of the UA to remodel in response to pregnancy. METHODS: We determined morphologic and cytologic changes in UA from nonpregnant and pregnant mice aged 12 weeks (young) and 40 weeks (old) and correlated them with their reproductive performance. RESULTS: In young mice, pregnancy induced an early increase in UA wall mass, which preceded lumen widening. These changes were not accompanied by altered densities of elastin and collagen in the ECM of the medial layer. Smooth muscle cell proliferation increased in midpregnancy and was paralleled by a transient decrease in smoothelin and smooth muscle alpha-actin expression. In old mice, these pregnancy-dependent changes in the UA wall were either absent or markedly reduced. Although by day 11 of pregnancy litter size did not differ between both age groups, the number of viable pups in old mice by day 17 of pregnancy and at birth was 25% and 60% less than in young mice. CONCLUSION: Outward hypertrophic remodeling of the UA during pregnancy in young mice is characterized by transient phenotypic modulation and proliferation of SMCs and alterations in the composition of the ECM. In contrast, in older mice, UA remodeling is markedly reduced and accompanied with a loss of viable fetuses near term pregnancy.