Genetic and pharmacologic blockade of central melanocortin signaling attenuates cardiac cachexia in rodent models of heart failure

The central melanocortin system plays a key role in the regulation of food intake and energy homeostasis. We investigated whether genetic or pharmacologic blockade of central melanocortin signaling attenuates cardiac cachexia in mice and rats with heart failure. Permanent ligation of the left coronary artery (myocardial infarction (MI)) or sham operation was performed in wild-type (WT) or melanocortin-4 receptor (MC4R) knockout mice. Eight weeks after surgery, WT-Sham mice had significant increases in lean body mass (LBM; P<0·05) and fat mass (P<0·05), whereas WT-MI did not gain significant amounts of LBM or fat mass. Resting basal metabolic rate (BMR) was significantly lower in WT-Sham mice compared to WT-MI mice (P<0·001). In contrast, both MC4-Sham and MC4-MI mice gained significant amounts of LBM (P<0·05) and fat mass (P<0·05) over the study period. There was no significant difference in the BMR between MC4-Sham and MC4-MI mice. In the second experiment, rats received aortic bands or sham operations, and after recovery received i.c.v. injections of either artificial cerebrospinal fluid (aCSF) or the melanocortin antagonist agouti-related protein (AGRP) for 2 weeks. Banded rats receiving AGRP gained significant amount of LBM (P<0·05) and fat mass (P<0·05) over the treatment period, whereas banded rats receiving aCSF did not gain significant amounts of LBM or fat mass. These results demonstrated that genetic and pharmacologic blockade of melanocortin signaling attenuated the metabolic manifestations of cardiac cachexia in murine and rat models of heart failure

Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells

Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment.Career Development Award: (#IK2 CX000538); U.S. Department of Veterans Affairs Clinical Sciences Research and Development Program (MJH); U.S.Department of Veterans Affairs Biomedical Laboratory Research and Development Program (DML) Merit Award: (#I01 BX000533); American Lung Association: (RT-350058)

Maternal High Fat Diet Is Associated with Decreased Plasma n–3 Fatty Acids and Fetal Hepatic Apoptosis in Nonhuman Primates

To begin to understand the contributions of maternal obesity and over-nutrition to human development and the early origins of obesity, we utilized a non-human primate model to investigate the effects of maternal high-fat feeding and obesity on breast milk, maternal and fetal plasma fatty acid composition and fetal hepatic development. While the high-fat diet (HFD) contained equivalent levels of n-3 fatty acids (FA's) and higher levels of n-6 FA's than the control diet (CTR), we found significant decreases in docosahexaenoic acid (DHA) and total n-3 FA's in HFD maternal and fetal plasma. Furthermore, the HFD fetal plasma n-6∶n-3 ratio was elevated and was significantly correlated to the maternal plasma n-6∶n-3 ratio and maternal hyperinsulinemia. Hepatic apoptosis was also increased in the HFD fetal liver. Switching HFD females to a CTR diet during a subsequent pregnancy normalized fetal DHA, n-3 FA's and fetal hepatic apoptosis to CTR levels. Breast milk from HFD dams contained lower levels of eicosopentanoic acid (EPA) and DHA and lower levels of total protein than CTR breast milk. This study links chronic maternal consumption of a HFD with fetal hepatic apoptosis and suggests that a potentially pathological maternal fatty acid milieu is replicated in the developing fetal circulation in the nonhuman primate

Activity-Dependent Dendritic Arborization Mediated by CaM-Kinase I Activation and Enhanced CREB-Dependent Transcription of Wnt-2

Members of the Wnt signaling family are important mediators of numerous developmental events, including activity-dependent dendrite development, but the pathways regulating expression and secretion of Wnt in response to neuronal activity are poorly defined. Here, we identify an NMDA receptor-mediated, Ca 2+-dependent signaling pathway that couples neuronal activity to dendritic arborization through enhanced Wnt synthesis and secretion. Activity-dependent dendritic outgrowth and branching in cultured hippocampal neurons and slices is mediated through activation by CaM-dependent protein kinase kinase (CaMKK) of the membrane-associated γ isoform of CaMKI. Downstream effectors of CaMKI include the MAP-kinase pathway of Ras/MEK/ERK and the transcription factor CREB. A serial analysis of chromatin occupancy screen identified Wnt-2 as an activity-dependent CREB-responsive gene. Neuronal activity enhances CREB-dependent transcription of Wnt-2, and expression of Wnt-2 stimulates dendritic arborization. This novel signaling pathway contributes to dynamic remodeling of the dendritic architecture in response to neuronal activity during development

Regulation of axonal extension and growth cone motility by calmodulin-dependent protein kinase I

Calcium and calmodulin (CaM) are important signaling molecules that regulate axonal or dendritic extension and branching. The Ca2+-dependent stimulation of neurite elongation has generally been assumed to be mediated by CaM-kinase II (CaMKII), although other members of the CaMK family are highly expressed in developing neurons. We have examined this assumption using a combination of dominant-negative CaMKs (dnCaMKs) and other specific CaMK inhibitors. Here we report that inhibition of cytosolic CaMKI, but not CaMKII or nuclear CaMKIV, dramatically decreases axonal outgrowth and branching in cultured neonatal hippocampal and postnatal cerebellar granule neurons. CaMKI is found throughout the cell cytosol, including the growth cone. Growth cones of neurons expressing dnCaMI or dnCaMKK, the upstream activator of CaMKI, exhibit collapsed morphology with a prominent reduction in lamellipodia. Live-cell imaging confirms that these morphological changes are associated with a dramatic decrease in growth cone motility. Treatment of neurons with 1,8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), an inhibitor of CaMKK, causes a similar change in morphology and reduction in growth cone motility, and this inhibition can be rescued by transfection with an STO-609-insensitive mutant of CaMKK or by transfection with constitutively active CaMKI. These results identify CaMKI as a positive transducer of growth cone motility and axon outgrowth and provide a new physiological role for the CaMKK-CaMKI pathway

Perinatal Exposure to a High-Fat Diet Is Associated with Reduced Hepatic Sympathetic Innervation in One-Year Old Male Japanese Macaques

<div><p>Our group recently demonstrated that maternal high-fat diet (HFD) consumption is associated with non-alcoholic fatty liver disease, increased apoptosis, and changes in gluconeogenic gene expression and chromatin structure in fetal nonhuman primate (NHP) liver. However, little is known about the long-term effects that a HFD has on hepatic nervous system development in offspring, a system that plays an important role in regulating hepatic metabolism. Utilizing immunohistochemistry and Real-Time PCR, we quantified sympathetic nerve fiber density, apoptosis, inflammation, and other autonomic components in the livers of fetal and one-year old Japanese macaques chronically exposed to a HFD. We found that HFD exposure <em>in-utero</em> and throughout the postnatal period (HFD/HFD), when compared to animals receiving a CTR diet for the same developmental period (CTR/CTR), is associated with a 1.7 fold decrease in periportal sympathetic innervation, a 5 fold decrease in parenchymal sympathetic innervation, and a 2.5 fold increase in hepatic apoptosis in the livers of one-year old male animals. Additionally, we observed an increase in hepatic inflammation and a decrease in a key component of the cholinergic anti-inflammatory pathway in one-year old HFD/HFD offspring. Taken together, these findings reinforce the impact that continuous exposure to a HFD has in the development of long-term hepatic pathologies in offspring and highlights a potential neuroanatomical basis for hepatic metabolic dysfunction.</p> </div

Quantification of the density of TH nerve fibers between CTR/CTR and HFD/HFD diet groups in the juvenile macaque liver.

<p>(A,C and E) Quantification of TH nerve fibers in the periportal region. (B,D and F) Quantification of TH nerve fibers in the hepatic parenchyma. TH immunofluorescence was acquired in each region by laser scanning confocal microscopy. The volume of TH immunoreactive fibers was normalized to the volume of hepatic tissue in each image. Data are expressed as the median normalized density for each juvenile diet group. (A-B) No differences were observed in the density of sympathetic innervation in juvenile liver between diet groups. CTR/CTR; n = 12, HFD/HFD; n = 9. (C-D) Quantification of the density of TH nerve fibers between CTR/CTR and HFD/HFD diet groups in the female juvenile macaque liver. A nonsignificant trend for higher sympathetic innervation was observed in the female juvenile liver between diet groups. CTR/CTR; n = 5, HFD/HFD; n = 4. (E-F) Quantification of the density of TH nerve fibers between CTR/CTR and HFD/HFD diet groups in the male juvenile macaque liver. Significantly reduced sympathetic innervation was observed between diet groups in both portal (E) and parenchymal regions (F) in the male juvenile liver. CTR/CTR; n = 7, HFD/HFD; n = 5. (* = <i>p</i><0.05, ** = <i>p</i><0.01). (G-J) Representative images of TH immunoreactivity in the portal region for CTR/CTR (G) and HFD/HFD (H) males. Representative images of TH immunoreactivity in the hepatic parenchyma for CTR/CTR (I) and HFD/HFD (J) males. Scale bar = 20 µm.</p

Peripheral Nervous System mRNA expression in Fetal Macaque Liver.¹

1<p>All values are means ± SEMs and are expressed as relative fold compared to CTR. n = 7 for CTR, n = 8 for HFD, n = 7 for REV.</p>2<p>Overall significance as determined by Kruskal-Wallis rank sum test.</p>a<p>Significantly different from CTR, p<.0167, Bonferroni adjusted α.</p>b<p>Significantly different from HFD, p<.0167, Bonferroni adjusted α.</p>c<p>Significantly different from REV, p<.0167, Bonferroni adjusted α.</p

Quantification of hepatic apoptosis between CTR/CTR and HFD/HFD juvenile macaques as determined by TUNEL staining.

<p>(A) Quantification of hepatic apoptosis between entire cohort of juvenile macaques. (B) Comparison of apoptosis in female juvenile macaque liver between CTR/CTR and HFD/HFD diet groups. (C) Quantification of apoptosis in male juvenile macaque liver between CTR/CTR and HFD/HFD diet groups. CTR/CTR n = 11, 6 males 5 females; HFD/HFD n = 10, 6 males, 4 females. (<b>* = </b><i>p</i><0.05, <b>**</b> = <i>p</i><0.01).</p

Peripheral Nervous System mRNA expression in Juvenile Macaque Liver.¹

1<p>All values are means ± SEMs and are expressed as relative fold compared to CTR/CTR. (n = 7−10 for CTR/CTR, n = 8−10 for HFD/HFD).</p>2<p>Overall significance as determined by Kruskal-Wallis rank sum test.</p