Adipose tissue is an endocrine organ, and its homeostatic mechanisms in normal weight, overweight and obese subjects must be elucidated. We sought to determine the basal adipose tissue biology of visceral (VIF) and subcutaneous (SQF) fat depots in 8 month old Sinclair minipigs, an animal that has been shown to be physiologically similar to humans. Metabolic analysis showed a decrease in LDL, white blood cells (WBC), and lymphocyte percentages as the minipigs aged from 6 to 8 months (p <0.0001 and = 0.0046 and 0.0165 respectively). There were no significant changes in triglycerides, HDL, VLDL, and neutrophil percentages. There was a trend in insulin increase (P=0.0722). Microarray analysis was performed to determine transcriptome differences between VIF and SQF. When VIF was compared to SQF, expression of a total of 788 transcript ID’s differed: were 240 up-regulated and 548 down-regulated. Examples included hydroxysteroid 11-beta dehydrogenase 2, fatty acid synthase, IL-18, and platelet factor 4 which were all up-regulated in VIF vs. SQF. The down-regulated transcripts included estrogen receptor 1, insulin-like growth factor binding protein 5, and platelet derived growth factor D. When SQF was compared to VIF, a total of 598 transcript IDs were up or down-regulated by more than a 2 fold difference (P<0.05). From this subset of the transcriptome, we found 471 IDs were up-regulated in SQ fat, and 127 were down- regulated. Interestingly, the up-regulated genes included prostaglandin F2 receptor negative regulator, estrogen receptor 1, thrombospondin 1, lipoprotein related receptor protein 2, and platelet derived growth factor D. Down-regulated genes in SQF compared to VIF included IL-18, platelet factor 4, cyclooxygenase, and fatty acid synthase. We found no significant difference in gene expression between SQF and VIF TNF alpha, TLR 4, and adiponectin in our. Immunofluorensce (IF) assay revealed that SQF expressed more CD 163 positive (alternatively activated) macrophages than VIF, and little to no CD 68 (classically activated) positive macrophages. Additionally, VIF expressed more CD 68 positive macrophages compared to SQF. The data from this study is consistent with the human and rodent literature which states that VIF is more metabolically active and pro-inflammatory compared to SQF.

Adipose tissue is an endocrine organ, and its homeostatic mechanisms in normal weight, overweight and obese subjects must be elucidated. We sought to determine the basal adipose tissue biology of visceral (VIF) and subcutaneous (SQF) fat depots in 8 month old Sinclair minipigs, an animal that has been shown to be physiologically similar to humans.Metabolic analysis showed a decrease in LDL, white blood cells (WBC), and lymphocyte percentages as the minipigs aged from 6 to 8 months (p <0.0001 and = 0.0046 and 0.0165 respectively). There were no significant changes in triglycerides, HDL, VLDL, and neutrophil percentages. There was a trend in insulin increase (P=0.0722).Microarray analysis was performed to determine transcriptome differences between VIF and SQF. When VIF was compared to SQF, expression of a total of 788 transcript ID’s differed: were 240 up-regulated and 548 down-regulated. Examples included hydroxysteroid 11-beta dehydrogenase 2, fatty acid synthase, IL-18, and platelet factor 4 which were all up-regulated in VIF vs. SQF. The down-regulated transcripts included estrogen receptor 1, insulin-like growth factor binding protein 5, and platelet derived growth factor D. When SQF was compared to VIF, a total of 598 transcript IDs were up or down-regulated by more than a 2 fold difference (P<0.05). From this subset of the transcriptome, we found 471 IDs were up-regulated in SQ fat, and 127 were down- regulated. Interestingly, the up-regulated genes included prostaglandin F2 receptor negative regulator, estrogen receptor 1, thrombospondin 1, lipoprotein related receptor protein 2, and platelet derived growth factor D. Down-regulated genes in SQF compared to VIF included IL-18, platelet factor 4, cyclooxygenase, and fatty acid synthase. We found no significant difference in gene expression between SQF and VIF TNF alpha, TLR 4, and adiponectin in our. Immunofluorensce (IF) assay revealed that SQF expressed more CD 163 positive (alternatively activated) macrophages than VIF, and little to no CD 68 (classically activated) positive macrophages. Additionally, VIF expressed more CD 68 positive macrophages compared to SQF. The data from this study is consistent with the human and rodent literature which states that VIF is more metabolically active and pro-inflammatory compared to SQF.

Lafora disease (LD) is a rare yet invariably fatal form of epilepsy characterized by progressive degeneration of the central nervous and motor systems and accumulation of insoluble glucans within cells. LD results from mutation of either the phosphatase laforin, an enzyme that dephosphorylates cellular glycogen, or the E3 ubiquitin ligase malin, the binding partner of laforin. Currently, there are no therapeutic options for LD, or reported methods by which the specific activity of glucan phosphatases such as laforin can be easily measured. To facilitate our translational studies, we developed an assay with which the glucan phosphatase activity of laforin as well as emerging members of the glucan phosphatase family can be characterized. We then adapted this assay for the detection of endogenous laforin activity from human and mouse tissue. This laforin bioassay will prove useful in the detection of functional laforin in LD patient tissue following the application of therapies to LD patients. We subsequently developed an in vitro readthrough reporter system in order to assess the efficacy of aminoglycosides in the readthrough of laforin and malin nonsense mutations. We found that although several laforin and malin nonsense mutations exhibited significant drug-induced readthrough, the location of the epitope tag used to detect readthrough products dramatically affected our readthrough results. Cell lines established from LD patients with nonsense mutations are thus required to accurately assess the efficacy of aminoglycosides as a therapeutic option for LD. Using hydrogen-deuterium exchange mass spectrometry (DXMS), we then gained insight into the molecular etiology of several point mutations in laforin that cause LD. We identified a novel motif in the phosphatase domain of laforin that shares homology with glycosyl hydrolases (GH) and appears to play a role in the interaction of laforin with glucans. We studied the impact of the Y294N and P301L LD mutations within this GH motif on glucan binding. Surprisingly, these mutations did not reduce glucan binding as expected, rather enhancing the binding of laforin to glucans. These findings elucidate the mechanism by which laforin interacts with and acts upon glucan substrates, providing a target for the development of therapeutic compounds.

Recent advancements have uncovered environmental contributions to obesity and diabetes etiology. In fact, perinatal malnutrition resulting in low birth weight (LBW) has been shown to correlate with later life obesity and impaired glucose tolerance in aged offspring. LBW can result from a myriad of developmental perturbations including macronutrient restriction, hypoxia, maternal stress and toxin exposure. Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that bioaccumulate in the food chain resulting in dietary exposure in humans. Maternal and cord blood PCB levels are inversely associated with birth weight, and recent studies indicate that perinatal exposures to PCBs contribute to gender-specific obesity development in children. PCBs have also been shown to enter breast milk resulting in direct exposure in early postnatal life. Therefore, we hypothesized that perinatal PCB exposure cause developmental blight resulting in decreased birth weight and increased adiposity and glucose intolerance with aging. We found that mice perinatally exposed to PCBs did not differ in birth weight, but exhibited sex-specific effects on adiposity. Females perinatally exposed to PCBs were significantly more obese at 7 weeks of age while male offspring exhibited no difference in fat mass but had decreased lean mass compared to controls. With aging, the differences in females dissipated while the male offspring decreased lean mass persisted. Male offspring perinatally exposed to PCBs displayed impaired glucose tolerance at 7 weeks of age but normalized over time, while the females were glucose intolerant only after 6 months of age. This impairment of glucose tolerance was not attributed to insulin resistance. These data illustrate time-dependent and sex-specific perturbations of maternal PCB exposure on offspring body composition and glucose homeostasis. As the liver is a major facilitator in glucose homeostasis and xenobiotic detoxification, we investigated PCB-induced alterations in hepatic gene expression and found attenuated expression of glycolytic genes and increased expression of detoxifying and antioxidant genes in both PCB-exposed maternal and offspring livers. Taken together, these data demonstrate a role for perinatal pollutant exposure in the etiology of glucose intolerance. Further studies are required to elucidate the mechanisms causing sex-specific modulation of body composition and glucose intolerance.

White adipose plays a major role in the regulation of whole body metabolism through the storage and hydrolysis of triglycerides and by secretion of adipokines. The function of endocrine cells is highly dependent on the unfolded protein response (UPR), a homeostatic signaling mechanism that balances the protein folding capacity of the endoplasmic reticulum (ER) with the cell's secretory protein load. Here we demonstrate that the adipocyte UPR pathway is necessary for its secretory functions, and can thus play a crucial role in the control of whole body energy homeostasis. ER protein folding capacity is dependent both on the number of available chaperones as well as on their activity, which requires a sufficient ATP supply. In 3T3-L1 adipocytes, mitochondrial biogenesis occurred in parallel with induction of the UPR; therefore, we tested whether it was necessary for efficient ER function. Inhibition of mitochondrial ATP synthesis through depletion of Tfam, a mitochondrial transcription factor, or treatment with inhibitors of oxidative phosphorylation, demonstrate that ER function is sensitive to acute changes in adenine nucleotide levels. In addition, adenylate kinase 2 (AK2), which regulates mitochondrial adenine nucleotide interconversion, is markedly induced during adipocyte and B cell differentiation. AK2 depletion impairs induction of the UPR and secretion in both cell types. Interestingly, cytosolic adenylate kinase 1 (AK1) does not have the same effect upon UPR induction. We show that adenine nucleotides promote proper ER function and alterations in specific aspects of ATP synthesis can impair UPR signaling. Understanding the complex energetic regulation of the UPR may provide insight into the relationship between UPR and disease.

In the mammalian genome, 5‟-CpG-3‟ dinucleotides are frequently methylated, correlating with transcriptional silencing. Genome-wide waves of demethylation are thought to occur only twice during development, in primordial germ cells and in the pre-implantation embryo. They are followed by de novo methylation, setting up a pattern that is inherited throughout development. No global methylation changes are thought to occur during further somatic development, although methylation does alter at gene-specific loci, contributing to tissue-specific patterns of gene expression. Here we studied DNA methylation in differentiating mouse erythroblasts in vivo using several approaches including genomic-scale, reduced representation bisulfite sequencing (RRBS). Surprisingly, demethylation at the erythroid-specific β-globin locus was coincident with a wave of global DNA demethylation at most genomic elements, including repetitive elements and genes silenced in erythropoiesis. Over 30% of total methylation is irreversibly lost during erythroid differentiation. Demethylation occurred through a passive mechanism, requiring the rapid DNA replication triggered with the onset of erythroid terminal differentiation. Global loss of DNA methylation was not associated with a global increase in transcription, as determined by GeneChip analysis. We propose that global demethylation is a consequence of cellular mechanisms required for the rapid demethylation and induction of β-globin and other erythroid genes. Our findings demonstrate that, contrary to previously held dogma, DNA demethylation can occur globally during somatic cell differentiation, providing a new experimental model for the study of global demethylation in development and disease.

The regulation of glucagon-like peptide-1 and peptide YY hormone levels are regulated based on different influential factors, but primarily levels are dependent upon ingested food content. As meals today become more fat-enriched, there is greater requirement for evaluation of these hormones that regulate insulin and satiety levels within the body. We have shown that the gene expression transcript production of glucagon-like peptide-1 and peptide YY are modulated by different concentrations, and times of short-chain fatty acids and long-chain fatty acids. Although the peptide hormone levels have the influential physiological role on effector tissue, the regulation of these hormones begins at the transcript levels. Recent research indicates that glucagon-like peptide-1 and peptide YY hormones are altered in response to different free-fatty acids. The present investigation generally demonstrated an overall decrease in both hormones after chronic exposure to fatty acids. Intestinal secretin tumor cell line (STC-1 cells) was used as a representative for intestinal L-cells. Quantitative real-time PCR analysis was used to determine the changes in RNA transcripts. Overall, there was a decrease in the 3-hour timeline, which continued to decrease in the 16-hour and 24-hour timelines for glucagon-like peptide-1. Peptide YY transcript expression in 3-hours increased significantly after exposure to propionate, a significant decrease after exposure to acetate, and no significant increase or decrease after exposure to butyrate. However, there was a significant decrease in peptide YY once reaching 24-hour exposure. It was determined there is a threshold for different concentrations of free-fatty acids to influence glucagon-like peptide-1 and peptide YY production, which was present in the different concentrations of butyrate. Lastly, exposure to both concentrations of linolenic acid caused a significant decrease in glucagon-like peptide-1 and peptide YY.

Metabolic dysfunction in skeletal muscle is a major contributor to the development of type 2 diabetes. Endurance exercise training has long been established as an effective means to directly restore skeletal muscle glucose and lipid uptake and metabolism. However, in addition to the direct effects of skeletal muscle on glucose and lipids, there is renewed interest in the ability of skeletal muscle to coordinate metabolic activity of other tissues, such as adipose tissue and liver. The purpose of this study was to examine the effects of endurance exercise on the expression level of two novel muscle-derived secreted factors, or myokines, Myonectin and Fibronectin type III domain containing 5 (FNDC5), the precursor for Irisin. Methods. We performed immunoblot analysis and quantitative real-time PCR analysis of Myonectin and FNDC5 in the diaphragm muscles of obese Zucker rat (OZR) and lean Zucker rat (LZR) with 9 weeks of aerobic training on a motorized treadmill. Results. We show that myonectin gene expression is increased in the OZR model of obesity and decreases with exercise in both lean and obese Zucker rats. Conversely, myonectin protein concentration was elevated with exercise. Similarly, FNDC5 mRNA levels are significantly higher in the OZR, however exercise training had no effect on the expression level of FNDC5 in either the LZR or OZR. We did not observe any difference in muscle protein content of Irisin with obesity or exercise. Conclusion. Our data shows that exercise training does not increase either FNDC5 or myonectin gene expression, indicating that increased transcriptional regulation of these myokines is not induced by exercise. However, our data also indicates a yet to be explored disconnect between myonectin gene expression and protein content. Further, this report highlights the importance of verifying reference genes when completing gene expression analysis. We found that many commonly used reference genes varied significantly by obesity and/or exercise and would have skewed the results of this study if used to normalize gene expression data. The unstable reference genes include: beta-Actin, beta-2-microglobulin, Non-POU domain containing, octamer-binding, Peptidylprolyl isomerase H, 18S ribosomal RNA, TATA box binding protein and Transferrin receptor.

The purpose of this investigation was to examine selected biochemical mechanisms known to influence contractility and energy metabolism in the myocardium, with particular emphasis placed on the regulatory role of protein phosphorylation in the ventricular myocardium. The investigation was conducted in three phases; initially the cardiac contraction cycle was examined to determine whether reported fluctuations in myocardial cAMP levels were associated with other biochemical events known to be cAMP-dependent. The second phase involved the determination of specific kinase activities and endogenous substrates in a highly purified cardiac sarcolemmal preparation. In the final phase, ventricular myocytes were utilized to examine the ability of adenosinergic and muscarinic agonists to influence the isoproterenol-induced increases in protein phosphorylation. Studies in the first phase examined cyclic AMP levels and selected kinase activities in hearts frozen at various stages of the cardiac cycle. An automated clamping device, capable of freezing a perfused rat heart in less than 50 msec, was utilized to separate the cardiac cycle into various phases. Three different timing schemes were employed to divide the cycle into 2 to 4 segments. These different timing schemes revealed no significant differences in cAMP during the cardiac cycle. Myocardial cAMP values ranged from 2.5 to 4.1 pmol/min/mg protein in all phases. However, in one scheme there was a tendency for cAMP to be elevated in early systole, with minimal values occurring diastole. There were also no significant differences seen for either glycogen phosphorylase or cAMP-dependent protein kinase (PKA) activity between various phases of the cardiac cycle. Since no significant fluctuations were observed in the levels of cAMP or the activities of PKA or glycogen phosphorylase during a single cardiac contraction cycle, it would appear that these agents do not exert their effects on cardiac function on a beat to beat basis. The second phase of study examined the nature and function of individual protein kinases in the myocardium. Using a highly purified cardiac sarcolemmal preparation, kinase specific, synthetic substrates were employed to quantify the activities of cAMP-dependent (PKA), calcium/calmodulin-dependent (PKCM), calcium/phospholipid-dependent (PKC) and cGMP-dependent (PKG) protein kinases. Additionally, endogenous protein substrates were examined in this preparation to provide possible insight as to the function of these kinases in the heart. The activities of PKA, PKG, PKCM, and PKC in nmol 32P/min/μg protein were as follows: PKA, 1606; PKG, 35.7; PKCM, 353; and PKC, 13.2. Three endogenous protein substrates of apparent molecular weights of 15kD, 28kD and 92kD were phosphorylated. While no endogenous protein phosphorylation was detectable as a result of cG-PK activity, all of the substrates were phosphorylated, to varying degrees, by both PKA and CACM-PK. PKC phosphorylated only the 15kD substrate. Even though several endogenous kinases are evident in the sarcolemmal preparation, cAMP-dependent protein kinase demonstrates the greatest degree of activity. This kinase also appeared to be the most abundant; however, there is some concern as to the source of these kinases in the membrane preparation since endothelial membranes as well as cardiac membranes appeared to be present. Evidence for endothelial contamination was provided by the finding that the membrane preparation contained appreciable amounts of angiotensin converting enzyme (ACE) activity, an enzyme felt to reside in the vascular endothelium. Since studies with this preparation could not exclude contribution of nonmuscle cell membranes a model consisting solely of dispersed ventricular myocytes was developed. The third phase of these studies examined protein phosphorylation in primary cultures of ventricular myocytes. Specifically, these studies examined protein phosphorylation induced by exposure to isoproterenol (ISO), a catecholamine known to effect changes in the phosphorylation state of proteins in the heart by means of a β-adrenergic-mediated/cAMP-dependent mechanism was examined. Additionally, the effects of phenylisopropy-ladenosine (PIA) and carbamyl choline chloride (CARB) were examined with regard to their anti-adrenergic role(s) in this process. Adherent, collagenase-dispersed, radiolabelled (32p) ventricular myocytes exposed to ISO demonstrated a dose and time dependent increase in 32p incorporation into several endogenous protein substrates. When the myocytes were exposed (60 sec) to either PIA or CARB prior to the exposure to ISO, ISO-induced 32p incorporation into protein substrates of apparent molecular weight of 6kD, 31kD and 155kD was reduced up to 67% when compared to the effects of ISO alone. Additionally, both PIA and CARB attenuated the ISO-induced increase in PKA activity in the myocyte, yet only CARB was seen to produce an inhibitory effect on the ISO-induced increase in cAMP levels in the myocytes. The effects of CARB were dose-dependent and inhibited the effects of ISO on 32p incorporation at all doses tested. PIA elicited biphasic effects: lower PIA concentrations were inhibitory in nature, while higher concentrations of PIA appeared to potentiate the increase in 32p incorporation induced by ISO. Based on electrophoretic mobilities (SDS/PAGE) of the 6kD and the 155kD substrates, these substrates have been tentatively identified as the monomeric form of the sarcoplasmic reticulum-associated protein, phospholamban, and the contractile filament-associated protein, C protein, respectively. The 31kD substrate has been identified, by means of immunoblot, as the contractile filament-associated protein, troponin I. The role of protein phosphorylation in the myocardium involves complex, inter-related mechanisms that encompass extracellular, transmembranal and cytoplasmic elements in the heart. It is well understood that certain mechanisms of the contraction cycle known to vary on a beat to beat basis, such as myosin ATPase, involve changes in protein phosphorylation. However, the nature of the various kinases and substrates examined in this study appear to influence longer-term events of myocardial contractility. Mechanisms coupled with hormone action, modulation of second messenger-dependent components, and factors associated with changes in contractility seen with aging and disease are more likely to exhibit changes similar to those described herein. A better understanding of the underlying biochemistry may provide greater insight into the importance of these metabolic changes.

Design and analysis of conceptually different cooling systems for the human heart preservation are numerically investigated. A heart cooling container with required connections was designed for a normal size human heart. A three-dimensional, high resolution human heart geometric model obtained from CT-angio data was used for simulations. Nine different cooling designs are introduced in this research. The first cooling design (Case 1) used a cooling gelatin only outside of the heart. In the second cooling design (Case 2), the internal parts of the heart were cooled via pumping a cooling liquid inside both the heart’s pulmonary and systemic circulation systems. An unsteady conjugate heat transfer analysis is performed to simulate the temperature field variations within the heart during the cooling process. Case 3 simulated the currently used cooling method in which the coolant is stagnant. Case 4 was a combination of Case 1 and Case 2. A linear thermoelasticity analysis was performed to assess the stresses applied on the heart during the cooling process. In Cases 5 through 9, the coolant solution was used for both internal and external cooling. For external circulation in Case 5 and Case 6, two inlets and two outlets were designed on the walls of the cooling container. Case 5 used laminar flows for coolant circulations inside and outside of the heart. Effects of turbulent flow on cooling of the heart were studied in Case 6. In Case 7, an additional inlet was designed on the cooling container wall to create a jet impinging the hot region of the heart’s wall. Unsteady periodic inlet velocities were applied in Case 8 and Case 9. The average temperature of the heart in Case 5 was +5.0oC after 1500 s of cooling. Multi-objective constrained optimization was performed for Case 5. Inlet velocities for two internal and one external coolant circulations were the three design variables for optimization. Minimizing the average temperature of the heart, wall shear stress and total volumetric flow rates were the three objectives. The only constraint was to keep von Mises stress below the ultimate tensile stress of the heart’s tissue.