Mice deficient in phosphofructokinase-M have greatly decreased fat stores.

Synthesis of triacylglycerol requires the glucose-derived glycerol component, and glucose uptake has been viewed as the rate-limiting step in glucose metabolism in adipocytes. Furthermore, adipose tissue contains all three isoforms of the glycolytic enzyme phosphofructokinase (PFK). We here report that mice deficient in the muscle isoform PFK-M have greatly reduced fat stores. Mice with disrupted activity of the PFK-M distal promoter were obtained from Lexicon Pharmaceuticals, developed from OmniBank OST#56064. Intra-abdominal fat was measured by magnetic resonance imaging of the methylene proton signal. Lipogenesis from labeled glucose was measured in isolated adipocytes. Lipolysis (glycerol and free fatty acid release) was measured in perifused adipocytes. Intra-abdominal fat in PFK-M-deficient female mice (5-10 months old) was 17 +/- 3% of that of wild-type littermates (n = 4; P < 0.02). Epididymal fat weight in 15 animals (7-9.5 months) was 34 +/- 4% of control littermate (P < 0.002), with 10-30% lower body weight. Basal and insulin-stimulated lipogenesis in PFK-M-deficient epididymal adipocytes was 40% of the rates in cells from heterozygous littermates (n = 3; P < 0.05). The rate of isoproterenol-stimulated lipolysis in wild-type adipocytes declined approximately 10% after 1 h and 50% after 2 h; in PFK-M-deficient cells it declined much more rapidly, 50% in 1 h and 90% in 2 h, and lipolytic oscillations appeared to be damped (n = 4). These results indicate an important role for PFK-M in adipose metabolism. This may be related to the ability of this isoform to generate glycolytic oscillations, because such oscillations may enhance the production of the triacylglycerol precursor alpha-glycerophosphate.

11.7 Tesla magnetic resonance microimaging of laryngeal tissue architecture.

OBJECTIVES/HYPOTHESIS: High-resolution imaging of vocal folds that distinguishes vocal fold (VF) layered microstructure and VF implants would provide a key experimental tool for translational research investigating biomaterial-based interventions to treat vocal fold scar. To establish proof of concept, we studied whether 11.7 Tesla (T) magnetic resonance (MR) microimaging provides the needed resolution to resolve vocal fold tissue architecture. STUDY DESIGN: We performed ex vivo MR microimaging of fixed ferret and canine larynges to determine whether changes in the layered architecture can be detected in the presence of scar and subsequent to biomaterial injections into the vocal folds. Serial section histological analyses were done to corroborate MR microimaging findings. METHODS: Multiple axial and transverse/coronal 300-microm slices were obtained using an 11.7 T MR spectrometer/500 MHz for proton with gradient-recalled echo and rapid acquisition with relaxation enhancement imaging sequences. RESULTS: High-resolution (39 microm/pixel) MR microimages distinguished VF epithelium, lamina propria, muscle, and cartilage in ferret and canine larynges. In ferret scarred VFs (n = 25), collagen-rich dense scar tissue was distinguishable from contralateral nonscarred VFs and from normal ferret VFs (n = 25), as confirmed on histology. MR microimaging accurately detected injected autologous fat, hyaluronic acid-based and polyethylene glycol (PEG)-based implants injected into both ferret and canine VFs. Importantly, MRI accurately showed resorption of PEG implants in ferrets and canines, as confirmed on histology. Additionally, ex vivo MR spectroscopy distinguished fat from PEG-based implants. CONCLUSIONS: Ex vivo 11.7 T MR microimaging provided high-resolution images of ferret and canine laryngeal tissue microstructure, although the superficial lamina propria could not be distinguished. Histology confirmed MR microimaging findings, indicating utility of MR microimaging of modeled scar, implant residence time, and tissue responses, thus providing integrative insight relevant to translational research.

Early-life sodium exposure unmasks susceptibility to stroke in hyperlipidemic, hypertensive heterozygous Tg25 rats transgenic for human cholesteryl ester transfer protein.

BACKGROUND: Early-life risk factor exposure increases aortic atherosclerosis and blood pressure in humans and animal models; however, limited insight has been gained as to end-organ complications. METHODS AND RESULTS: We investigated the effects of early-life Na exposure (0.23% versus 0.4% NaCl regular rat chow) on vascular disease outcomes using the inbred, transgenic [hCETP](25) Dahl salt-sensitive hypertensive rat model of male-predominant coronary atherosclerosis, Tg25. Rather than the expected increase in coronary heart disease, fetal 0.4% Na exposure (< or =2 g of Na per 2-kcal/d diet) induced adult-onset stroke in both sexes (ANOVA P<0.0001), with earlier stroke onset in Tg25 females. Analysis of later onset of 0.4% Na exposure resulted in decreased stroke risk and later stroke onset despite longer 0.4% Na exposure durations, which indicates increasing risk with earlier onset of 0.4% Na exposure. Histological analysis of stroke-positive rat brains revealed cerebral cortical hemorrhagic infarctions, microhemorrhages, neuronal ischemia, and microvascular injury. Ex vivo MRI of stroke-positive rat brains detected cerebral hemorrhages, microhemorrhages, and ischemia with middle cerebral artery distribution and cerebellar noninvolvement. Ultrasound microimaging detected carotid artery disease. Prestroke analysis detected neuronal ischemia and decreased mass of isolated cerebral but not cerebellar microvessels. CONCLUSIONS: Early-life Na exposure exacerbated hypertension and unmasked stroke susceptibility, with greater female vulnerability in hypertensive, hyperlipidemic Tg25 rats. The reproducible modeling in stroke-prone Tg25 rats of carotid artery disease, cerebral hemorrhagic infarctions, neuronal ischemia, microhemorrhages, and microvascular alterations suggests a pathogenic spectrum with causal interrelationships. This "mixed-stroke" spectrum could represent paradigms of ischemic-hemorrhagic transformation and/or a microangiopathic basis for the association of ischemic lesions, microhemorrhages, and strokes in humans. Together, the data reveal early-life Na exposure to be a significant modifier of hypertension and stroke disease course and hence a potentially modifiable prevention target that deserves systematic study.

Effects of dihydrotestosterone on differentiation and proliferation of human mesenchymal stem cells and preadipocytes.

UNLABELLED: The mechanisms by which androgens regulate fat mass are poorly understood. Although testosterone has been reported to increase lipolysis and inhibit lipid uptake, androgen effects on proliferation and differentiation of human mesenchymal stem cells (hMSCs) and preadipocytes have not been studied. Here, we investigated whether dihydrotestosterone (DHT) regulates proliferation, differentiation, or functional maturation of hMSCs and human preadipocytes from different fat depots. DHT (0-30 nM) dose-dependently inhibited lipid accumulation in adipocytes differentiated from hMSCs and downregulated expression of aP2, PPARgamma, leptin, and C/EBPalpha. Bicalutamide attenuated DHT's inhibitory effects on adipogenic differentiation of hMSCs. Adipocytes differentiated in presence of DHT accumulated smaller oil droplets suggesting reduced extent of maturation. DHT decreased the incorporation of labeled fatty acid into triglyceride, and downregulated acetyl CoA carboxylase and DGAT2 expression in adipocytes derived from hMSCs. DHT also inhibited lipid accumulation and downregulated aP2 and C/EBPalpha in human subcutaneous, mesenteric and omental preadipocytes. DHT stimulated forskolin-stimulated lipolysis in subcutaneous and mesenteric preadipocytes and inhibited incorporation of fatty acid into triglyceride in adipocytes differentiated from preadipocytes from all fat depots. CONCLUSIONS: DHT inhibits adipogenic differentiation of hMSCs and human preadipocytes through an AR-mediated pathway, but it does not affect the proliferation of either hMSCs or preadipocytes. Androgen effects on fat mass represent the combined effect of decreased differentiation of fat cell precursors, increased lipolysis, and reduced lipid accumulation.