Mariana Cifuentes

Overweight Postmenopausal Women Lose Bone With Moderate Weight Reduction and 1 g/day Calcium Intake

Overweight postmenopausal women may be more susceptible to bone loss with weight reduction than previously studied obese women. The influence of energy restriction and Ca intake on BMD was assessed in 66 individuals. Weight reduction resulted in bone loss at several sites in women consuming 1 g Ca/day and was mitigated with higher calcium intake at 1.7 g/day.

Osteopontin facilitates bone resorption, decreasing bone mineral crystallinity and content during calcium deficiency.

Osteopontin null-mice were previously shown to have bones containing more mineral and larger mineral crystals. These bones were independently seen to be resistant to ovariectomy-induced remodeling. To separate the physicochemical effects of osteopontin, which is an in vitro inhibitor of mineral crystal formation and growth, from effects of osteopontin on in vivo bone remodeling, this study examined mature (5-month-old) osteopontin-null (Opn−/−) and wildtype (WT) mice given a calcium-deficient diet. Biochemical parameters were measured during 4 weeks of Ca deficiency, followed by 1 week of refeeding adequate Ca. Ca deficiency caused a transiently greater rise in bone resorption in WT than Opn−/− mice (P = 0.01), whereas only the Opn−/− mice tended to increase Ca absorption (P = 0.08), yet both groups showed elevated levels of parathyroid hormone (PTH) (P < 0.001). The rise in markers of bone formation due to Ca deficiency was similar in both groups during Ca deficiency. Fourier transform infrared microspectroscopy assessed mineral properties at 20 µm spatial resolution in different anatomic regions of the bone. The Ca-deficient Opn−/− animals had slightly increased mineral content as compared to the WT, and there was a significant increase in the mineral content of older (endosteal) bone, implying that osteoclast recruitment was impaired. Crystallinity in the Ca-deficient Opn−/− bones was increased relative to the Ca-deficient WT at all sites except adjacent to the periosteum (younger mineral). These data suggest that osteopontin has both a physicochemical effect (inhibiting crystal growth and crystal proliferation) and a role in osteoclast recruitment, and in its absence, extraskeletal organs maintain calcium homeostasis.

The anti-adipogenic effect of angiotensin II on human preadipose cells involves ERK1,2 activation and PPARG phosphorylation

Despite the importance of adipocyte formation for adipose tissue physiology, current knowledge about the mechanisms that regulate the recruitment of progenitor cells to undergo adipogenic differentiation is limited. A role for locally generated angiotensin II emerged from studies with human and murine cells. Preadipose cells from different human fat depots show reduced response to adipogenic stimuli when exposed to angiotensin II. This investigation sought to gain an insight into the intracellular mechanisms involved in the anti-adipogenic response of human preadipose cells from omental fat to angiotensin II. Its effect was evaluated on cells stimulated to adipogenic differentiation in vitro, by assessment of glycerol-3-phosphate dehydrogenase activity and expression of early markers of adipogenesis. Extracellular signal-regulated kinase(1,2) (ERK(1,2)) pathway activation was inferred from the phosphorylated to total ERK(1,2) ratio determined by western blot. Exposure to angiotensin II throughout the 10-day differentiation period resulted in a reduced adipogenic response. A similar anti-adipogenic effect was observed when this hormone was present during the first 48 h of induction to differentiation. Angiotensin II treatment had no consequences on CCAAT/enhancer-binding protein beta and peroxisome proliferator-activated receptor gamma (PPARG) induction, but increased the phosphorylated form of the key adipogenic regulator PPARG. Upon angiotensin II exposure, a raise of phosphorylated ERK(1,2) was determined, which was more prominent 8-20 h after induction of adipogenesis (when controls reached negligible values). Chemical inhibition of ERK(1,2) phosphorylation prevented angiotensin II-dependent reduction in adipogenesis. These results support the participation of the mitogen-activated protein kinase/ERK(1,2) pathway in the anti-adipogenic effect of angiotensin II on preadipose cells from human omental adipose tissue.

Precise and accurate determination of calcium isotope ratios in urine using HR-ICP-SFMS

Precise and accurate determination of calcium isotope ratios in urine is imperative in limiting the expense of enriched isotopes used in human metabolic tracer studies. The calcium isotope spectrum obtained from an ICP-MS (using conventional spray chamber), operated in low resolution mode (LR = 300), is subject to numerous polyatomic, isobaric and doubly charged interferences. The most severe polyatomic interfences can be resolved at resolutions greater than 3500, whereas, isobaric and doubly charged interferences are easily corrected. Precise (<0.1% 1σ) isotope ratios, however, are not routinely obtained in medium resolution (MR = 4300). Here we use a HR-ICP-MS in MR to identify and determine methods to reduce polyatomic interferences. Using oxalate precipitation of urinary calcium and desolvation sample introduction, interferences can be reduced sufficiently to allow determination of the 42, 43, 44, 46 and 48 isotopes of calcium with the precision of LR. Isotope ratio precision and accuracy is < ±0.1% (1σ) for analysis of three isotopes (42, 43, and 44), as required for clinical studies. Propagating this level of uncertainty indicates that >4% enrichments provide < ±10% (2σ) uncertainty in the calculated fractional calcium absorption values. Minimizing unnecessary enrichments can increase the number of subjects in studies without increasing the substantial cost of enriched isotopes. This new analytical method is applied to our on-going clinical trials where 0.017 mg kg−1 of 42Ca (90.8%; intravenous, IV) and 0.012 mg kg−143Ca (52.1%;oral) administered to postmenopausal women show average 24 h (pooled) urinary enrichments of 5.0% and 5.1% from the intravenous and oral doses, respectively.

ER-to-mitochondria miscommunication and metabolic diseases

Eukaryotic cells contain a variety of subcellular organelles, each of which performs unique tasks. Thus follows that in order to coordinate these different intracellular functions, a highly dynamic system of communication must exist between the various compartments. Direct endoplasmic reticulum (ER)-mitochondria communication is facilitated by the physical interaction of their membranes in dedicated structural domains known as mitochondria-associated membranes (MAMs), which facilitate calcium (Ca(2+)) and lipid transfer between organelles and also act as platforms for signaling. Numerous studies have demonstrated the importance of MAM in ensuring correct function of both organelles, and recently MAMs have been implicated in the genesis of various human diseases. Here, we review the salient structural features of interorganellar communication via MAM and discuss the most common experimental techniques employed to assess functionality of these domains. Finally, we will highlight the contribution of MAM to a variety of cellular functions and consider the potential role of MAM in the genesis of metabolic diseases. In doing so, the importance for cell functions of maintaining appropriate communication between ER and mitochondria will be emphasized.

Calcium sensing receptor activation elevates proinflammatory factor expression in human adipose cells and adipose tissue.

The proinflammatory status of adipose tissue has been linked to the metabolic and cardiovascular consequences of obesity. Human adipose cells express the calcium sensing receptor (CaSR), and its expression is elevated in inflammatory states, such as that associated with obesity. Given the CaSRs association with inflammation in other tissues, we evaluated its role elevating the adipose expression of inflammatory factors. The CaSR activation by the calcimimatic cinacalcet (5μM) in adipose tissue and in vitro cultured LS14 adipose cells elicited an elevation in the expression of the proinflammatory cytokines IL6, IL1β, TNFα, and the chemoattractant CCL2. This was in part reverted by SN50, an inhibitor of the inflammatory mediator nuclear factor kappa B (NFκB). Our observations suggest that CaSR activation elevates cytokine and chemokine production, partially mediated by NFκB. These findings support the relevance of the CaSR in the pathophysiology of obesity-induced adipose tissue dysfunction, with an interesting potential for pharmacological manipulation.

Energy Restriction Is Associated with Lower Bone Mineral Density of the Tibia and Femur in Lean but Not Obese Female Rats

Energy restriction decreases bone mineral density (BMD), and epidemiological studies suggest that the risk of weight loss-induced bone loss is greater in lean than in heavier individuals. Our goal in this study was to determine how bone density and geometry respond to energy restriction in mature obese rats compared with lean rats. At 6 mo of age, 36 diet-induced obese and lean female Sprague-Dawley rats were allocated to control (CTL; ad libitum; n = 18) and energy-restricted (EnR; 40% restriction; n = 18) diets. After 10 wk of dietary intervention, obese EnR rats lost more weight (-91 +/- 34 g) than lean EnR rats(-61 +/- 14 g) (P < 0.02), [corrected] whereas body weight did not change significantly in the 2 CTL groups (14 +/- 23 g). Only the lean EnR (and not obese EnR) rats showed lower BMD compared with CTL rats at the tibia, distal, and proximal femur and femoral neck, and trabecular bone volume (P < 0.05). Serum estradiol declined in lean EnR rats compared with baseline (P < 0.05) but not in the obese EnR rats. In addition, the final serum 25-hydroxyvitamin D (25OHD) concentration was higher (P < 0.05) in obese than in lean EnR rats. Serum parathyroid hormone decreased (P < 0.05) from baseline to final in lean and obese CTL, but not EnR rats. These data support the hypothesis that energy restriction in lean rats compared with obese rats is more detrimental to bone, and it is possible that the greater decline in estrogen and lower levels of 25OHD contribute to this effect.

Antilipolytic effect of calcium-sensing receptor in human adipocytes

The extracellular calcium-sensing receptor (CaSR), a seven transmembrane G-protein-coupled receptor, was cloned in 1993. Its activation was first associated to the regulation of calcium homeostasis; however, the presence in tissues unrelated with this role has revealed its participation in numerous other cell functions. We previously described CaSR expression in human adipocytes, and here we investigated the effect of its activation on adipocyte lipolytic activity by measuring glycerol release to the incubation medium. Treatment of adipocytes with CaSR agonists elicited an inhibitory effect on basal lipolysis, which was prevented by a CaSR antagonist. To further corroborate the antilipolytic effect of CaSR activation, lipolysis was evaluated under conditions that interfere with main antilipolytic regulatory pathways. Cells were preincubated with pertussis toxin (PT, a Giα protein inhibitor), the phosphatidylinositol 3 kinase (PI3K) inhibitors wortmannin and LY-294002 as well as the cAMP analog 8Br-cAMP, all of which influenced the antilipolytic effect of CaSR stimulation. In light of the current view of adipose tissue as an organ involved in whole-body metabolic control, the role of the CaSR modulating basal lipolysis elicits great interest, given its metabolic sensing capabilities due to the variety of ligands that regulate its activity, and its potential cross-talk with insulin and adipose tissue-secreted factors.

Larger Anti-adipogenic Effect of Angiotensin II on Omental Preadipose Cells of Obese Humans

Objective: The ability to form new adipose cells is important to adipose tissue physiology; however, the mechanisms controlling the recruitment of adipocyte progenitors are poorly understood. A role for locally generated angiotensin II in this process is currently proposed. Given that visceral adipose tissue reportedly expresses higher levels of angiotensinogen compared with other depots and the strong association of augmented visceral fat mass with the adverse consequences of obesity, we studied the role of angiotensin II in regulating adipogenic differentiation in omental fat of obese and non‐obese humans.

Obesity-associated proinflammatory cytokines increase calcium sensing receptor (CaSR) protein expression in primary human adipocytes and LS14 human adipose cell line ☆

Obesity-associated health complications are thought to be in part due to the low-grade proinflammatory state that characterizes this disease. The calcium sensing receptor (CaSR), which is expressed in human adipose cells, plays an important role in diseases involving inflammation. To assess the relevance of this protein in adipose pathophysiology, we evaluated its expression in adipocytes under obesity-related proinflammatory conditions. As in primary adipose cells, we established that LS14, a recently described human adipose cell line, expresses the CaSR. Differentiated LS14 and primary adipose cells were exposed overnight to cytokines typically involved in obesity-related inflammation (interleukin (IL)1beta, IL6 and tumor necrosis factor (TNF)alpha). The cytokines increased CaSR abundance in differentiated adipocytes. We incubated LS14 cells with medium previously conditioned (CM) by adipose tissue from subjects with a wide range of body mass index (BMI). Cells exposed to CM from subjects of higher BMI underwent a greater increase in CaSR protein, likely resulting from the greater proinflammatory cytokines secreted from obese tissue. Our observations that proinflammatory factors increase CaSR levels in adipocytes, and the reported ability of CaSR to elevate cytokine levels, open new aspects in the study of obesity inflammatory state pathophysiology, providing a potential novel therapeutic prevention and treatment target.