Evolution of an invasive ductal carcinoma to a small cell carcinoma of the breast: A case report.

RATIONALE: Small cell carcinoma (SCC) is a rare subtype of breast cancer and presents a complex diagnostic and treatment challenge, due to paucity of data. To the best of our knowledge, most cases of breast SCC reported in the literature describe a de novo breast primary. Our case is unique as it describes the evolution of an invasive ductal carcinoma after treatment into a SCC of the breast. PATIENT CONCERNS AND DIAGNOSIS: We report a case of a 53-year-old female, lifelong non-smoker, who initially presented with breast mass noted on self examination. Breast and axillary lymph node biopsy demonstrated a hormone receptor positive invasive ductal carcinoma with a metastatic T3 lesion. INTERVENTION: She was treated with first-line palbociclib/letrozole with initial clinical response, and at progression was switched to capecitabine with no response. Repeat biopsy of the axillary lesion showed evolution of the tumor into a triple negative breast cancer. She was then treated with third-line paclitaxel and radiation therapy with good initial response. She eventually had further disease progression and presented with a new mediastinal lymphadenopathy causing SVC syndrome. Biopsy of this showed a small cell variant of breast neuroendocrine carcinoma. Due to the evolution of histology in this case, a retrospective review of her initial breast specimen as well as the second biopsy from the axilla was conducted which confirmed that the mediastinal lymphadenopathy was metastatic from the original breast tumor. OUTCOMES AND LESSONS: We speculate that the initial treatment allowed a minority of treatment-resistant neuroendocrine cells to grow and become the dominant face of the tumor. Our patient had an excellent response to carboplatin/etoposide and consolidative locoregional radiotherapy but presented with an early intracranial recurrence. This is a similar pattern of metastases as seen in lung SCC and highlights a potential role for prophylactic cranial irradiation in breast SCC. Further studies are needed to better understand the biology and treatment of breast SCC which continues to present a challenge for clinicians.

Mothers are more sensitive to infant cues after breastfeeding compared to bottle-feeding with human milk.

The belief that breastfeeding promotes maternal bonding is widely held by both the public and professional health organizations. Yet to our knowledge, all research examining the link between breastfeeding and maternal behavior in humans has been correlational, limiting our ability to draw causal conclusions. In many mammals, the hormone prolactin, which is central to milk production, rises in response to each breastfeeding session and promotes maternal sensitivity, yet there is a dearth of research in human mothers. To fill these research gaps, we randomly assigned 28 breastfeeding mothers to either breastfeed in the lab or feed their infants previously expressed breastmilk in a bottle before participating in a video-recorded free play session with their infant. Plasma prolactin was measured 40 min after the start of the feeding session and video observations were coded for maternal sensitivity. We found that women randomly assigned to breastfeed were more sensitive to infant cues than women randomly assigned to bottle-feed. Prolactin levels did not differ between feeding groups, although prolactin was positively correlated with maternal sensitivity. Our results suggest that feeding milk directly from the breast (compared to bottle-feeding) increases maternal sensitivity towards infants, at least in the short term.

Regulation of metabolism during hibernation in brown bears (Ursus arctos): Involvement of cortisol, PGC-1α and AMPK in adipose tissue and skeletal muscle.

The purpose of this study was to investigate changes in expression of known cellular regulators of metabolism during hyperphagia (Sept) and hibernation (Jan) in skeletal muscle and adipose tissue of brown bears and determine whether signaling molecules and transcription factors known to respond to changes in cellular energy state are involved in the regulation of these metabolic adaptations. During hibernation, serum levels of cortisol, glycerol, and triglycerides were elevated, and protein expression and activation of AMPK in skeletal muscle and adipose tissue were reduced. mRNA expression of the co-activator PGC-1α was reduced in all tissues in hibernation whereas mRNA expression of the transcription factor PPAR-α was reduced in the vastus lateralis muscle and adipose tissue only. During hibernation, gene expression of ATGL and CD36 was not altered; however, HSL gene expression was reduced in adipose tissue. During hibernation gene expression of the lipogenic enzyme DGAT in all tissues and the expression of the FA oxidative enzyme LCAD in the vastus lateralis muscle were reduced. Gene and protein expression of the glucose transporter GLUT4 was decreased in adipose tissue in hibernation. Our data suggest that high cortisol levels are a key adaptation during hibernation and link cortisol to a reduced activation of the AMPK/PGC-1α/PPAR-α axis in the regulation of metabolism in skeletal muscle and adipose tissue. Moreover, our results indicate that during this phase of hibernation at a time when metabolic rate is significantly reduced metabolic adaptations in peripheral tissues seek to limit the detrimental effects of unduly large energy dissipation.

Resistance Training Threshold for Elevating Bone Mineral Density in Growing Female Rats.

The purpose of this study was to determine the minimum amount of resistance exercise that would stimulate bone formation yielding an elevation in bone mineral density (BMD) during the growth period in female rats. Female rats were randomly divided into: Control (Con, n=8), 3 ladder climb resistance-trained group (3LC, n=8), 4 ladder climb resistance-trained group (4LC, n=8), 5 ladder climb resistance-trained group (5LC, n=8), and 6 ladder climb resistance-trained group (6LC, n=8). All exercised groups were conditioned to climb a vertical ladder with weights appended to their tail 3 days/wk for a total of 6 wks. After 6 wks, left tibia BMD (g/cm2) was significantly greater for 4LC (0.197±0.003), 5LC (0.200±0.004) and 6LC (0.202±0.003) when compared to Con (0.185±0.006). Left femur BMD (g/cm2) was significantly greater for 4LC (0.260±0.005), 5LC (0.269±0.004) and 6LC (0.272±0.006) when compared to Con (0.244±0.006). There were no significant differences in tibia and femur BMD between 4LC, 5LC, and 6LC groups. The results suggest that during growth, a high volume of resistance exercise was required to elicit an elevation in BMD in females.

Intracoronary Thrombus Formation Following Carbon Monoxide Poisoning.

Thromboembolic events in the context of carbon monoxide (CO) exposure have been well described in the literature. Six cases of clinically significant coronary thrombosis following CO exposure were previously reported. However, factors affecting the development of coronary thrombus in CO exposure are poorly understood, and the significance of this finding in a forensic context is not clear. This article discusses a case of coronary thrombosis found at autopsy following a death in which CO poisoning was suspected. A 67-year-old man was found dead in his garage with four vehicles with their ignition in the "on" position and their tanks empty. At autopsy, severe coronary atherosclerosis and an acute nonocclusive coronary thrombus were found. Given the dissimilarities among cases and the presence of CO exposure, it was suggested that the coronary artery thrombosis is likely due to the inherent prothrombotic mechanism of CO, the only common denominator in all the cases.

Osteoblast-derived FGF9 regulates skeletal homeostasis.

FGF9 has complex and important roles in skeletal development and repair. We have previously observed that Fgf9 expression in osteoblasts (OBs) is regulated by G protein signaling and therefore the present study was done to determine whether OB-derived FGF9 was important in skeletal homeostasis. To directly test this idea, we deleted functional expression of Fgf9 gene in OBs using a 2.3kb collagen type I promoter-driven Cre transgenic mouse line (Fgf9OB-/-). Both Fgf9 knockout (Fgf9OB-/-) and the Fgf9 floxed littermates (Fgf9fl/fl) mice were fully backcrossed and maintained in an FBV/N background. Three month old Fgf9OB-/- mice displayed a significant decrease in cancellous bone and bone formation in the distal femur and a significant decrease in cortical thickness at the TFJ. Strikingly, female Fgf9OB-/- mice did not display altered bone mass. Continuous treatment of mouse BMSCs with exogenous FGF9 inhibited mouse BMSC mineralization while acute treatment increased the proliferation of progenitors, an effect requiring the activation of Akt1. Our results suggest that mature OBs are an important source of FGF9, positively regulating skeletal homeostasis in male mice. Osteoblast-derived FGF9 may serve a paracrine role to maintain the osteogenic progenitor cell population through activation of Akt signaling.

IL-15 Mediates Mitochondrial Activity through a PPARδ-Dependent-PPARα-Independent Mechanism in Skeletal Muscle Cells.

Molecular mediators of metabolic processes, to increase energy expenditure, have become a focus for therapies of obesity. The discovery of cytokines secreted from the skeletal muscle (SKM), termed "myokines," has garnered attention due to their positive effects on metabolic processes. Interleukin-15 (IL-15) is a myokine that has numerous positive metabolic effects and is linked to the PPAR family of mitochondrial regulators. Here, we aimed to determine the importance of PPARα and/or PPARδ as targets of IL-15 signaling. C2C12 SKM cells were differentiated for 6 days and treated every other day with IL-15 (100 ng/mL), a PPARα inhibitor (GW-6471), a PPARδ inhibitor (GSK-3787), or both IL-15 and the inhibitors. IL-15 increased mitochondrial activity and induced PPARα, PPARδ, PGC1α, PGC1ß, UCP2, and Nrf1 expression. There was no effect of inhibiting PPARα, in combination with IL-15, on the aforementioned mRNA levels except for PGC1ß and Nrf1. However, with PPARδ inhibition, IL-15 failed to induce the expression levels of PGC1α, PGC1ß, UCP2, and Nrf1. Further, inhibition of PPARδ abolished IL-15 induced increases in citrate synthase activity, ATP production, and overall mitochondrial activity. IL-15 had no effects on mitochondrial biogenesis. Our data indicates that PPARδ activity is required for the beneficial metabolic effects of IL-15 signaling in SKM.

AMPK Phosphorylates Desnutrin/ATGL and Hormone-Sensitive Lipase To Regulate Lipolysis and Fatty Acid Oxidation within Adipose Tissue.

The role of AMP-activated protein kinase (AMPK) in promoting fatty acid (FA) oxidation in various tissues, such as liver and muscle, has been well understood. However, the role of AMPK in lipolysis and FA metabolism in adipose tissue has been controversial. To investigate the role of AMPK in the regulation of adipose lipolysis in vivo, we generated mice with adipose-tissue-specific knockout of both the α1 and α2 catalytic subunits of AMPK (AMPK-ASKO mice) by using aP2-Cre and adiponectin-Cre. Both models of AMPK-ASKO ablation show no changes in desnutrin/ATGL levels but have defective phosphorylation of desnutrin/ATGL at S406 to decrease its triacylglycerol (TAG) hydrolase activity, lowering basal lipolysis in adipose tissue. These mice also show defective phosphorylation of hormone-sensitive lipase (HSL) at S565, with higher phosphorylation at protein kinase A sites S563 and S660, increasing its hydrolase activity and isoproterenol-stimulated lipolysis. With higher overall adipose lipolysis, both models of AMPK-ASKO mice are lean, having smaller adipocytes with lower TAG and higher intracellular free-FA levels. Moreover, FAs from higher lipolysis activate peroxisome proliferator-activated receptor delta to induce FA oxidative genes and increase FA oxidation and energy expenditure. Overall, for the first time, we provide in vivo evidence of the role of AMPK in the phosphorylation and regulation of desnutrin/ATGL and HSL and thus adipose lipolysis.

IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells.

Myokines are specialized cytokines that are secreted from skeletal muscle (SKM) in response to metabolic stimuli, such as exercise. Interleukin-15 (IL-15) is a myokine with potential to reduce obesity and increase lean mass through induction of metabolic processes. It has been previously shown that IL-15 acts to increase glucose uptake in SKM cells. However, the downstream signals orchestrating the link between IL-15 signaling and glucose uptake have not been fully explored. Here we employed the mouse SKM C2C12 cell line to examine potential downstream targets of IL-15-induced alterations in glucose uptake. Following differentiation, C2C12 cells were treated overnight with 100 ng/ml of IL-15. Activation of factors associated with glucose metabolism (Akt and AMPK) and known downstream targets of IL-15 (Jak1, Jak3, STAT3, and STAT5) were assessed with IL-15 stimulation. IL-15 stimulated glucose uptake and GLUT4 translocation to the plasma membrane. IL-15 treatment had no effect on phospho-Akt, phospho-Akt substrates, phospho-AMPK, phospho-Jak1, or phospho-STAT5. However, with IL-15, phospho-Jak3 and phospho-STAT3 levels were increased along with increased interaction of Jak3 and STAT3. Additionally, IL-15 induced a translocation of phospho-STAT3 from the cytoplasm to the nucleus. We have evidence that a mediator of glucose uptake, HIF1α, expression was dependent on IL-15 induced STAT3 activation. Finally, upon inhibition of STAT3 the positive effects of IL-15 on glucose uptake and GLUT4 translocation were abolished. Taken together, we provide evidence for a novel signaling pathway for IL-15 acting through Jak3/STAT3 to regulate glucose metabolism.

Negative Skeletal Effects of Locally Produced Adiponectin.

Epidemiological studies show that high circulating levels of adiponectin are associated with low bone mineral density. The effect of adiponectin on skeletal homeostasis, on osteoblasts in particular, remains controversial. We investigated this issue using mice with adipocyte-specific over-expression of adiponectin (AdTg). MicroCT and histomorphometric analysis revealed decreases (15%) in fractional bone volume in AdTg mice at the proximal tibia with no changes at the distal femur. Cortical bone thickness at mid-shafts of the tibia and at the tibiofibular junction was reduced (3-4%) in AdTg mice. Dynamic histomorphometry at the proximal tibia in AdTg mice revealed inhibition of bone formation. AdTg mice had increased numbers of adipocytes in close proximity to trabecular bone in the tibia, associated with increased adiponectin levels in tibial marrow. Treatment of BMSCs with adiponectin after initiation of osteoblastic differentiation resulted in reduced mineralized colony formation and reduced expression of mRNA of osteoblastic genes, osterix (70%), Runx2 (52%), alkaline phosphatase (72%), Col1 (74%), and osteocalcin (81%). Adiponectin treatment of differentiating osteoblasts increased expression of the osteoblast genes PPARγ (32%) and C/ebpα (55%) and increased adipocyte colony formation. These data suggest a model in which locally produced adiponectin plays a negative role in regulating skeletal homeostasis through inhibition of bone formation and by promoting an adipogenic phenotype.

AMPK-α2 is involved in exercise training-induced adaptations in insulin-stimulated metabolism in skeletal muscle following high-fat diet.

AMP-activated protein kinase (AMPK) has been studied extensively and postulated to be a target for the treatment and/or prevention of metabolic disorders such as insulin resistance. Exercise training has been deemed a beneficial treatment for obesity and insulin resistance. Furthermore, exercise is a feasible method to combat high-fat diet (HFD)-induced alterations in insulin sensitivity. The purpose of this study was to determine whether AMPK-α2 activity is required to gain beneficial effects of exercise training with high-fat feeding. Wild-type (WT) and AMPK-α2 dominant-negative (DN) male mice were fed standard diet (SD), underwent voluntary wheel running (TR), fed HFD, or trained with HFD (TR + HFD). By week 6, TR, irrespective of genotype, decreased blood glucose and increased citrate synthase activity in both diet groups and decreased insulin levels in HFD groups. Hindlimb perfusions were performed, and, in WT mice with SD, TR increased insulin-mediated palmitate uptake (76.7%) and oxidation (>2-fold). These training-induced changes were not observed in the DN mice. With HFD, TR decreased palmitate oxidation (61-64%) in both WT and DN and increased palmitate uptake (112%) in the WT with no effects on palmitate uptake in the DN. With SD, TR increased ERK1/2 and JNK1/2 phosphorylation, regardless of genotype. With HFD, TR reduced JNK1/2 phosphorylation, regardless of genotype, carnitine palmitoyltransferase 1 expression in WT, and CD36 expression in both DN and WT. These data suggest that low AMPK-α2 signaling disrupts, in part, the exercise training-induced adaptations in insulin-stimulated metabolism in skeletal muscle following HFD.

Laboratory tests, interpretation, and use of resources: a program to introduce the basics.

PROBLEM ADDRESSED: The overuse of laboratory testing has increased rapidly and is contributing to the financial strain on the health care system in Canada. Moreover, a substantial proportion of ordered tests are unnecessary. In a search of all the Canadian family physician residency programs, none lists laboratory training as mandatory or as an optional elective in its curriculum. OBJECTIVE OF PROGRAM: To introduce family medicine residents to appropriate and efficient use of laboratory tests. PROGRAM DESCRIPTION: The program was run as a series of identical 4-hour small group sessions to facilitate discussion and laboratory tours. The curriculum focused on 7 key topics: problems associated with laboratory testing, sources of laboratory errors, definitions of normal and abnormal test results, appropriate use of laboratory requisition forms, laboratory quality assurance methods, laboratory collection processes, and costs of common laboratory tests. Residents were taken to a patient specimen collection site for a tour and introduction, followed by approximately 2 hours of didactic sessions, and ending with a tour of a large tertiary care testing facility. CONCLUSION: The program was very well received by family medicine residents and resulted in a substantial increase in residents' self-assessed knowledge of the 7 topics covered in the curriculum. It is hoped that this program will fill an important gap in residency training and support residents' competency in the "selectivity" domain of training.

Desnutrin/ATGL activates PPARδ to promote mitochondrial function for insulin secretion in islet ß cells.

Excessive caloric intake leading to obesity is associated with insulin resistance and dysfunction of islet ß cells. High-fat feeding decreases desnutrin (also called ATGL/PNPLA2) levels in islets. Here we show that desnutrin ablation via RIP-Cre (ßKO) or RIP-CreER results in hyperglycemia with impaired glucose-stimulated insulin secretion (GSIS). Due to decreased lipolysis, islets have higher TAG content but lower free FA levels. ßKO islets exhibit impaired mitochondrial respiration and lower production of ATP required for GSIS, along with decreased expression of PPARδ target genes involved in mitochondrial oxidation. Furthermore, synthetic PPARδ, but not PPARα, agonist restores GSIS and expression of mitochondrial oxidative genes in ßKO mice, revealing that desnutrin-catalyzed lipolysis generates PPARδ ligands. Finally, adenoviral expression of desnutrin in ßKO islets restores all defects of ßKO islet phenotype and function, including GSIS and mitochondrial defects, demonstrating the critical role of the desnutrin-PPARδ-mitochondrial oxidation axis in regulating islet ß cell GSIS.

Constitutive protein kinase A activity in osteocytes and late osteoblasts produces an anabolic effect on bone.

Osteocytes have been implicated in the control of bone formation. However, the signal transduction pathways that regulate the biological function of osteocytes are poorly defined. Limited evidence suggests an important role for the Gs/cAMP pathway in osteocyte function. In the present study, we explored the hypothesis that cAMP-dependent kinase A (PKA) activation in osteocytes plays a key role in controlling skeletal homeostasis. To test this hypothesis, we mated mice harboring a Cre-conditional, mutated PKA catalytic subunit allele that encodes a constitutively active form of PKA (CαR) with mice expressing Cre under the control of the osteocyte-specific promoter, DMP1. This allowed us to direct the expression of CαR to osteocytes in double transgenic progeny. Examination of Cre expression indicated that CαR was also expressed in late osteoblasts. Cortical and trabecular bone parameters from 12-week old mice were determined by µCT. Expression of CαR in osteocytes and late osteoblasts altered the shape of cortical bone proximal to the tibia-fibular junction (TFJ) and produced a significant increase in its size. In trabecular bone of the distal femur, fractional bone volume, trabecular number, and trabecular thickness were increased. These increases were partially the results of increased bone formation rates (BFRs) on the endosteal surface of the cortical bone proximal to the TFJ as well as increased BFR on the trabecular bone surface of the distal femur. Mice expressing CαR displayed a marked increase in the expression of osteoblast markers such as osterix, runx2, collagen 1α1, and alkaline phosphatase (ALP). Interestingly, expression of osteocyte marker gene, DMP1, was significantly up-regulated but the osteocyte number per bone area was not altered. Expression of SOST, a presumed target for PKA signaling in osteocytes, was significantly down-regulated in females. Importantly, no changes in bone resorption were detected. In summary, constitutive PKA signaling in osteocytes and late osteoblasts led to a small expansion of the size of the cortical bone proximal to the TFJ and an increase in trabecular bone in female mice. This was associated with down-regulation of SOST and up-regulation of several osteoblast marker genes. Activation of the PKA pathway in osteocytes and late osteoblasts is sufficient for the initiation of an anabolic skeletal response.

Increased bone mass in mice lacking the adipokine apelin.

Adipose tissue plays an important role in skeletal homeostasis, and there is interest in identifying adipokines that influence bone mass. One such adipokine may be apelin, a ligand for the Gi-G protein-coupled receptor APJ, which has been reported to enhance mitogenesis and suppress apoptosis in MC3T3-E1 cells and primary human osteoblasts (OBs). However, it is unclear whether apelin plays a physiological role in regulating skeletal homeostasis in vivo. In this study, we compared the skeletal phenotypes of apelin knockout (APKO) and wild-type mice and investigated the direct effects of apelin on bone cells in vitro. The increased fractional cancellous bone volume at the distal femur was observed in APKO mice of both genders at 12 weeks of age and persisted until the age of 20. Cortical bone perimeter at the femoral midshaft was significantly increased in males and females at both time points. Dynamic histomorphometry revealed that APKO mice had increased rates of bone formation and mineral apposition, with evidences of accelerated OB proliferation and differentiation, without significant alteration in osteoclast activity. An in vitro study showed that apelin increased proliferation of primary mouse OBs as well as suppressed apoptosis in a dose-dependent manner with the maximum effect at 5nM. However, it had no effect on the formation of mineralized nodules. We did not observed significantly altered in osteoclast parameters in vitro. Taken together, the increased bone mass in mice lacking apelin suggested complex direct and paracrine/endocrine effects of apelin on bone, possibly via modulating insulin sensitivity. These results indicate that apelin functions as a physiologically significant antianabolic factor in bone in vivo.

Contraction-induced signaling: evidence of convergent cascades in the regulation of muscle fatty acid metabolism.

The regulation of fatty acid utilization during muscle contraction and exercise remains to be fully elucidated. Evidence suggests that the metabolic responses of skeletal muscle induced by the contraction-induced changes in energy demand are mediated by the activation of a multitude of intracellular signaling cascades. This review addresses the roles played by 3 intracellular signaling cascades of interest in the regulation of fatty acid uptake and oxidation in contracting skeletal muscle; namely, the AMP-activated protein kinase (AMPK), calcium/calmodulin-dependent protein kinases (CaMKs), and the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling cascades. Data delineating the potential role of AMPK in cross-talk with CaMKII, CaMK kinase (CaMKK), and ERK1/2 are presented. Collectively, data show that in perfused rodent muscle, regulation of fatty acid uptake and oxidation occurs via (i) CaMKII signaling via both AMPK-dependent and -independent cascades, (ii) CaMKK signaling via both AMPK-dependent and -independent cascades, (iii) AMPK signaling in a time- and intensity-dependent manner, and (iv) ERK1/2 signaling in an intensity-dependent manner.

AMP-activated protein kinase α2 is an essential signal in the regulation of insulin-stimulated fatty acid uptake in control-fed and high-fat-fed mice.

Owing to its critical role in the regulation of skeletal muscle metabolism, AMP-activated protein kinase (AMPK) remains a central focus of research for the treatment of insulin resistance. The purpose of the present study was to determine the role of AMPKα2 activity in the regulation of glucose uptake and fatty acid (FA) metabolism in insulin-resistant skeletal muscle. Male C57BL/6 mice were divided into groups fed a control diet (CD) or high-fat (60%) diet (HFD) for 6 weeks and were either wild-type (WT) or possessed an AMPKα2 dominant negative transgene (DN). After 6 weeks, hindlimbs of CD (n = 10) and HFD mice (n = 10) were perfused with or without 450 µU ml(-1) insulin. Muscles of CD (n = 8) and HFD mice (n = 8) were used for measurement of basal protein expression. In CD mice, low AMPKα2 activity did not affect basal FA uptake (FAU), but it increased basal FA oxidation (FAO) by 28% and prevented the typical insulin-mediated increase in FAU and decrease in FAO. In HFD-fed mice, low AMPKα2 activity increased basal FAU by 147% (P < 0.05). In both WT and DN mice, HFD abolished the typical insulin-mediated increase in FAU and decrease in FAO. In HFD-fed mice, low AMPKα2 activity increased SIRT1 activity and decreased Protein Tyrosine Phosphatase 1B (PTP1B) expression and Akt(Thr308) phosphorylation (P < 0.05). Adipose tissue protein expression of interleukin-6 and tumour necrosis factor α was increased by HFD in WT mice but not in DN mice (P < 0.05). Skeletal muscle interleukin-15 expression was decreased in both feeding conditions in the DN mice (P < 0.05). The data from this study suggest that in insulin-resistant conditions low AMPKα2 activity impacts the regulation of skeletal muscle FA metabolism via changes in SIRT1 activity, PTP1B expression and Akt phosphorylation and the expression of adipose tissue pro-inflammatory markers.

Desnutrin/ATGL is regulated by AMPK and is required for a brown adipose phenotype.

While fatty acids (FAs) released by white adipose tissue (WAT) provide energy for other organs, lipolysis is also critical in brown adipose tissue (BAT), generating FAs for oxidation and UCP-1 activation for thermogenesis. Here we show that adipose-specific ablation of desnutrin/ATGL in mice converts BAT to a WAT-like tissue. These mice exhibit severely impaired thermogenesis with increased expression of WAT-enriched genes but decreased BAT genes, including UCP-1 with lower PPARα binding to its promoter, revealing the requirement of desnutrin-catalyzed lipolysis for maintaining a BAT phenotype. We also show that desnutrin is phosphorylated by AMPK at S406, increasing TAG hydrolase activity, and provide evidence for increased lipolysis by AMPK phosphorylation of desnutrin in adipocytes and in vivo. Despite adiposity and impaired BAT function, desnutrin-ASKO mice have improved hepatic insulin sensitivity with lower DAG levels. Overall, desnutrin is phosphorylated/activated by AMPK to increase lipolysis and brings FA oxidation and UCP-1 induction for thermogenesis.

AMPKα2 deficiency uncovers time dependency in the regulation of contraction-induced palmitate and glucose uptake in mouse muscle.

AMP-activated protein kinase (AMPK) is a fuel sensor in skeletal muscle with multiple downstream signaling targets that may be triggered by increases in intracellular Ca(2+) concentration ([Ca(2+)]). The purpose of this study was to determine whether increases in intracellular [Ca(2+)] induced by caffeine act solely via AMPKα(2) and whether AMPKα(2) is essential to increase glucose uptake, fatty acid (FA) uptake, and FA oxidation in contracting skeletal muscle. Hindlimbs from wild-type (WT) or AMPKα(2) dominant-negative (DN) transgene mice were perfused during rest (n = 11), treatment with 3 mM caffeine (n = 10), or muscle contraction (n = 11). Time-dependent effects on glucose and FA uptake were uncovered throughout the 20-min muscle contraction perfusion period (P < 0.05). Glucose uptake rates did not increase in DN mice during muscle contraction until the last 5 min of the protocol (P < 0.05). FA uptake rates were elevated at the onset of muscle contraction and diminished by the end of the protocol in DN mice (P < 0.05). FA oxidation rates were abolished in the DN mice during muscle contraction (P < 0.05). The DN transgene had no effect on caffeine-induced FA uptake and oxidation (P > 0.05). Glucose uptake rates were blunted in caffeine-treated DN mice (P < 0.05). The DN transgene resulted in a greater use of intramuscular triglycerides as a fuel source during muscle contraction. The DN transgene did not alter caffeine- or contraction-mediated changes in the phosphorylation of Ca(2+)/calmodulin-dependent protein kinase I or ERK1/2 (P > 0.05). These data suggest that AMPKα(2) is involved in the regulation of substrate uptake in a time-dependent manner in contracting muscle but is not necessary for regulation of FA uptake and oxidation during caffeine treatment.

Short-term AMP-regulated protein kinase activation enhances insulin-sensitive fatty acid uptake and increases the effects of insulin on fatty acid oxidation in L6 muscle cells.

Evidence shows that exercise increases insulin-sensitive glucose uptake and that exercise-induced AMP-regulated protein kinase (AMPK) activation is a likely candidate to mediate this metabolic adaptation. The purpose of this study was to determine whether repeated AMPK activation can similarly enhance insulin-sensitive fatty acid (FA) metabolism. L6 myotubes were incubated under the following conditions: repeated plus acute 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) treatment (RAA; 1 mmol/L AICAR for 5 h/d for 5 days plus 1 mmol/L AICAR for 60 min on day 6), repeated AICAR (RA; 1 mmol/L AICAR for 5 h/d for five days) or acute AICAR (AA; 1 mmol/L AICAR for 60 min) and were compared with control cells that were not treated with AICAR. On day six, cells from each group were incubated with or without 100 nmol/L insulin. AICAR treatment and insulin stimulation independently increased (P < 0.05) palmitate uptake in all groups. RAA potentiated the insulin-induced increase in palmitate uptake by 97% (P < 0.05) as compared with control cells. RA and AA treatments prevented the insulin-induced decrease in palmitate oxidation, while RAA treatment restored the sensitivity of the cells to insulin action on palmitate oxidation. Total peroxisome proliferator-activated receptor-gamma co-activator-1 alpha, atypical protein kinase C-zeta, cytochrome C and CD36 protein content was increased (P < 0.05) by RA treatment, but unaffected by insulin. These results indicate that repeated AMPK activation induces improvements in insulin-sensitive FA uptake and oxidation and that this occurs partly via changes in the expression of proteins linked to insulin signaling and FA uptake and oxidation capacity.